Markers of Cardiovascular Diseases and Metabolic Syndrome

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Markers of Cardiovascular Diseases and Metabolic Syndrome Troponin ProBNP, BNP and NT-proBNP Myoglobin Pregnancy Associated Plasma Protein A (PAPP-A) Myeloperoxidase (MPO) Soluble CD40 Ligand (sCD40L)

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MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

119

ready play a major role, but the demand for more effective and sensitive cardiac markers that can be used not only for the diagnosis but also for the prognosis of different cardiac pathologies is widely recognized across the entire clinical world.

Metabolic syndrome (MS) is a combination of medical disorders including increased blood pressure, abdominal obesity, abnormal cholesterol levels, insulin resistance and glucose intolerance. People with the metabolic syndrome are at an increased risk of developing cardiovascular diseases and type 2 diabetes. The dominant underlying risk factors for this syndrome appear to be abdominal obesity and insulin resistance. In cases of insulin resistance, the body cannot use insulin efficiently, hence the metabolic syndrome is also called the insulin resistance syndrome. Although its exact frequency is not known, the condition is widespread among the adult population in developed nations, and increases in frequency with age. It is also starting to appear in affluent adults from developing nations who have adopted Western diets and lifestyles, as well as afflicting an increasing number of children and adolescents as the worldwide epidemic of obesity spreads throughout the age groups.

For more than 15 years, HyTest specialists have been involved in the development and production of high quality products applicable for research and in vitro diagnostic activities (IVD) in the cardiovascular field.

And we can proudly say that HyTest is the leading producer of different reagents for cardiac diagnostics. Our reagents are widely used for the development of sensitive and precise immunoassays, while some of our cardiac protein preparations were selected as international standards. We are constantly widening our product range and encouraging further contacts with our customers.

Introduction Cardiovascular diseases (CVD) are the most lethal diseases known to the Western world and as the standard of living continues to grow CVD will , soon become the world s leading cause of mortality.

This means more demanding challenges are being placed on scientific communities to research and develop new and more specific diagnostic approaches to unstable angina, acute myocardial infarction (AMI) and other life threatening cardiovascular pathologies. In this field, different cardiac markers al-

One feature clearly distinguishes HyTest products from similar products developed by other companies. The majority of antibodies described in this catalogue were tested not only with purified antigen, but also with the antigen that could be found in human blood, urine and different tissues. For the development of sandwich immunoassays, we are recommending selected pairs of antibodies. Our task is to minimize customers’ efforts and time spent in the development of immunoassays. Therefore, we deliver products that can be used by our customers without the need for additional research work.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

3

Table of Contents

4

I PROTEINS OF TROPONIN COMPLEX 1. Troponin I 1.1. Human cardiac troponin I (cTnI) 1.2. Human cardiac troponin complex (I-T-C) 1.3. Artificial I-T-C and I-C complexes 1.4. Troponin I Diversity kit 1.5. Troponin I Calibrator set 1.6. Cardiac troponin I free serum 1.7. Human skeletal troponin I (skTnI) 2. Troponin T 2.1. Human cardiac troponin T (cTnT) 2.2. Human skeletal troponin T (skTnT) 3. Troponin C 3.1. Human cardiac troponin C (cTnC) 4. Troponin antigens from different animal species

7 7 7 8 10 10 10 11 11 11 11 12 12 12 13

II TROPONIN-SPECIFIC ANTIBODIES 1. Anti-cTnI monoclonal antibodies 1.1. Applications 1.1.1. High sensitivity cTnl assay concept 1.1.2. cTnl quantitative sandwich immunoassay 1.1.3. Immunoassays for the detection of cTnI from different animal species 1.1.4. Detection of binary cTnI-TnC complex 1.1.5. Detection of binary cTnI-cTnT complex 1.1.6. Detection of dephosphorylated cTnI 1.1.7. Detection of cTnI or cTnI fragments by Western blotting 2. Anti-cTnT monoclonal antibodies 2.1. Detection of cTnT from different animal species 3. Anti-TnC monoclonal antibodies 4. MAbs specific to human cardiac troponin complex 4.1. Applications 4.1.1. cTnl quantitative sandwich immunoassay

14 14 15 15 19 20 20 20 21 21 22 23 24 24 24 24

III HUMAN proBNP AND proBNP-DERIVED PEPTIDES (BNP AND NT-proBNP) 1. Human recombinant proBNP and NT-proBNP 1.1. Human recombinant (not glycosylated) proBNP and NT-proBNP expressed in E. coli 1.2. Glycosylated human recombinant proBNP 1.2.1. Glycosylated proBNP as a stable standard for BNP and proBNP immunoassays 2. Anti-BNP monoclonal antibodies 2.1. Applications 2.1.1. BNP/proBNP sandwich immunoassay 2.1.2. BNP and proBNP immunodetection in Western blotting 2.1.3. proBNP sandwich immunoassay 3. Antibodies for New Type of BNP immunoassay – “Single Epitope Sandwich” assay (SES) New data!

4. Anti-NT-proBNP monoclonal antibodies 4.1. Applications 4.1.1. Direct ELISA 4.1.2. NT-proBNP quantitative sandwich immunoassays 4.1.3. Stability studies of endogenous NT-prpBNP 4.1.4. NT-proBNP and proBNP immunodetection in Western blotting 5. ProBNP quantitative sandwich immunoassays 6. BNP and NT-proBNP free plasma

27 29 29 30

IV MYOGLOBIN 1. Human myoglobin 2. Anti-myoglobin monoclonal antibodies 3. Myoglobin free serum

45 45 46 46

V PREGNANCY ASSOCIATED PLASMA PROTEIN A (PAPP-A) 1. Heterotetrameric PAPP-A/proMBP complex (htPAPP-A) 2. htPAPP-A and total PAPP-A specific monoclonal antibodies 2.1. Applications 2.1.1. Total PAPP-A and htPAPP-A sandwich immunoassays 2.1.2. PAPP-A immunodetection in Western blotting 3. Recombinant homodimeric PAPP-A (dPAPPA-A)

47 48 48 49 49 49 50

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

31 32 32 32 34 34 35 38 38 38 39 41 41 43 44

Table of Contents 4. Anti-dPAPP-A monoclonal antibodies 4.1. Applications 4.1.1. Selective dPAPP-A sandwich immunoassay New data!

4.1.2. Selective dPAPP-A immunodetection in Western blotting New data!

51 51 51 53

VI MYELOPEROXIDASE (MPO) 1. Human myeloperoxidase 2. Anti-MPO monoclonal antibodies 2.1. Applications 2.1.1. Sandwich assay for quantitative MPO immunodetection 2.1.2. MPO immunodetection in Western blotting 3. MPO free serum

54 54 55 55 55 56 57

VII SOLUBLE CD40 LIGAND (sCD40L) 1. Anti-sCD40L monoclonal antibodies 1.1. Applications 1.1.1. sCD40L immunodetection in Western blotting 1.1.2. Sandwich immunoassay for quantitative sCD40L immunodetection

58 58 59 59 59

VIII HUMAN CYSTATIN C 1. Human cystatin C antigens 1.1. Human recombinant cystatin C 1.2. Cystatin C purified from human blood NEW! 2. Anti-cystatin C monoclonal antibodies 2.1. Applications 2.1.1. Cystatin C quantitative sandwich immunoassays 2.1.2. Cystatin C immunodetection in Western blotting 2.1.3. Cross-reaction with sera from different animal species New data!

3. Polyclonal anti-cystatin C antibodies NEW! 4. Cystatin C free serum

61 62 62 62 63 64 64 65 65 66 66

IX HUMAN SERUM ALBUMIN (HSA) 1. Anti-human serum albumin monoclonal antibodies 1.1. Applications 1.1.1. Anti-albumin MAbs for the development of sandwich immunoassay 1.1.2. Albumin immunodetection in Western blotting 1.1.3. MAbs for albumin extraction from human serum (plasma)

67 67 68 68 68 68

X UROTENSIN II 1. Anti-urotensin II monoclonal antibodies 1.1. Applications 1.1.1. Urotensin II competitive immunoassay

69 69 70 70

XI PROCALCITONIN (PCT) 1. Anti-procalcitonin monoclonal antibodies 2. Anti-calcitonin monoclonal antibodies 3. Anti-katacalcin monoclonal antibodies 4. Anti-N-terminal PCT monoclonal antibodies

71 71 73 73 74

XII D-DIMER AND HIGH MOLECULAR WEIGHT FIBRIN DEGRADATION PRODUCTS 1. Human D-dimer 2. Anti-D-dimer monoclonal antibodies 2.1. Antibodies recognizing D-dimer cross-linked region 2.2. Antibodies specific to D-dimer and to high molecular weight fibrin degradation products 2.3. Antibodies recognizing epitopes common for D-dimer, fibrinogen and D-monomer 2.4. Applications 2.4.1. One-step D-dimer immunoassay 2.4.2. Two-step D-dimer immonoassay 2.4.3. Detection of D-dimer in Western blotting New data!

76 76 77 77

XIII THROMBIN ACTIVATABLE FIBRINOLYSIS INHIBITOR (TAFI) 1. Human TAFI 2. Anti-TAFI monoclonal antibodies

80 80 81

XIV FIBRINOGEN 1. Anti-fibrinogen monoclonal antibodies

82 82

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

77 77 78 78 78 78

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Table of Contents XV FIBRINOPEPTIDE A 1. Synthetic fibrinopeptide A 2. Anti-fibrinopeptide A monoclonal antibodies

84 84 84

XVI FATTY ACID BINDING PROTEIN (FABP) 1. Human FABP 2. Anti-FABP monoclonal antibodies 3. FABP free serum

85 85 85 86

XVII GLYCOGEN PHOSPHORYLASE, BB ISOENZYME (GPBB) 1. Human GPBB 2. Anti-GPBB monoclonal antibodies

87 87 87

XVIII SERUM AMYLOID A (SAA) 1. Anti-SAA monoclonal antibodies 1.1. Applications 1.1.1. Sandwich assay for quantitative SAA immunodetection and Western blotting

89 89 90 90

XIX HIGH SENSITIVITY HUMAN C-REACTIVE PROTEIN (hsCRP) 1. Human CRP 2. Anti-CRP monoclonal antibodies 2.1. Applications 2.1.1. Direct ELISA 2.1.2. CRP immunodetection in Western blotting 2.1.3. High sensitivity CRP sandwich immunoassay 2.1.4. Affinity information 3. CRP free serum

91 92 92 92 92 93 94 95 96

XX LECTIN-LIKE OXIDIZED LDL RECEPTOR 1 (LOX-1) 1. Recombinant antigens 1.1. LOX-1, extracellular domain (not glycosylated) 1.2. LOX-1, extracellular domain (glycosylated) 2. Anti-sLOX-1 monoclonal antibodies

97 97 97 97 98

XXI RETINOL-BINDING PROTEIN 4 (RBP4) 1. Purified endogenous RBP4 2. Anti-human RBP4 monoclonal antibodies 2.1. Applications 2.1.1. Sandwich immunoassay for RBP4 detection in human plasma 2.1.2. Immunoprecipitation 2.1.3. Western blotting

99 100 101 101 101 102 102

XXII HUMAN ADIPONECTIN 103 1. Native purified adiponecting antigen NEW! 104 2. Anti-human adiponectin monoclonal antibodies 105 2.1. Applications 105 105 2.1.1. Adiponectin sandwich immunoassay New data!

2.1.2. Assays detecting total, HMW or LMW forms of human adiponectin New data! 107 2.1.3. Western blotting 107

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XXIII LEPTIN 1. Anti-leptin monoclonal antibodies

109 109

XXIV PROINSULIN, INSULIN AND C-PEPTIDE

110

XXV BRAIN S-100 PROTEIN 1. Human brain S-100 protein 2. Anti-S-100 monoclonal antibodies

112 112 113

XXVI SPECIAL PRODUCTS 1. Immunosorbents 2. Labeled Antibodies 3. Fab and F(ab)2 Fragments of Monoclonal Antibodies

114 114 114 114

XXVII ABBREVIATIONS USED IN THE CATALOG

115

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

I Proteins of Troponin Complex Troponin complex is a heteromeric protein that plays an important role in the regulation of skeletal and cardiac muscle contraction. Troponin complex consists of three different subunits – troponin T (TnT), troponin I (TnI) and troponin C (TnC) and each of those subunits is responsible for a part of the troponin complex function. TnT is a tropomyosin-binding subunit that regulates the interaction of the troponin complex with thin filaments; TnI inhibits ATP-ase activity of acto-myosin; TnC is a Ca 2+– binding subunit, which plays the predominant role in Ca 2+ dependent regulation of muscle contraction (1).

TnT and TnI in cardiac muscle are presented by forms that are different from those in skeletal muscles. Two isoforms of TnI and two isoforms of TnT are expressed in human skeletal muscle tissue (skTnI and skTnT). Only one tissue-specific isoform of TnI is described for cardiac muscle tissue (cTnI), whereas the existence of several cardiac specific isoforms of TnT (cTnT) are described in the literature. No cardiac specific isoform is known for human TnC, and TnC in human cardiac muscle tissue is presented by an isoform typical for slow skeletal muscle. Another form of

TnC, fast skeletal TnC isoform, is more typical for fast skeletal muscles (2). cTnI is expressed only in myocardium and there are no examples known of cTnI expression in healthy or injured skeletal muscle or in other tissue types. cTnT is probably less cardiac specific and expression of cTnT in skeletal tissue of patients with chronic skeletal muscle injuries has been described (3).

Firstly cTnI (4) and latterly cTnT (5) were used as markers of cardiac cell death. Both proteins are now widely used for the diagnosis of acute myocardial infarction (AMI), unstable angina, post-surgery myocardium trauma, as well as some other diseases related to cardiac muscle injury.

Both markers can be detected in the patient’s blood 3-6 hours after the onset of the chest pain, reaching the peak level within 16–30 hours. Elevated concentration of cTnI and cTnT in blood samples can be detected even 5–8 days following the onset of the symptoms, in turn meaning that both proteins are also useful for the late diagnosis of AMI (6).

1. Troponin I 1.1. Human cardiac troponin I (cTnI) Source: Purity: Presentation: Storage: Applications: Remarks:

Human cardiac muscle tissue. Blood sample from the tissue donors was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis >98% Lyophilized from 0.01 M HCl At -70°C in lyophilized form. After reconstitution into the recommended buffer the solution should be stored at -70 °C Mass cTnI standard, immunogen for antibody production, cTnI biochemical and immunochemical studies cTnI has limited solubility in buffers with physiological salt concentration and neutral pH It is recommended to reconstitute the product in urea/Tris buffer, pH 7.5 (7 M urea, 5 mM EDTA, 15 mM mercaptoethanol, 20 mM Tris) to around 1 mg/ml concentration

Human troponin I is presented in cardiac muscle tissue by a single isoform with a molecular weight of 23876 Da and it consists of 209 amino acid residues.

The theoretical pI of cTnI is 9.87. cTnI molecule contains two serines in the 22 and 23 positions. Both amino acid residues can be phosphorylated in vivo by protein kinase A, meaning that four

forms of protein – one dephospho, two monophospho and one bisphospho – can coexist in the cell.

Phosphorylation of cTnI changes the conformation of the protein and modifies its interaction with other troponins, as well as the interaction with anti-TnI antibodies. According to the latest findings, a significant part of cTnI released into the patient’s bloodstream is phosphorylated (7).

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

7

For over 15 years, cTnI has been considered a reliable marker of cardiac muscle tissue injury. It is deemed to be more sensitive and significantly more specific in the diagnosis of the myocardial infarction than the “golden marker” of the last few decades – CK-MB, as well as myoglobin and LDH isoenzymes.

HyTest’s cTnI is purified from human cardiac muscle tissue by the immunoaffinity method followed by additional ion-exchange chromatography. Preparation contains some amount (<5%) of cTnI proteolytical fragments retaining troponin’s immunological activity. According to immunological and mass spectral studies, cTnI is acetylated (from N-terminal part of

the molecule) and partially mono- and biphosphorylated. cTnI isolated from the cardiac muscle tissue and containing posttranslational modifications represents a more natural form the of the protein when compared to highly purified recombinant cTnI. On SDS-PAGE, cTnI is presented by a single band with an apparent molecular weight of 29 kDa (Fig. 1, lane 3).

Completely phosphorylated (biphosphorylated by protein kinase A) and partly phosphorylated (dephosphorylated by alkaline phosphatase) cTnI can be prepared at the request of the customer.

Ordering information:

Product

Cat. #

Purity

Source

Human cardiac TnI Human cardiac TnI (phosphorylated) Human cardiac TnI (dephosphorylated)

8T53 8T53ph 8T53dp

>98% >95% >95%

Human cardiac muscle Human cardiac muscle Human cardiac muscle

1.2. Human cardiac troponin complex (I-T-C) Source: Composition: Presentation: Storage: Application: Remarks:

Human cardiac muscle tissue. Blood sample from the tissue donors was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis Ternary heterocomplex consisting of cTnI, cTnT and TnC Frozen solution in 150 mM NaCl, 5 mM CaCl2 , 20 mM Tris pH 7.5 At -70°C. Avoid repeated freezing and thawing. It is recommended to aliquot the product after the first thawing Stabilized form of natural cTnI, best for the calibration of the assays and standard preparation, immunogen for antibody production, troponin biochemical and immunochemical studies Purification was performed in mild conditions. Troponin complex is native and corresponds to the presentation of troponin complex in serum of AMI patients For better stability it is recommended to prepare stock solutions of troponin complex in normal human serum with final concentration of cardiac troponin I not less than 1000 ng/ml

Inside the cardiac troponin complex the strongest interaction between molecules has been demonstrated for cTnI – TnC binary complex, particularly in the presence of Ca 2+ (Ka = 1.5x10 -8 M-1) (8). This interaction is very important for cTnI immunodetection and should be taken into consideration by assay manufacturers. TnC, forming a complex with cTnI, changes the conformation of cTnI molecule and shields part of its surface, therefore affecting the interaction of some anti-cTnI antibodies with the antigen. Hence, the immunological properties of cTnI in troponin complex are considerably different to the

8

properties of the free protein. Consequently, some MAbs recognizing free (isolated) cTnI do not interact with cTnI in cTnI-TnC binary or cTnI-cTnT-TnC ternary complexes and vice versa. According to the latest data, cTnI is released in the bloodstream of the patient in the form of a binary complex with TnC or ternary complex with cTnT and TnC (9). cTnI-TnC complex formation plays an important, positive role in improving the stability of the cTnI molecule. cTnI, which is extremely unstable in its free form, and demonstrates significantly better stability

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

in complex with TnC or in ternary cTnI-cTnT-TnC complex. These two forms of the protein are preferable as material for standard and calibrator preparation.

In the troponin complex supplied by HyTest, cTnI is presented in the same form as it can be detected in the blood of AMI patients. Purification of the troponin complex is performed in mild conditions without treatment with urea containing buffers (as it usually is done when preparing individual troponin components). The concentration is precisely determined for

each of the three components of the complex (Table 1). It was demonstrated that stability of cTnI in native complex is significantly better than the stability of the purified form of the protein or the stability of cTnI in artificial troponin complexes combined from purified proteins (Fig. 2).

On SDS-PAGE, troponin complex is presented by three main bands – cTnT, cTnI and TnC – with corresponding apparent molecular weights of 39, 29 and 18 kDa (Fig. 1, lanes 1 and 5).

Table 1. Human cardiac troponin complex Lot 03/01 (example).

Concentration (mg/ml)

Molar ratio

cTnI cTnC cTnT

1.00 0.91 0.88

1.00 1.18 0.61

% of Tn I immunological activity

Troponin subunit

120 100 80 60 40 20 0

0

2

4

6

8

10

12

14

16

Incubation time (days) Figure 1. SDS gel electrophoresis of human cardiac troponin complex under reducing conditions. Lanes 1 and 5: cardiac troponin complex, lane 2: TnC, lane 3: cTnI and lane 4: cTnT.

Figure 2. Comparison of the stability of different forms of cTnI dissolved in normal human serum and incubated for different time periods at +4°C. Native troponin complex (V), artificial troponin complex combined from individual cTnI, cTnT and TnC (O ), purified cTnI (Q).

Advantages of native troponin complex over purified cTnI or artificial complex include: - antigen is in the same form as in AMI blood samples t VODIBOHFE UFSUJBSZ TUSVDUVSF t VODIBOHFE BOUJCPEZ CJOEJOH TJUFT t CFTU UP VTF GPS DBMJCSBUPST BOE TUBOEBSE preparation - high cTnI stability

HyTest’s Troponin I-T-C Complex has been chosen by the AACC cTnI Standardization Subcommittee for international reference material. The standard reference material (SRM® 2921) is available from the National Institute of Standards and Technology. Additional information can be found at www.nist.gov.

HyTest holds patents for ‘Method and kit for the diagnosis of troponin I’ (US7285418 and EP0938678).

Ordering information: Product

Cat. #

Purity

Source

Human cardiac troponin complex (I-T-C)

8T62

N/A

Human cardiac muscle

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

9

1.3. Artificial I-T-C and I-C complexes Source: Composition: Presentation: Storage: Application:

Human cardiac muscle tissue. Blood sample from the tissue donors was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV, and syphilis Ternary or binary heterocomplex consisting of cTnI, cTnC and TnT (in the case of ternary complex) Frozen solution in 50 mM Tris-HCl, pH 7.5, 0.15 M KCl, 5 mM CaCl2, 15 mM B-mercaptoethanol, pH 7.5 -70°C, avoid repeated freezing and thawing. It is recommended to aliquot the product after the first thawing Stabilized form of natural cTnI, best for the calibration of the assays and standard preparation, immunogen for antibody production, troponin biochemical and immunochemical studies, for better stability it is recommended to prepare stock solutions of troponin complex in normal human serum with final concentration of cardiac troponin I not less than 1 μg/ml

Artificial ternary (I-T-C) and binary (I-C) complexes that are assembled from highly purified proteins can be prepared at the request of the customer.

Ordering information: Product

Cat. #

Purity

Source

Artificial I-T-C complex Artificial I-C complex

8T62a 8IC63

N/A N/A

Highly purified proteins Highly purified proteins

1.4. Troponin I Diversity kit Source: Presentation: Storage:

Human cardiac muscle tissue. Blood sample from the tissue donors was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV, and syphilis Lyophilized from solution in pooled normal human serum (Scantibodies) -70°C, avoid repeated freezing and thawing after reconstitution

Multiple cardiac troponin I (cTnI) forms can be detected in human blood. Such posttranslational modifications as phosphorylation, proteolysis and complex formation with troponin C (TnC) were described in literature for cTnI molecule (10). The antibodies used for the assay development should recognize different cTnI forms presented in the blood with the same efficiency.

HyTest cTnI Diversity kit includes four pairs of antigen preparations (normal/modified) to be used in the

testing of assay susceptibility to different cTnI modifications - complex formation, phosphorylation, proteolysis, and presence of heparin in the sample. The kit includes: 1. Free and complexed (in vitro) forms of cTnI 2. In vitro partially phosphorylated and dephosphorylated Tn complex 3. Native troponin complex before and after partial proteolytic degradation 4. cTnl without heparin and cTnl in complex with heparin

Ordering information: Product

Cat. #

Purity

Remarks

Troponin I Diversity kit

K01

N/A

Different forms of human cTnI

1.5. Troponin I Calibrator set Source: Presentation: Storage:

Human cardiac muscle tissue. Blood sample from the tissue donors was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV, and syphilis Lyophilized from solution in pooled normal human serum (Scantibodies) -70°C, avoid repeated freezing and thawing after reconstitution

cTnI calibrators are based on an isolated native troponin complex reconstituted in normal human serum. The calibrator set covers a concentration range

from 0 to 100 ng/ml. cTnI calibrators with different antigen concentrations can be prepared at the request of the customer.

Ordering information:

10

Product

Cat. #

Purity

Remarks

Troponin I Calibrator set

8T60

N/A

Troponin complex in normal human serum

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

1.6. Cardiac troponin I free serum Prepared from: Method of purification: Delivery form: Storage:

Pooled normal human serum. Blood samples from donors were tested and found to be negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis Immunoaffinity chromatography Frozen liquid -20°C

cTnI free serum is prepared from pooled normal human serum by immunoaffinity chromatography. The affinity sorbent utilizes several MAbs with different epitope specificity to eliminate not only intact cTnI molecule from serum, but also its proteolytical frag-

ments. Based on ELISA testing, cTnI level in cTnI free serum is not higher than 0.03 ng/ml. cTnI free serum could be used as a matrix for standard and calibrator preparations.

Ordering information: Product

Cat. #

Source

cTnI free serum

8TFS

Pooled normal human serum

1.7. Human skeletal troponin I (skTnI) Source: Purity: Composition: Presentation: Application: Remarks: Storage:

Human skeletal muscle. Blood sample from the tissue donor was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis >95% Slow and fast skTnI Lyophilized from 0.01 M HCl Negative control for cTnI specific antibodies, immunogen for antibody production, calibrator and standard preparation, troponin biochemical and immunochemical studies skTnI has limited solubility in buffers with physiological salt concentration and neutral pH, it is recommended to reconstitute the product in urea/Tris buffer, pH 7.5 (7 M urea, 5 mM EDTA, 15 mM mercaptoethanol, 20 mM Tris) -70°C

Antibodies utilized in human cardiac TnI assays should not have a cross-reaction with skeletal isoforms of troponin I. If cross-reaction occurs in cases of testing blood samples from patients with acute or chronic cardiac injuries, wrong positive results are possible. With this in mind, it is important to test the assay or antibodies used for assay development to be insensitive to the presence of skeletal TnI in the sample. HyTest’s skTnI is purified from human skeletal tissue by the immunoaffinity method followed by additional ion-exchange chromatography. On SDS-

PAGE, it is presented by two bands, fast and slow skeletal isoforms of the protein.

Skeletal isoforms of troponin I were suggested to be used as markers of acute and chronicle skeletal muscle injuries (11). In human skeletal muscles troponin I is presented by two forms; slow skeletal (186 amino acid residues, MW 21561, theoretical pI 9.61) and fast skeletal (181 amino acid residues, MW 21207, theoretical pI 8.88).

Ordering information: Product

Cat. #

Purity

Source

Human skeletal TnI

8T25

>95%

Human skeletal muscle

2. Troponin T 2.1. Human cardiac troponin T (cTnT) Source: Purity: Presentation: Application: Remarks: Storage:

Human cardiac muscle tissue. Blood sample from the tissue donor was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis >98% Lyophilized from 0.01 M HCl Immunogen for the antibody production, mass cTnT standard, troponin biochemical and immunochemical studies It is recommended to reconstitute the product in urea/Tris buffer, pH 7.5 (7 M urea, 5 mM EDTA, 15 mM mercaptoethanol, 20 mM Tris) -70°C. After reconstitution in recommended buffer the solution should be stored at -70°C

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

11

As well as cTnI, cardiac isoform of TnT is widely used as a marker of myocardial cell death. As a marker, , cTnT has the same kinetics of release into patients bloodstreams and the same sensitivity for minor myocardial events as cTnI, but it is considered to be less cardiac specific (12).

cTnT is purified from human cardiac muscle tissue by the immunoaffinity method followed by additional ion-exchange chromatography and it is presented by a single band on SDS-PAGE with an apparent molecular weight of 39 kDa (Fig. 1, lane 4).

Ordering information: Product

Cat. #

Purity

Source

Human cardiac TnT

8T13

>98%

Human cardiac muscle

2.2. Human skeletal troponin T (skTnT) Source: Purity: Composition: Presentation: Application: Storage:

Human skeletal muscle. Blood sample from the tissue donor was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis >95% Slow and fast skTnT Lyophilized from essentially salt-free distilled water Immunogen for antibody production, troponin biochemical and immunochemical studies -70°C, after reconstitution in recommended buffer the solution should be stored at -70°C

Two isoforms of TnT are expressed in human skeletal muscles. One form is typical for slow skeletal muscle (277 amino acid residues, MW 32817, theoretical pI 5.86), while the other is for fast skeletal muscle (257

amino acid residues, MW 30465, theoretical pI 6.07).

Isolated human skeletal troponin T consists of both isoforms and on SDS-PAGE it is presented by two bands.

Ordering information: Product

Cat. #

Purity

Source

Human skeletal TnT

8T24

>95%

Human skeletal muscle

3. Troponin C 3.1. Human cardiac troponin C (cTnC) Source: Purity: Presentation: Application: Remarks: Storage:

Human cardiac muscle tissue. Blood sample from the tissue donor was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis >98% Lyophilized from 0,01 M HCl Immunogen for antibody production, stabilizer of TnI in solutions, troponin biochemical and immunochemical studies It is recommended to reconstitute the product in 50 mM Tris-HCl, pH 7.5, 150 mM KCl, 5 mM CaCl2 -70°C, after reconstitution in recommended buffer the solution should be stored at -70°C

Two forms of troponin C (TnC) are expressed in human muscles. One is typical for slow skeletal muscles while the other is typical for fast skeletal muscles (2). In myocardium, TnC is presented by the slow skeletal isoform consisting of 161 amino acid residues, MW 18416 and theoretical pI 4.05.

TnC forms high affinity complexes with cTnI. It was demonstrated that cTnI is presented mainly as a complex with TnC in the blood stream of AMI patients

(9). In the binary cTnI-TnC complex TnC protects cTnI from protease cleavage. Therefore, TnC can be used as a natural stabilizer of cTnI in water solutions (13).

TnC is purified from human cardiac muscle tissue by the immunoaffinity method followed by additional ion-exchange chromatography. On SDS-PAGE, TnC is presented by a single band with an apparent molecular weight of 18 kDa (Fig. 1, lane 2).

Ordering information:

12

Product

Cat. #

Purity

Source

Human cardiac TnC

8T57

>98%

Human cardiac muscle

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

4. Troponin antigens from different animal species Troponin I and troponin T are sensitive and specific markers of myocardial tissue damage. Therefore, they are extensively used in animal preclinical safety tests of new drugs. For assay calibration HyTest

offers troponin complex and isolated troponins I, T, and C puriďŹ ed from canine, rat, bovine, mouse, rabbit and porcine cardiac or skeletal muscle tissues.

Ordering information: Product

Cat. #

Source

Cardiac troponin I Skeletal troponin I Cardiac troponin T Skeletal troponin T Troponin C Troponin complex Cardiac troponin I Skeletal troponin I Cardiac troponin T Skeletal troponin T Troponin C Troponin complex Cardiac troponin I Skeletal troponin I Cardiac troponin T Skeletal troponin T Troponin C Troponin complex Cardiac troponin I Skeletal troponin I Cardiac troponin T Skeletal troponin T Troponin C Troponin complex Cardiac troponin I Skeletal troponin I Cardiac troponin T Skeletal troponin T Troponin C Troponin complex Cardiac troponin I Skeletal troponin I Cardiac troponin T Skeletal troponin T Troponin C Troponin complex

8T53c 8T25c 8T13c 8T24c 8T57c 8T62c 8T53r 8T25r 8T13r 8T24r 8T57r 8T62r 8T53b 8T25b 8T13b 8T24b 8T57b 8T62b 8T53m 8T25m 8T13m 8T24m 8T57m 8T62m 8T53rb 8T25rb 8T13rb 8T24rb 8T57rb 8T62rb 8T53p 8T25p 8T13p 8T24p 8T57p 8T62p

Canine cardiac muscle Canine skeletal muscle Canine cardiac muscle Canine skeletal muscle Canine cardiac muscle Canine cardiac muscle Rat cardiac muscle Rat skeletal muscle Rat cardiac muscle Rat skeletal muscle Rat cardiac muscle Rat cardiac muscle Bovine cardiac muscle Bovine skeletal muscle Bovine cardiac muscle Bovine skeletal muscle Bovine cardiac muscle Bovine cardiac muscle Mouse cardiac muscle Mouse skeletal muscle Mouse cardiac muscle Mouse skeletal muscle Mouse cardiac muscle Mouse cardiac muscle Rabbit cardiac muscle Rabbit skeletal muscle Rabbit cardiac muscle Rabbit skeletal muscle Rabbit cardiac muscle Rabbit cardiac muscle Porcine cardiac muscle Porcine skeletal muscle Porcine cardiac muscle Porcine skeletal muscle Porcine cardiac muscle Porcine cardiac muscle

Information about antibodies for the detection of troponin antigens from different animal species can be found from the part: II TROPONIN SPECIFIC ANTIBODIES, 1.1.3. Immunoassays for the detection of cTnI from other species.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

13

II Troponin-specific Antibodies

1. Anti-cTnI monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Specificity: Epitope specificity: Purification method: Presentation:

Mice Balb/c Sp2/0 Free human cardiac Troponin I, native human cardiac Troponin complex, or human cardiac TnI peptides Specific to human cTnI Determined Protein A affinity chromatography MAb solution in PBS, pH 7.4, with 0.1% sodium azide

Hybridoma clones have been derived from hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with free human cardiac Troponin I, native human cardiac Troponin complex, or human cardiac TnI peptides.

All MAbs react with free cardiac Troponin I (cTnI) and cTnI forming complexes with other Troponin components. None of the MAbs (except M46, 267, 596, 472 and C5) have cross-reaction with skeletal muscle Troponin I. Cross-reactivity with skeletal Troponin I

10B11 22B11 P4-14G5, P4-9F6

23C6

The epitope specificity of all MAbs was precisely determined either by the SPOT technique or by other methods utilizing different peptide libraries (Fig. 3).

We currently have available antibodies specific to almost all parts of cTnI molecule and these antibodies could be used for both scientific studies and the development of new generations of cTnI assays.

M155 228 19C7

3C7

M18 10

4C2

is less than 10% for MAbs M46, 267 and 596, and more than 50% for MAbs C5 and 472. MAb 247 reacts only with free cTnl.

20

30

40

50

60

70

80

ADGSSDAAREPRPAPAP I RRRSSNYRAYA T EPHAKKKSK I SASRKLQLKT L L LQI A KQE L EREAEERRGEKGRALST RCQ 8E10, 16A11

84

560

90

100

110

415

120

130

581

M46

140

150

441

160

PLE LTG LGF AELQDLCRQLHARVDKVDEERYD I EAKVT KN I TE I A DLTQK I FDL RGK FKRPT L RRVR I SADAMMQAL LA 267,458,596,625

170

MF4

C5

180

190

p45-10

200

RAKESLDL RAHLKQVKKEDTEKENREVGDWRKN I DA LSGMEGRKKKFES

Figure 3. Epitope mapping of anti-cTnI monoclonal antibodies.

14

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

In Table 2 you can find data demonstrating the crossreactivity of some of the MAbs with cTnI from several animal species. The majority of the presented mono-

clonal antibodies can be used for immunodetection of cTnI from different animal species.

Table 2. Cross-reaction of anti-cTnI MAbs with antigens from different animal species

MAb 10B11 4C2 19C7 8E10 16A11 C5 MF4 22B11 247

Cross-reaction in Western blotting Human + ++ ++ + + ++ + ++ ++

Bovine ++ ++ + + ++ + ++

Porcine + ++ + + + ++ + + ++

Goat ++ ++ + + ++ + ++

Canine + ++ + + + ++ + ++

Rabbit + ++ ++ + + ++ +

Cat + + ++ + + ++ + ++

Rat + ++ ++ ++ + ++

Mouse ++ + ++ + ++

Fish ++ -++ N/A

1.1. Applications 1.1.1. High sensitivity cTnl assay concept In 2007, scientific and medical societies were celebrating the 20th anniversary of the first troponin I assay described in literature (Cummins B, Auckland ML, Cummins P. Cardiac-specific troponin-I radioimmunoassay in the diagnosis of acute myocardial infarction. Am Heart J. 1987 113(6):1333-44.). We can now ascertain that preceding twenty years or so, the concept of troponin I assay has been greatly transformed. Current generations of commercially available assays have sensitivity of almost 1000-fold higher (10 pg/ml vs. 10 ng/ml) than that of the experimental assay described by Dr. Cummins in 1987. New monoclonal antibodies specific to several selected epitopes are able to recognize all known modifications (free and complexed, phosphorylated, partially digested by proteases and others) of the antigen circulating in blood. Special forms of the antigen used as standards or calibrators helped to improve correlations between different assays by more than 10-fold. All of these changes make troponin I assay a more precise and reliable tool in clinical practice.

High sensitivity of new generations of cTnI assays helps to register even minor events resulting in necrosis or apoptosis of myocardial tissue. However, this is not the case for every sample. The presence of cTnI-

specific autoantibodies (Eriksson S, Hellman J, Pettersson K. Autoantibodies against cardiac troponins.

N Engl J Med. 2005 6;352(1):98-100.) in the blood of some patients makes detection of the antigen almost impossible. Autoantibodies, competing with the assay antibodies for the binding sites on the antigen surface, make troponin I “invisible” for the diagnostic assays. And the effect of autoantibodies becomes even more prominent in cases of minor events, when antigen concentration in human blood is very low. By testing multiple blood samples, our specialists were able to discriminate between the major epitopes recognized by autoantibodies. The data obtained enabled us to select monoclonal antibodies that could be used for the development of cTnI assays, which are entirely or almost entirely unaffected by the presence of autoantibodies in the sample.

Therefore, it becomes increasingly evident that it is necessary to keep in mind that highly sensitive troponin I assay should utilize antibodies specific to the epitopes that are not affected by numerous modifications, which could occur with the antigen in human blood. Such an assay concept is described in the following pages.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

15

Cardiac isoform of Troponin I (cTnI) is nowadays a golden marker of cardiac muscle cell damage and death. Different diagnostic platforms were designed for quantitative measurements of cTnI in human blood and are used extensively in large hospitals and small diagnostic laboratories. But still there is no between assay agreement and it is often the case that one and the same blood sample gives different values when being analyzed by different cTnI assays.

The most common reason for the discrepancy in the assay measurements is the difference in the epitope specificity of the antibodies used in different assays.

We now know that multiple factors are influencing cTnI measurements, among which are posttranslational modifications (proteolytic degradation (16), phosphorylation (17)), complexing with other molecules (troponin C (9), heparin (17)) and cTnI-specific autoantibodies circulating in patients’ blood (18).

Different mono- and polyclonal antibodies, utilized in assays, are sensitive to these factors in different degrees.

HyTest specialists have been involved in cTnI studies for over 15 years. We have both generated and tested several thousand monoclonal antibodies specific to different regions of cTnI molecule; we tried several hundred different two-site MAb combinations to find the best one for precise cTnI immunoassay. Following the results of our studies, we can conclude that it is currently impossible to have one antibody pair (one capture and one detection antibody), which would be totally insensitive to all known cTnI modifications and interferences. Based on our opinion, MAb combinations, which could be used for the development of precise cTnI immunoassay, should utilize two monoclonal antibodies for capture (plate or particle coating) and two MAbs for detection (conjugated with the specific label). We call such an approach a 2+2 concept. In these assays monoclonal antibodies should be selected in such a way that if one of the MAbs (capture or detection) is sensitive to the presence of some factor in the sample, then the other MAb should be insensitive to the same factor.

Therefore the effect of negative or positive interference is minimized. In addition, one important parameter should be considered: antibodies utilized in the

16

assay should be specific to the cardiac isoform of the protein and should not have cross-reaction with the two skeletal isoforms.

Currently, HyTest can suggest several combinations of monoclonal antibodies that are useful for the development of cTnI assays according to the 2+2 concept. Such assays would be cardiac specific and almost insensitive to all known factors that could affect cTnI measurements. Moreover, while selecting antibodies we also considered the fact that the new generation of cTnI assays should display high sensitivity and antibody combinations could be used in point-of-care diagnostics platforms. Consequently, assays described in Table 3 have good kinetics and they recognize standard preparation of antigen (cTnI in troponin complex) with very high sensitivity.

Table 3. The best HyTest 2+2 combinations of monoclonal antibodies.

Capture antibodies

Detection antibodies

MF4 + 560

19C7 + 267

M18 + 560

19C7 + MF4

M18 + 560

19C7 + 267

Epitope specificity of antibodies: M1818-28 , 19C741-49 , 56083-93 , 267169-178 , MF4190-196.

Factors that influence cTnI measurements are schematically presented in Fig. 4. Antibodies specific to different parts of the molecule are sensitive to these factors in different degrees. For instance, it is a wellknown fact that purified cTnI is highly susceptible to proteolytic degradation. However, in troponin complex, the central part of the cTnI closely interacts with troponin C (TnC) and TnC protects cTnI from proteolytic degradation. Consequently, the epitopes located on the central part of the cTnI are significantly more stable than the epitopes located at the terminal parts of the molecule. Meanwhile TnC competes with antibodies for binding with cTnI and only few antibodies, specific to the central part of cTnI, can recognize complexed form of the antigen.

In Figs. 5–9 we are comparing new assays designed in accordance with the 2+2 concept with conventional assays sensitive to different factors, therefore illustrating the insensitivity of new assays to these factors.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

Pi Pi

Pi

TnC

cTnI

Sites of phosphorylation Troponin C Autoantibodies Heparin Sites of proteolysis

TnC

Figure 4. Factors influencing cTnI measurements.

A. Cardiospecificity 300000.00 250000.00 200000.00 150000.00

CPS

Three isoforms of cTnI are described for humans – one is expressed in cardiac muscle (cardiac TnI), while the two others are essential for slow and fast skeletal muscles (slow skTnI and fast skTnI, respectively). Identity in sequence between cTnI and slow skTnI is approximately 40% and less, although still significant, for fast skTnI. Therefore, all antibodies selected for cTnI assay should be tested to ensure no cross-reaction with skeletal isoforms of the protein. As can be seen from Fig. 5, being tested even with high concentrations of skTnI (50 ng/ml), new assays produce no response, therefore indicating their extremely high cardiospecificity.

100000.00 50000.00 0.00 MF4+56019C7+267

M18+56019C7+MF4

M18+56019C7+267

Tn I card. 50 ng/ml Tn I skel. 50 ng/ml Figure 5. Testing of 2+2 assays with cardiac and skeletal isoforms of TnI.

B. Sensitivity to heparin 120.00 100.00

% of cTn I imm.activity

cTnI is a highly basic molecule with pI ~9.9 that easily forms complexes with molecules with negative charge. Heparin is widely used in clinical practice as an anticoagulant and almost all patients with suspected acute myocardial infarction (AMI) receive heparin during the first minutes following admission.

It was demonstrated that some anti-cTnI MAbs are sensitive to the presence of heparin in the sample and give lower response with the samples containing heparin (MAb 228 in the pair 228-MF4 in Fig. 6).

MAbs, utilized in 2+2 concept are not sensitive to the presence of heparin.

80.00 60.00 40.00 20.00 0.00 228-MF4

MF4+56019C7+267

M18+56019C7+MF4

M18+56019C7+267

W/o heparin +5 IU/ml heparin

Figure 6. MAb combinations tested with cTnI (concentration 50 ng/ml) in the presence (5 IU/ml) and absence of heparin.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

17

C. Sensitivity to phosphorylation (protein kinase A)

180.00 180.00 140.00 120.00

% of cTn I imm. activity

Two serines in the 22 and 23 positions could be phosphorylated by protein kinase A in vivo, so four forms of protein (one dephospho-, two monophospho- and one bisphospho-) can coexist in the cell.

Phosphorylation of cTnI changes the conformation of the protein and modifies its interaction with other troponins, as well as the interaction with some anticTnI antibodies (MAbs 1G11 and 22B11 in pairs 19C71G11 and 19C7-22B11 in Fig. 7). According to the latest findings, a significant part of cTnI released into the patient’s bloodstream is phosphorylated. MAbs utilized in 2+2 concept are not sensitive to the phosphorylation status of cTnI.

100.00 80.00 60.00 40.00 20.00 0.00

19C7-1G11 19C7-22B11 MF4+560- M18+560- M18+56019C7+267 19C7+MF4 19C7+267

Native Tn complex Tn complex –Pi Tn complex +Pi Figure 7. Testing of new assays with native troponin complex, artificially phosphorylated troponin complex and artificially dephosphorylated troponin complex (Note: MAb 1G11 recognizes only phosphorylated cTnI and MAb 22B11 is specific only to the dephosphorylated atigen).

D. Sensitivity to proteolytic degradation 120.00 100.00

% of cTn I imm. activity

cTnI is known to be an extremely unstable molecule.

We incubated native troponin complex for 116 hours with endogenous tissue protease cocktail and tested it in 2+2 assays. In all 2+2 assays, antigen demonstrated significantly greater stability in comparison to the control assay utilizing antibodies 9F6 and p45-10 with the epitopes located at the terminal parts of the molecule (assay is sensitive to the proteolytic degradation of the antigen).

80.00 60.00 40.00 20.00 0.00 9F6-p45-10

MF4+56019C7+267

M18+56019C7+MF4

M18+56019C7+267

0h 116 h Figure 8. Testing of new assays with degraded antigen.

E. Sensitivity to the presence of autoantibodies 40.00 35.00

% of cTn I imm. activity

Recent studies revealed that some blood samples (5-10% of total population) contain high level of autoantibodies specific to cTnI. Antigen concentration in such samples can be significantly underestimated or antigen becomes undetectable (in cases of low concentration of cTnI in the sample). In the 2+2 concept, we utilized at least one capture and one detection MAb, which are in less degrees sensitive to the presence of autoantibodies. As a result, new assays can detect antigen even in the samples with a high content of autoantibodies.

30.00 25.00 20.00 15.00 10.00 5.00 0.00

19C7-8E10

MF4+56019C7+267

M18+56019C7+MF4

M18+56019C7+267

50 ng/ml

Figure 9. Antigen was spiked into the serum sample with a high level of autoantibodies and the signal was compared with the antigen spiked in the same concentration (50 ng/ml) into buffer (100% of activity) (Note: Assay 19C7-8E10 is sensitive to the presence of autoantibodies in the sample).

18

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

All cTnI specific MAbs were tested in sandwich fluoroimmunoassay as capture and detection antibodies.

Table 4. Antibody combinations for cTnI quantitative sandwich immunoassay.

Capture

Detection

19C7 560 625 560 19C7 + M18

16A11 19C7 19C7 458 MF4 + 16A11

M18 + 560 228 + 560 M18 + 560 MF4 + 560 19C7 + MF4

MF4 + 19C7 19C7 + 458 19C7 + 267 19C7 + 267 7B9 (Anti-TnC MAb; Cat. # 4T27)

The pairs demonstrate high sensitivity (Fig.10), good kinetics, low background and high reproducibility.

Using combinations of MAbs (2–3 antibodies with different epitope specificity) for capture and 2–3 MAbs for detection, the sensitivity of sandwich immunoassays can be increased by twofold or fourfold.

Immunoassays, based on these recommended antibodies, recognize complexed and free forms of the antigen with same sensitivity. It was recently demonstrated that during incubation in the necrotic muscle following AMI, cTnI is cleaved by endogenous proteases. Consequently, a mixture of intact cTnI molecule and its proteolytic fragments can be detected in the bloodstream several hours after the onset of the chest pain. Different parts of cTnI molecule display different stability, with the most stable being the fragment located between 30 and 110 amino acid residues, possibly due to of its protection by TnC. For improved sensitivity and reproducibility in assay designing, we recommend using antibodies that recognize the stable part of the molecule or combination of antibodies (>1 MAbs for capture and for detection), specific to different parts of the molecule. Even when you are using a combination of MAbs for capture and detection it is desirable to have among these antibodies one which is specific to the stable part of the molecule.

As the vast majority (>95%) of cTnI in human blood is presented as a binary cTnI-TnC complex, the antibodies utilized in the assay should recognize cTnI in complex with TnC. Furthermore, due to the complexed nature of circulating cTnI, it is possible to utilize TnC specific MAbs in pairs with cTnI specific MAbs in cTnI assay. Such an approach helps to improve the sensitivity and reproducibility of cTnI immunoassays.

For additional information regarding other MAb combinations tested in cTnI immunoassay please contact the HyTest customer service department.

In assay designing it should be taken into consideration that the assay should not be affected by partial proteolytic degradation of cTnI molecule, oxidationreduction, phosphorylation, complex formation with TnC, presence of heparin in the samples and negative interference (15). The antibody combinations that we recommend are not affected by any of these factors. The antibodies are validated for traditional types of sandwich immunoassays as well, as for immunochromatography methods.

100000

10000

CPS

1.1.2. cTnI quantitative sandwich immunoassay

1000

100 0.1

1

10

TnI concentration (ng/ml)

Figure 10. cTnI calibration curve: One step assay in streptavidin coated plates. Biotinylated 19C7 MAbs for capture (200 ng/well) and Eu-labeled 16A11 MAbs for detection (200 ng/well). Antigen: cTnI (native troponin complex). Incubation time: 20 min. Temperature: 20 °C.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

19

1.1.3. Immunoassays for the detection of cTnI from different animal species

M155 – 19C7

100000

10000

CPS

New drug and evaluation of new surgery approaches are carried out on experimental animals. The effects of the new technologies on cardiac function and on cardiac myocyte viability are important and could be studied by cTnl measurements in animal blood. As is shown in Table 2, the majority of anti-cTnI MAbs has wide specificity and recognizes not only human cTnI but also cardiac TnI from other species. Several combinations of MAbs were tested and could be used for quantitative detection of cTnI in blood of different species. In Fig. 11, we are presenting the calibration curve for the assay utilizing MAb M155 for capture and MAb 19C7 for detection. This MAb combination gives equal response with antigens from different animal species. Other MAb pairs, with epitopes located in different parts of troponin molecule, can also be used for the same purpose.

1000 Human cTnl Rat cTnl Canine cTnl Mouse cTnl

100 0.1

1

(in native complex) (in native complex) (in native complex) (in native complex)

10

cTnl concentration (ng/ml)

Figure 11. cTnI calibration curve: One step assay in streptavidin coated plates.

Capture MAb: M155 (biotinylated), 200 ng/well.

Detection MAb: 19C7 (Eu-labeled), 200 ng/well.

Antigens: Human, Rat, Canine, Mouse (native troponin complex).

Incubation time: 20 min.

Temperature: 20 °C.

1.1.4. Detection of binary cTnI-TnC complex

1.1.5. Detection of binary cTnI-cTnT complex

For the quantitative measurements of binary cTnI – TnC complex, we recommend using a principle of mixed sandwich immunoassay (Fig. 12 A, B). The detection MAb in such an assay is cTnI specific (e.g.

19C7), whereas capture antibody (e.g. 7B9) recognizes TnC. As the main part of cTnI in the bloodstream of AMI patients is presented in the form of cTnI – TnC complex, results of measurements by this assay in AMI serum correlate well with results received by cTnI assay (Fig. 12 C).

A similar approach that was recommended for the quantitative detection of cTnI-TnC complex can also be used for cTnI-cTnT complex detection. We recommend using anti-cTnI MAb 19C7 for capture and anticTnT MAb 1F2 for detection. Such an assay has no cross-reaction with free forms of cTnI, cTnT and TnC, and recognizes only cTnI-cTnT binary and cTnI–cTnT– TnC ternary complexes.

100000

Tn I concentration (ng/ml)

10000

CPS

TnC cTnI

AntiTnC

250

1000

AnticTnI

B

1

10

100

Tn I concentration (ng/ml)

Figure 12. Measurement of cTnI-TnC complex by mixed assay.

A. Scheme of the assay.

B. Calibration curve (ternary native complex as a standard).

C. cTnI concentration in the form of cTnI - TnC complex (light blue; measured by ”mixed” assay – Biotinylated 19C7 MAbs for capture (200 ng/well) and Eu-labeled 7B9 – anti-TnC MAbs - for detection (200 ng/well)) and total cTnI concentration (free and complexed – dark blue) measured in serial serum samples of AMI patient.

20

150

100

50

0

100

A

200

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

0

20

C

Hours after onset of chest pain

40

60

80

1.1.6. Detection of dephosphorylated cTnI cTnI is phosphorylated in vivo and it can also be phosphorylated in vitro by cAMP-dependent protein kinase A. Sites of phosphorylation are the serines in the 22nd and 23rd position (14). Some of the HyTest MAbs recognize the epitopes containing these two amino acid residues. It was demonstrated that MAb 22B11 recognizes only the dephospho-form of cTnI and does not react with the mono- or biphospho-

rylated form of the antigen (Fig. 13). 22B11 can be used for qualitative or semi-quantitative immunodetection of dephosphorylated cTnI in Western blotting and for quantitative measurements of dephosphorylated cTnI in sandwich immunoassay (Fig. 14). Using such immunoassay, we demonstrated that a significant part of cTnI in the patient’s blood is partially phosphorylated (Fig. 15).

-Pi

+Pi

A

-Pi

+Pi

B

-Pi

cTn I Immunological activity

100

+Pi

C

80

cTnI-Pi cTnI+Pi

60

40

20

0

19C7-10B11

B

A Figure 13. Monoclonal antibodies 22B11(A), 10B11(B) and 8E10 (C), recognizing dephospho- and phospho- forms of human cardiac TnI.

19C7-22B11

Figure 14. Two immunoassays recognizing dephospho- (light blue columns) and biphospho- (dark blue columns) forms of cTnI.

1.1.7. Detection of cTnI or cTnI fragments by Western blotting

16 14

Tn I concentration (ng/

8E10-21B11 12

8E10-10B11

10 8 6

All anti-cTnI MAbs recognize human cTnI or cTnI fragments in Western blotting (Fig. 16). For improved sensitivity in Western blotting we recommend using MAbs 19C7, 16A11 and MF4.

4 2 0 0

20

40

60

80

Time after admission (h)

Figure 15. cTnl measurements: total (red) and dephosphorylated (black) cTnl in serial blood samples from AMI patient.

1

2

3

4

Figure 16. Detection of cTnI by Western blotting. After SDS-PAGE cTnI was transferred to nitrocellulose membrane and probed by 16A11 MAbs and then by HRP-conjugated goat anti-mouse IgG antibodies. Substrate - diaminobenzidine. Lane 1: 3 ng cTnI, lane 2: 10 ng cTnI, lane 3: 30 ng cTnI, lane 4: 100 ng cTnI.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

21

Ordering information: MAb

Cat. #

Specificity

Subclass

Epitope

Application

P4-14G5 P4-9F6 23C6 10B11 M18

4T21 4T21 4T21 4T21 4T21

cTnI cTnI cTnI cTnI cTnI

IgG1 IgG1 IgG2a IgG1 IgG1

1 – 15 a.a.r.

1 – 15 a.a.r.

15 – 25 a.a.r.

16 – 20 a.a.r.

18 – 28 a.a.r.

EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay

4C2

4T21

cTnI

IgG2a

23 – 29 a.a.r.

(capture, detection), WB EIA, Sandwich immunoassay

(capture, detection), WB 3C7 228 M155 10F4

4T21 4T21 4T21 4T21

cTnI cTnI cTnI cTnI

IgG1 IgG1 IgG1 IgG2a

25 – 40 a.a.r.

26 – 35 a.a.r.

26 – 35 a.a.r.

34 – 37 a.a.r.

19C7

4T21

cTnI

IgG2b

41 – 49 a.a.r.

247

4T21

cTnI

IgG1

65 – 74 a.a.r.

EIA, Sandwich immunoassay (detection), WB EIA, Sandwich immunoassay (capture), WB EIA, Sandwich immunoassay (detection), WB EIA, Sandwich immunoassay (capture, detection), WB EIA, Sandwich immunoassay

(capture, detection), WB EIA, Sandwich immunoassay (capture), WB,

free cTnl only 560

4T21

cTnl

IgG1

83 – 93 a.a.r.

EIA, Sandwich immunoassay

(capture), WB 16A12 8E10

4T21 4T21

cTnI cTnI

IgG1 IgG1

86 – 90 a.a.r.

86 – 90 a.a.r.

16A11

4T21

cTnI

IgG1

86 – 90 a.a.r.

EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay

(capture, detection), WB EIA, Sandwich immunoassay

(capture, detection), WB 17F3

4T21

cTnI

IgG1

87 – 90 a.a.r.

84

4T21

cTnl

IgG1

415 M46

4T21 4T21

cTnl cTnI

IgG1 IgG1

117– 119 a.a.r. EIA, Sandwich immunoassay, (capture), WB 104 – 119 a.a.r. EIA, Sandwich immunoassay, (capture), WB

EIA, Sandwich immunoassay (capture), WB

581 441

4T21 4T21

cTnl cTnl

IgG1 IgG1

625

4T21

cTnI

IgG1

130 – 145 a.a.r. EIA, Sandwich immunoassay, WB, <10% c/r with skeletal Tnl 143 – 152 a.a.r. EIA, Sandwich immunoassay, (capture), WB 148 – 158 a.a.r. EIA, Sandwich immunoassay, (capture, detection) WB 169 – 178 a.a.r. EIA, Sandwich immunoassay

458

4T21

cTnI

IgM

169 – 178 a.a.r. EIA, Sandwich immunoassay

596

4T21

cTnI

IgG1

267

4T21

cTnI

IgG2a

472

4T21

cTnI

IgG3

C5 MF4 p45-10

4T21 4T21 4T21

cTnI cTnI cTnI

IgG2b IgG1 IgG

1G11 22B11

4T45 4T46

Phosphorylated cTnl Dephosphorylated cTnl

IgG2b IgG2b

169 – 178 a.a.r. EIA, Sandwich immunoassay (detection), WB, <10% c/r with skeletal Tnl 169 – 178 a.a.r. EIA, Sandwich immunoassay, WB, <10% c/r with skeletal Tnl 182 – 191 a.a.r. EIA, (capture, detection), >50% c/r with skeletal Tnl 186 – 192 a.a.r. EIA, WB, >50% c/r with skeletal Tnl 190 – 196 a.a.r. EIA, Sandwich immunoassay, WB 195 – 209 a.a.r. EIA, Sandwich immunoassay (capture, detection), WB N/A EIA, Sandwich immunoassay, WB 20 – 24 a.a.r.

EIA, Sandwich immunoassay, WB

(capture, detection), WB (detection), WB

2. Anti-cTnT monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Epitope specificity: Purification method: Presentation:

Mice Balb/c Sp2/0 Human or bovine cTnT Partially determined Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide

Hybridoma clones have been derived from hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with free human cTnT, bovine TnT or human Tn complex. MAbs were tested 22

to have no cross-reaction with cTnI and TnC. The epitope specificity of some MAbs was precisely determined by the SPOT technique or other methods utilizing cTnT peptides. For MAbs not interacting with

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

SPOT peptides, epitopes were localized using cTnT BrCN peptides. Cross-reaction of anti-human cTnT

MAbs with cTnTs from other species was studied in Western blotting with crude extracts of cardiac tissue from different species (Table 5).

Table 5. Anti-cTnT monoclonal antibodies

MAb Cross-reaction in Western blotting Human ++ + + + ++ ++ ++ ++ +

7F4 1F2 7G7 3D6 2F3 1A11 2G3 1F11 7A9

Bovine N/A + + + + ++ + ++ +

Porcine ++ ++ ++ + ++ +

Goat N/A + + ++ ++ + ++ +

Canine + + + + + + +

Rabbit + + + + + + +

Cat + + + + + -

Rat N/A + + + + -

Mouse N/A + + ++ + + -

Fish + + + + + -

2.1. Detection of cTnT from different animal species cTnT as a sensitive and reliable marker of myocardial cell death is widely used in preclinical studies of new drugs, experimental cardiology and other studies performed on the experimental animals to demonstrate the effect of artificial intervention on the viability of myocardial cells.

HyTest provides a combination of two monoclonal antibodies suitable for the development of sandwich immunoassay for quantitative detection of human, canine, rat and mouse cTnTs in blood samples (Fig. 17). The antibodies were chosen on the basis of their reproducibility, reliability and reactivity with cTnTs from different animal species. MAb 1C11 has epitope located in the 95-181 region of cTnT sequence. This part of the cTnT molecule is very conservative and has a high degree of similarity between human, rat and mouse TnTs. Therefore MAb 1C11 reacts equally well with these antigens.

CPS

100 000

10 000

1000 10

TnT concentration (ng/ml)

100

Figure 17. Calibrating curves for sandwich cTnT fluoroimmunoassay with different animal TnTs as antigen: (Q) human, (O) canine, (V) rat, (W) mouse MAb 1C11 for capture (1 μg/well) and MAb 7E7 for detection (200 ng/well). Assay time: 30 min. Room temperature.

Hybridoma cell line producing MAb 7E7 was raised after hybridization of myeloma cell line Sp2/0 with splenocytes of mice immunized with human tertiary Tn complex. Thus MAb 7E7 demonstrates slight cross-reaction with cTnI (about 5% based on ELISA data).

Ordering information: MAb

Cat. #

Specificity Subclass

Epitope

Application

9G6 7F4 1F2 7G7 3D6 2F3 1A11 1C11 2G3 1F11 7A9 7E7

4T19 4T19 4T19 4T19 4T19 4T19 4T19 4T19 4T19 4T19 4T19 4T19

cTnT cTnT cTnT cTnT cTnT cTnT cTnT cTnT cTnT cTnT cTnT cTnT

1 – 60 a.a.r.

60 – 70 a.a.r.

60 – 71 a.a.r.

60 – 71 a.a.r.

71 – 95 a.a.r.

95 – 181 a.a.r.

95 – 181 a.a.r.

95 – 181 a.a.r.

95 – 181 a.a.r.

146 – 160 a.a.r.

180 – 287 a.a.r.

N/A

EIA, Sandwich immunoassay (detection), WB EIA, Sandwich immunoassay (detection), WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay (capture), WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay (detection), WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay (capture, detection), WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay (detection), WB

IgG1 IgG2b IgG1 IgG1 IgG1 IgG2b IgG2b IgG1 IgG2b IgG2b IgG1 IgG1

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

23

3. Anti-TnC monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation:

Mice Balb/c Sp2/0 Human cardiac troponin complex or isolated slow skeletal isoform of TnC Protein A affinity chromatography MAb solution in PBS with 0.1 % sodium azide

Hybridoma clones have been derived from hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with isolated slow skeletal Ordering information:

(”cardiac”) form of TnC or by native cardiac troponin complex. MAbs were tested to have no cross-reaction with cTnT and cTnI. All MAbs recognize TnC in binary complex with cTnI.

MAb

Cat. #

Specificity

Subclass

Application

1A2

4T27

TnC

IgG2a

EIA, Sandwich immunoassay (capture), WB

7B9

4T27

TnC

IgG1

EIA, Sandwich immunoassay (detection), WB

12G3

4T27

TnC

IgG2b

EIA, Sandwich immunoassay, WB

4. MAbs specific to human cardiac troponin complex Host animal: Cell line used for fusion: Antigen: Purification method: Presentation:

Mice Balb/c Sp2/0 Human cardiac troponin complex (native) Protein A affinity chromatography MAb solution in PBS with 0.1 % sodium azide

Hybridoma clones have been derived from hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with native troponin complex, isolated in mild conditions from human cardiac tissue. MAbs were shown to recognize ternary troponin complex or binary I-C complex and have no crossreaction with free cTnT, cTnI and TnC subunits.

4.1. Applications 4.1.1 cTnI quantitative sandwich immunoassay In the blood of AMI patients, the majority of cTnI is presented as a binary I-C complex or ternary complex. Concentration of free cTnI subunit is incomparably lower than concentration of complexed cTnI. Therefore new HyTest anti-hcTn complex MAbs 20C6 and Tcom8 could be used in cTnI sandwich immunoassay in pairs with MAbs specific to human cTnI (Cat. # 4T21) or TnC (Cat. # 4T27), see Table 6 and Fig. 18.

Such assays demonstrate very high sensitivity (0.01 ng/ml, Fig. 19) for purified and endogenous antigen, good kinetics, low background, high reproducibility and have significantly lower susceptibility to the presence of anti-cTnI autoantibodies in the sample (Fig. 20). A comparison of calibration curves of new assay (Tcom8 – 7B9) with two conventional assays (19C7560 and 2+2 assay) is presented in Fig. 19.

24

Table 6. Antibody combinations for cTnI quantitative sandwich immunoassay:

Capture

Detection

19C7 (Anti-TnI MAb; Cat. # 4T21)

20C6

19C7+560 (Anti-TnI MAb; Cat. # 4T21)

20C6

Tcom8

7B9 (Anti-TnC MAb; Cat # 4T27)

Eu3+

TnT Tnl TnC MAb 7B9 MAb Tcom8

Figure 18. Scheme of new cTnI assay.

Capture antibody – Tn complex specific MAb Tcom8 Detection antibody – TnC specific MAb 7B9

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

tween TnC and TnI subunits in troponin complex by changing conformation of TnC molecule. This possibly results in decreased affinity of interaction of MAb Tcom8 with the antigen. And that observation makes EDTA undesirable in the samples to be measured by new cTnI assay.

Calibration curves

100 000

CPS

10 000

1000 19C7-560-Eu 2+2(M18+560-19C7-Eu+MF4-Eu) Tcom8-7B9-Eu

140

100

19C7-560-Eu M18+560-19C7-Eu-MF4-Eu Tcom8-789-Eu

120 0,1

1

10

100

cTnl concentration (ng/ml)

Figure 19. Calibration curves (ternary native complex as a standard) for new cTnI assay (MAbs Tcom8 – 7B9) and for immunoassays of two previous generations (19C7-560 and 2+2 assay).

The new assay has less susceptibility to the negative interference of autoantibodies. When native troponin complex was spiked in the donor’s blood sample, containing a high level of anti-cTnI autoantibodies, significantly improved recovery was observed in the case of the new assay than in cases of cTnI assays of previous generations, utilizing all MAbs specific to cTnI molecule. (Fig. 20)

% of cTnl immunological activity

0,01

80 60 40 20 0

Oh W/o additions

+EDTA

+Heparin

+Pi

+Pi

116h

Proteolysis

Figure 21. Effect of different additives and cTnI modifications on the antigen (Tn complex, 30 ng/ml) measurements by three assays – new assay Tcom8 – 7B9 and by two conventional: 19C7-560 and 2+2 immunoassays. +EDTA – 5 mM EDTA was added to the sample; +heparin – 5 IU/ml added to the sample; +/- Pi – Tn complex was phosphorylated by kinase A or dephosphorylated; proteolysis 116 h – Tn complex was incubated in the presence of endogeneous proteases for 116 h.

19C7-560

120

M18+560-19C7-Eu-MF4-Eu TCom8-789-Eu

cTnl immunological activity (%)

100

80

60

40

20

Preliminary clinical trials have revealed that in blood samples from the patients with minor cardiac events the new assay is able to detect more cTnI than the best tested conventional cTnI assays (Fig. 22). That fact makes the new assay preferable for AMI early diagnosis or for diagnosis of minor cardiac events associated with minor myocardial tissue necrosis.

0 Buffer

4.00

if serum

Tnl in AMI patient’s sera

M18+560-19C7-Eu-MF4-Eu Tcom8-7B9-Eu

Figure 20. Cardiac Tn complex spiked (30 ng/ml) in buffer and in serum sample with high level of anti- TnI autoantibodies and measured by new assay (Tcom8-7B9) and by two conventional assays (19C7 – 560 and 2+1 assay M18+560 – MF4).

Different additives and antigen modifications did not significantly influence the antigen recognition by the new assay, with only the one exception – the presence of EDTA in the sample (Fig. 21). EDTA is known to change (decrease) the affinity of interaction be-

cEtW concentration (ng/ml)

3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00

9 8 7 6 5 4 3 2 1 12 11 10 m m m m m m m m m m m m ru ru ru ru ru ru ru ru ru ru ru ru e e e Se Se Se Se Se Se Se Se Se S S S

Figure 22. TnI concentration in sera of patients with acute myocardial infarction or unstable angina, measured by “2+2” and new assays

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

25

Ordering information: MAb

Cat. #

Specificity

Subclass

Application

20C6 Tcom8

4TC2 4TC2

Tn complex TnC

IgG2b IgG1

EIA, Sandwich immunoassay EIA, Sandwich immunoassay

References: 1. Gomes, A.V, Potter,J.D., Szczesna-Cordary, D. (2002) The role of Troponin in muscle contraction. Life, 54: 323-333.

2. Marston,S.B., Redwood,C.S. (2003) Modulation of thin filament activation by breakdown or isoform switching of thin filament Proteins. Circ. Res., 93: 11701178.

3. Sarko J, Pollack CV Jr. (2002) Cardiac troponins. J Emerg Med. 2002, 23(1), pp. 57-65.

4. Cummins B, Auckland ML, Cummins P. (1987) Cardiac-specific troponin-I radioimmunoassay in the diagnosis of acute myocardial infarction. Am Heart J. 113(6): 1333-1344.

5. Katus HA, Remppis A, Looser S, Hallermeier K, Scheffold T, Kubler W. (1989) Enzyme linked immunoassay of cardiac troponin T for the detection of acute myocardial infarction in patients. J Mol Cell Cardiol. 21(12): 1349-1353.

6. Hamm CW. (2001) Acute coronary syndromes. The diagnostic role of troponins. Thromb Res., 103 Suppl 1:S63-9.

7. Labugger R, Organ L, Collier C, Atar D, Van Eyk JE. (2000) Extensive troponin I and T modification detected in serum from patients with acute myocardial infarction. Circulation. 102(11): 1221-1226.

8. Reiffert SU, Jaquet K, Heilmeyer LM Jr, Herberg FW (1998) Stepwise subunit interaction changes by mono- and bisphosphorylation of cardiac troponin I. Biochemistry, 37(39): 13516-13525.

9. Katrukha AG, Bereznikova AV, Esakova TV, Pettersson K, Lovgren T, Severina ME, Pulkki K, Vuopio-Pulkki LM, Gusev NB. (1997) Troponin I is released in bloodstream of patients with acute myocardial infarction not in free form but as complex. Clin. Chem. 43(8): 1379-1385.

10. Wu AH, Feng YJ, Moore R, Apple FS, McPherson PH, Buechler KF, Bodor G. (1998) Characterization of cardiac troponin subunit release into serum after acute myocardial infarction and comparison of assays for troponin T and I. American Association for Clinical Chemistry Subcommittee on cTnI Standardization. Clin. Chem., 44(6): 1198-1208.

26

11. Onuoha GN, Alpar EK, Dean B, Tidman J, Rama D, Laprade M, Pau B.(2001) Skeletal troponin-I release in orthopedic and soft tissue injuries. J Orthop Sci. 6(1): 11-15.

12. Bertinchant JP, Robert E, Polge A, Marty-Double C, Fabbro-Peray P, Poirey S, Aya G, Juan JM, Ledermann B, de la Coussaye JE, Dauzat M. (2000) Comparison of the diagnostic value of cardiac troponin I and T determinations for detecting early myocardial damage and the relationship with histological findings after isoprenalineinduced cardiac injury in rats. Clin Chim Acta. 298(12): 13-28.

13. Katrukha AG, Bereznikova AV, Filatov VL, Esakova TV, Kolosova OV, Pettersson K, Lovgren T, Bulargina TV, Trifonov IR, Gratsiansky NA, Pulkki K, Voipio-Pulkki LM, Gusev NB. (1998) Degradation of cardiac troponin I: implication for reliable immunodetection. Clin. Chem. 44(12): 2433-2440.

14. Jaquet K, Thieleczek R, Heilmeyer LM Jr. (1995) Pattern formation on cardiac troponin I by consecutive phosphorylation and dephosphorylation. Eur J Biochem. 231(2): 486-90.

15. Katrukha AG. (2003) Antibody selection strategies in cardiac troponin assays.

Cardiac Markers, 2nd edition, Edited by Alan HB. Wu. 173-185.

16. Katrukha AG, Bereznikova AV, Filatov VL, Esakova TV, Kolosova OV, Pettersson K, Lovgren T, Bulargina TV, Trifonov IR, Gratsiansky NA, Pulkki K, Voipio-Pulkki LM, Gusev NB. Degradation of cardiac troponin I: implication for reliable immunodetection. Clin Chem. 1998; 44(12):2433-40.

17. Katrukha A, Bereznikova A, Filatov V, Esakova T. Biochemical factors influencing measurement of cardiac troponin I in serum. Clin Chem Lab Med. 1999;37(11-12):1091-5.

18. Eriksson S, Halenius H, Pulkki K, Hellman J, Pettersson K. Negative interference in cardiac troponin I immunoassays by circulating troponin autoantibodies. Clin Chem. 2005, 51(5):839-47.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

III Human proBNP and proBNP–derived peptides (BNP and NT-proBNP) Brain natriuretic peptide (BNP) is a peptide hormone with natriuretic, vasodilatory and renin inhibitory properties (1, 2, 3). BNP belongs to a family of structurally similar peptide hormones that also includes atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and urodilatin. These peptides are characterized by a 17 amino acid ring structure with a disulfide bond between two cystein residues. The ring structure shows a high identity level between different natriuretic peptides (11 out of 17 amino acid residues are identical for all group representatives).

BNP molecule is composed of 32 amino acid residues (a.a.r.) with a disulfide bond located between the residues Cys10 and Cys26 (Fig. 23).

M

D

R

I

S S

K

S

R

S

G

G

F

NH2

L

C S

P

K

M

V Q

G

S

G

G C K

V

L

R

R

H

COOH

Figure 23. Schematic representation of human BNP sequence and structure.

BNP is the product of proteolytic processing of the precursor molecule preproBNP (Fig. 24). PreproBNP is composed of 134 a.a.r. and it is synthesized in cardiac myocytes. The removal of signal peptide (a.a.r. 1-26) re-

1

134 PreproBNP

1

26

Signal peptide

S-S

27 (1)

134 (108) ProBNP

R-S77 S-S 76 Proprotein convertase

1

76 NT-proBNP

77

108 BNP S-S

Figure 24. Schematic representation of preproBNP processing. Removal of the signal peptide (26 a.a.r.) from the N-terminus of preproBNP results in the proBNP molecule (108 a.a.r.) which is then processed in a convertase-dependent reaction to form bioactive BNP (32 a.a.r.) and the N-terminal part of the proBNP, namely NT-proBNP (76 a.a.r.).

sults in the appearance of the proBNP molecule (a.a.r.

27-134). Then proBNP (108 a.a.r.) is processed in convertase-dependent reaction and forms two peptides, the BNP (a.a.r. 77-108) and the N-terminal part of the proBNP (NT-proBNP, a.a.r. 1-76). Both BNP (biologically active molecule) and NT-proBNP (physiological activity is not clarified) as well as unprocessed proBNP are secreted into the bloodstream and circulate in human blood. The reason(s) for this incomplete proBNP processing were unknown until recently. Amino acid sequences, isoelectric points and molecular weights of all three peptides are presented in Table 7.

Recently, some new data regarding proBNP biochemistry has been published. Schellenberger et al. (4) demonstrated that proBNP from HF patients’ plasma is an O-linked glycoprotein. Seven sites of O-glyco-

Table 7. Amino acid sequences, isoelectric points and molecular weights of BNP, proBNP and NT-proBNP.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

27

1

108

ProBNP

1

O-glycosylation

108

ProBNP

1 T71

108

ProBNP

T71 Cleavage by convertase

1

NT-proBNP

76 T71

1

32

BNP

Circulation

Figure 25. New scheme of proBNP processing. After translation proBNP is glycosylated at several sites in its N-terminal. Two pools of proBNP different in the status of T71 glycosylation are formed: non-glycosylated at T71 and molecules glycosylated at this site. Glycosylation suppress subsequent processing of proBNP. Non-processed proBNP is released in blood. Only proBNP that is not glycosylated at T71 could be effectively processed into BNP and NT-proBNP.

sylation (T36, S37, S44, T48, S53, T58 and T71) were identified for recombinant proBNP expressed in CHO cells. According to HyTest specialists novel findings (5), glycosylation of the region located close to the proBNP cleavage site and especially glycosylation of T71 residue is crucial for further proBNP processing. Glycosylation of T71 suppresses the subsequent processing of proBNP and intact proBNP is released in blood. ProBNP molecules that are not glycosylated at T71 could be effectively processed into BNP and NT-proBNP (Fig. 25). Consequently, the major part of proBNP molecules in human blood have glycosylated T71 residue, whereas in NT-proBNP T71 it is not glycosylated.

In healthy adults, plasma levels of BNP lie in the range 13.9 - 63.7 pg/ml (BNP Triage assay package insert) while the corresponding values for NT-proBNP are 68243 pg/ml (Roche proBNPII assay package insert). It was established that proBNP synthesis increases in response to cardiac wall stretch, which leads to increased BNP and proBNP concentrations in blood. An elevated level of both peptides was described for patients with different cardiac pathologies – heart failure (HF), acute coronary syndromes (ACS), left ventricular hypertrophy, cardiomyopathy, valvular heart diseases, atrial fibrillation and cardiac amyloidosis. The BNP concentration in HF patients plasma increases up to several ng/ml, whereas NT-proBNP concentration increases up to several tens of ng/ml.

28

The blood concentration of both analytes in HF patients correlates with the severity of disease. It has been reported that both peptide concentrations are already elevated in asymptomatic patients during the very early stage of heart failure (NYHA I stage according to the New York Heart Association classification).

NYHA classes II and III, and especially class IV patients demonstrate significantly elevated concentrations of BNP and NT-proBNP in their blood. Therefore, peptide measurements in human blood are widely used for the evaluation of patients with suspected HF and when assessing the severity of the disease.

BNP and NT-proBNP measurements are also useful for the risk stratification of the patients with different cardiac pathologies. It was shown that patients who develop complications are characterized by significantly higher BNP and NT-proBNP concentrations than patients without complications. In patients with congestive HF, high BNP (NT-proBNP) levels predict death and are relevant to cardiovascular risk, whereas in patients with ACS elevated levels of both peptides are predictive for mortality and severe heart failure.

At present, both analytes are used in clinical practice as it was demonstrated that their diagnostic and prognostic values are similar (6, 7). Recent data regarding proBNP biochemistry, such as proBNP/NT-proBNP glycosylation or incomplete proBNP processing, could significantly influence the current approach to both BNP and NT-proBNP measurements. As HyTest researchers demonstrated, NT-proBNP glycosylation negatively affects its recognition by some antibodies (8). The central part of the NT-proBNP molecule (a.a.r. 28-56) is scarcely accessible for antibodies due to O-glycosylation, whereas regions 13-27 and 61-76 are well recognized by antibodies. The degree of NT-proBNP glycosylation varies significantly from patient to patient and NT-proBNP concentration could be seriously underestimated in the case of the high level of glycosylation of NT-proBNP molecules in human blood. Therefore, we suggest that antibodies specific to the regions that are not affected by glycosylation should be selected for the development of NT-proBNP immunoassays. HyTest offers several MAbs specific to unmodified (not glycosylated) regions of NT-proBNP. MAbs were tested with HF patients blood samples and it was shown that antibodies are able to recognize glycosylated protein with high efficiency.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

The other important observation concerns proBNP measurements in human blood. Several groups have reported that significant amounts of proBNP can be , found in HF patients blood samples (9, 10, 11). According to the data recently published by HyTest specialists, proBNP is the predominant form displaying BNP immunoreactivity in patients with HF and the proBNP/ BNP ratio is not constant and varies from patient to patient, ranging from 1.8 to 32.6 (9, 12). For this reason our specialists suggest that proBNP measurements by assays, utilizing one antibody specific to the BNP and another to the NT-proBNP part of the molecule, could be of the same clinical value as the BNP measurements.

ProBNP studies in human blood revealed that endogenous proBNP, as well as NT-proBNP, is glycosylated in the region 28-56 and is poorly recognized by antibodies specific to this region. However, in contrast to NT-proBNP, proBNP is also glycosylated in the region 61-76. Therefore, in the development of proBNP immunoassays, our specialists recommend using one antibody specific to region 13-27 (which is not occupied with oligosaccharides) and another one that is specific to the BNP portion of proBNP (residues 77-108).

HyTest offers a set of high-affinity monoclonal antibodies that are specific to different epitopes of BNP

and NT-proBNP molecules. A wide range of MAb combinations is available for the development of sensitive and reliable BNP, NT-proBNP and proBNP immunoassays.

HyTest also currently offers monoclonal antibodies for the development of the new type of BNP assay – “Single Epitope Sandwich”. Such an assay utilizes one MAb, 24C511-17 specific to the BNP molecule, and another MAb, Ab-BNP2, which recognizes the immune complex formed by BNP and MAb 24C5 (for additional information see pages 35–38).

We supply our customers with detailed additional information regarding different MAbs applications – the development of quantitative sandwich BNP, NT-proBNP and proBNP immunoassays, immunodetection of antigens in direct ELISA or Western blotting.

HyTest also offers recombinant proBNP and NT-proBNP antigens expressed in E. coli and recombinant glycosylated proBNP expressed in eucaryotic cell line. Recombinant proteins could be used as standards and calibrators in immunoassays.

You can also find in our catalogue BNP/NT-proBNP/ proBNP free plasma, which could be used as a matrix for standard or calibrator preparation (see page 44).

1. Human recombinant proBNP and NT-proBNP 1.1. Human recombinant (not glycosylated) proBNP and NT-proBNP expressed in E. coli Source: Purity: Application: Storage:

Recombinant protein expressed in E. coli >95% according to Tricine-SDS-PAGE NT-proBNP and proBNP calibrators and standards -70°C

Human recombinant NT-proBNP (a.a.r. 1-76) and human recombinant proBNP (a.a.r. 1-108) are expressed in Escherichia coli. Both polypeptides have the same sequence as natural proteins with the only difference being additional Met residue at the Nterminus of the molecule. Antigens are recognized by monoclonal antibodies, specific to different parts of NT-proBNP (Cat. # 4NT1). Recombinant proBNP is also recognized by BNP-specific antibodies (Cat. # 4BNP2). Data showing proBNP stability studies (measured by proBNP immunoassay) can be found in Fig. 45).

26 17 14 6.5

1

2

3

Figure 26. Tricine-SDS-PAGE in reducing conditions of recombinant NT-proBNP and proBNP expressed in E. coli.

Lane 1: low molecular weight standards (Bio-Rad) Lane 2: proBNP, 3 μg Lane 3: NT-proBNP, 3 μg Gel staining: Coomassie brilliant blue R-250

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

29

Both NT-proBNP and proBNP are highly purified, with purity exceeding 95% according to Tricine-SDSPAGE (Fig. 26) and HPLC studies. Antigens could be used as calibrators or standards in NT-proBNP or proBNP assays. Recombinant proBNP can also be used as a calibrator or standard in BNP assays. The

calibration curve for the NT-proBNP-specific assay with recombinant NT-proBNP used as a calibrator is presented in Fig. 39, and with proBNP as a calibrator for ”Single Epitope Sandwich” assay in Fig. 33 and for proBNP immunoassay in Fig. 44.

Ordering information: Product

Cat. #

Purity

Human recombinant NT-proBNP (E. coli) Human recombinant proBNP (E. coli)

8NT2 8PRO9

>95% >95%

1.2. Glycosylated human recombinant proBNP Source: Purity: Application: Storage:

Recombinant protein expressed in mammalian cell line >95% according to Tricine-SDS-PAGE Calibrator or standard in immunoassays, immunogen for antibody production -70°C

As well as endogenous proBNP, also recombinant proBNP expressed in human embryonic kidney epithelial cell line is glycosylated. SDS–PAGE of recombinant peptide shows diffuse bands typical for glycoproteins with an apparent molecular mass of approximately 30 kDa for proBNP (Fig. 27).

Fig. 28 shows gel after Tricine-SDS PAGE stained with the GelCode Glycoprotein Staining Kit (Pierce) which specifically detects glycoproteins.

202

202

133

133 71

71 41.8

41.8

30.6

30.6 17.8

17.8

6.9

1

2

3

4

Figure 27. “Tricine-SDS-PAGE” in reducing conditions of glycosylated human recombinant proBNP. Lane 1: kaleidoscope prestained standards (Bio-Rad).

Lanes 2 and 3: glycosylated proBNP, 3 and 5 μg, respectively.

Lane 4: proBNP from E. coli, 2 μg.

Gel staining: Coomassie brilliant blue R-250.

30

Figure 28. Tricine-SDS-PAGE in reducing conditions of recombinant glycosylated proBNP. Staining of glycoproteins.

Lane 1: molecular weight standards (Bio-Rad) Lane 2: glycosylated proBNP, 4 μg Lane 3: proBNP expressed in E. coli, 2 μg Lane 4: horseradish peroxidase (as positive control), 5 μg Gel staining: GelCode Glycoprotein Staining Kit (Pierce). As indicated in the picture, glycosylated proBNP is stained with the kit in contrast to proBNP expressed in E. coli.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

HyTest’s recombinant glycosylated proBNP was tested in sandwich immunoassays utilizing MAbs, specific to the different regions of proBNP molecule (Cat. # 4NT1, 4BNP2). These MAbs are able to recognize recombinant proBNP expressed in E. coli which is non-glycosylated with high sensitivity. It was demonstrated that MAbs specific to the region 1-27 recognized recombinant proBNP expressed in mammalian cell line with high efficiency, whereas MAbs specific to the central region (fragment 28-56) hardly

recognized proBNP molecules due to glycosylation.

In cases of the antibodies specific to the region 6176, glycosylated proBNP was almost “invisible” for such antibodies. Whereas BNP part of the proBNP molecule was fully accessible for antibodies.

Recombinant glycosylated proBNP could be used as a calibrator or standard in proBNP and BNP immunoassays.

1.2.1. Glycosylated proBNP as a stable standard for BNP and proBNP immunoassays

HyTest specialists recently demonstrated that BNPimmunoreactivity in human blood mainly belongs to the proform of BNP – proBNP (9). It was also shown that endogenous proBNP is a glycoprotein (4).

Based on these data we suggested using recombinant proBNP as a calibrator in BNP immunoassays.

Stability studies of endogenous proBNP, purified from pooled plasma of HF patients and recombinant glycosylated proBNP (expressed in mammalian cell line), which is the most similar to the endogenous one have revealed that both proBNP forms have comparable stability (Fig. 29). Antigens were reconstituted in pooled normal human plasma and then samples were incubated at room temperature for different time periods up to 24 hours. After that proBNP-immunoreactivity was measured by BNP immunoassay. About 75% of initial proBNP-immunoreactivity was observed after 24 hours of incubation in case of endogenous as well as recombinant proBNP. Detailed description of the used BNP immunoassay see on pages 32-33.

100

Immunological activity, %

Synthetic BNP is currently used as a standard in BNP immunoassays. However, it is known that synthetic BNP demonstrates low stability being reconstituted in plasma or some other proteases-containing matrixes. Due to low stability, the use of synthetic BNP as a standard in BNP assays is limited.

90 80 70 60 50 40 30 20 10 0 0

4

8

12

16

20

24

Incubation time, hours

Figure 29. proBNP stability studies. Recombinant glycosylated proBNP (from mammalian cell line) (- -) and endogenous proBNP, purified from pooled HF patients plasma (- -) were reconstituted in pooled normal human EDTA-plasma and incubated at RT for different time periods. Immunological activity was measured by immunoassay, utilizing MAb 50E126-32 as capture and MAb 24C511-17 as detection.

Taking into account the results obtained in stability studies and the prevalence of proBNP in human blood, HyTest specialists suggest using proBNP, expressed in eukaryotic cells, as a stable standard or calibrator in BNP immunoassays.

For using recombinant glycosylated proBNP as a standard or calibrator for proBNP immunoassays, see page 43.

Ordering information: Product

Cat. #

Glycosylated human recombinant proBNP

8GOB2

Purity >95%

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

31

2. Anti-BNP monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Specificity: Purification method: Presentation: Application:

Balb/c mice Sp2/0 Human BNP and synthetic peptide 11–22, conjugated to carrier proteins Human BNP and proBNP Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide BNP and proBNP immunoassay, BNP and proBNP immunodetection in Western blotting

Hybridoma cell lines producing MAbs were derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with human synthetic BNP (whole molecule) or synthetic BNP peptide 11FGRKMDRISSSS22 (for MAbs 24C5 and 26E2) conjugated with carrier protein. Following precise epitope mapping, the exact epitope of MAb

24C5 was determined as a.a.r. 11-17.

All antibodies recognize BNP and proBNP circulating in human blood, as well as synthetic BNP (Bachem, Peptide Institute) and recombinant proBNPs expressed in E. coli (Cat. # 8PRO9) and in mammalian cells (Cat. # 8GOB2).

2.1. Applications 2.1.1. BNP/proBNP sandwich immunoassay

Recommended pairs for sandwich immunoassay (capture – detection): 50E126–32 – 24C511–17 50E126–32 – 26E211–22 24C511–17 – 50B726–32 24C511–17 – 57H326–32 In Fig. 30, we present the calibration curve for sandwich fluoroimmunoassay utilizing MAb 50E126-32 as the capture and MAb 24C511-17 as the detection antibody. The analytical sensitivity of this immunoassay is better than 0.5 pg/ml (synthetic BNP, Bachem). The 50E126-32–24C511-17 immunoassay recognizes three

BNP-comprising polypeptides – BNP, recombinant proBNP (non-glycosylated, E. coli), and recombinant proBNP (glycosylated, mammalian cell line) with the same efficiency. A detailed description of this immunoassay has recently been published (9).

10000000 1000000 100000

CPS

We recommend several MAb combinations for sandwich immunoassay. All of the combinations were tested with plasma samples of HF patients and could be used for the development of highly sensitive, rapid sandwich immunoassays suitable for quantitative measurements of BNP and proBNP in human blood.

10000 1000 100 10 0,1

10

1000

100000

1E+07

BNP concentration, pg/ml

Figure 30. Calibration curve for BNP 50E126–32 – 24C511–17 sandwich fluoroimmunoassay.

Capture antibody: 50E126–32 (biotinylated) Detection antibody: 24C511–17 (Eu3+ - labeled) Antigen: synthetic BNP (Bachem) Incubation: mixture of antibodies (50 μl) and antigen (50 μl) is incubated for 30 minutes at room temperature in streptavidin-coated plate.

32

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

Analysis of antigens, purified from plasma of HF patients revealed that this immunoassay was able to detect both endogenous BNP and glycosylated proBNP. Moreover, proBNP was the major form contributing to immunological activity measured by this immunoassay (Fig. 31) (9, 12). Fig. 31 shows BNP immunoreactivity measurements in fractions following separation of one representative plasma sample of HF patient on Superdex Peptide gel-filtration column (GE-Healthcare). The BNP assay detected two picks of BNP immunoreactivity, the first corresponding to the proBNP form and the second corresponding to the BNP form.

Results of the stability studies of endogenous proBNP, purified from pooled plasma of HF patients and recombinant glycosylated proBNP (form mammalian cell line), which is the most similar to the endogenous one, being measured by 50E126-32-24C511-17 immunoassay see page 31.

For MAb 24C511-17 application in the “Single Epitope Sandwich” assay (SES assay) see pages 35-38 and reference 12.

proBNP

1

Ag concentration, ng/ml

0,9 0,8 0,7 0,6 0,5 0,4

BNP

0,3 0,2 0,1 0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

Fraction number

Figure 31. Gel-filtration studies of endogenous proBNP and BNP. BNP immunoreactivity measurements in fractions after proteins from plasma of HF patients were separated by gel-filtration on Superdex Peptide column (GEHealthcare). One representative plasma sample. BNP immunoreactivity was measured by 50E126-32-24C511-17 assay (- -). The first peak corresponds to proBNP, the second corresponds to BNP form.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

33

2.1.2. BNP and proBNP immunodetection in Western blotting All MAbs recognize synthetic BNP and recombinant proBNP (E. coli, mammalian cell line) in Western blotting

following antigen transfer onto nitrocellulose membrane (Fig. 32).

A

B

1

2

3

4

5

6

26.6 kDa

26.6 kDa

16.9 kDa 14.4 kDa 6.5 kDa

16.9 kDa 14.4 kDa 6.5 kDa

7

1

Figure 32. Immunodetection of human synthetic BNP (A), recombinant proBNP (E. coli) (B) and recombinant proBNP (mammalian cell line) (C) in Western blotting by different monoclonal antibodies after TricineSDS-PAGE under reducing conditions.

A, B. Lanes: 1- MAb 24C511-17, 2- MAb 26E211-22, 3- MAb 2G9, 4- MAb 43B1226-32, 5- MAb 50B726-32, 6- MAb 50E126-32, 7- MAb 57H326-32.

C. Lanes: 1 – MAb 24C511-17; 2 – 50B726-32; 3 – 57H326-32.

2

3

4

5

6

7

C

proBNP (mammalian cell line)

1

2

2.1.3. ProBNP sandwich immunoassay For more information see page 43.

Ordering information: MAb

Cat. #

Specificity

Subclass

Epitope

Application

24C5

4BNP2

BNP, proBNP

IgG1

a.a.r. 11-17

26E2

4BNP2

BNP, proBNP

IgG1

a.a.r. 11-22

43B12

4BNP2

BNP, proBNP

IgG2a

a.a.r. 26-32

50E1

4BNP2

BNP, proBNP

IgG1

a.a.r. 26-32

50B7

4BNP2

BNP, proBNP

IgG2a

a.a.r. 26-32

57H3

4BNP2

BNP, proBNP

IgG2a

a.a.r. 26-32

2G9

4BNP2

BNP, proBNP

IgG1

N/A

EIA, Sandwich immunoassay (capture, detection), WB EIA, Sandwich immunoassay (detection), WB EIA, Sandwich immunoassay (detection), WB EIA, Sandwich immunoassay (capture, detection), WB EIA, Sandwich immunoassay (capture, detection), WB EIA, Sandwich immunoassay (capture, detection), WB EIA, Sandwich immunoassay (detection), WB

34

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

3

3. Antibodies for New Type of BNP immunoassay – “Single Epitope Sandwich” (SES) assay New data!

MAb 24C5 Antigen: Host animal: Cell line used for fusion: Antigen specificity: Epitope specificity: Purification method: Presentation: Application:

Synthetic peptide, corresponding to human BNP sequence 11FGRKMDRISSSS22, conjugated with carrier protein.

Balb/c mice Sp2/0 Human BNP or proBNP Fragment 11-17 of human BNP molecule Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide Capture MAb in Single Epitope Sandwich assay

MAb Ab-BNP2 Antigen: Host animal: Cell line used for fusion: Specificity: Purification method: Presentation: Application:

Immune complex consisting of MAb 24C5 and human synthetic BNP Balb/c mice Sp2/0 Immune complex of BNP-specific MAb 24C5 with human BNP or proBNP Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide Detection MAb in Single Epitope Sandwich assay

Brain natriuretic peptide (BNP) is an acknowledged marker of heart failure (HF) that is widely used in clinical practice for HF diagnosis and patient management.

wich BNP immunoassay. This feature provides additional advantages to the SES assay over conventional BNP assays in terms of a higher apparent stability of BNP antigen in the sample or bloodstream.

BNP is known as an unstable molecule (13, 14). Several recent studies have revealed that BNP is presented by multiple forms in HF patients’ plasma, truncated from both N- and C-termini and only a small portion of BNP circulates as a full-size BNP32 molecule (15).

The majority of commercially available BNP assays are designed as sandwich-type immunoassays utilizing two MAbs specific to distantly located epitopes.

At least one of these two antibodies is specific to the ring structure, while the other one is usually specific to the C-terminus of the BNP molecule. Recent data regarding BNP instability in circulation suggests that immunoassays utilizing at least one MAb specific to the terminal epitope could underestimate the real BNP content in the blood sample.

Sensitivity of the SES assay. The prototype assay, designed by HyTest specialists to evaluate antibodies and the principle of the single epitope sandwich is a one-step assay utilizing biotinylated capture MAb 24C5 and detection MAb Ab-BNP2 labeled with stable Eu3+ chelate. Both MAbs and antigen are simultaneously incubated in streptavidin-coated plates and the assay time is 35 min. The limit of detection of the SES assay is 0.4 pg/ml (human synthetic BNP, Peptide Institute, Japan). This is the highest sensitivity described in literature for all commercial and experimental BNP assays. A detailed description of the prototype SES BNP assay has recently been published (16).

HyTest specialists have recently developed antibodies for a brand new type of BNP immunoassay – the “Single Epitope Sandwich” immunoassay (SES assay) - which differs from all commercially available “conventional”- type sandwich BNP assays (16). In the SES assay the capture antibody (MAb 24C5, epitope 11-17), which is specific to the relatively stable ring part of BNP molecule recognizes antigen. The detection antibody is specific only to the complex of the capture antibody with the BNP (or proBNP) and does not recognize these two molecules, (capture antibody and BNP) separately. Therefore only a single epitope of BNP molecule is needed for this novel type of sand-

Interaction with BNP and proBNP forms. According to the recent studies, the major portion of BNP immunoreactivity in the patient’s blood is not presented by BNP, but in fact is presented by proBNP (9, 17). To be precise in measurements of BNP immunoreactivity in the sample, assay antibodies should recognize BNP and proBNP with the same efficiency.

The SES assay recognizes three forms displaying BNP immunoreactivity – BNP, non-glycosylated proBNP and glycosylated proBNP – with the same efficiency (Fig. 33). When the SES BNP assay was tested with plasma samples of HF patients, it was shown to be suitable for precise quantification of circulating BNP and proBNP molecules.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

35

10000000 1000000

CPS

100000 10000 1000 100

0,0001

0,001

0,01

0,1

1

10

100

Ag concentration, pmol/ml

Antigen stability studies. As it was mentioned above, BNP is known as a very unstable molecule that is easily cleaved by endogenous proteases in human blood. However, in SES assay, in which MAbs need the single relatively stable central epitope for sandwich formation, BNP displays significantly higher stability, than when measured in conventional sandwich assays, comprising antibodies with distant epitopes.

In Fig. 34 results of stability studies of synthetic BNP and endogenous antigen from plasma of HF patients are presented. Stability was assessed by SES assay and conventional type assay prototype utilizing MAbs with distant epitopes – MAb 50E126-32 as capture and

A

Figure 33. Recognition of different antigen forms displaying BNP-immunoreactivity by SES assay. Calibration curves for three different antigens: – – synthetic BNP, – – recombinant proBNP (glycosylated, expressed in mammalian cell line; HyTest Cat.# 8GOB2), – – recombinant proBNP (non glycosylated, expressed in E. coli).

MAb 24C511-17 as detection. Samples were incubated at room temperature for different time periods lasting up to 24 hours. Compared to the conventional sandwich assay, the apparent stability of the synthetic antigen is significantly higher when measured by SES assay. While approximately 95% of BNP immunoreactivity was observed with SES assay after 24 hours of incubation, only 62% of initial BNP immunoreactivity was detected when the conventional sandwich immunoassay was used. Furthermore, the apparent stability of the endogenous peptide was also higher when BNP immunoreactivity in individual HF plasma sample incubated for different time periods was measured by the SES assay.

B

120

100

BNP-immunocreactivity, %

BNP-immunocreactivity, %

100

120

80 60 40 20 0 0

4

8

12

16

20

24

80 60 40 20 0 0

Incubation time, hours

12

24

Incubation time, hours

Figure 34. Stability studies of BNP. (A) Synthetic BNP (Peptide Institute, Japan) reconstituted in individual normal human EDTA-plasma and (B) Individual HF patient EDTA-plasma were incubated at RT (24 ºC) for different time periods. BNP immunoreactivity was tested in the SES assay (- -) and conventional type BNP assay utilizing two MAbs 50E126-32 and 24C511-17 specific to the distant epitopes (- -).

BNP measurements in HF plasma. BNP immunoreactivity measurements (BNP and proBNP) in individual EDTA-plasma samples of HF patients performed by two types of BNP assays also revealed the superiority of the single epitope principle over the conventional one. BNP concentration in 94 HF plasma samples was quantified by SES assay prototype and by commercially available conventional type Siemens ADVIA Centaur BNP immunoassay. The Siemens assay utilizes one MAb specific to the C-terminus (epitope 2732) and another MAb specific to the ring structure of 36

BNP molecule (epitope 14-21) (18). Both assays were calibrated with recombinant proBNP, expressed in E. coli. In all plasma samples, when compared with the results obtained by Siemens BNP immunoassay, SES assay detected significantly more BNP; from 1.2 to 7.2-fold; 2.1±0.9 (mean±SD). BNP concentration in seven plasma samples (7.4% of a total of 94 samples) when measured by SES assay was from 3 to 7.2-fold higher than that measured by Siemens assay. Results of the BNP measurements in 94 HF patients plasma are presented in Fig. 35.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

9,2 8,8 8,4 8 7,5 7,2 6,8

BNP concentration, ng/ml

6,4 5 5,8 5,2 4,8 4,4 4 3,6 3,2 2,8 2,4 2 1,8 1,2 0,8 0,4 93

91

89

87

85

83

81

79

77

75

73

71

69

67

65

63

61

59

57

55

53

51

49

47

45

43

41

39

37

35

33

31

29

27

25

23

21

19

17

15

13

9

11

7

5

3

1

0

HF patient samples

Figure 35. BNP measurements in 94 HF patients’ plasma samples. BNP concentration was measured by the SES (green bars) and Siemens (brown bars) BNP assays. Cases when the concentrations measured by SES assay were 3 to 7.2-fold higher than those measured by Siemens assay are marked by red ovals.

As can be seen from Fig. 35 Siemens assay underestimates circulating BNP concentrations in HF patients. This observation can at least be explained by the fact that one of the MAbs, utilized in the Siemens assay, is specific to the epitope 27-32 and cannot recognize BNP forms truncated from the C-terminus. The HyTest SES assay, being significantly less sensitive to the proteolytic degradation of the antigen, is capable of detecting all forms displaying BNP-immunoreactivity: intact and terminus-truncated antigens. The SES assay appears to be a preferable assay for the absolute BNP quantification in human blood.

The true BNP values are required to make a correct diagnosis for patients admitted to emergency depart-

ment (ED) with symptoms of HF. Specific “rule out” (BNP<100 pg/ml) and “rule in” (BNP>400 pg/ml) values are currently used by cardiologists to make the most accurate diagnosis in ED (19). Fig. 36 represents the difference in BNP content for four selected patients measured by SES and Siemens assays. Being measured by the Siemens BNP assay these patients (especially patient #4) could be misclassified (uncertainty zone: concentration range from 100 pg/ml to 400 pg/ml) and could therefore be mistakenly diagnosed.

When measured by the SES assay, the same patients undoubtedly belong to the “rule in” zone. This example confirms the idea that the SES assay approach is a preferable for the precise BNP quantification.

HF is definitely present

800

600 500 400

Uncertainty zone

BNP concentration, pg/ml

700

300 200 100

1

2

3

HF patient samples

4

Figure 36. BNP measurements in plasma of four selected HF patients by two BNP assays. BNP concentration was measured by the SES (green bars) and Siemens (red bars) assays. When measured by the Siemens assay these patients may have unconfirmed HF (BNP concentrations in the range of 100400 pg/ml), whereas measured by the SES assay - confirmed HF (more than 400 pg/ml). The lower limit of the uncertainty zone or unconfirmed HF (100 pg/ml) is marked as a green dash line; the lower limit of the zone where HF is definitely present (400 pg/ml) and is marked as red dash line.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

37

P: HyTest Ltd. WO 2008/125733 A1 IMMUNOASSAY FOR QUANTIFICATION OF AN UNSTABLE ANTIGEN SELECTED FROM BNP AND PROBNP

Ordering information: MAb

Cat. #

Specificity

Isotype

Epitope

Application

24C5

4BNP2

BNP, proBNP

IgG1

a.a.r. 11-17

SES assay (capture)

Ab-BNP2

4BFab5

Immune complex

IgG2a

N/A

SES assay (detection)

(MAb 24C5/BNP/proBNP)

Ordering information: Product

Cat. #

Purity

Application

Human recombinant proBNP (E. coli)

8PRO9

>95%

BNP assay standard or calibrator

Glycosylated human recombinant proBNP

8GOB2

>95%

BNP assay standard or calibrator

4. Anti-NT-proBNP monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Specificity: Purification method: Presentation:

Mice Balb/c Sp2/0 Synthetic peptides, corresponding to different regions of human NT-proBNP, conjugated with carrier protein Human NT-proBNP and proBNP Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide

Hybridomas producing MAbs were generated after immunization of Balb/c mice with synthetic peptides, corresponding to different parts of NT-proBNP sequence, conjugated to carrier protein. All antibodies

were checked on their ability to recognize recombinant NT-proBNP and proBNP expressed in E. coli (Cat.# 8NT2, 8PRO9) in direct ELISA, sandwich immunoassay and Western blotting.

4.1. Applications 3

All MAbs recognize recombinant human NT-proBNP and proBNP expressed in E. coli in direct ELISA (Fig. 37).

A 490 nm

4.1.1. Direct ELISA 2

1

0 0,00001

0,0001

0,001

0,01

0,1

1

MAb μg/well

Figure 37. Titration curves of different MAbs in ELISA. MAbs: 5B6 ( ), 13G12 (V), 11D1 (O), 15F11(Q) Antigen: human recombinant NT-proBNP expressed in E. coli, 0.01 μg/well

38

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

4.1.2. NT-proBNP quantitative sandwich immunoassays All HyTest NT-proBNP monoclonal antibodies specific to different parts of the molecule were tested in sandwich immunoassay as capture and detection antibodies with recombinant NT-proBNP and proBNP expressed in E. coli as well as with serum/plasma samples from HF patients. It was demonstrated that antibody pairs using at least one of the MAbs specific to the very N-terminal region (a.a.r. 1-12) or the mid fragment of NT-proBNP (a.a.r. 28-56) were unable to recognize endogenous NT-proBNP well. In contrast, the same pairs that were not able to recognize endogenous protein detected recombinant NT-proBNP or proBNP expressed in prokaryotic cells (E. coli) with high sensitivity. It was shown that antibodies specific to the very N-terminal part of the molecule were unable to recognize endogenous NT-proBNP due to its proteolytic degradation. Meanwhile glycosylation was the major reason for antibodies specific to the central region of

NT-proBNP being unable to recognize endogenous protein. MAb pairs with one of the antibodies specific to region 5-12 or 13-27 and another antibody specific to region 61-76 demonstrated the highest signal with endogenous NT-proBNP (8).

Therefore, to ensure precise quantitative NT-proBNP measurements in human blood we recommend using two-site antibody combinations utilizing antibodies specific to the N- or C-terminal parts of NT-proBNP molecule (Fig. 38). The best recommended pairs for precise NT-proBNP sandwich immunoassay are (capture – detection): 15C463-71 -13G1213-20 15C463-71 -29D125-12 15F1113-24-24E1167-76 15C463-71 -18H513-20

oligosaccharide residues

76

1 Epitopes recognized by MAbs

5-12 a.a.r.

13-27 a.a.r.

61-76 a.a.r.

Figure 38. Epitope location of MAbs, which are recommended for the development of NT-proBNP sandwich immunoassays.

10000000 1000000 100000

CPS

All assays utilizing recommended MAb combinations demonstrate high sensitivity (10–15 pg/ml) and good kinetics. A representative calibration curve for the assay 15C463-71–13G1213-20 is shown in Fig. 39 and a detailed description of this immunoassay has recently been published (9). All of the best MAbs combinations were tested with plasma/serum samples from HF patients to demonstrate the ability of antibodies to recognize the antigen circulating in human blood.

10000 1000 100 10

Figure 39. Calibration curve for NT-proBNP 15C4-13G12 assay: Capture antibody: 15C463-71 (biotinylated) Detection antibody: 13G1213-20 (Eu3+ - labeled) Antigen: human recombinant NT-proBNP expressed in E. coli Incubation: mixture of both antibodies (50 μl) and antigen (50 μl) is incubated for 30 minutes at room temperature in streptavidin-coated plate.

0,001

0,01

0,1

1

10

100

1000

NT-proBNP concentration (ng/ml)

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

39

A

80 60 40 20 0 1

2

3

4

5 HF patients

6

7

B

100 NT-proBNP concentration, ng/ml

NT-proBNP concentration, ng/ml

100

80 60 40 20 0 1

8

2

3

4 5 HF patients

6

7

8

Figure 40. Comparison of endogenous NT-proBNP immunoreactivity before and after deglycosylation in several representative samples. Concentration of endogenous NT-proBNP before (green columns) and after (violet columns) deglycosylation were measured by sandwich immunoassays: 15C463-7113G1213-20 (A), and 11D131-39-13G1213-20 (B). Monoclonal antibodies used in the former assay are specific to the N- or C-terminal parts of the molecule (not glycosylated). MAb 11D131-39 utilized in the latter assay as a capture antibody, is specific to the central glycosylated region.

To elucidate how glycosylation influences measurements of NT-proBNP, assay 15C463-71 -13G1213-20 was tested with NT-proBNP extracted from 52 HF patients plasma samples and then treated by a mixture of enzymes removing O-linked oligosaccharides (8). As a control, the 11D131-39 -13G1213-20 assay, which is highly susceptible to NT-proBNP glycosylation, was used. Immunoreactivity measured by assay 11D131-39 -13G1213-20 in the samples after deglycosylation in some cases was up to 40 fold higher than in the case of untreated NT-proBNP. It was observed that changes of measurable concentrations after deglycosylation were different in different blood samples. In some cases, measured concentrations increase only

40

2-3 fold, while in other cases it was 10 fold or even >20 fold. It was concluded that NT-proBNP is glycosylated in all analyzed patients and that the level of NT-proBNP glycosylation is different in the blood of individual patients. The assay 15C463-71-13G1213-20 using MAbs specific to the terminal parts of the molecule, was not as sensitive to deglycosylation of endogenous protein from individual blood samples.

Only a small growth of the signal was observed following deglycosylation (mean 1.37 fold). It was therefore concluded that antibodies specific to the N- and C-terminal parts of the NT-proBNP molecule (but not to the very N-terminal) are the best choice for the development of the precise NT-proBNP assay.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

4.1.3. Stability studies of endogenous NT-proBNP 120 100 Immunological activity, %

Stability of endogenous NT-proBNP after incubation of patients’ serum samples at two temperatures (+4°C and +20°C) for different time periods, was analyzed by antigen measurements in sandwich immunoassay 15C463-71-13G1213-20 (Fig. 41). Less than 10% of the initial immunological activity was lost following 72 hours of incubation at +4°C and approximately 10–15% following incubation of the sample after 24 hours at room temperature. Therefore serum samples could be stored in refrigerator (or even at room temperature) for a relatively long period until NT-proBNP concentration is measured by immunoassays, utilizing MAbs specific to the region 13-24 and 61-76.

80 60 40 20 0 0

12

24

36

48

60

72

Incubation time, hours Figure 41. Stability studies of endogenous NT-proBNP being measured in 15C4-13G12 sandwich immunoassay. Pooled blood serum from patients with HF was incubated at +4ºC ( ) and at room temperature ( ) for 72 hours.

4.1.4. NT-proBNP and proBNP immunodetection in Western blotting All MAbs recognize human NT-proBNP and proBNP expressed in E. coli in Western blotting studies following antigen transfer onto nitrocellulose membrane (Fig. 42). MAbs specific to N-terminal region (1-27) recognize

recombinant proBNP expressed in mammalian cell line after transfer onto nitrocellulose membrane (Fig. 43) in contrast to antibodies specific to the region 28-76.

proBNP (mammalian cell line)

NT-proBNP (E. coli)

1

2

3

4

1

2

3

4 5 6 7 8

9 10

5

Figure 42. Detection of human recombinant NT-proBNP expressed in E. coli in Western blotting by different monoclonal antibodies after Tricine-SDS gel electrophoresis.

Lanes: 1 – MAb 5B61-12; 2 – 15F1113-24; 3 – 11D131-39; 4 – 15D748-56; 5 – 24E1167-76.

Antigen: recombinant NT-proBNP expressed in E. coli, 2.5 μg/well.

Figure 43. Detection of human recombinant proBNP expressed in mammalian cells in Western blotting by different monoclonal antibodies after Tricine-SDS gel electrophoresis.

Lanes: 1 – MAb 5B61-12; 2 – 29D125-12; 3 – 15F1113-24; 4 – 13G1213-20; 5 – 18H513-20; 6 – 16F313-20; 7 – 11D131-39; 8 – 15D748-56; 9 – 15C463-71; 10 – 24E1167-76.

Antigen: recombinant proBNP expressed in mammalian cells (Cat.# 8GOB2), 0.15 μg/line.

MAbs 11D131-39, 15D748-56, 15C463-71 and 24E1167-76 does not recognize glycosylated proBNP expressed in the mammalian cell line.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

41

Ordering information: Antigen recognition MAb

Cat. #

5B6

4NT1

29D12

4NT1

13G12

4NT1

18H5

4NT1

16F3

4NT1

15F11

4NT1

7B5

4NT1

11D1

4NT1

16E6

4NT1

15D7

4NT1

15C4

4NT1

24E11

4NT1

28F8

4NT1

42

Specificity

Human NT-proBNP Human NT-proBNP Human NT-proBNP Human NT-proBNP Human NT-proBNP Human NT-proBNP Human NT-proBNP Human NT-proBNP Human NT-proBNP Human NT-proBNP Human NT-proBNP Human NT-proBNP Human NT-proBNP

Human recombinan NT-proBNP, proBNP (E. coli)

Human endogenous NT-proBNP, (glycosylated)

Human endogenous proBNP, (glycosylated)

Subclass

Epitope, a.a.r.

IgG1

1-12

+

–

–

IgG2a

5-12

+

+

+

IgG2a

13-20

+

+

+

IgG1

13-20

+

+

+

IgG1

13-20

+

+

+

IgG2b

13-24

+

+

+

IgG1

13-24

+

+

+

IgG1

31-39

+

–

–

IgG1

34-39

+

–

–

IgG1

48-56

+

–

–

IgG2b

63-71

+

+

–

IgG2a

67-76

+

+

–

IgG2a

67-76

+

+

–

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

Application

Sandwich immunoassays, EIA, WB Sandwich immunoassays, EIA, WB Sandwich immunoassays, EIA, WB Sandwich immunoassays, EIA, WB Sandwich immunoassays, EIA, WB Sandwich immunoassays, EIA, WB Sandwich immunoassays, EIA, WB Sandwich immunoassays, EIA, WB Sandwich immunoassays, EIA, WB Sandwich immunoassays, EIA, WB Sandwich immunoassays, EIA, WB Sandwich immunoassays, EIA, WB Sandwich immunoassays, EIA, WB

5. ProBNP quantitative sandwich immunoassays 10000000 1000000 100000 10000 CPS

According to recent studies carried out by HyTest, proBNP is the predominant form displaying BNP immunoreactivity in the blood of HF patients (9). Therefore, proBNP measurements by assays utilizing one antibody specific to the BNP and another specific to the NT-proBNP part of the molecule could be of the same clinical value as BNP measurements by conventional BNP assays, utilizing both MAbs specific to the BNP peptide. For the development of proBNP assays HyTest specialists recommend using one MAb specific to the region 13-27 (which is not occupied with polysaccharide residues) of NT-proBNP (Cat. # 4NT1) and another MAb specific to region 11-22 or 2632 of BNP (Cat. # 4BNP2) (Fig. 46). In contrast to NTproBNP immunodetection, antibodies specific to the region 61-76 could not be used for the development of proBNP assay. This is because this region is glycosylated in endogenous proBNP molecules, in contrast to NT-proBNP (5).

100 10 0,1

As a calibrator or standard for proBNP immunoassays HyTest specialists recommend using glycosylated proBNP, expressed in eukaryotic cells. It is better than antigen expressed in E. coli since it better represents the endogenous protein and has higher stability (Fig. 45).

100000

10000000

120 100 80 60 40 20 10 0

Analytical sensitivity of the assay 50E1BNP 26-32 – 16F3NT-proBNP 13-20 (recombinant proBNP expressed in E. coli used as calibrator) is better than 3 pg/ml (Fig. 44).

1000

Figure 44. Calibration curve for proBNP 50E1-16F3 sandwich immunoassay.

Capture antibody: 50E1BNP 26–32 (biotinylated) Detection antibody: 16F3NT-proBNP 13–20 (Eu3+labeled).

Antigen: Recombinant proBNP expressed in E. coli Incubation: Mixture of antibodies (50 μl) and antigen (50 μl) is incubated for 30 minutes at room temperature in streptavidin-coated plate.

Immunological activity, %

These pairs demonstrate high sensitivity, good kinetics and recognize recombinant proBNP expressed in E. coli and in mammalian cell line, as well as proBNP in HF patients’ blood.

10

ProBNP concentration, pg/ml

Recommended pairs for sandwich immunoassay are (capture – detection) : 50E1BNP 26-32 – 16F3NT-proBNP 13-20 50E1BNP 26-32 – 18H5NT-proBNP 13-20 7B5NT-proBNP 13-20 – 2G9BNP

1000

12

24

36

48

72

72

64

Figure 45. Stability studies of recombinant proBNP expressed in E. coli (- -) and in mammalian cell line (- -). Antigens were reconstituted in normal human citrate plasma and incubated at +4°C for 96 hours. Immunological activity was measured in sandwich immunoassay utilizing MAb 50E1BNP 26–32 as capture and MAb 16F3NT-proBNP13–20 as detection antibody.

oligosaccharide residues

11–22 a.a.r.

108

1 Figure 46. Epitope location of MAbs, which are recommended for the development of proBNP sandwich immunoassays.

Epitopes recognized by MAbs

96

Incubation time, hours

5–12 a.a.r.

26 –32 a.a.r.

13 –27 a.a.r.

NT-proBNP part

BNP part

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

43

6. BNP and NT-proBNP free plasma Source:

Method of purification : Delivery form : Storage:

Prepared from pooled normal human K3-EDTA plasma. Blood samples from donors were tested and found to be negative for HBs Ag, HIV-1 and HIV-2 antibodies, HCV and syphilis Immunoaffinity chromatography Frozen liquid -20ºC

BNP and NT-proBNP free plasma is prepared from pooled normal human K3-EDTA plasma by immunoaffinity chromatography. The affinity sorbent utilizes several MAbs with different epitope specificity

to eliminate not only intact proBNP and proBNP-derived molecules from plasma but also proBNP-proteolytic fragments. BNP and NT-proBNP free plasma could be used as a matrix for standard and calibrator preparation.

Ordering information: Product

Cat. #

Source

BNP and NT-proBNP free plasma

8BFP

Pooled normal human plasma

References: 1. Mair J, Hammerer-Lercher A, Puschendorf B. The impact of cardiac natriuretic peptide determination on the diagnosis and management of heart failure. Clin Chem Lab Med 2001;39:571-88.

2. Cowie MR, Mendez GF. BNP and congestive heart failure. Prog Cardiovasc Dis 2002;44:293-321.

3. Pandey KN. Biology of natriuretic peptides and their receptors. Peptides 2005;26:901-32.

4. Schellenberger U, O’Rear J, Guzzetta A, Jue RA, Protter AA, Pollitt NS. The precursor to B-type natriuretic peptide is an O-linked glycoprotein. Arch Biochem Biophys 2006;451:160-6.

5. Semenov AG, Postnikov AB, Tamm NN, Seferian KR, Karpova NS, Bloshchitsyna MN, Koshkina EV, Krasnoselsky MI, Serebryanaya DV, Katrukha AG. Processing of proBNP is suppressed by O-glycosylation in the region close to the cleavage site. Clin Chem 2009; 55(3): 489-98.

6. Emdin M, Passino C, Prontera C, Fontana M, Poletti R, Gabutti A, Mammini C, Giannoni A, Zyw L, Zucchelli G, Clerico A. Comparison of brain natriuretic peptide (BNP) and amino-terminal ProBNP for early diagnosis of heart failure.Clin Chem 2007;53:1289-97.

7. Mueller T, Gegenhuber A, Poelz W, Haltmayer M. Diagnostic accuracy of B type natriuretic peptide and amino terminal proBNP in the emergency diagnosis of heart failure. Heart 2005;91:606-12.

8. Seferian KR, Tamm NN, Semenov AG, Tolstaya AA, Koshkina EV, Krasnoselsky MI, Postnikov AB, Serebryanaya DV, Apple FS, Murakami MM, Katrukha AG. Immunodetection of glycosylated NT-proBNP circulating in human blood. Clin Chem 2008;54:866-73.

9. Seferian KR, Tamm NN, Semenov AG, Mukharyamova KS, Tolstaya AA, Koshkina EV, Kara AN, Krasnoselsky MI, Apple FS, Esakova TV, Filatov VL, Katrukha AG. The brain natriuretic peptide (BNP) precursor is the major immunoreactive form of BNP in patients with heart failure. Clin Chem 2007; 53:866-73.

10. Giuliani I, Rieunier F, Larue C, Delagneau JF, Granier C, Pau B, Ferrière M, Saussine M, Cristol JP, Dupuy AM, Merigeon E, Merle D, Villard S. Assay for measurement of intact B-type natriuretic peptide prohormone in blood. Clin Chem 2006;52:1054-61.

44

11. Liang F, O’Rear J, Schellenberger U, Tai L, Lasecki M, Schreiner GF, Apple FS, Maisel AS, Pollitt NS, Protter AA. Evidence for functional heterogeneity of circulating B-type natriuretic peptide. J Am Coll Cardiol 2007;49:1071-8.

12. Tamm NN, Seferian KR, Semenov AG, Mukharyamova KS, Koshkina EV, Krasnoselsky MI, Postnikov AB, Serebryanaya DV, Apple FS, Murakami MM, Katrukha AG. Novel immunoassay for quantification of brain natriuretic peptide and its precursor in human blood. Clin Chem 2008;54:1511-8.

13. Belenky A, Smith A, Zhang B, Lin S, Despres N, Wu AH, Bluestein BI. The effect of class- specific protease inhibitors on the stabilization of B-type natriuretic peptide in human plasma. Clin Chim Acta 2004;340:163-72.

14. Murdoch DR, Byrne J, Farmer R, Morton JJ. Disparity between studies of the stability of BNP in blood: comparison of endogenous and exogenous peptide.

Heart 1999;81:212 15. Niederkofler EE, Kiernan UA, O’Rear J, Menon S, Saghir S, Protter AA, Nelson RW, Schellenberger U. Detection of endogenous B-type natriuretic peptide at very low concentrations in patients with heart failure. Circ Heart Fail.

2008 Nov;1(4):258-64.

16. Tamm NN, Seferian KR, Semenov AG, Mukharyamova KS, Koshkina EV, Krasnoselsky MI, Postnikov AB, Serebryanaya DV, Apple FS, Murakami MM, Katrukha AG. Novel immunoassay for quantification of brain natriuretic peptide and its precursor in human blood. Clin Chem 2008;54:1511-8.

17. Nishikimi T et.al, Diversity of molecular forms of plasma brain natriuretic peptide in heart failure-different proBNP-108 to BNP-32 ratios in atrial and ventricular overload. Heart. 2010;96(6):432-9.

18. Wu AH et al. Analytical and clinical evaluation of the Bayer ADVIA Centaur automated B-type natriuretic peptide assay in patients with heart failure: a multisite study. Clin Chem. 2004;50(5):867-73.

19. Maisel A. et al. State of the art: Using natriuretic peptide levels in clinical practice. European Journal of Heart Failure 2008; 10; 824-839.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

IV Myoglobin Myoglobin is a small heme-containing protein (153 amino acid residues, molecular weight (w/o heme) 17053 Da and theoretical pI=7.29), that is responsible for the oxygen deposition in muscle tissues. Only one form of myoglobin is expressed in cardiac and skeletal muscles.

Myoglobin is known as a marker of myocardial damage and it has been used for more than three decades (1). Nowadays, it continues to be is very commonly used in clinical practice as an early marker of AMI (2). It appears in patients’ blood 1 – 3 hours following onset of the symptoms, reaching peak level within 8 – 12 hours (3). Myoglobin is not so cardiac

specific as cTnI or cTnT. Due to high myoglobin concentration in skeletal muscle tissue, even minor skeletal muscle injury results in the significant increase of myoglobin concentration in blood (4). Therefore, myoglobin is used together with cTnI or cTnT in clinical practice for improved specificity in AMI diagnosis.

Myoglobin is purified from human cardiac tissue by several chromatographic steps including gel-filtration and anion-exchange chromatography. After SDS-PAGE in reducing conditions myoglobin is presented by a single band with apparent molecular mass of 17 kDa (Fig. 47).

Figure 47. SDS-PAGE of human myoglobin in reducing conditions.

Lane 1: Molecular weight standards, Pharmacia (94, 67, 43, 30, 20 and 14.4) Lanes 2 and 3: Human myoglobin 3 μg and 6 μg, respectively.

Gel staining: Coomassie brilliant blue R-250.

1

2

In 2001 HyTest’s myoglobin preparation was selected by International Federation of Clinical Chemistry as an International Standard Material.

3

1. Human myoglobin Source: Purity: Presentation: Application: Storage:

Human cardiac tissue. Blood sample from the tissue donors was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis >98% Lyophilized Immunogen for antibody production, immunological and mass myoglobin standard, myoglobin biochemical and immunochemical studies -20°C

Ordering information: Product

Cat. #

Purity

Source

Human myoglobin

8M50

>98%

Human cardiac muscle

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

45

2. Anti-myoglobin monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation:

Mice Balb/c Sp2/0 Human myoglobin Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide

Hybridoma clones have been derived from hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with purified human myoglobin. Different combinations of monoclonal antibod-

ies can be used for the immunoassay development.

The best MAb combinations for sandwich immunoassay are: 4E2 – 7C3 7C3 – 4E2

Ordering information: MAb

Cat. #

Specificity

Subclass

Application

4E2 7C3

4M23 4M23

Myoglobin Myoglobin

IgG1 IgG1

EIA, Sandwich immunoassay (capture) EIA, Sandwich immunoassay (detection)

3. Myoglobin free serum Pooled normal human serum Immunoaffinity chromatography Frozen liquid –20°C

Myoglobin free serum is prepared from pooled normal human serum by immunoaffinity chromatography. The matrix for affinity sorbent utilizes several monoclonal antibodies with different epitope specificity. Based on ELISA testing the level of myoglobin in myoglobin free serum is below 0.1 ng/ml (Fig. 48).

2500

2000

CPS

Prepared from: Method of purification: Delivery form: Storage:

1500

1000

Myoglobin free serum is used as a matrix for standard and calibrator preparations.

500

0 1

2

3

Figure 48. Myoglobin level in normal human serum and in myoglobin free serum detected in sandwich-immunoassay. Column 1: Buffer, column 2: Pooled normal human serum before myoglobin extraction (4 ng/ml of myoglobin) and column 3: Residual signal in myoglobin free serum.

Ordering information: Product

Cat. #

Source

Myoglobin free serum

8MFS

Pooled normal human serum

References: 1. Kagen L, Scheidt S, Butt A. (1977) Serum myoglobin in myocardial infarction: the “staccato phenomenon.” Is acute myocardial infarction in man an intermittent event? Am J Med. 62(1): 86-92.

2. Penttila K. et al. (2002) Myoglobin, creatine kinase MB isoforms and creatine kinase MB mass in early diagnosis of myocardial infarction in patients with acute chest pain. Clin Biochem. 35(8): 647-53.

46

3. Srinivas VS. et al. (2001) Myoglobin levels at 12 hours identify patients at low risk for 30-day mortality after thrombolysis in acute myocardial infarction: a Thrombolysis in Myocardial Infarction 10B substudy. Am Heart J. 142(1): 29-36.

4. Van Nieuwenhoven FA. Et al. (1995) Discrimination between myocardial and skeletal muscle injury by assessment of the plasma ratio of myoglobin over fatty acid-binding protein. Circulation. 92(10): 2848-54.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

V Pregnancy Associated Plasma Protein A (PAPP-A) Pregnancy-associated plasma protein-A (PAPP-A) was first identified as a high-molecular weight constituent in human pregnancy serum. PAPP-A is a metalloprotease that belongs to the metzincin superfamily of zinc peptidases. The insulin-like growth factor binding protein 4 (IGFBP-4), as well as IGFBP-5 have been found to be specific substrates for PAPP-A in vitro (1, 2). Cleavage of IGFBP-4 and IGFBP-5 by PAPP-A causes release of bound IGF, which plays an important role in promoting cell differentiation and proliferation.

PAPP-A exists as a covalent (disulfide bridged) heterotetrameric complex in the blood of pregnant women and consists of two 200 kDa PAPP-A subunits and two 50-90 kDa subunits of the proform of eosinophil major basic protein (proMBP). Such a complex is denoted as heterotetrameric PAPP-A (PAPP-A/ proMBP or htPAPP-A) and it was shown that proMBP has inhibitory properties against protease activity of PAPP-A in htPAPP-A heteromeric complex (2). htPAPP-A is a widely recognized biochemical marker of Down syndrome (DS) that is used in the first trimester of pregnancy. htPAPP-A level in maternal serum increases with gestational age until term. In cases of DS pregnancy, htPAPP-A concentration in the first trimester is markedly decreased (3). Therefore, htPAPP-A is currently used as a biochemical marker of DS in the first trimester of pregnancy in combination with free b-subunit of chorionic gonadotropin (bhCG) and nuchal translucency. Before this combination of markers was established, biochemical screening for DS was only performed in the second trimester (16-18 gestational weeks). This new approach in the DS screening allows starting medical intervention significantly earlier in comparison to previously used methods.

It was recently shown that another form of protein – homodimeric PAPP-A (dPAPP-A) with a molecular mass of approximately 400 kDa, is abundantly expressed in unstable coronary atherosclerotic plaques (4). Further-

more, it has been demonstrated that the blood level of dPAPP-A is significantly elevated in patients with unstable angina or acute myocardial infarction, as compared to patients with stable angina and control subjects (5, 6). In addition, dPAPP-A has also been shown to be a strong independent marker of risk stratification for patients with acute coronary syndrome (ACS) (7). The ACS-related form of PAPP-A, presumably originating from ruptured plaque, is not complexed with proMBP subunit, as it is in htPAPP-A. Therefore, it is supposed that in atherosclerotic plaque dPAPP-A functions as an active protease and can promote IGF release. Therefore, one may speculate that dPAPP-A influences the transformation of a stable atherosclerotic plaque into an unstable one. Such involvement of dPAPP-A in the pathophysiology of ACS suggests that it may serve as a marker of plaque destabilization and PAPP-A measurements in the patient’s blood could prove to be very helpful in identifying patients at the very beginning of the process of plaque disruption.

Structural difference of dPAPP-A from pregnancy related htPAPP-A makes possible immunochemical discriminations of these two PAPP-A forms (8).

Antibodies, specific to dPAPP-A that have no crossreaction with htPAPP-A could be used for the development of immunoassays for precise selective dPAPP-A measurements in human blood.

HyTest is the largest global supplier of htPAPP-A antigen purified from retroplacental blood. And HyTest is now able to offer a new product - recombinant dPAPP-A. Furthermore, we also now offer PAPP-A and proMBP specific monoclonal antibodies suitable for the development of highly sensitive and rapid sandwich-type PAPP-A immunoassays for quantitative detection of htPAPP-A in maternal blood, as well as for detection of dPAPP-A in the blood of ACS patients. We also present here a new generation of dPAPP-A specific monoclonal antibodies that only recognize recombinant and atherosclerotic dPAPP-A and have no crossreaction with the htPAPP-A form of antigen.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

47

Heterotetrameric form, htPAPP-A

Homodimeric form, dPAPP-A

Structure

PAPP-A

SS SS SS

SS SS

SS SS

SS

proMBP subunit

Molecular weight, kDa

500

400

Enzyme (proteolytic) activity

Inhibited by proMBP

Presumably active

Diagnostic application

Down syndrome

Myocardial infarction

Sandwich immunoassay

Total PAPP-A, htPAPP-A sandwich immunoassays (see page 49)

Selective dPAPP-A sandwich immunoassays (see page 51)

1. Heterotetrameric PAPP-A/proMBP complex (htPAPP-A) Source: Purification method: Presentation: Application: Storage:

Pooled retroplacental blood Combination of various chromatographic methods Lyophilized Calibrator or standard for PAPP-A immunoassays –20°C

212 -

HyTest’s htPAPP-A is purified from the pooled retroplacental blood and purity is over 85% according to SDS-PAGE (Fig. 49). htPAPP-A is recognized by monoclonal antibodies specific to different parts of PAPP-A or proMBP (Cat # 4P41). Antigen can be used as a calibrator for total PAPP-A and htPAPP-A sandwich immunoassays (see pages 49 and 51).

Figure 49. SDS-gel electrophoresis of htPAPP-A in reducing conditions.

Lane 1: molecular weight standards Lanes 2, 3: human htPAPP-A Antigen loaded: 5 μg Gel staining: A: Coomassie brilliant blue R-250, B: Stains all (staining of glycosylated proteins).

Comments: proMBP subunit migrates in gel as a diffuse spot with molecular mass about 50-90 kDa and is not stained by Coomassie brilliant blue because of high degree of glycosylation (~40%).

- PAPPA-A, subunit

170 -

116 -

]

76 -

- proMBP, subunit

53 -

1

2 A

3 B

Ordering information: Product

Cat. #

Purity

Source

PAPP-A, heterotetrameric form (htPAPP-A)

8P64

>85%

Pooled retroplacental blood

2. htPAPP-A and total PAPP-A specific monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Specificity: Presentation: Application:

Mice Balb/c Sp2/0 Human htPAPP-A (Cat # 8P64) Protein A affinity chromatography MAbs recognize either PAPP-A or proMBP subunits of htPAPP-A heterotetrameric complex MAbs solution in PBS with 0.1% sodium azide htPAPP-A and dPAPP-A immunoassays, htPAPP-A and dPAPP-A immunoprecipitation, PAPP-A subunit immunodetection in Western blotting

Hybridoma clones producing MAbs have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with puri-

48

fied human htPAPP-A antigen (Cat.# 8P64). PAPP-A subunit specific MAbs recognize both of the known endogenous forms of the antigen - htPAPP-A or dPAPP-A.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

2.1. Applications 2.1.1. Total PAPP-A and htPAPP-A sandwich immunoassays All MAbs were tested in pairs in sandwich fluoroimmunoassay as capture and detection antibodies with both forms of the antigen - htPAPP-A and dPAPP-A. For quantitative PAPP-A immunodetection we recommend the use of several two-site antibody combinations.

A

100000

Table 8. Recommended pairs for sandwich immunoassay:

Detection of human htPAPP-A antigen (capture – detection) 10E2 – 5H9

10E2 – 10E1

5H9 – 10E2

4G11 – 3C8

5H9 – 7A6

4G11 – 10H9

10E1 – 11E4

10E1 – 7A6

100000

B

PAPPA-A 4G11-3C8

Detection of total PAPP-A (htPAPP-A and/or dPAPP-A (capture – detection)

PAPPA-A 4G11-10H9

10000

CPS

CPS

10000

1000

1000

1

10

1

100

10

100

PAPPA-A, ng/ml

PAPPA-A, ng/ml

Figure 50. Calibration curves for two PAPP-A sandwich immunoassays. (A) 4G11 – 3C8 and (B) 4G11 – 10H9.

Capture MAb: 4G11 (biotinylated) Detection MAbs: 3C8 or 10H9 (labeled with stable Eu3+ -chelate) Antigen: htPAPP-A Mixture of antibodies and antigen was incubated for 30 minutes at room temperature in streptavidin-coated plates.

2.1.2. PAPP-A immunodetection in Western blotting - 500 kDa

- 200 kDa

MAbs 18A10, 3C8 and 7A6 recognize PAPP-A subunit, whereas MAbs 5H9 and 11E4 recognize the proMBP subunit of htPAPP-A in Western blotting after SDS-gel electrophoresis in reducing and non-reducing conditions.

MAbs 4G11, 10A5 and 10E1 recognize htPAPP-A in Western blotting only after electrophoresis in nonreducing conditions (Fig. 51).

1

2

3

4

5

6

7

8

9

Figure 51. Detection of human PAPP-A and proMBP subunits of htPAPP-A by monoclonal antibodies in Western blotting.

Lane 1: 18A10 Lane 7: 10A5 Lane 2: 7A6 Lane 8: 3C8 Lane 3: 3C8 Lane 9: 10E1 Lane 4: 5H9 (proMBP-specific) Lanes 1-5: after SDS-PAGE in reduction conditions.

Lane 5: 11E4 (proMBP-specific) Lanes 6-9: Non-reducing conditions. Heterotetrameric Lane 6: 7A6 complex was detected by anti-PAPP-A MAbs

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

49

Ordering information: MAb 10E1 10E2 5H9 4G11 7A6 10A5 3C8 10H9 18A10 11E4

Cat.# 4P41 4P41 4P41 4P41 4P41 4P41 4P41 4P41 4P41 4P41

Specificity PAPP-A subunit PAPP-A subunit proMBP subunit PAPP-A subunit PAPP-A subunit PAPP-A subunit PAPP-A subunit PAPP-A subunit PAPP-A subunit proMBP subunit

Subclass IgG2b IgG2b IgG2b IgG2a IgG2a IgG2a IgG2a IgG2a IgG2a IgG2b

Application EIA (capture, detection), WB EIA (capture, detection) EIA (capture, detection), WB EIA (capture), WB EIA (detection), WB WB EIA (detection), WB EIA (detection) WB EIA (capture, detection), WB

PAPP52

4P41

PAPP-A subunit

IgG1

EIA (capture)

3. Recombinant homodimeric PAPP-A (dPAPP-A) Source: Purification method: Presentation: Application: Storage:

Expressed in mammalian cell line Combination of various chromatographic methods Frozen Calibrator or standard for dPAPP-A immunoassays -70°C

Recombinant human dPAPP-A is expressed in mammalian cells as a full-length protein (~200 kDa). Immunoreactivity of recombinant dPAPP-A is similar to immunoreactivity of endogenous protein (found in atherosclerotic plaques) and can therefore be used as a calibrator or standard in dPAPP-A immunoassays.

250250250-

The similarity of immunochemical properties of PAPP-A subunits of recombinant dPAPP-A – dPAPP-A purified from atherosclerotic arteries and htPAPP-A – was also confirmed by Western blotting analysis with MAbs specific to PAPP-A subunit (Fig. 52B).

130130-

130-

Moreover, identity of recombinant dPAPP-A to PAPP-A from human pregnancy blood was also corroborated by liquid chromatography/tandem mass spectrometry analysis (MS-MS).

9572-

9572-

5555-

55-

3628-

3628-

1

2 A

3

1

2

3

B

Figure 52.

A – SDS-PAGE analysis in reducing conditions of human recombinant dPAPP-A and tissue (purified from atherosclerotic plaques) dPAPP-A. Lane 1: human htPAPP-A (Cat.# 8P64) Lane 2: recombinant dPAPP-A (Cat.# 8P97) Lane 3: tissue dPAPP-A Gel staining: Coomassie brilliant blue R-250 Comments: proMBP subunit released from htPAPP-A upon reduction is not stained by Coomassie brilliant blue because of high degree of glycosylation.

50

Apparent molecular mass of PAPP-A subunit of recombinant protein in SDS-PAGE studies in reducing conditions was equal to molecular masses of PAPP-A purified from atherosclerotic tissue and PAPP-A from pregnancy serum – approximately 200 kDa (Fig. 52A).

B – Western blotting analysis of PAPP-A purified from atherosclerotic tissue. For immunostaining mixture of anti-PAPP-A MAbs (18A10, 7A6 and 3C8) (Cat.# 4P41) was used. Lanes are noted as in Fig.4A

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

It was also demonstrated that recombinant dPAPP-A is an active protease that cleaves IGFBP-4. Reaction of PAPP-A proteolytic activity was assayed as described in (9): 100 ng IGFBP-4 were incubated over night at 37°C in 2 mM CaCl2, 50 mM Tris, pH 7.5, contained 5 nM IGF-II with standard protease inhibitors, with either the presence or absence of 2 ng of recombinant dPAPP-A. Reaction was analyzed by SDS-PAGE with Western blotting detection using polyclonal anti-IGFBP-4 antibodies (Fig. 53).

553628-

IGFBP-4

17IGFBP-4 fragments

11-

1

2

Figure 53. A – Detection of protease activity of recombinant dPAPP-A. dPAPP-A cleaves recombinant IGFBP-4.

Lane 1 IGFBP-4, incubated with recombinant dPAPP-A (Cat.# 8P97) Lane 2: IGFBP-4 incubated w/o recombinant dPAPP-A

Ordering information: Product

Cat. #

Purity

Source

PAPP-A, homodimeric form (dPAPP-A)

8P97

>90%

Recombinant, expressed in mammalian cells

4. Anti-dPAPP-A monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Purified human dPAPP-A from atherosclerotic tissue Protein A affinity chromatography MAbs solution in PBS with 0.1% sodium azide Selective dPAPP-A immunoassay with no cross-reactivity to htPAPP-A, dPAPP-A immunoprecipitation, selective Western blotting detection

Hybridomas producing MAbs were generated after Balb/c mice immunization with dPAPP-A purified from human atherosclerotic arteries. Four anti-dPAPP-A MAbs (PAPP2, PAPP7, PAPP8 and PAPP30) were select-

ed based on to their specificity and affinity of interaction with recombinant and atherosclerotic tissue dPAPP-A. None of these three antibodies cross-react with the tetrameric form of the antigen and only recognize dPAPP-A.

4.1. Applications 4.1.1. Selective dPAPP-A sandwich immunoassay New We recommend several MAbs combinations for the development of dPAPP-A sandwich immunoassay.

One of the antibodies (capture) in such an assay is dPAPP-A specific, while another one (detection) can recognize all known forms of PAPP-A. All recommended combinations were tested with dPAPP-A purified from atherosclerotic coronary arteries, as well as with human recombinant dPAPP-A and htPAPP-A. All MAb combinations were able to recognize dimeric forms of the antigen with high specificity and had no cross-reaction with htPAPP-A.

data!

These MAb combinations could be used for the development of highly sensitive, rapid sandwich immunoassays that are suitable for the selective quantitative measurements of dPAPP-A in human blood.

Recommended pairs for sandwich immunoassay (capture – detection): PAPP52 – PAPP30 PAPP2 – 7A6

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

51

100000

O

O Atherosclerotic tissue dPAPPA-A Recombinant dPAPP-A Heterotetrameric PAPP-A

CPS

In Fig. 54, we have presented the calibration curve for the sandwich fluoroimmunoassay utilizing MAb PAPP2 as capture and MAb 7A6 as detection MAbs. The detection limit of such an immunoassay is better than 0.3 ng/ml with human recombinant dPAPP-A (Cat.# 8P97) used as a calibrator. This assay revealed no cross-reactivity with the heterotetrameric form of PAPP-A.

O

O

10000 O

Our new PAPP52-PAPP30 (capture-detection) sandwich immunoassay was developed for specific dPAPP-A measurement (Fig. 55). In this assay one antibody, PAPP30, is specific to dPAPP-A (without cross-reaction with htPAPP-A), whereas another one, PAPP52, is able to recognize all forms of PAPP-A. Detection MAb PAPP30 was labeled with stable Eu3+ chelate. The assay recognized endogenous and recombinant dPAPP-A and demonstrated very low cross-reactivity (<1%) with htPAPP-A. The analytical detection limit of the assay was less than 1 ng/mL when purified recombinant dPAPP-A (Cat.# 8P97) was used as a calibrator. dPAPP-A levels in plasma samples of 43 ACS patients (acute myocardial infarction, unstable angina, 3-20 hours following the onset of the chest pain) and 34 non-ACS patients (control group) were also measured (Fig. 56).

O O

1000 1 10 PAPP-A concentration, ng/ml

0,1

100

Figure 54. Calibration curves for new dPAPP-A immunoassay.

Capture MAb: PAPP2 Detection MAb: 7A6 (labeled by Eu3+ chelate) Incubation volume 100 μl. Incubation time: 30 min at room temperature.

The dPAPP-A levels in plasma from ACS patients were 2.77-fold higher than in plasma samples of the control group, measured by PAPP52-PAPP30 assay (P<0.0005). Therefore, the immunoassay was suitable for the direct measurement of dPAPP-A in patients’ plasma.

100000

Concentration of dPAPP-A, ng/ml

40

CPS

10000

1000

100 1

10

100

30

20

10

0 ACS

dPAPP-A concentration, ng/ml

Figure 55. Calibration curves for new PAPP52-PAPP30 dPAPP-A-specific sandwich immunoassay.

Capture MAb: PAPP52 Detection MAb: PAPP30 (labeled with Eu3+ chelate) Incubation volume: 100 μl.

Incubation time: 30 min at room temperature.

52

Normal

Figure 56. dPAPP-A concentration in plasma samples of 43 ACS patients (ACS) and 34 non-ACS patients control group (Normal) measured by PAPP52 - PAPP30 sandwich immunoassay (mean+/-SD).

Capture MAb: PAPP52 Detection MAb: PAPP30 (labeled with Eu3+ chelate) Incubation volume: 100 μl.

Incubation time: 30 min at room temperature.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

4.1.2. Selective dPAPP-A immunodetection in Western blotting New data!

MAbs PAPP2, PAPP7 and PAPP8 recognize dPAPP-A in non-reducing conditions following antigen transfer onto nitrocellulose membrane after SDS-gel electrophoresis (Fig. 57). However, they have no cross-reaction with human htPAPP-A. Therefore it is possible to make a selective Western blotting detection of the dimeric form of PAPP-A.

Figure 57. Detection of recombinant dPAPP-A and htPAPP-A by antidPAPP-A monoclonal antibodies in Western blotting.

Tested antibodies recognize dPAPP-A and have no cross-reaction with htPAPP-A. A: Recombinant dPAPP-A B: Human htPAPP-A Immunochemical staining by: Lane 1: MAb PAPP2 Lane 2: MAb PAPP7 Lane 3: MAb PAPP8.

1

2

3

A

1

2

3

B

Ordering information: MAb

Cat. #

Specificity

Subclass

Application

PAPP2 PAPP7 PAPP8

4PD4 4PD4 4PD4 4PD4

Human dPAPP-A Human dPAPP-A Human dPAPP-A Human dPAPP-A

IgG1 IgG1 IgG1 IgG1

EIA (capture) EIA (capture) EIA (capture) EIA (detection)

PAPP30

References: 1. Oxvig, C., Sand, O., Kristensen, T., Gleich GJ., Circulating human pregnancy associated plasma protein-A is disulfide-bridged to the proform of eosinophil major basic protein.// J Biol Chem 268:12243-6(1993).

2. Overgaard, MT., Haaning, J., Boldt, HB., Olsen, IM., Laursen, LS., et al. Expression of recombinant human pregnancy-associated plasma protein-A and identification of the proform of eosinophil major basic protein as its physiological inhibitor.// J Biol Chem; 275:31128-33 (2000).

3. Palomaki GE, Lambert-Messerlian GM, Canick JA A summary analysis of Down syndrome markers in the late first trimester.// Adv Clin Chem. 2007;43:177210.

4. Bayes-Genis, A., Conover, C. A., Overgaard, M. T., Bailey, K. R., Christiansen, M., Holmes, D. R. Jr, et al. Pregnancy-associated plasma protein A as a marker of acute coronary syndromes.// N Engl J Med, 345 (14), 1022-9 (2001).

5. Heeschen C, Dimmeler S, Hamm CW, Fichtlscherer S, Simoons ML, Zeiher AM. Pregnancy-associated plasma protein-A levels in patients with acute coronary syndromes Comparison with markers of systemic inflammation, platelet activation, and myocardial necrosis. //J Am Coll Cardiol 2005;45:229-37.

6. Hájek P, Macek M, Hladíková M, Houbová B, Alan D, Durdil V, Fiedler J, Malý M, Ostádal P, Veselka J, Krebsová A. Pregnancy-associated plasma protein A and proform eosinophilic major basic protein in the detection of different types of coronary artery disease.// Physiol Res. 2008;57(1):23-32.

7. Qin, Q.P.,Laitinen, P., Majamaa-Voltti, K., Eriksson, S., Kumpula, E.K., and Petterson, K. Release Patterns of Pregnancy Associated Plasma Protein A (PAPP-A) in Patients with Acute Coronary Syndroms.//Scand Cardiovasc J, 36(6), 358-61(2002).

8. Qin QP, Kokkala S, Lund J, Tamm N, Voipio-Pulkki LM, Pettersson K. Molecular distinction of circulating pregnancy-associated plasma protein A in myocardial infarction and pregnancy.// Clin Chem 2005;51:75-83.

9. Michael T. Overgaard, Jesper Haaning, Henning B. Boldt, Inger M. Olsen, Lisbeth S. Laursen, Michael Christiansen, Gerald J. Gleich, Lars Sottrup-Jensen, Cheryl A. Conoveri, and Claus Oxvig. Expression of Recombinant Human Pregnancy-associated Plasma Protein-A and Identification of the Proform of Eosinophil Major Basic Protein as Its Physiological Inhibitor.//The journal of biological chemistry; 2000;275, 40:31128-31133.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

53

VI Myeloperoxidase (MPO) Myeloperoxidase (MPO) is a hemoprotein that is abundantly expressed in polymorphonuclear leukocytes (neutrophils) and secreted during their activation. The presence of a peroxidase in the cytoplasmic granules of leukocytes was first suggested at the beginning of 20th century but it was only in the early 1940s that it was purified for the first time. Native MPO is a covalently bound tetrameric complex of two glycosylated alpha chains (MW 59 – 64 kDa) and two unglycosylated beta chains (MW 14 kDa) with total MW approximately 150 kDa and theoretical pI 9.2 (1).

MPO plays an important role in neutrophil microbicidal action through catalyzing chloride ion oxidation to hypochlorous acid, which is a potent antimicrobial agent. On the other hand, it was demonstrated that MPO causes oxidative modification of low density lipoprotein (LDL) to a high uptake form that is considered to be a key event in the promotion of atherogenesis (2). For this reason, MPO is believed to participate in the initiation and progression of cardiovascular diseases. MPO possesses potent proinflammatory properties and may directly contribute to tissue injury. In addition, MPO is demonstrated to be involved in pathogenesis of lung cancer (3), Alzheimer’s disease (4) and multiple sclerosis (5).

MPO is now believed to be one of the most promising cardiac markers. It was recently demonstrated that an increased MPO level in patient’s blood serves as a risk marker for atherosclerosis (6) and coronary

artery disease (7). It predicts the early risk of myocardial infarction, as well as the risk of other major adverse cardiac events in patients with chest pain in the ensuing 30-day and 6-month periods (8, 9).

The value of MPO as a marker is that MPO predicts these outcomes independently of other known laboratory tested risk factors, including troponins, creatine kinase MB isoform (CK-MB), C-reactive protein (CRP) and lipid profile. Moreover, unlike troponins I and T, CK-MB, and CRP, MPO makes it possible to identify patients at risk for cardiac events in the absence of myocardial necrosis (8). All of these factors make MPO measurements in patients an indispensable procedure to reveal patients with chest pain that are at an increased risk of cardiovascular complications.

There are some autoimmune diseases connected with the development of autoantibodies against MPO. MPO is a main target of anti-neutrophil cytoplasm antibodies (ANCA) - serological markers for certain systemic vasculitides, e.g. periarteriitis nodosa, microscopic polyarteriitis and pulmonary eosinophilic granulomatosis (Churg-Strauss syndrome) (10). Low to moderate anti-MPO autoantibody levels are also reported in rheumatoid arthritis.

HyTest has been producing human MPO for many years and our anti-MPO monoclonal antibodies are suitable for the development of quantitative MPO immunoassay. Sandwich immunoassays utilizing these antibodies demonstrated excellent results in precise MPO immunodetection in patients’ blood samples.

1. Human myeloperoxidase Source: Purity: Presentation: Application: Storage:

Human leukocyte mass. Blood sample from the tissue donors was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis >90% Lyophilized Antigen in the MPO-specific autoantibodies assays, immunological MPO standard, immunogen for antibody production, MPO biochemical and immunochemical studies -20°C

Human MPO is purified from human leukocyte mass and can be used as an antigen in immunoassays designed for the detection of MPO-specific autoantibodies; as an immunological MPO standard, as an immunogen for antibody production and in MPO biochemical and immunochemical studies. On gel elec-

54

trophoresis under reducing conditions MPO is presented by two main bands with apparent molecular masses of 60 kDa (MPO A-chain) and 15 kDa (MPO B-chain); and with minor band with apparent molecular mass about 40 kDa, revealing immunological activity of MPO A-chain and most likely representing A-chain degradation product (Fig. 58).

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

1

2

94 kDa 67 kDa MPO A-chain 43 kDa

MPO A-chain degradation product

30 kDa

Figure 58. SDS-PAGE of human MPO under reducing conditions.

Lane 1: Molecular weight standards (Pharmacia) Lane 2: Human MPO, 4 μg

20 kDa MPO B-chain

14.4 kDa

Ordering information:

Product

Cat. #

Purity

Source

Human myeloperoxidase

8M80

>90%

Human leukocyte mass

2. Anti-MPO monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human MPO Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide MPO immunoassay, MPO immunoaffinity purification, MPO immunodetection in Western blotting

Hybridoma clones have been derived from hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with purified human MPO. All

MAbs were tested with purified antigen in direct ELISA, Western blotting and sandwich immunoassay.

2.1. Applications 2.1.1. Sandwich assay for quantitative MPO immunodetection The best of HyTest’s combinations of monoclonal antibodies for MPO sandwich immunoassays were selected from several hundred MAb combinations and tested for better sensitivity, specificity and kinetics.

As was mentioned previously, the blood of patients suffering certain systemic vasculitides, pulmonary eosinophilic granulomatosis and some other pathologies, contains anti-MPO autoantibodies. Presence of MPO autoantibodies in the tested sample could dramatically influence the results of MPO immunodetection. We have tested our most sensitive two site

MAb combinations with several blood samples with high titers of MPO-specific autoantibodies and selected those combinations, which are in the less degree sensitive to the presence of autoantibodies.

The best MAb combinations for sandwich immunoassay are (capture – detection): 16E3 18B7 18B7 19G8

– – – –

18B7 16E3 4B3 16E3

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

55

1000000

1000000

B

A 100000

CPS

CPS

100000

10000 10000

1000 1000

1

10

100

1000

1

10000

10

100

1000

10000

MPO concentration (ng/ml)

MPO concentration (ng/ml)

Figure 59. Calibration curves for two MPO sandwich immunoassays. A: 16E3-18B7, B: 18B7-16E3

2.1.2. MPO immunodetection in Western blotting Several monoclonal antibodies – 4A4, 16G6, 18B7, and 19H2 - could be used for MPO immunodetection in Western blotting (Fig. 60).

MPO A-chain MPO degraded A-chain

1

2

3

Figure 60. MPO staining by anti-MPO MAbs in Western blotting after gel electrophoresis under reducing conditions.

Lane 1: 16G6, lane 2: 18B7, lane 3: 19H2.

Ordering information: MAb

Cat. #

Specificity

Subclass

Application

2A11 4A4 16G6 18B7 19H2 4B3 16E3 17G2 19G8

4M43 4M43 4M43 4M43 4M43 4M43 4M43 4M43 4M43

MPO MPO MPO MPO MPO MPO MPO MPO MPO

IgG2a IgG2b IgG1 IgG1 IgG1 IgG1 IgG1 IgG2b IgG1

Sandwich immunoassay Sandwich immunoassay, WB Sandwich immunoassay, WB Sandwich immunoassay, WB Sandwich immunoassay, WB Sandwich immunoassay Sandwich immunoassay Sandwich immunoassay Sandwich immunoassay

56

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

3. MPO free serum Prepared from: Method of purification: Delivery form: Storage:

Pooled normal human serum Immunoaffinity chromatography Frozen liquid -20°C

MPO free serum is prepared from pooled normal human serum by the immunoaffinity chromatography method. The affinity sorbent utilizes several immobi-

lized MAbs with different epitope specificities. MPO free serum can be used as a matrix for standard and calibrator preparation.

Ordering information:

Product

Cat. #

Source

MPO free serum

8MPFS

Pooled normal human serum

References: 1. Nauseef WM et al.(1988): Biosynthesis and processing of myeloperoxidase – a marker for myeloid cell differentiation. Eur J Haematol. 40(2): 97-110.

2. Klebanoff SJ. (2005): Myeloperoxidase: friend and foe. J Leukos Biol. 77: 598625.

3. Chevrier I et al. (2003): Myeloperoxidase: new polymorphisms and relation with lung cancer risk. Pharmacogenetics. 13(12): 729-39.

4. Reynolds WF et al. (2000): MPO and APOEepsilon4 polymorphisms interact to increase risk for AD in Finnish males. Neurology. 55(9): 1284-90.

5. Nagra RM et al. (1997): Immunohistochemical and genetic evidence of myeloperoxidase involvement in multiple sclerosis. J Neuroimmunol. 78(1-2): 97107.

6. Nambi V. (2005): The use of myeloperoxidase as a risk marker for atherosclerosis. Curr Atheroscler Rep. 7(2): 127-31.

7. Zhang R. et al. (2001): Association between myeloperoxidase levels and risk of coronary artery disease. JAMA. 286(17): 2136-42.

8. Brennan ML et al. (2003): Prognostic value of myeloperoxidase in patients with chest pain. New Eng J Med. 349(17): 1595-1604.

9. Baldus S. et al. (2003): Myeloperoxidase serum level predicts risk in patients with acute coronary syndromes. Circulation 10: 1440-1445.

10. Choi HK et al. (2001): Diagnostic performance of antineutrophil cytoplasmic antibody tests for idiopathic vasculitides: meta-analysis with a focus on antimyeloperoxidase antibodies. J Rheumatol. 28(7): 1584-1590.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

57

VII Soluble CD40 Ligand (sCD40L) Soluble CD40 ligand (sCD40L) is a soluble part of transmembrane protein named CD40-ligand (CD40L), which is a member of the tumor necrosis factor-A family of proteins. CD40L is a 33 kDa type II membrane glycoprotein expressed on the surface of CD4+ T-cells, basophiles, platelets and mast cells.

Soluble CD40L is an 18 kDa soluble product of CD40L cleavage that releases from stimulated cell surface.

CD40L was originally identified on T-lymphocytes and further on the activated platelets (1, 2). It is expressed rapidly on the surface of activated CD4+ Tcells where it mediates functional T- and B-cells interaction. CD40L interacts with CD40 presented on the surface of B cells, macrophages, monocytes, endothelial cells and vascular smooth muscle cells, where it implicates in various inflammatory responses and thrombus formation (3). The CD40-CD40L interaction plays an important pathogenic role in different inflammatory processes including sclerosis and some autoimmune diseases. It has been recently shown that CD40-CD40L interaction may be involved in atherogenesis and plaque destabilization, triggering the development of acute coronary syn-

drome and myocardial infarction (4). It was suggested that CD40 signaling can be a new potential therapeutic target in some inflammatory disorders. It was demonstrated that blocking of CD40-CD40L interaction considerably limits atherogenesis and such experimental autoimmune diseases as collagen arthritis, multiple sclerosis, thyroiditis etc (5, 6, 7). The correlation between a raised level of sCD40L and some cardiac disorders was demonstrated in multiple studies. Higher concentration of sCD40L was determined in patients with either acute or chronic heart failure. Moreover, an elevated level of sCD40L was significantly correlated with the severity of disease (8). In some investigations sCD40L was suggested as a new marker that predicts increased risk for future cardiovascular events in patients with acute coronary syndromes. It should be noted that sCD40L as a marker is independent of other predictive markers such as cardiac TnI or CRP (9).

HyTest proposes a new generation of monoclonal antibodies suitable for the development of sandwich immunoassay for sensitive and specific sCD40L immunodetection in human blood samples.

1. Anti-sCD40L monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human recombinant sCD40L Protein A affinity chromatography MAb solution in PBS with 0.1% of sodium azide sCD40L immunoassays, Western blotting

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with recombinant human soluble CD40 Ligand / TRAP Analog conjugated

58

with BSA. The ability to recognize sCD40L in human blood samples was tested with all MAbs in sandwich immunoassay.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

1.1. Applications 1.1.1. sCD40L immunodetection in Western blotting MAbs ability to recognize the human recombinant sCD40L was tested in Western blotting (Fig. 61). All MAbs can detect sCD40L after Western blotting, but for the best sensitivity we recommend using MAbs 2B10 and 30B4.

31 kDa

21.5 kDa 14.4 kDa 1H4

2A3

2B10

10A6

30B4

Figure 61. Immunodetection of human recombinant sCD40L using specific monoclonal antibodies in Western blotting after PAGE in reducing conditions. Anti-mouse IgG conjugated with HRP were used for MAbsCD40L complex visualization.

1.1.2. Sandwich immunoassay for quantitative sCD40L immunodetection 10000000

1000000

100000

CPS

Several of HyTest’s best combinations of monoclonal antibodies for sCD40L immunodetection in sandwich immunoassays were selected. Calibration curves for the pairs 1H4-2A3 and 10A6-2A3 are shown in Fig. 62.

1H4-2A3Eu 10A6-2A3Eu

10000

1000

100 1

10

100

1000

10000

100000

1000000

sCD40L human recombinant, pg/ml

Figure 62. Calibration curves for human sCD40L immunodetection using anti-sCD40L monoclonal antibodies.

Capture MAbs: 1H4 and 10A6 (biotinylated), 0.2 μg/well.

Detection MAb: 2A3 (Eu-labeled), 0.1 μg/well.

Antigen: Recombinant sCD40L (Cell Science, USA) One step assay.

Incubation time 40 minutes.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

59

MAbs two-site combinations demonstrating high response with human recombinant antigen were also tested on their ability to detect native sCD40L in human serum. Results of sCD40L immunodetection in human serum using the sandwich immunoassay are presented in Fig. 63.

100000

10000

CPS

1H4-2A3Eu 10A6-2A3Eu

For specific and sensitive sCD40L immunodetecti-on in different biological samples we recommend using MAbs 1H4, 2B10, 10A6 and 30B4 as the capture and MAb 2A3 as the detection antibodies.

1000

100 1

1:2

1:4

1:8

1:18

1:32

1:64 1:128 1:256 1:512 1:1024

Human serum dilutions

Figure 63. Titration curves of human blood samples for two sCD40L sandwich immunoassays.

Capture MAbs: 1H4 and 10A6 (biotinylated), 1 μg/well.

Detection MAb: 2A3 (Eu-labeled), 0.1 μg/well.

Serum sample: 75 μl/well.

Incubation time 40 minutes.

Ordering information: MAb

Cat. #

Specificity

Subclass

Application

30B4 1H4 2A3 2B10 10A6

4CD40 4CD40 4CD40 4CD40 4CD40

sCD40L sCD40L sCD40L sCD40L sCD40L

IgG1 IgG1 IgG1 IgG1 IgG1

EIA, Sandwich immunoassay (capture), WB EIA, Sandwich immunoassay (capture) EIA, Sandwich immunoassay (detection) EIA, Sandwich immunoassay (capture), WB EIA, Sandwich immunoassay (capture)

References: 1. Hollenbaugh D, Grosmaire LS, Kullas CD, Chalupny NJ, Braesch- Andersen S, Noelle RJ, Stamenkovic I, Ledbetter JA, Aruffo A. (1992) The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity. EMBO J. 11:4313–4321.

2. Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller- Berghaus G, Kroczek RA. (1998) CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature 391:591–594.

3. Mach, F., Schonbeck, U., Bonnefoy, J.-Y., Pober, J. S.,Libby, P. (1997) Activation of monocyte/macrophage functions related to acute atheroma complication by ligation of CD40. Induction of collagenase, stromelysin, and tissue factor. Circulation 96:396–399.

4. Garlichs CD, Eska. S, Raaz D, Schmidt A, Ludwig J, Herrmann M, Klinghamer L, Daniel WG, Schmeisser A. (2001) Patients with acute coronary syndromes express enhanced CD40 ligand/CD154 on platelets. Heart 86:649–655.

60

5. Durie FH, Fava RA, Foy TM, Aruffo A, Ledbetter JA, Noelle RJ. (1993) Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40.

Science 261:1328–1330.

6. Gerritse K, Laman JD, Noelle RJ, Aruffo A, Ledbetter JA, Boersma WJ, Claassen E. (1996) CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Proc. Natl Acad. Sci. USA 93:2499–2504.

7. Francois Mach, Uwe Schonbeck, Galina K. Sukhova, Elizabeth Atkinson, Peter Libby. (1998) Reduction of atherosclerosis in mice by inhibition of CD40signalling. Nature. 394(9):200-203.

8. Thor Ueland, Pal Aukrust, Arne Yndestad, Kari Otterdal, Stig S. Froland, Kenneth Dickstei4, John Kjekshus, Lars Gullestad, Jan K. Damas. (2005) Soluble CD40 ligand in acute and chronic heart failure. Eur. Heat J. 26:1101-1107.

9. Varo N, de Lemos JA, Libby P, Morrow DA, Murphy SA, Nuzzo R, Gibson CM, Cannon CP, Braunwald E, Schonbeck U. (2003) Soluble CD40L: risk prediction after acute coronary syndromes. Circulation. 108(9):1049-52.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

VIII Human Cystatin C Cystatin C is a low molecular weight (13.4 kDa) protein that functions as an inhibitor of various cysteine proteases in the bloodstream. It inhibits both endogenous proteases, such as lysosomal cathepsins, and proteases of parasites and microorganisms.

Cystatin C binds to the target molecule in μM to the sub pM range in a competitive reversible manner (1).

Due to its important function, cystatin C is expressed at the stable levels by most of the nucleated cells.

Cystatin C consists of 120 amino acid residues encoded by a 7.3 kb gene located in chromosome 20 (2). The Leu68Gln mutation in the cystatin C protein sequence is directly linked to the development of hereditary cystatin C amyloid angiopathy (HCCAA) in which the patients suffer from repeated cerebral hemorrhages (3).

Cystatin C is known in clinical practice as a well-described serum marker of renal failure that is not dependent on age, sex or lean muscle mass (4, 5). At the same time, cystatin C is becoming acknowledged as a marker of elevated risk of death from cardiovascular complications – myocardial infarction and stroke (5). A stable production rate and free filtration by the renal glomeruli due to the low molecular weight, and positive charge (pI 9.3) are strong advantages of cystatin C as a serum marker of renal function in comparison to other analytes that are used today in clinical practice. Creatinine-based equations to estimate the glomerular filtration rate (GFR) are sensitive to some non-renal factors, such as age, sex, race and lean muscle mass. There is a growing number of reports demonstrating that cystatin C is more preferable than creatinine for the measurement of GFR, so long as it does not depend on all of these fac-

tors (5). Cystatin C is also a more sensitive marker of mild renal dysfunction than creatinine (6). The concentrations of plasma (serum) cystatin C in healthy individuals range from 0.8 to 1.2 mg/l, depending on measurement methods (7). Increased cystatin C serum levels are almost exclusively associated with a reduction in GFR. Serum concentrations of cystatin C are increased approximately 2-fold during various renal disorders (7). An elevated serum cystatin C level is also a strong predictor of the risk of death and cardiovascular events in elderly persons (5).

The urinary concentrations of cystatin C are low (100 μg/l for healthy subjects) since the protein is metabolized by the proximal tubule after filtration in the renal glomerulus. However, the concentrations of cystatin C in urine from patients with renal tubular disorders are raised by approximately 200-fold (8). Cystatin C that is purified from human urine can be partially truncated, which potentially complicates the application of the urine protein as a standard for immunoassays (9).

HyTest offers everything you need for the development of the cystatin C immunoassay - human recombinant cystatin C, native human cystatin C purified from human blood, anti-cystatin C polyclonal antibodies, as well as a set of high-affinity monoclonal antibodies that are specific to different epitopes of human cystatin C molecule. We also supply our customers with information regarding the best MAb combinations to be used in sandwich immunoassays for quantitative measurements of cystatin C in body fluids.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

61

1. Human cystatin C antigens 1.1. Human recombinant cystatin C Source: Purity: Presentation: Application: Storage:

Recombinant, expressed in E. coli >95% Lyophilized Standard or calibrator for cystatin C immunoassays, cystatin C biochemical and immunochemical studies -20°C

1.2. Cystatin C purified from human blood NEW! Source: Purity: Presentation: Application: Storage:

HyTest offers recombinant human cystatin C expressed in E. coli as a full length peptide with additional methionine residue at the N-terminus. The protein is purified to homogeneity using several chromatography methods (Fig. 64).

Pooled human serum >95% Lyophilized Standard or calibrator for cystatin C immunoassays, cystatin C biochemical and immunochemical studies -20°C

HyTest offers native human cystatin C purified from pooled normal human serum. The protein is purified to homogeneity using several chromatography methods (Fig. 65).

Cystatin C Cystatin C

1

2

Figure 64. SDS-PAGE of human recombinant cystatin C expressed in E. coli, reducing conditions.

Lane 1: Molecular weight standards, Fermentas (250, 130, 92, 75, 55, 36, 28, 17, and 11 kDa) Lane 2: Human recombinant cystatin C from E. coli, 5 μg.

Gel staining: Coomassie brilliant blue R-250.

62

1

2

Figure 65. SDS-PAGE of cystatin C from pooled human serum, reducing conditions.

Lane 1: Molecular weight standards, Fermentas (250, 130, 92, 75, 55, 36, 28, 17, and 11 kDa) Lane 2: Cystatin C from pooled human serum, 5 μg.

Gel staining: Coomassie brilliant blue R-250.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

Immunochemical properties of human recombinant cystatin C expressed in E. coli, cystatin C purified from pooled human serum, and cystatin C purified from human urine (RDI) were analyzed by seven HyTest prototype cystatin C immunoassays (Fig. 66).

HyTest’s human recombinant cystatin C and cystatin C purified from pooled human serum had very similar immunochemical activity with the antigen in hu-

600000

man serum in cases of all tested assays. However, cystatin C purified from human urine had significantly lower immunochemical activity when measured by four out of seven tested immunoassays. It can be explained by possible truncation of cystatin C purified from human urine. This data suggests that recombinant and purified antigens from human blood serve better as standards or calibrators in cystatin C immunoassays than protein purified from human urine.

Cystatin C from human urine Cystatin C from human serum

500000

Human recombinant Cystatin C (E. coli) Pooled normal human serum

CPS

400000

300000

Figure 66. Immunochemical properties of three forms of cystatin C protein, in comparison with antigen from pooled normal human serum.

Cystatin C preparations (all at concentration 10 ng/ml) and diluted pooled normal human serum were analyzed.

Sandwich type fluoroimmunoassay was used to measure cystatin C: Capture MAbs: Cyst13, Cyst28 and Cyst24.

Detection MAbs: Cyst10, Cyst16, Cyst13, Cyst19 and Cyst28 are Eu3+-labeled.

200000

100000

0 Cyst13Cyst10

Cyst28Cyst10

Cyst28Cyst16

Cyst24Cyst19

Cyst24Cyst10

Cyst24Cyst13

Cyst24Cyst28

Ordering information: Product

Cat.#

Purity

Source

Cystatin C, human, recombinant

8CY5

>95%

E. coli

Cystatin C, human, endogenous

8CN4

>95%

Pooled human serum

2. Anti-cystatin C monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human cystatin C Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide Cystatin C immunoassays, cystatin C immunodetection in Western blotting

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with cystatin C purified

from human urine. Eight anti-cystatin C MAbs were selected in regard to their specificity and high-affinity interaction with the cystatin C molecule.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

63

2.1. Applications 2.1.1. Cystatin C quantitative sandwich immunoassays

Cyst24 – Cyst19 Cyst24 – Cyst28 Cyst23 – Cyst13

10000000

Cyst24 – Cyst19 Cyst24 – Cyst28 Cyst24 – Cyst13

1000000

100000

CPS

All selected MAbs were tested in sandwich fluoroimmunoassay as capture and detection antibodies with purified human antigen and pooled serum samples (Fig. 67 and 68). The best recommended pairs (capture - detection) are:

10000

These pairs demonstrate high sensitivity and perfect antigen recognition in blood samples.

1000

The best MAb combinations can be used for antigen detection even at 100,000-fold serum dilution (Fig. 68). For these assays we observed high degree of parallelism between titration curve of purified human cystatin C and the curves of serial dilutions of pooled serum sample.

Affinity constants for Cyst10 and Cyst24 MAbs (human cystatin C from RDI used as an antigen) were measured using the Biacore technique:

10

100

1000

Cystatin C, ng/ml

Figure 67. Calibration curves of the best immunoassays.

One-step fluoroimmunoassay in streptavidin coated plates.

Capture MAbs: Cyst24 and Cyst23 are biotinylated (200 ng/well).

Detection MAbs: Cyst19, Cyst28 or Cyst13 are Eu3+-labeled (200 ng/ml).

Incubation volume 100 μl.

Incubation time: 30 min at room temperature.

10000000

Cyst24 – Cyst19 Cyst24 – Cyst28 Cyst24 – Cyst13

1000000

100000

CPS

Cyst24 - 1.09x10 -8 M Cyst10 - 5.55x10 -8 M

1

10000

1000

100

10 10

100

1000

10000

100000

Serum dilution, times

Figure 68. Titration curves of pooled normal human serum in Cyst24 – Cyst19, Cyst24 – Cyst28, and Cyst23 – Cyst13 (capture – detection) sandwich fluoroimmunoassays.

64

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

2.1.2. Cystatin C immunodetection in Western blotting Several monoclonal antibodies – Cyst13, Cyst18 and Cyst19 – could be used for cystatin C immunodetection in Western blotting (Fig. 69).

Cystatin C

Figure 69. Detection of human cystatin C in Western blotting by different monoclonal antibodies after Tricine-SDS-PAGE in reducing conditions.

Lane 1: MAb Cyst13 Lane 2: MAb Cyst18 Lane 3: MAb Cyst19 Antigen: Cystatin C purified from human urine (RDI), 0.2 μg/lane.

1

2.1.3 Cross-reaction with sera from different animal species New data!

Among all possible sandwich combinations of anticystatin C MAbs produced by using human antigen, we have defined the set of pairs with significant crossreactivity with dog, cat or horse serum (Table 9).

2

3

Table 9. Cross-reaction of anti-cystatin C MAbs with sera from different animal species. Sandwich type fluoroimmunoassay was used to measure cross-reaction; capture-detection MAb pairs are shown in the table. No cross-reaction (–), 7-30% cross-reaction (+), or 30-90% cross-reaction (++) are indicated in comparison with pooled normal human serum.

Dog

Cat

Horse

Cyst29 - Cyst11

+

+

–

Cyst29 - Cyst16

+

++

–

Cyst11 - Cyst20

++

+

–

Cyst29 - Cyst20

+

++

++

Cyst11 - Cyst29

+

+

–

Cyst16 - Cyst29

+

+

–

Cyst20 - Cyst29

–

+

++

Cyst20 - Cyst13

–

–

++

Cyst29 - Cyst13

–

–

++

Ordering information: MAb

Cat. #

Specificity

Subclass

Application

Cyst10

4CC1

Human Cystatin C

IgG3

EIA, Sandwich immunoassay

Cyst11

4CC1

Human Cystatin C, c/r with dog and cat serum

IgG1

EIA, Sandwich immunoassay

Cyst13

4CC1

Human Cystatin C, c/r with horse serum

IgG1

EIA, Sandwich immunoassay, WB

Cyst16

4CC1

Human Cystatin C, c/r with dog and cat serum

IgG1

EIA, Sandwich immunoassay

Cyst18

4CC1

Human Cystatin C

IgG1

EIA, Sandwich immunoassay, WB

Cyst19

4CC1

Human Cystatin C

IgG1

EIA, Sandwich immunoassay, WB

Cyst20

4CC1

Human Cystatin C, c/r with dog, cat, and horse serum

IgG1

EIA, Sandwich immunoassay

Cyst23

4CC1

Human Cystatin C

IgG1

EIA, Sandwich immunoassay

Cyst24

4CC1

Human Cystatin C

IgG1

EIA, Sandwich immunoassay

Cyst28

4CC1

Human Cystatin C

IgG1

EIA, Sandwich immunoassay

Cyst29

4CC1

Human Cystatin C, c/r with dog, cat, and horse serum

IgG2a

EIA, Sandwich immunoassay

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

65

3. Polyclonal anti-cystatin C antibodies NEW! Host animal: Antigen: Purification method: Presentation:

Rabbit Human recombinant cystatin C Affinity chromatography utilizing human recombinant cystatin C-agarose PAb solution in PBS with 0.1% sodium azide

Polyclonal anti-cystatin C antibodies were obtained through the immunization of rabbits with highly purified (>95%) human recombinant cystatin C expressed in E. coli. Affinity chromatography utilizing

human recombinant cystatin C-agarose makes it possible to produce highly purified anti-cystatin C polyclonal antibodies that are free from rabbit serum proteins and non-specific immunoglobulins.

Ordering information: Product

Cat.#

Host

Remarks

Polyclonal anti-cystatin C

PCC2

Rabbit

EIA, WB, IP

4. Cystatin C free serum Source: Purification method: Delivery form: Storage:

Pooled normal human serum Immunoaffinity chromatography Frozen liquid -20°C

Cystatin C free serum is prepared from pooled normal human serum by immunoaffinity chromatography method. Cystatin C free serum can be used as a matrix for standard and calibrator preparation.

Serum sample

Cystatin C concentration (ng/ml)

Pooled serum (before cystatin C depletion)

800

Cystatin C free serum

0.4

Ordering information: Product

Cat.#

Source

Cystatin C free serum

8CCFS

Pooled Normal Human Serum

References: 1. Turk B, Turk D and Salvesen GS: Regulating Cysteine Protease Activity: Essential Role of Protease Inhibitors As Guardians and Regulators. Current Pharmaceutical Design, 2002, 8, 1623-1637.

2. Schnittger S, Rao VV, Abrahamson M, Hansmann I: Cystatin C (CST3), the candidate gene for hereditary cystatin C amyloid angiopathy (HCCAA), and other members of the cystatin gene family are clustered on chromosome 20p11.2.

Genomics. 1993; 16(1):50-5.

3. Palsdottir A, Snorradottir AO, Thorsteinsson L: Hereditary cystatin C amyloid angiopathy: genetic, clinical, and pathological aspects. Brain Pathol. 2006; 16(1):55-9.

4. Séronie-Vivien S, Delanaye P, Piéroni L, Mariat C, Froissart M, Cristol JP: Cystatin C: current position and future prospects. Clin Chem Lab Med. 2008; 46(12):1664-86.

66

5. Naruse H, Ishii J, Kawai T, Hattori K, Ishikawa M, Okumura M, Kan S, Nakano T, Matsui S, Nomura M, Hishida H, Ozaki Y: Cystatin C in Acute Heart Failure Without Advanced Renal Impairment. Am J Med. 2009 Apr [Epub ahead of print] 6. Artunc FH, Fisher IU, Risler T, Erley CM: Improved estimation of GFR by serum cystatin C in patients undergoing cardiac catheterization. Int J Cardiol. 2005; 102(2):173-8.

7. Roos JF, Doust J, Tett SE, Kirkpatrick CM: Diagnostic accuracy of cystatin C compared to serum creatinine for the estimation of renal dysfunction in adults and children-A meta-analysis. Clin Biochem. 2007; 40(5-6): 383-91.

8. Uchida K, Gotoh A: Measurement of cystatin-C and creatinine in urine. Clin Chim Acta. 2002; 323(1-2): 121-128.

9. Popovi T, Brzin J, Ritonja A, Turk V: Different forms of human cystatin C. Biol Chem Hoppe Seyler. 1990; 371(7):575-80.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

IX Human serum albumin (HSA) Albumin is a main protein of blood plasma that has multiple functions. It is synthesized by the liver and secreted into the bloodstream as a 67 kDa singlechain protein. Its tertiary structure is sustained by 17 disulfide bonds and albumin concentration in plasma is approximately 40 mg/ml, which is more than 50% of the total plasma protein concentration. Albumin regulates filtration and absorption of fluid across capillary walls as up to 80% of blood osmotic pressure is determined by albumin. It also binds and transports lots of substances in blood, including fatty acids, steroid hormones, bilirubin, tryptophan, calcium ions, as well as drugs such as penicillin, aspirin, dicumarin, or sulfonamides.

Albumin concentration in blood usually decreases either because of liver diseases leading to a derangement of protein synthesis or due to its loss with urine in renal diseases. Albumin excretion with urine is normally less than 20 μg/min and an increase in albumin concentration in urine is called albuminuria. The urinary excretion of albumin within 20 – 200 μg/min (30 – 300 mg per day) is defined as microalbuminuria, while the albumin excretion that exceeds 200 μg/min is called clinical albuminuria. The latter usually reflects overt nephropathy (glomerular affection) and could be detected by a sulfosalicylic acid method or a dipstick method.

Microalbuminuria has been considered to be an early marker of nephropathy and reflects microvascular damage over the blood circulating system. Microalbuminuria is a risk marker of cardiovascular complications in patients with diabetes mellitus (1).

It predicts renal disease, left ventricular dysfunction, stroke, myocardial ischemia and infarction (2). In addition, microalbuminuria has been found in primary hypertensive patients, as well as in patients with atrial fibrillation, carotid and femoral atherosclerosis, hepatitis C infection (3).

The presence of albumin in urine can be detected by several methods, although the immunochemical approach is the most sensitive and specific. Immunoassays, utilizing albumin-specific antibodies could be used for albumin quantifications not only in urine, but also in other biological fluids. To select antibodies that are most suitable for microalbuminuria diagnosis, HyTest’s specialists have tested several dozen albumin-specific monoclonal antibodies and several hundred two-site MAb combinations. A sensitive, precise, and rapid assay for reliable quantitative albumin immunodetection in human urine was our goal, which we succeeded in achieving.

1. Anti-human serum albumin monoclonal antibodies Mice Balb/c Sp2/0 Human serum albumin Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide Albumin immunoassay, albumin immunodetection in Western blotting

Over 30 hybridoma clones were derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with human serum albumin. Monoclonal antibodies that are presented here are albumin specific and do not have cross-reactivity with other tested human proteins, such as bovine serum albumin or human alpha fetoprotein.

MAb

Cat. #

Specificity

Subclass

Application

1C8 15C7 1A9 6B11 14E7 HSA11 HSA20

4T24 4T24 4T24 4T24 4T24 4T24 4T24

HSA HSA HSA HSA HSA HSA HSA

IgG1 IgG2b IgG2a IgG2a IgG2b IgG1 IgG1

EIA Sandwich immunoassay, WB Sandwich immunoassay, WB Sandwich immunoassay, WB Sandwich immunoassay, WB Sandwich immunoassay, WB Sandwich immunoassay, WB

Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Ordering information:

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

67

1.1. Applications 1.1.1. Anti-albumin MAbs for the development of sandwich immunoassay

1.1.2. Albumin immunodetection in Western blotting

All of HyTest’s albumin-specific MAbs were tested in sandwich immunoassay as capture and detection antibodies to select the best two-site MAb combination for the development of quantitative sandwich immunoassay. The selected MAb combinations are:

All antibodies presented here could be used for the detection of human serum albumin in Western blotting after gel electrophoresis under reducing and non-reducing conditions (Fig. 71).

HSA20 15C7 15C7 HSA11 1A9

– – – – –

14E7 1A9 6B11 15C7 15C7 Figure 71. Albumin staining by five albumin-specific MAbs in Western blotting after electrophoretic separation of human plasma proteins under reducing conditions.

Lane 1: MAb 6B11 Lane 2: MAb HSA11 Lane 3: MAb 14E7 Lane 4: MAb 15C7

3.0 2.5

A

A490

2.0

1

1.5

2

3

4

1.1.3. MAbs for albumin extraction from human serum (plasma)

1.0 0.5 0.0 10-8

10-7

10-6

10-5

10-4

10-3

Serum dilution

Anti-albumin MAbs could be used for the preparation of immunosorbents to deplete albumin from serum, which is a necessary stage of the proteomic studies of blood proteins.

3.0 2.5

B

A490

2.0 1.5 1.0 0.5 0.0 10

-8

10

-7

10

-6

10

-5

10

-4

10

-3

Serum dilution

References: 1. Bennett PH et al. Screening and management of microalbuminuria in patients with diabetes mellitus: recommendations to the Scientific Advisory Board of the National Kidney Foundation from an ad hoc committee of the Council on Diabetes Mellitus of the National Kidney Foundation. Am J Kidney Dis. 1995, 25(1), pp.107-112.

2. Salmasi AM et al. The degree of albuminuria is related to left ventricular hypertrophy in hypertensive diabetics and is associated with abnormal left ventricular filling: a pilot study. Angiology. 2003, 54(6), pp. 671-678.

3. Wachtell K et al. Albuminuria and cardiovascular risk in hypertensive patients with left ventricular hypertrophy: the LIFE study. Ann Intern Med. 2003, 139(11), pp.901-906.

Figure 70. Titration of human serum in two albumin sandwich immunoassays.

A: 15C7(capture)– 1A9 (detection) B: HSA20(capture) – 14E7(detection)

68

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

X Urotensin II Urotensin II (U-II) is a ubiquitous vasoactive hormone (1) with pleiotropic effects. U-II exerts its activity by binding to a G-protein-coupled receptor termed UT (2). U-II and its receptor are highly expressed in the cardiovascular system. Increased U-II plasma levels have been reported in patients with cardiovascular diseases of varying etiologies.

Expression of U-II and UT is low in healthy cardiomyocytes, but are upregulated in the myocardial infarction animal model and in patients with heart failure. U-II system appears to be involved in cardiac hypertrophy as an adaptive response in injured myocardium (3). Recent studies have demonstrated elevated plasma U-II concentrations in patients with essential hypertension, coronary artery disease, diabetes mellitus, atherosclerosis, renal dysfunction and cirrhosis. Some authors suggest that increased levels of U-II may play a crucial role in the development of carotid atherosclerosis in hypertensive patients (4).

Urotensin II in vivo is derived from a large precursor molecule (pre-pro-urotensin II) by enzymatic cleavage. Mature peptide consists of 11 amino acid residues and has a highly conserved C-terminal cyclic disulfide-linked hexapeptide structure, which con-

W F E

T

P

N-terminus

E

C

K Y C

V

Cyclic active component

Figure 72. Mature urotensin II (U-II) peptide sequence from human/ monkey (2).

fers most of the biological activity (Fig. 72). The Nterminus of the peptide varies in length and structure between animal species (2).

Immunochemical detection of human U-II is mainly possible in competitive assay, which has a limited range of measurable concentrations and requires the utilization of antibodies with high specificity.

HyTest offers a set of monoclonal antibodies, specific to the human urotensin II molecule. We also supply our customers with detailed information regarding various applications; the development of quantitative competitive U-II immunoassay and immunodetection of antigen in direct immunoassay.

1. Anti-urotensin II monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human urotensin II Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide Urotensin II immunoassay, competitive human urotensin II immunoassay

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with human urotensin II conjugated with the carrier protein.

Three anti-urotensin II MAbs recognize urotensin II in direct immunoassay and one of them was selected as an antibody that can be used in the competitive immunoassay.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

69

1.1. Applications 1.1.1. Urotensin II competitive immunoassay In the competitive immunoassay, an antigen preadsorbed on plate, and free antigen in sample, compete for Eu-labeled MAb. UII-9 MAb is the best antibody that can be used in urotensin II competitive immunoassay (Fig. 73).

600000

UII-9

CPS

500000 400000 300000 200000 100000 0 0,1

1

10

100

U-II, ng/ml

Figure 73. Calibration curve for Urotensin II competitive immunoassay with Eu-labeled UII-9.

Ordering information MAb

Cat.#

SpeciďŹ city

Subclass

Application

UII-6 UII-9 UII-13

4UT2 4UT2 4UT2

Human urotensin II Human urotensin II Human urotensin II

IgG1 IgG1 IgG1

EIA EIA, competitive immunoassay EIA

References: 1. Kwok Leung Ong, Karen S.L. Lam, and Bernard M.Y. Cheung. Urotensin II: Its Function in Health and Its Role in Disease. Cardiovasc. Drugs Ther. 2005, Jan; 19(1): 65-75.

2. Onan, D. et.al. UrotensinII: the old kid in town. TRENDS in Endocrinology and Metabolism, 2004, Vol.15(4): 175-182.

3. Tzanidis A. et al. Direct actions of urotensin II on the heart. Implications for cardiac ďŹ brosis and hypertrophy. Circ. Res. 2003; 93: 246-253.

4. Toshiaki Suguro, Takuya Watanabe et al. Increased Human Urotensin II Levels Are Correlated With Carotid Atherosclerosis in Essential Hypertension. Am. J. Hypertens., 2007, Feb, Vol. 20 (2): 211-217

70

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

XI Procalcitonin (PCT) Procalcitonin (PCT) is a small protein that comprises 116 amino acid residues (a.a.r.) with a molecular weight of approximately 13 kDa (Fig. 74). The amino acid sequence of PCT was described for the first time by Moullec et al. in 1984 (1). PCT belongs to a family of related proteins (the CAPA peptides family), which includes calcitonin gene-related peptides I and II, amylin, adrenomodulin and calcitonin. Like other peptides of the CAPA family, PCT appears from the precursor molecule - pre-procalcitonin comprising 141 a.a.r. by the removal of 25 a.a.r. from the Nterminus.

PCT is produced normally in C-cells of the thyroid glands. It undergoes successive cleavages to form three molecules: N-terminal fragment (N-terminal PCT, 57 a.a.r.), calcitonin (32 a.a.r.) and katacalcin (21 a.a.r.) (Fig. 74).

In 1993, the elevated level of PCT in patients with a system infection of bacterial origin was reported (2) and PCT is now considered to be the main marker of disorders accompanied by systemic inflammation and sepsis. The diagnostic value of

PCT is important due to the close correlation between PCT concentration and the severity of inflammation (3). It was shown that “inflammatory” PCT is not produced in C-cells. Cells of neuroendocrine origin are presumably the source of PCT during inflammation (4).

Raising PCT concentration may in some cases be induced by factors independent of sepsis and infection. Surgery, polytrauma, heat shock, burn injures and cardiogenic shock also lead to an increase in the PCT level (3). The mechanism of the increasing level of PCT in these cases is not clearly defined. The importance of monitoring PCT level changes following cardiac surgery or heart transplantation for differentiating acute graft rejection from bacterial or fungal infections was confirmed in multiple studies (4).

Therefore, the diagnostic value of PCT is very high and the usefulness of PCT quantification in patients’ blood using specific monoclonal antibodies is very perspective and quite obvious (4, 5).

PROCALCITONIN, 116 a.a.r.

N-terminal PCT, 57 a.a.r.

Calcitonin, 32 a.a.r.

Katacalcin, 21 a.a.r.

APFRSALESSPADPATLSEDEARLLLAALVQDYVQMKASELEQEQEREGSSLDSPRS -KR-CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP-GKKR-DMSSDLERDHRPHVSMPQNAN

Figure 74. Amino acid sequence of human Procalcitonin.

1. Anti-procalcitonin monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Specificity: Purification method: Presentation:

Mice Balb/c Sp2/0 Calcitonin or N-terminal PCT or katacalcin conjugated with carrier protein Specific to different epitopes of PCT Protein A affinity chromatography Solution in PBS with 0.1% sodium azide

HyTest offers monoclonal antibodies that are specific to different fragments of the PCT molecule: Nterminal fragment of PCT, calcitonin and katacalcin.

MAbs can be used for the detection of the unprocessed PCT molecule or partially processed PCT us-

ing pairs of antibodies specific to different parts of PCT. The specificity of antibodies is shown in Fig 75.

Furthermore, pairs of antibodies recommended for the development of the PCT sandwich immunoassay are also presented on page 72 Fig. 78.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

71

N-terminal PCT

Calcitonin

Katacalcin

MAbs: 6F10 27A3 38F11 44D9 42

MAbs: 13B9 13F2 13G11 14A2 16B5 24B2

MAbs: 14C12 18B7 22A11

Figure 75. Epitope specificity of anti-PCT MAbs.

The most sensitive immunoassay for PCT detection in blood samples was developed using the following pair of MAbs: MAb 16B5 (calcitonin-specific, capture) and MAb 42 (N-terminal PCT, detection) (Fig. 76). However, several other combinations of MAbs could be used for PCT immunodetection in sandwich immunoassay (Figs. 76 and 78).

Assays were tested for their ability to detect native PCT in human serum. Different serum samples from septic patients and healthy individuals were analyzed using different combinations of anti-PCT MAbs. Serum titration curves for the assay 16B5(capture) – 42 (detection) are shown in Fig. 77.

10000000 1000000

Patient 1

1000000

Patient 2 100000

Normal serum

CPS

CPS

100000

16B5-42

10000

14C12-14A2

10000

27A3-22A11 1000 0,01

0,1

1

10

100

1000

1000 1

PCT concentration (ng/ml)

1:25

1:125

1:625

Serum dilutions

Figure 76. Calibration curves for three human PCT sandwich fluoroimmunoassays utilizing antibodies with different epitope specificity.

Capture MAbs – 1 μg/well Detection MAbs (Eu-labeled) – 0.1 μg/well.

Antigen – PCT human recombinant.

Incubation time – 30 min.

HyTest offers several PCT-specific MAbs that detect native PCT in human blood samples with high sensitivity. Most of the MAbs can be used in different

Figure 77. Titration of human serum samples taken from patients with sepsis of bacterial origin and healthy individual. Pair of MAbs 16B5-42 (capture-detection) was used in sandwich fluoroimmunoassay.

Capture MAb 16B5 – 1 μg/well Detection MAb 42 (Eu-labeled) – 0.1 μg/well.

Incubation time – 45 min.

combinations for PCT immunodetection. The best pairs recommended for PCT sandwich type immunoassay are presented in Fig. 78.

16B5 - 42 27A3 - 14A2

14C12 - 14A2

N-terminal PCT

Calcitonin

Katacalcin

6F10, 27A3, 38F11, 44D9, 42

13B9, 13F2, 13G11, 14A2, 16B5, 24B2

14C2, 18B7, 22A11

18B7 - 44D9 38F11 - 22A11 Figure 78. Pairs of MAbs recommended for PCT sandwich immunoassay (capture-detection).

72

1.5

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

2. Anti-calcitonin monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Specificity: Purification method: Presentation:

Mice Balb/c Sp2/0 Synthetic calcitonin conjugated with carrier protein Specific to human calcitonin Protein A affinity chromatography Solution in PBS with 0.1% sodium azide

Calcitonin, a product of enzymatic cleavage of Procalcitonin, is a peptide that comprises 32 amino acid residues with a molecular weight of approximately 3.4 kDa, and theoretical pI 6.72. Calcitonin is a hormone that participates in calcium and phosphorus metabolism.

HyTest offers MAbs that are highly specific to different epitopes of the calcitonin molecule. The titration curve of anti-calcitonin MAb 13B9 is shown in Fig. 79.

Some anti-calcitonin MAbs are also recommended for PCT immunodetection in combinations with antibodies specific to N-terminal PCT and Katacalcin.

Antibodies were also tested for their ability to detect PCT using Western blotting. All MAbs recognize PCT in blotting after SDS-electrophoresis in reducing conditions (Fig. 80)

kDa 200 116 67

10000

A, 490 mm

1000

31 21 100

14 0.010 0.1

1

10

100

1000

6.5

MAb 13B9 concentration (ng/ml)

Figure 79. Titration curve of anti-Calcitonin MAb 13B9 in indirect ELISA. Antigen: Calcitonin human recombinant - 0.02 μg/well.

1

2

3

4

5

6

Figure 80. Detection of human recombinant PCT in Western blotting by monoclonal antibodies specific to calcitonin following 15% SDS-PAGE in reducing conditions. Antigen loaded - 100 ng/track.

1 – 13B9 2 – 13F2 3 – 13G11 4 – 14A2 5 – 16B5 6 – 24B2

3. Anti-katacalcin monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Specificity: Purification method: Presentation:

Mice Balb/c Sp2/0 Synthetic katacalcin conjugated with carrier protein Specific to human katacalcin Protein A affinity chromatography Solution in PBS with 0.1% sodium azide

Katacalcin is a C-terminal part of the procalcitonin molecule which comprises 21 amino acid residues with a molecular weight of approximately 2.4 kDa, and theoretical pI 5.26. The physiological role of katacalcin is unknown.

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73

10000

1000

A, 490 mm

Antibodies to katacalcin could be used for the specific and sensitive detection of PCT in human blood.

It is recommended to use antibodies to katacalcin mostly as the capture antibodies in PCT immunoassays. The titration curve of anti-katacalcin MAb 14C12 is shown in Fig. 81.

0.100

0.010 0.1

0.1

1

10

100

1000

MAb 14C12 concentration (ng/ml)

Figure 81. Titration curve of anti-katacalcin MAb 14C12 in indirect ELISA. Antigen: Katacalcin conjugated with BSA - 0.02 μg/well.

Antibodies were also tested for their ability to detect PCT in Western blotting. All MAbs recognize PCT in Western blotting after SDS-electrophoresis in reducing conditions (Fig. 82).

200 116 67 31 21

14 Figure 82. Detection of human recombinant PCT by monoclonal antibodies specific to katacalcin after 15% SDS-PAGE in reducing conditions. Antigen loaded - 100 ng/track.

1 – 14C12 2 – 18B7 3 – 22A11

6.5

1 2

3

4. Anti-N-terminal PCT monoclonal antibodies Mice Balb/c Sp2/0 Synthetic N-terminal fragment of procalcitonin conjugated with carrier protein Specific to N-terminal fragment of human procalcitonin Protein A affinity chromatography Solution in PBS with 0.1% sodium azide

The N-terminal fragment of procalcitonin is a peptide that comprises 57 amino acid residues. MAbs specific to N-terminal PCT are recommended for PCT immunodetection in combination with anti-calcitonin or anti-katacalcin antibodies. Anti-N-terminal PCT antibodies work better as the detection antibodies in sandwich immunoassay.

10000

1000

A, 490 mm

Host animal: Cell line used for fusion: Antigen: Specificity: Purification method: Presentation:

0.100

The titration curve for anti-N-terminal PCT MAb 42 is shown on Fig. 83.

Figure 83. Titration curve of anti-N-terminal PCT MAb 42 in direct ELISA. Antigen: N-terminal PCT conjugated with BSA - 0.02 μg/well.

0.010 0.1

1

10

100

MAb 42 concentration (ng/ml)

74

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

1000

Antibodies were also tested for their ability to detect PCT in Western blotting. All MAbs recognize PCT in blotting after SDS-electrophoresis in reducing conditions (Fig. 84).

200 116 67 31 21

14

Figure 84. Detection of human recombinant PCT by monoclonal antibodies specific to N-terminal fragment of PCT after 15% SDS-PAGE in reducing conditions. Antigen loaded - 100 ng/track.

1 – 6F10 2 – 27A3 3 – 28F11 4 – 44D9 5 – 42

6.5

1

2

3

4

5

Ordering information MAb 6F10 27A3 38F11 44D9 42 13B9 24B2 13F2 13G11 14A2 16B5 14C12 22A11 18B7

Cat. # 4PC47 4PC47 4PC47 4PC47 4PC47 4C10 4C10 4C10 4C10 4C10 4C10 4PC47 4PC47 4PC47

Specificity N-terminal fragment N-terminal fragment N-terminal fragment N-terminal fragment N-terminal fragment Calcitonin (central part) Calcitonin (central part) Calcitonin (central part) Calcitonin (central part) Calcitonin (central part) Calcitonin (central part) Katacalcin (C-terminal part) Katacalcin (C-terminal part) Katacalcin (C-terminal part)

References: 1. Le Moullec J.M., et al. The complete sequence of human procalcitonin. // FEBS Lett., 167 (1), 93-97 (1984).

2. Assicot M, et al. High serum procalcitonin concentrations in patients with sepsis and infection. // Lancet, 341, 515-18 (1993).

3. Meisner M., Reinhart K. Is procalcitonin really a marker of sepsis? // Int. J. Intensive Care, 8 (1), 15-25. (2001).

Isotype IgG1 IgG2a IgG1 IgG2a IgG2a IgG2a IgG1 IgG1 IgG1 IgG1 IgG2b IgG1 IgG1 IgG1

Remarks EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB

4. Sponholz C. et al. Diagnostic value and prognostic implications of serum procalcitonin after cardiac surgery: a systematic review of the literature. Critical Care, 10:R145 (2006).

5. Meisner M. Pathobiochemistry and clinical use of procalcitonin. // Clin. Chim.

Acta., 323, 17-29 (2002).

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75

XII D-dimer and High Molecular Weight Fibrin Degradation Products Fibrinogen is a main protein of the blood coagulation system. It consists of two identical subunits, each of which contains three polypeptide chains: A, B and G. The process of blood coagulation results in the conversion of fibrinogen into fibrin by thrombin and subsequent fibrin polymerization. The fibrin clot is then digested by plasmin and fibrin degradation products with different molecular weights are released into the bloodstream.

D-dimer is a final product of fibrin degradation (MW 180 kDa, Fig. 85). It consists of the remnants of all three chains that are cross linked by disulfide bonds. The dimeric structure is held by two isopeptide bonds between the C-terminal parts of G-chains forming a cross-linked region.

The D-dimer level in healthy individuals is less than 0.5 Mg/ml. Elevated levels of D-dimer were found in the blood of patients with pulmonary thromboembolism, deep vein thromboses, atherosclerosis and other cardiovascular diseases. The elevated level of D-dimer in blood indicates a risk of myocardial infarction and is believed to be a reliable marker of pathological coagulation that underlies pathogenesis of most cardiovascular diseases (1, 2). It is widely used to exclude deep vein thrombosis (3). Furthermore, elevated levels of D-dimer without thrombotic symptoms makes it possible to suspect malignancy.

For the accurate determination of D-dimer, the assays must not detect fibrinogen and its degration products (D-monomer). HyTest offers D-dimer produced from clotted fibrinogen by means of plasmin digestion and anti-D-dimer MAbs.

1. Human D-dimer Source: Purity: Presentation: Application: Storage:

Human plasma >90% Lyophilized Immunological D-dimer standard, biochemical and immunochemical studies -20°C

220 kDa 156 kDa

D-dimer

116 kDa

For more than 10 years, HyTest has been one of the world’s leading suppliers of the D-dimer antigen, which is utilized by assay manufacturers as either a standard or a calibrator. We offer a highly purified D-dimer that is prepared from a human plasma clot.

76 kDa

53 kDa Figure 85. SDS gel electrophoresis of D-dimer under non-reducing conditions.

Lane 1: Molecular weight standards Lane 2: D-dimer (3 μg) Gel staining: Coomassie brilliant blue R-250

Ordering information: Product

Cat.#

Purity

Source

Human D-dimer

8D70

>90%

Human plasma

76

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

2. Anti-D-dimer monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Fibrin clot, fibrin degradation peptides, D-dimer Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide D-dimer immunoassay, cross-linked fibrin degradation products and D-dimer immunodetection in Western blotting

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells

of Balb/c mice immunized with homogenized clot, fibrin degradation peptides or D-dimer.

2.1. Antibodies recognizing D-dimer cross-linked region HyTest offers a new, unique MAb DD93 that recognizes a cross-linked region of D-dimer. It detects crosslinked gamma-chains of D-dimer in Western blotting under reducing and non-reducing conditions.

2.2. Antibodies specific to D-dimer and to high molecular weight fibrin degradation products

1,8 1,6 1,4 1,2

A490

HyTest offers MAbs DD1, DD2, DD3, DD22, DD41, DD44, and DD46, which are specific to D-dimer.

These MAbs show no cross-reactivity with fibrinogen.

MAbs DD1, DD2, and DD3 do not cross-react with D-monomer. MAbs DD22, DD41, DD44, and DD46 show no cross-reaction (or very low cross-reaction) with D-monomer in direct ELISA (with D-monomer used for plate coating) and some cross-reaction with D-monomer in sandwich immunoassay. The level of cross-reactivity with D-monomer in the sandwich is dependent on the specificity of secondary MAbs used in a pair.

2,0

1,0 0,8 0,6 0,4 0,2 0,0 1

10

100

1000

Antibody dilution Figure 86. Titration curves of D-dimer specific monoclonal antibody DD1. Antigen for coating: 200 ng/well ( Q - D-dimer, V - fibrinogen, O - D-monomer). Initial DD1 antibody concentration: 100 μg/ml.

2.3. Antibodies recognizing epitopes common for D-dimer, fibrinogen and D-monomer MAbs DD4, DD5 and DD6 recognize D-dimer and high molecular weight fibrin degradation products but also cross-react with fibrinogen and have a low crossreaction with D-monomer. These MAbs could only be used as detection antibodies in a two-step sandwich protocol for D-dimer quantification.

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77

2.4. Applications

2.4.2. Two-step D-dimer immunoassay

2.4.1. One-step D-dimer immunoassay

The following pairs can be used in a two-step sandwich immunoassay for D-dimer detection in human plasma:

HyTest recommends two MAb combinations to be used in one-step immunoassays for D-dimer quantification in human plasma:

DD1 – DD6 DD2 – DD6 DD1 – DD5 DD2 – DD5 DD1 – DD4 DD2 – DD4

DD2 – DD41 DD2 – DD44 The recommended pairs are specific to cross-linked material (D-dimer and high molecular weight fibrin degradation products) and do not show cross-reactivity with fibrinogen, fibrinogen degradation products or D-monomer (Fig. 87).

1000000

CPS

800000 600000

DD DM Fg

400000 200000 0 0,01

0,1

1

10

The recommended pairs are specific to cross-linked material in samples and do not detect fibrinogen, fibrinogen degradation products or D-monomer.

Due to cross-reactivity of all detection antibodies with fibrinogen, we strongly recommend using these pairs in a two-step assay. In the first step, one should incubate capture antibodies with plasma samples and in the second step one should incubate the immune complex (capture MAb-antigen) with the detection antibodies to form a sandwich. To be analyzed in a sandwich immunoassay, plasma must be diluted at least two-fold with 10 mM Tris-HCl buffer, pH 7.5, containing 1 M NaCl and 0.1% Tween 20, to avoid non-specific binding.

Protein concentration (μg/ml)

Figure 87. Detection of D-dimer, D-monomer and fibrinogen by the DD2–DD41 assay.

2.4.3. Detection of D-dimer in Western blotting

New data!

Anti-D-dimer antibodies can be used in Western blotting. MAb DD1 recognizes D-dimer and does not recognize fibrinogen, D-monomer, or D-dimer treated with mercaptoethanol (Fig. 88). Detailed information on the detection of D-dimer by different MAbs in Western blotting is summarized in Table 10.

Figure 88. D-dimer, D-monomer, and fibrinogen immunodetection

by MAb DD1 in Western blotting.

Lane 1: D-dimer Lane 2: D-monomer Lane 3: fibrinogen Lane 4: D-dimer treated with mercaptoethanol

D-dimer

1 2 34

78

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

Table 10. Detection of D-dimer and fibrinogen by anti-D-dimer MAbs in Western blotting.

MAb

D-dimer (non-reducing conditions)

D-dimer (reducing conditions)

Fibrinogen (non-reducing conditions)

Fibrinogen (reducing conditions)

DD1

+

-

-

-

DD2

+

-

+

-

DD3

+

-

-

-

DD4

+

-

+

-

DD5

+

-

+

-

DD6

+

-

+

-

DD22

+

+ (beta-chain)

+

+

DD41

+

+ (beta-chain)

+

+

DD44

+

+ (beta-chain)

-/+

+

DD46

+

+ (beta-chain)

+

+

DD93

+

+ (gamma-chain)

-

-

Ordering information: MAb

Cat. #

Specificity

Subclass

Application

DD1

4D30

IgG2a

EIA, Sandwich immunoassay (capture), WB

DD2

4D30

IgG2b

EIA, Sandwich immunoassay (capture), WB

DD3

4D30

IgG2b

EIA, Sandwich immunoassay (capture), WB

DD4

4D30

IgG2b

EIA, Sandwich immunoassay (detection), WB

DD5

4D30

IgG2b

EIA, Sandwich immunoassay (detection), WB

DD6

4D30

IgG2a

EIA, Sandwich immunoassay (detection), WB

DD22

4D30

IgG2a

EIA, sandwich immunoassay, WB

DD41

4D30

IgG2a

EIA, sandwich immunoassay, WB

DD44

4D30

IgG2b

EIA, sandwich immunoassay, WB

DD46

4D30

IgG2a

EIA, sandwich immunoassay, WB

DD93

4D30

N/cr with D-monomer and fibrinogen N/cr with D-monomer and fibrinogen N/cr with D-monomer and fibrinogen Low C/R with D-monomer, C/R with fibrinogen Low C/R with D-monomer, C/R with fibrinogen Low C/R with D-monomer, C/R with fibrinogen N/cr with D-monomer and fibrinogen (in direct ELISA) N/cr with D-monomer and fibrinogen (in direct ELISA) N/cr with D-monomer and fibrinogen (in direct ELISA) N/cr with D-monomer and fibrinogen (in direct ELISA) N/cr with D-monomer and fibrinogen

IgG1

EIA, sandwich immunoassay (detection), WB

References: 1. Bounameaux H, de Moerloose P, Perrier A, Reber G. Plasma measurement of D-dimer as diagnostic aid in suspected venous thromboembolism: an overview. Thromb Haemost. 1994; 71: 1-6.

2. Rowbotham BJ, Carroll P, Whitaker AN, Bunce IH, Cobcroft RG, Elms MJ, Masci PP, Bundesen PG, Rylatt DB, Webber AJ. Measurement of crosslinked fibrin derivatives - use in the diagnosis of venous thrombosis. Thromb Haemost. 1987; 57: 59-61.

3. Righini M, Perrier A, De Moerloose P, Bounameaux H. D-Dimer for venous thromboembolism diagnosis: 20 years later. J Thromb Haemost. 2008; 6:1059-71.

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XIII Thrombin Activatable Fibrinolysis Inhibitor (TAFI) Thrombin activatable fibrinolysis inhibitor (TAFI; synonyms: procarboxypeptidase B, procarboxypeptidase U, procarboxypeptidase R) belongs to a family of Zn-containing metallocarboxypeptidases specific to C-terminal lysine and arginine residues. It circulates in plasma as a zymogen with a molecular weight of 55 kDa (401 amino acid residues; pI 5.0, Fig. 89). Becoming activated by thrombin-thrombomodulin complex during blood coagulation, it exerts carboxypeptidase activity. Activated TAFI removes C-terminal lysine residues from fibrin, which are necessary for plasminogen binding to fibrin, which in turn prevents plasminogen from activation into plasmin and retards the lysis of a fibrin clot.

The concentration of TAFI in plasma of healthy people is 5 – 10 μg/ml. High plasma levels of TAFI were found in patients with stable angina pectoris, angiographically verified coronary artery disease and in ischemic stroke. Elevated TAFI concentration in blood is considered to be a risk factor for venous thrombosis. A deficiency of TAFI might contribute to the severity of bleeding disorders in hemophilias A and B. TAFI level is decreased in cases of chronic liver disease. All of these facts make TAFI an important diagnostic parameter in cardiovascular diseases.

HyTest’s TAFI is purified from human plasma by affinity chromatography.

1. Human TAFI Source: Purity: Presentation: Application:

Human plasma >90% Liquid or frozen Immunological and mass TAFI standard

220 kDa 116 kDa 76 kDa TAFI 53 kDa

Figure 89. SDS gel electrophoresis of TAFI under non-reducing conditions.

Lane 1: molecular weight standards Lane 2: human TAFI (5 μg) Gel staining: Coomassie brilliant blue R-250

Ordering information: Product

Cat. #

Purity

Source

Human TAFI

8TA1

>90%

Human plasma

80

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

2. Anti-TAFI monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human TAFI Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide TAFI immunoassay, TAFI immunoaffinity purification and TAFI immunodetection in Western blotting

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with purified human TAFI. Specificity of antibodies was confirmed by ELISA and Western blotting. All antibodies recognize TAFI in ELISA (Fig. 90). MAbs 13H4, 16C5 and 19E2 recognize TAFI in Western blotting after SDS gel electrophoresis under non-reducing conditions (Fig. 91).

1,8

TAFI Figure 91. Detection of TAFI by MAb 13H4 in Western blotting (non-reducing conditions).

A

1,6 1,4

Figure 90. Titration curves of purified TAFI (A) and TAFI in plasma (B) using antibody pair 13D5 – 13H4.

MAb 13D5 for capture: 200 ng/well MAb 13H4 for detection: conjugated with HRP Room temperature

A490

1,2 1,0 0,8 0,6 0,4 0,2 0,0 5000

1000

100

10

1

0,1

Recommended pairs to be used for TAFI detection in human plasma by sandwich immunoassay:

TAFI concentration (ng/ml)

1,8

13D5 13D5 15A10 15A10 16C5

B

1,6 1,4

A490

1,2

– – – – –

13H4 19E2 13H4 19E2 13D5

1,0 0,8 0,6 0,4 0,2 0,0 1

10

100

1000

10000

0,1

Plasma dilution

References: 1. Van Tilburg N.H. et al. Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis. Blood 2000; 95: 2855-9.

2. Silveira A. et al. Plasma procarboxypeptidase U in men with symptomatic coronary artery disease. Thromb Haemost 2000; 84: 364-8.

3. Schroeder V. et al. Thrombin activatable fibrinolysis inhibitor (TAFI) levels in patients with coronary artery disease investigated by angiography. Thomb. Haemost. 2002; 88(6): 1020-5.

4. Ladenvall C. et al. Thrombin activatable fibrinolysis inhibitor activation peptide shows association with all major subtypes of ischemic stroke and with TAFI gene variation. Arterioscler. Thromb. Vasc. Biol. 2007; 27(4): 955-62.

Ordering information: MAb

Cat. #

Specificity

Subclass

Application

13C3 13D5 13H4 15A10 19E2 16C5

4TA1 4TA1 4TA1 4TA1 4TA1 4TA1

TAFI TAFI TAFI TAFI TAFI TAFI

IgG2a IgG2a IgG1 IgG2a IgG1 IgG1

EIA, Sandwich immunoassay EIA, Sandwich immunoassay EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay EIA, Sandwich immunoassay, WB EIA, Sandwich immunoassay, WB

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81

XIV Fibrinogen Fibrinogen is a main component of the blood coagulation system. It is a large protein (MW 340 kDa) consisting of two identical subunits, with each subunit containing three polypeptide chains: A, B and G. All chains are connected to one another by a number of disulfide bonds. In the process of fibrin formation, fibrinopeptides A (1 – 16 amino acid residues) and B (1 – 17 amino acid residues) are released by thrombin from the N-terminal parts of A- and B-chains, respectively. Resulting fibrin-monomers polymerize to form a fibrin clot. As fibrin is the main substrate for thrombus formation, fibrinogen clotting underlies pathogenesis of MI, thromboembolism and throm-

boses of arteries and veins. Fibrin formation is also involved in pathogenesis of inflammation, tumor growth and many other diseases.

The normal fibrinogen concentration in plasma is approximately 3 mg/ml. The elevated level of fibrinogen in the patient’s blood is regarded as an independent risk factor for cardiovascular diseases. An increase in blood fibrinogen concentration was shown to be a strong predictor of coronary heart disease (1, 2). All of these facts make fibrinogen an important parameter in the diagnosis of cardiovascular diseases.

1. Anti-fibrinogen monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human fibrin degradation products Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide Fibrinogen immunoassay and fibrinogen immunodetection in Western blotting

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with purified human fibrin degradation products.

Fg

Specificity of antibodies was confirmed by ELISA and Western blotting. All antibodies recognize fibrinogen in ELISA. MAbs 1F3, 6G12, 15E11, 27C8 and 40F11 recognize fibrinogen in Western blotting after SDS gel electrophoresis under non-reducing conditions; MAbs 15H12 and 41D9 recognize fibrinogen in Western blotting after electrophoresis under both reducing and non-reducing conditions (Fig. 92).

Antibodies recommended for sandwich immunoassay to detect fibrinogen together with fibrin degradation products are: 1F3 – 27C8 40F11 – 1F3

82

1

2

3

Figure 92. Detection of fibrinogen by anti-fibrinogen antibodies in Western blotting (non-reducing conditions).

Lane 1: MAb 1F3 Lane 2: MAb 6G12 Lane 3: MAb 15H12

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

Ordering information: MAb

Cat. #

Specificity

Subclass Application

1F3

4F1

Fibrinogen, Fibrin degradation products

IgG2b

6G12

4F1

Fibrinogen, Fibrin degradation products

IgG2b

15H12

4F1

Fibrinogen, Fibrin degradation products

IgG2b

27C8

4F1

Fibrinogen, Fibrin degradation products

IgG2a

40F11

4F1

Fibrinogen, Fibrin degradation products

IgG2b

EIA, Sandwich immunoassay (capture, detection), WB EIA, Sandwich immunoassay (capture, detection), WB EIA, Sandwich immunoassay (capture, detection), WB EIA, Sandwich immunoassay (capture, detection), WB EIA, Sandwich immunoassay (capture, detection), WB

References: 1. Lowe G. et al. Blood rheology, cardiovascular risk factors, and cardiovascular disease: the West of Scotland Coronary Prevention Study. Thromb Haemost, 2000; 84:553-8.

2. Danesh J. et al. Association of fibrinogen, C-reactive protein, albumin, or leukocyte count with coronary heart disease: meta-analyses of prospective studies. JAMA, 1998; 279:1477-82.

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83

XV Fibrinopeptide A Fibrinogen consists of two identical subunits that contain three polypeptide chains: A, B and G. The process of fibrinogen clotting begins with the removal of N-terminal peptides from its A-chains (fibrinopeptide A) and B-chains (fibrinopeptide B) by thrombin that is formed by a set of cascade reactions. An increase in the fibrinopeptide A level is a direct marker of fibrinogen clotting. Elevated levels of fibrinopeptide A were found in patients with myocardial infarction, coronary

heart disease and other disorders accompanied by the activation of the blood coagulation system.

Fibrinogen can only be specifically detected by immunological methods if capture antibodies recognize fibrinopeptides A or B in intact fibrinogen. Otherwise fibrin degradation products will be measured together with fibrinogen.

1. Synthetic fibrinopeptide A Source: Purity: Presentation: Application: Storage:

Amino acid synthesis >99% Lyophilized Immunoassay standard and calibrator +4°C

Fibrinopeptide A is synthesized from amino acids according to the sequence ADSGEGDFLAEGGGVR that is 1–16 amino acid residues of the A-chain of fi-

brinogen. The identity and purity of fibrinopeptide A was confirmed by mass spectroscopy and the purity was found to be more than 99%.

Ordering information: Product

Cat. #

Purity

Source

Synthetic fibrinopeptide A

8FP1

>99%

Amino acid synthesis

2. Anti-fibrinopeptide A monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Fibrinopeptide A Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide Fibrinogen and fibrinopeptide A immunoassay

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with fibrinopeptide A conjugated with a carrier protein. Antibodies recognize free fibrinopeptide A and fibrinopeptide A region of fibrinogen A-chain.

For specific fibrinogen detection by sandwich immunoassay we recommend using anti-fibrinopeptide A MAbs for capture and anti-fibrinogen MAbs for detection. Recommended pairs: 49D2 (anti-fibrinopeptide A) – 40F11 (anti-fibrinogen) 1F7 (anti-fibrinopeptide A) – 1F3 (anti-fibrinogen) 26E7 (anti-fibrinopeptide A) – 27C8 (anti-fibrinogen)

Ordering information: MAb

Cat. #

Specificity

Subclass

Application

1F7 26E7 49D2

4FP1 4FP1 4FP1

Fibrinopeptide A Fibrinopeptide A Fibrinopeptide A

IgG2a IgG2b IgG2a

EIA, Sandwich immunoassay (capture), WB EIA, Sandwich immunoassay (capture), WB EIA, Sandwich immunoassay (capture), WB

References: 1. Sonel A. et al. Prospective Study Correlating Fibrinopeptide A, Troponin I, Myoglobin, and Myosin Light Chain Levels With Early and Late Ischemic Events in Consecutive Patients Presenting to the Emergency Department With Chest Pain. Circulation. 2000; 102:1107-13.

84

2. Rapold H.J. et al. Fibrin formation and platelet activation in patients with myocardial infarction and normal coronary arteries. Eur Heart J. 1989; 10:323-33.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

XVI Fatty Acid Binding Protein (FABP) Fatty Acid Binding Protein (FABP) is a small cytosolic protein that is responsible for the transportation and deposition of fatty acids inside the cell. Several different isoforms of FABP are expressed in different tissue types. Cardiac isoform of FABP (cFABP) is expressed mainly in cardiac muscle tissue and in significantly lower concentration in skeletal muscles.

Cardiac isoform of FABP consists of 132 amino acid residues with molecular weight 14727 Da and theoretical pI 6.34.

It was recently demonstrated that cFABP can be used as an early marker of myocardial infarction. cFABP has the same kinetics of liberation into patients blood as myoglobin and as cFABP concentration is significantly lower in skeletal muscle (as compared to myoglobin) the concentration of cFABP in

the blood of healthy donors is also significantly lower (6 – 10 ng/ml for cFABP and 40 – 60 ng/ml for myoglobin). This fact means that cFABP is a more sensitive and reliable early marker of myocardial cell death. Recent studies have demonstrated that FABP can also be useful for the early detection of minor myocardial events such as unstable angina. Switching clinical studies from myoglobin to cFABP can be helpful for the improvement of early AMI diagnosis.

FABP is purified from human cardiac tissue by several chromatographic methods, including gel-filtration and ion-exchange chromatography. After SDSPAGE in reducing conditions, cFABP is presented as a single band with an apparent molecular weight of 15 kDa.

1. Human FABP Source: Purity: Presentation: Application: Storage:

Human cardiac tissue. Blood sample from the tissue donors was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis >95% Lyophilized Immunogen for antibody production, immunological and mass FABP standard, FABP biochemical and immunochemical studies -20°C

Ordering information: Product

Cat. #

Purity

Source

Human fatty acid binding protein

8F65

>95%

Human cardiac muscle

2. Anti-FABP monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Epitope specificity: Purification method: Presentation:

Mice Balb/c Sp2/0 Human FABP Partially determined Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide

Hybridoma clones have been derived from hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with purified human FABP. The best combination for sandwich immunoassay is MAb 9F3 used as a capture antibody and MAb 10E11

as a detection antibody (Fig. 93). The immunoassay utilizing these monoclonal antibodies was evaluated in clinical studies and demonstrated high sensitivity, good kinetics and good recognition of the antigen in patients samples (Fig. 94).

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

85

1000000

Increasement over cut-off value

200

CPS

100000

10000

1000

175 150 125 100 Human cardiac Tnl FABP

75 50 25 0

1

10

100

0

1000

10

20

30

40

50

60

70

Hours after onset of chest pain

FABP (ng/ml)

Figure 93. Calibration curve of FABP sandwich immunoassay.

Figure 94. Time-dependent changes of FABP (blue) and cTnI (green) concentrations in the blood of representative AMI patient.

Ordering information: MAb

Cat. #

Specificity

Subclass

Application

5B5 9F3 10E1 6B6

4F29 4F29 4F29 4F29

FABP FABP FABP FABP

IgG1 IgG1 IgG1 IgG2b

EIA, Sandwich immunoassay (capture, detection) EIA, Sandwich immunoassay (capture, detection) EIA, Sandwich immunoassay (capture, detection) WB, A/C

3. FABP free serum Prepared from: Method of purification: Delivery form: Storage:

Pooled normal human serum Immunoaffinity chromatography Frozen liquid -20°C

FABP free serum is prepared from pooled normal human serum by immunoaffinity chromatography. The matrix for affinity sorbent utilizes three monoclonal antibodies with different epitope specificity.

FABP free serum does not contain more than 0,5 ng/ ml of human FABP (according to ELISA) and can be used as a matrix for standard and calibrator preparations.

Ordering information: Product

Cat. #

Source

FABP free serum

8FFS

Pooled normal human serum

86

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

XVII Glycogen Phosphorylase, BB Isoenzyme (GPBB) Glycogen phosphorylase BB is an enzyme that plays an important role in the glycogen turnover. Three known isoforms of glycogen phosphorylase are expressed in brain and cardiac muscle tissue (GPBB), skeletal muscles (GPMM) and the liver (GPLL). GPBB is a homodimer that consists of two subunits with molecular mass 96682 Da (843 amino acid residues) and theoretical pI 6.26.

In 1987, GPBB was suggested for the first time as a marker of acute myocardial ischemia and acute myocardial infarction. It is considered to be an early marker of myocardial cell death and its kinetics of release closely resemble those of myoglobin and

FABP. Recent studies have demonstrated that GPBB can be useful in the diagnosis of myocardial tissue damage, e.g. in patients with bypass surgery and unstable angina. Whilst all of these features make GPBB a very promising marker of myocardial cell injury, its cardio-specificity should still be estimated.

GPBB is purified from human cardiac tissue by several chromatographic methods, including gel-filtration and ion-exchange chromatography. After SDSPAGE in reducing conditions, GPBB is presented as a single band with apparent molecular mass 92 kDa. Preparation does not contain more than 2% of glycogen phosphorylase MM isoenzyme.

1. Human GPBB Source: Purity: Presentation: Application: Storage:

Human cardiac tissue. Blood sample from the tissue donors was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis

>95% Solution in buffer, containing 1 mM B-glyserophosphate, 1 mM EDTA, 15 mM ME, 0.5 M NaCl, and 50% glycerol, pH 7.8 Immunogen for antibody production, immunological and mass GPBB standard, GPBB enzymatic, biochemical and immunochemical studies -20°C

Ordering information: Product

Cat. #

Purity

Source

Human GPBB

8G67

> 95 %

Human cardiac muscle

2. Anti-GPBB monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human GPBB Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide GPBB immunoassay, GPBB immunoaffinity purification, GPBB immunodetection in Western blotting

Hybridoma clones have been derived from hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with purified human GPBB. Five MAbs (7B9, 8G7, 6G5, 11C10 and 1G6) are specific to the BB isoenzyme, while others also recognize MM isoenzyme.

The best combinations to be used in sandwich immunoassay are: 1G6 1G6 6F1 17B6

– – – –

6F1 9F5 7B9 10H5

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

87

Ordering information: MAb

Cat. #

SpeciďŹ city

Subclass

Application

6G5 8G7 1G6

4GP31 4GP31 4GP31

BB isoenzyme BB isoenzyme BB isoenzyme

IgG1 IgG1 IgG2b

7B9

4GP31

BB isoenzyme

IgG1

10D12 3G1 10H5

4GP31 4GP31 4GP31

BB and MM isoenzyme BB and MM isoenzyme BB and MM isoenzyme

IgG1 IgG1 IgG2a

9F5

4GP31

BB and MM isoenzyme

IgG1

17B6

4GP31

BB and MM isoenzyme

IgG1

6F1

4GP31

BB and MM isoenzyme

IgG1

WB WB EIA, Sandwich immunoassay (capture), WB EIA, Sandwich immunoassay (detection), WB WB WB EIA, Sandwich immunoassay (detection), WB EIA, Sandwich immunoassay (detection), WB EIA, Sandwich immunoassay (capture, detection), WB EIA, Sandwich immunoassay (capture, detection), WB

88

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

XVIII Serum Amyloid A (SAA) Serum amyloids A form a family of apolipoproteins containing several groups of proteins with a molecular weight of approximately 11-12 kDa. In human blood SAA are mostly associated with high density lipoprotein (HDL). One group of SAA, the so called constitutive SAA proteins (namely SAA4 and SAA5), are synthesized in the liver and almost not secreted into the bloodstream. Another group, acute-phase SAA, includes SAA1 and SAA2, which are also produced by hepatocytes and secreted into the blood following interleukin-6 stimulation in response to infection, inflammation, injury or stress (1).

Acute phase SAA is the major SAA component in human plasma. During inflammation apolipoprotein (apo)A-1 in HDL is partially displaced by SAA. This process results in the reduction of the anti-atherogenetic properties of HDL, altering HDL-mediated cholesterol delivery to cells (2). The physiological functions of SAA in lipoproteins other than HDL remain unclear.

The elevated level of plasma SAA was originally described in patients with different types of amyloidosis (SAA is a precursor of protein deposits, typical for amyloid A amyloidosis), rheumatic diseases, Alzheimer’s disease and neoplastic disorders (3). It was demonstrated that in cases of viral infections and kidney allograft rejection, SAA could prove to be an

even more useful marker than other inflammatoryassociated proteins (1).

Recent studies have revealed the association of inflammatory response with the etiology and clinical course of cardiovascular disorders, although exact mechanism for this association remains unknown.

In this context acute phase proteins are intensively studied as potential markers that can predict the development of disease. Among other inflammation markers, SAA was proven to be a prognostic marker for patients with acute coronary syndrome (4) and stable coronary artery disease (5). Moreover, population-based studies have clarified the value of this marker in the prediction of the future development of coronary artery disease in healthy people (6, 7). SAA levels were strong predictors of 3-year cardiovascular events (7). In contrast to other markers of CVD, SAA directly demonstrates intravascular inflammation, reflecting unstable atheromatous plaques eroding. Although other non-specific inflammatory markers such as CRP also correlate with cardiovascular disease, the wider dynamic range and more rapid response of SAA suggests, that it could be an even better marker of disease progress.

HyTest has developed anti-SAA monoclonal antibodies that are suitable for the development of quantitative SAA immunoassay.

1. Anti-SAA monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human recombinant SAA Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide SAA immunoassay, SAA immunoprecipitation, SAA immunodetection in Western blotting

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with human recombinant

SAA, conjugated with carrier protein. All MAbs were tested with recombinant antigen in direct ELISA, Western blotting and sandwich immunoassay.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

89

1.1. Applications 1.1.1. Sandwich assay for quantitative SAA immunodetection and Western blotting

SAA1 SAA6 SAA15 SAA6 SAA7 SAA1

– – – – – –

SAA15 SAA15 SAA1 SAA1 SAA15 SAA6

1000000

100000 CPS

The best of HyTest’s combinations of monoclonal antibodies for SAA sandwich immunoassays were selected from 400 tested two-site MAb combinations on the basis of their sensitivity, specificity and kinetics. The best two-site MAb combinations are (capture - detection):

SAA1-SAA15Eu* SAA6-SAA15Eu*

10000

1000 0,1

1

10

100

1000

SAA, ng/ml

Figure 95. Calibration curves for human recombinant SAA detection using anti-SAA monoclonal antibodies.

Capture MAbs: 0.2 μg/well Detection MAbs: Eu-labeled, 0.1 μg/well.

Antigen: Recombinant human SAA (Cell science, USA)

All selected anti-SAA MAbs recognize SAA in Western blotting as a single band of 12 kDa.

Ordering information: MAb

Cat. #

Specificity

Subclass

Application

SAA1 SAA6 SAA11 SAA12 SAA14 SAA15

4SA11 4SA11 4SA11 4SA11 4SA11 4SA11

SAA SAA SAA SAA SAA SAA

IgG1 IgG1 IgG2b IgG2a IgG1 IgG1

Sandwich immunoassay, WB Sandwich immunoassay, WB Sandwich immunoassay, WB Sandwich immunoassay, WB Sandwich immunoassay, WB Sandwich immunoassay, WB

References: 1. Malle E et al (1993): Serum amyloid A (SAA): an acute phase protein and apolipoprotein. Atherosclerosis, 102: 131-146.

2. Marhaug G et al. (1982): Characterization of amyloid related protein SAA compleed with serum lipoprotein (apoSAA). Clin Exp Immunol 50: 382-389.

3. Benson MD, Cohen AS. (1979): Serum amyloid A protein in amyloidosis, rheumatic, and enoplastic diseases. Arthritis Rheum. 22(1): 36-42.

4. Liuzzo G et al. (1994): The prognostic value of C-reactive protein and serum amyloid A protein in severe unstable angina. N Engl J Med 331: 417-424.

90

5. Ogasawara K et al (2004): A serum amyloid A and LDL complex as a new prognostic marker in stable coronary artery disease. Atherosclerosis, 174: 349356.

6. Ricker PM et al. (2000): C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 342: 836843.

7. Johnson BD et al (2004): Serum amyloid A as a predictor of coronary artery disease and cardiovascular Outcome in women. Circulation, 109:726-732.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

XIX High Sensitivity Human C-Reactive Protein (hsCRP) C-reactive protein (CRP) was originally discovered by Tillett and Francis in 1930 as a substance in the serum of patients with acute inflammation that reacted with the C polysaccharide of pneumococcus. Human CRP belongs to a very conservative family of proteins, named “pentraxins”, which is characterized by calcium dependent ligand binding and radial symmetry of five monomers forming a ring around central pore (1).

CRP is a 224 residue protein with a monomer molecular mass of approximately 25 kDa and pI 6.4 (2-5).

Monomers are noncovalently associated into pentameric structure.

For decades, CRP was known as liver-derived protein.

However, recent data has shown a significant level of CRP expression in other tissues, such as blood vessel wall and coronary artery smooth muscle cells, where CRP is supposed to be presented as a monomer (mCRP), while native pentameric protein (nCRP) is predominantly found in plasma (2-5).

The exact function of CRP in human organism remains under discussion. This protein has been shown to participate in inflammatory as well as innate immunity processes. Important bioactivities of CRP are determined by its ability to bind a variety of ligands, such as damaged cell membranes, apoptotic cells, fibronectin, etc, with highest affinity to phosphocholine residues. When CRP is ligand-bound, it could be recognized by complement component C1q, whereby classical complement pathway is activated. Via interaction with complement factor H, CRP regulates the alternative complement pathway (7).

C-reactive protein is accepted in clinical use as the major, although rather non-specific, marker of inflammation. In generally healthy subjects, CRP levels are usually less than 5 mg/L. In pathology, CRP concentration has an enormous, 10,000-fold dynamic range (approximately 0.05–500 mg/L) (8). The highest levels of CRP (above 30 mg/L) are observed in bacterial infection, such as septic arthritis, meningitis and pneumonia. Mildly elevated CRP, approximately 1040 mg/L was described following myocardial infarction and other types of tissue damage.

In 2003, the Centers for Disease Control and Prevention (CDC) and the American Heart Association (AHA) issued a statement that identified CRP as the inflammatory marker best suited for use in current clinical practice to assess cardiovascular risk (9). Many epidemiologic studies have indicated that CRP is a strong independent predictor of future cardiovascular events, including myocardial infarction, ischemic stroke, peripheral vascular disease, and sudden cardiac death without known cardiovascular disease (as reviewed by Clearfield (10)). The CDC/AHA guidelines support the use of CRP in primary prevention and set cutoff points according to relative risk categories: low risk (<1.0 mg/L), average risk (1.0-3.0 mg/L), and high risk (>3.0 mg/L). This is why present day high sensitivity CRP (hsCRP) assays are aimed at nanogram per milliliter (ng/ml) CRP level distinction, and abbreviation hsCRP now is the accepted name of the detected protein in such assays. The significant relationship between plasma hsCRP and the risk of death in patients with the acute coronary syndrome was shown (11). Furthermore, Koenig et al. (12) reported that increased circulating hsCRP concentrations are associated with an increased risk of death from several widespread chronic diseases.

Novel immunometric assays, utilizing unique monoclonal antibodies produced by HyTest, achieve excellent sensitivity with linear detection range from 0.025 mg/L to 2.5 mg/L in magnetic biosensor assay (13); from 0.01 mg/L to 50 mg/L in the immunochemiluminometric assay (14) (with detection limit of 0.004 mg/L in both assays). In a solid-phase sandwich fluorescence immunoassay using nanocrystals, a detection limit of 0.0011 mg/L was reached (15). Our best pairs C2-C6 and C5-CRP135 and several others provide 10,000-fold linearity in experimental immunofluorometric assays. These antibody combinations could be used for the development of hsCRP assays for different diagnostic platforms. In conventional CRP assays turbidimetry and competitive assay techniques are often applied and relatively low affinity of utilized MAbs is usually preferable in such assays. For the convenience of our customers, we derived monoclonal antibodies with different affinity, enabling them to be used in different types of immunoassays.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

91

1. Human CRP Source: Purity: Presentation: Application: Storage:

Human pleural/ascitic fluid or plasma. Blood sample from the tissue donors was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis >95% Solution in 0.3 M NaCl, 0.05% sodium azide, 20 mM Tris, pH 8.0 Immunogen for antibody production, immunological and mass CRP standard, CRP biochemical and immunochemical studies +4°C

Ordering information: Product

Cat. #

Purity

Source

Human C-reactive protein

8C72

>95%

Human pleural/ascitic fluid or plasma

2. Anti-CRP monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human CRP Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide Different types of quantitative CRP immunoassays, CRP immunodetection in Western blotting, CRP immunoaffinity purification, immunohistochemistry.

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with purified human CRP.

2.1. Applications In native CRP molecule each protomer has two coordinated Ca2+ ions (17). HyTest offers anti-CRP MAbs which are either sensitive or insensitive to the absence of Ca 2+ in the solution. Some of our antibodies recognize antigen only in the presence of Ca 2+ (MAbs C3, C4). The majority of HyTest MAbs do not depend on Ca2+ presence in sandwich immunoas-

say and are able to efficiently recognize antigen even in the presence of EDTA in the tested sample (MAbs C1, C2, C5, C6, C7, CRP11, CRP30, CRP36, CRP103, CRP135, CRP169).

All HyTest anti-CRP MAbs were tested in different immunological applications.

2.1.1. Direct ELISA All HyTest anti-CRP MAbs were tested in direct ELISA and all of them recognize native CRP with high sensitivity.

92

Most of the antibodies recognize nCRP both in the presence and absence of Ca2+, while MAb C3 binds to CRP only in Ca2+ presence (Fig. 96 and Fig. 97).

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

A490

3,50 3,00

CRP+2mM Ca2+

2,50

CRP+5mM EDTA

2,00 1,50 1,00 0,50 0,00 0,0002 0,0005 0,0015 0,0016 0,0137 0,0412 0,1225 0,3704

1,1111

MAb CRP11, μg/well

Figure 96. Interaction of MAb CRP11 with human native CRP in direct ELISA. 100 ng of native CRP (HyTest) per well was coated onto wells in Tris-buffered saline, containing 2 mM CaCl2 or 5 mM EDTA.

3,500 3,000

CRP+2mM Ca2+ CRP+5mM EDTA

A490

2,500 2,000 1,500 1,000 0,500 0,000 0,0002 0,0005 0,0015 0,0016 0,0137 0,0412 0,1225 0,3704 1,1111 MAb C3, μg/well

Figure 97. Interaction of MAb C3 with human native CRP in direct ELISA. 100 ng of native CRP (HyTest) per well was coated onto wells in Tris-buffered saline, containing 2 mM CaCl2 or 5 mM EDTA.

2.1.2. CRP immunodetection in Western blotting MAbs C1, CRP11, CRP36 and CRP169 recognize human CRP in Western blotting after antigen transfer onto nitrocellulose membrane. Results of experiments illustrating CRP immunodetection in Western blotting by MAbs CRP36 and CRP169 are presented in Fig. 98.

kDa 130

nCRP

95

kDa 130

72

95 72 55

55

36 28 mCRP

Figure 98. Immunodetection of C-reactive protein using anti-CRP MAbs in Western blotting after SDS gel electrophoresis.

36

Native CRP was loaded onto gel in non-reducing (A) or reducing (B) conditions. After electrophoresis protein was transferred from the gel onto nitrocellulose membrane and probed with MAbs CRP36 and CRP169.

28

A: CRP in non-reducing conditions after SDS gel electrophoresis according to Taylor and van der Berg (16).

B: CRP after SDS gel electrophoresis in reducing conditions.

For visualization of MAb-CRP complex anti-mouse IgG conjugated with HRP and 3,3-Diaminobenzidine tetrahydrochloride (DAB) as HRP substrate were used.

17 36 169

A

36 169

B

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

93

2.1.3. High sensitivity CRP sandwich immunoassay All MAbs were tested in sandwich fluoroimmunoassay as capture and detection antibodies with normal human serum (NHS) in the presence and absence of Ca2+ ions. The best pairs recommended for use are (outlined as capture - detection): C2 C5 C7 C5 CRP30 C3 C2

– – – – – – –

The representative calibration curves for pairs C2-C6 and C5-CRP135 are shown on Fig. 99 and Fig. 100, respectively. HyTest’s MAbs recognize CRP antigen with excellent sensitivity and good kinetics; the linearity range is over four orders of magnitude.

C6 C6 C6 CRP135 CRP135 C6 (Ca2+-sensitive assay) C4 (Ca2+- sensitive assay) Figure 99. Immunodetection of CRP standard in sandwich immunoassay by MAb pair C2-C6.

MAb C2 is biotinylated, MAb C6 is labeled with stable Eu3+ chelate.

Mixture of antibodies and antigen samples (100 μl) was incubated for 10 min at room temperature in streptavidin coated plates.

10000000 1000000 C2blot-C6Eu 100000

cps

10000 1000 100 10 0,01

0,1

1

10 CRP, ng/ml

100

1000

10000

Figure 100. Immunodetection of CRP in sandwich immunoassay by MAb pair C5-CRP135.

MAb C5 is biotinylated, MAb CRP135 is labeled with stable Eu3+ chelate.

Mixture of antibodies and antigen samples (100 μl) was incubated for 30 min at room temperature in streptavidin coated plates.

10000000 1000000 C5blot-CRP135Eu cps

100000 10000 1000 100 0,01

0,1

1

10

100

1000

10000

CRP, ng/ml

Several of HyTest’s MAbs pair recommendations are sensitive to the presence of EDTA in the solution, whereas others are not affected by this presence (Fig. 101). The pair C5-CRP135 as well as some others could be used both in the presence and absence of Ca2+ ions. The C3-C6 MAbs combination is strongly calcium-dependent.

6000000 NHS+2mM Ca2+

5000000

NHS+5mM EDTA

4000000 3000000 2000000 1000000

Figure 101. Influence of EDTA on CRP measurements by C3-C6 and C5CRP135 immunoassays.

Normal human serum (with 2 mM CaCl2 or 5 mM EDTA added) was used as source of the antigen.

94

0

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

C3-C6

C5-CRP135

2.1.4. Affinity information

For some applications, such as turbidimetry, nephelometry and competitive immunoassay, affinity constants of utilized antibodies have to be estimated. HyTest offers a panel of MAbs with different affinity (Fig. 102 and Table 11) and we have estimated affinity constants for some of HyTest anti-CRP MAbs

by using the Biacore® technique. Biacore methodology is based on the surface-enhanced plasmon resonance effect. It enables the assessment of interaction between two partners in real-time. Rate constants of associations and dissociation could be visualized and affinity constant could therefore be derived.

CRP 100nM 160 C5 140 C2 120 CRP30 100 CRP103

RU

80

CRP135

60 40 20

7DEOH $I¿QLW\ FRQVWDQWV RI VHOHFWHG +\7HVW DQWL &53 0$EV

MAb C2 C5 CRP30 CRP103 CRP135

Kd (M) 1.93×10-9 1.7×10-8 4.3×10-8 5.2×10-8 4.4×10-9

0 -200

200

400

600

800

1000

-20 -40

Time, s

Sensograms were obtained using MAbs immobilized onto chip and native CRP in solution.

Figure 102. Biacore X sensorgrams for selected MAbs C2, C5, CRP30, CRP103 and CRP135 at 100 nM CRP concentration.

Diluted nCRP was exposed to the chip-immobilized MAbs in HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% polysorbate 20, pH 7.4)

Ordering information: MAb

Cat.#

Specificity

Subclass

Application

C1 C2 C3 C4 C5 C6 C7

4C28 4C28 4C28 4C28 4C28 4C28 4C28

C-Reactive Protein C-Reactive Protein C-Reactive Protein C-Reactive Protein C-Reactive Protein C-Reactive Protein C-Reactive Protein

IgG2b IgG1 IgG1 IgG1 IgG2a IgG2a IgG1

CRP11 CRP30

4C28 4C28

C-Reactive Protein C-Reactive Protein

IgG1 IgG1

CRP36 CRP103

4C28 4C28

C-Reactive Protein C-Reactive Protein

IgG2a IgG2b

CRP135

4C28

C-Reactive Protein

IgG1

CRP169

4C28

C-Reactive Protein

IgG2a

EIA, WB EIA, sandwich immunoassay (capture) EIA, sandwich immunoassay (capture), EIA, sandwich immunoassay (detection), EIA, sandwich immunoassay (capture) EIA, sandwich immunoassay (detection) EIA, sandwich immunoassay (capture, detection) EIA, WB Low affinity MAb, EIA, sandwich immunoassay (capture) EIA, WB Low affinity MAb, EIA, sandwich immunoassay (capture) EIA, sandwich immunoassay (capture, detection) EIA, WB

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

95

3. CRP free serum Pooled normal human serum Immunoaffinity chromatography Frozen liquid -20°C

CRP free serum is prepared from pooled normal human serum by immunoaffinity chromatography. The matrix for affinity sorbent utilizes three monoclonal antibodies with different epitope specificity. According to ELISA testing the level of the antigen in CRP free serum is below 0.02 ng/ml (Fig. 103). CRP free serum can be used as a matrix for standard and calibrator preparations.

60000

50000 40000 CPS

Prepared from: Method of purification: Delivery form: Storage:

30000 20000

Figure 103. CRP level in normal human serum and in CRP free serum detected in sandwich-immunoassay.

1. Buffer 2. Initial signal in normal human serum 25 fold diluted (corresponds to 4 μg/ ml of CRP) 3. Residual signal in CRP free serum

10000 0 1

2

3

Ordering information: Product

Cat.#

Source

C-Reactive Protein free serum

8CFS

Pooled normal human serum

References: 1. Hirschfield G.M., Pepys M.B. “C-reactive protein and cardiovascular disease: new insights from an old molecule.” Q J Med 2003; 96:793-807.

2. Yasojima Koji et al. ” Generation of C-Reactive Protein and Complement Components in Atherosclerotic Plaques.” Am J Pathol. 2001 March; 158(3): 1039– 1051.

3. Kobayashi S, Inoue N, et al. “Interaction of oxidative stress and inflammatory response in coronary plaque instability: important role of C-reactive protein.” Arterioscler Thromb Vasc Biol 2003, 23:1398–1404.

4. Ciubotaru I., Potempa L.A., Wander R.C. “Production of Modified C-Reactive Proteinin U937-Derived Macrophages”.

5. Diehl E. E. et al.”Immunohistochemical Localization of modified C-reactive protein antigen in normal vascular tissue.” American Journal of the Medical Sciences 2000; 319(2):79.

6. Potempa L.A. et al. “Expression, detection and assay of a neoantigen (NeoCRP) associated with a free, human C-reactive protein subunit.” Molec. Immun 1987; 24(5), 531-541.

7. Bıro A. et al. ”Studies on the interactions between C-reactive protein and complement proteins” Immunology 2007 May;121(1):40-50.

8. Lowe G.D.O., Pepys M.B. ”C-Reactive Protein and Cardiovascular Disease: Weighing the Evidence” Current Atherosclerosis Reports 2006, 8:421–428.

9. Ridker P.M. “C-reactive protein: a simple test to help predict risk of heart attack and stroke” Circulation. 2003; 108:e81-e85.

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10. Clearfield M.B. “C-reactive protein: a new risk assessment tool for cardiovascular disease” JAOA 2005; 105(9):409-416.

11. Scirica B.M., Morrow D.A. et al “Clinical Application of C-Reactive Protein Across the Spectrum of Acute Coronary Syndromes” Clinical Chemistry 2007; 53: 1800-1807.

12. Koenig W, Khuseyinova N., Baumert J. and Meisinger C. “Prospective Study of High-Sensitivity C-Reactive Protein as a Determinant of Mortality: Results from the MONICA/KORA Augsburg Cohort Study, 1984–1998.” Clinical Chemistry 2008; 54:2; 335–342.

13. Meyer M.H. et al. “CRP determination based on a novel magnetic biosensor.”

Biosens Bioelectron 2007 Jan 15; 22(6):973-9.

14. Shiesh S.C. et al. “Determination of C-reactive protein with an ultra-sensitivity immunochemiluminometric assay”. J Immunol Methods 2006 Apr 20;311(12):87-95.

15. Sin K.K. et al.“Fluorogenic nanocrystals for highly sensitive detection of C-reactive protein.” IEE Proc Nanobiotechnol 2006 Jun;153(3):54-8.

16. Karolina E. Taylor and Carmen W. van den Berg “Structural and functional comparison of native pentameric, denatured monomeric and biotinylated C-reactive protein.” Immunology 2006; 120, 404–411.

17. Thompson D., Pepys M.B., Wood S.P. “The physiological structure of human Creactive protein and its complex with phosphocholine.” Structure 1999; 7:169177.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

XX Lectin-like oxidized LDL receptor 1 (LOX-1) Acute coronary syndrome (ACS) is a leading cause of mortality and morbidity in the developed world.

It is believed that ACS is started by rapture of lipidladen atheromatous plague in the coronary vessel wall and thrombus formation over the site of rupture.

Vessel lumen gets occluded by thrombus and tissue supplied by blood from this vessel suffers from a lack of oxygen and nutrients (1). None of the existing biomarkers indicates plague rupture before myocardial suffering and damage becomes evident.

Binding of oxidized low-density lipoproteins (LDL) to specific cell surface receptors on vascular endothelium serves as one of earliest events leading to en-

dothelial dysfunction and atheromatous plaque formation. Several oxidized LDL receptors have been identified so far and among them LOX-1 is characterized as a major receptor of oxidized LDL in endothelial cells of large arteries (2). LOX-1 is a type II transmembrane protein and its molecular weight is approximately 50 kDa. LOX-1 belongs to C-type lectin family + and is able to bind carbohydrates in Ca 2 -dependent manner. LOX-1 is glycosylated in several sites. It has been shown that LOX-1 is expressed on the surface of endothelial cells and could be proteolytically cleaved with formation of the soluble form of LOX-1 (sLOX-1) (3). The level of sLOX-1 in human blood may serve as an early and reliable predictor of future ACS (4).

1. Recombinant antigens

1

2

92 kDa 67 kDa

1.1. LOX-1, extracellular domain (not glycosylated) HyTest offers recombinant sLOX-1 expressed in E. coli. The protein contains part of the LOX-1 primary sequence restricted from 58 to 273 residues (5). It is expressed in E. coli as a His-tagged protein and is further purified to homogeneity using metalchelating chromatography (Fig. 104). Recombinant protein has the same sequence as the native protein with only one difference – additional Met residue at the N-terminus of the molecule.

43 kDa 30 kDa

sLOX-158-273

20 kDa

Figure 104. SDS-PAGE of sLOX-158-273 recombinant, expressed in E. coli.

Lane 1: Molecular weight markers, Lane 2: Isolated sLOX-158-273, 5 μg.

1.2. LOX-1, extracellular domain (glycosylated)

42 kDa Glycosylated

HyTest also offers recombinant LOX-1 antigen expressed in human cell line transfected with vector containing DNA coding for 58-273 part of human LOX-1 sequence. LOX-1 expressed in human cell line is recommended for use as a calibrator for sLOX-1 immunoassay.

Ordering information:

sLOX-158-273

30 kDa

20 kDa 2

1

Figure 105. SDS-PAGE of sLOX-158-273 glycosylated, expressed in human cell line. Lane 1: Isolated sLOX-158-273, 2 μg. Lane 2: Molecular weight numbers.

Product

Cat.#

Purity

Source

LOX-1, extracellular domain LOX-1, extracellular domain, glycosylated

8SLX1 8GSL1

>92% >92%

Recombinant Recombinant

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

97

2. Anti-sLOX-1 monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Specificity: Purification method: Presentation:

Balb/c mice Sp2/0 Recombinant human sLOX-158-273, expressed in E. coli Human recombinant LOX-158-273 Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with recombinant sLOX-1 expressed in E. coli.

3,0 2,5

A 490

HyTest offers several MAbs, recognizing with good sensitivity (in direct ELISA) recombinant sLOX-1 expressed in E. coli (Fig. 106) and in eukaryotic cells.

3,5

2,0 1,5 1,0

All antibodies could be used for the immunodetection of recombinant LOX-1 in Western blotting studies.

0,5 0 0,10

1,00

10,00

100,00

1000,00

10000,00

MAb LOX20-7, ng/ml

Figure 106. Titration curve of monoclonal antibody LOX20-7.

Antigen: recombinant sLOX-1 (E. coli).

Ordering information: MAb

Cat #

Specificity

Subclass

Application

LOX19-22 LOX20-4 LOX20-7 LOX20-13 LOX20-15 LOX20-20

4LOX1 4LOX1 4LOX1 4LOX1 4LOX1 4LOX1

Recombinant sLOX-1 Recombinant sLOX-1 Recombinant sLOX-1 Recombinant sLOX-1 Recombinant sLOX-1 Recombinant sLOX-1

IgG1 IgG1 IgG2a IgG1 IgG1 IgG1

EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB

References: 1. Libby, P. Current concepts of the pathogenesis of acute coronary events. 2001, Circulation, 104, 365-372.

2. Sawamura, T., et al., An endothelial receptor for oxidized low density lipoprotein. 1997. Nature, 386, 73-77.

3. Mehta, J., et al., Lectin-like, oxidized low-density lipoprotein receptor-1 (LOX1): A critical player in the development of atherosclerosis and related disorders. 2006, Cardiovasc.Res., 69, 36-45.

98

4. Hayashida, K., Serum Soluble Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Levels Are Elevated in Acute Coronary Syndrome: A Novel Marker for Early Diagnosis. 2005, Circulation, 112, 812-818.

5. Tatsuguchi, M., et al., Oxidized LDL receptor gene (OLR1) is associated with the risk of myocardial infarction. 2003, Biochem. Biophys. Res. Commun. 303, 247-250.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

XXI Retinol-binding protein 4 (RBP4) Retinol-binding protein 4 (RBP4) belongs to a lipocalin protein family and functions as a carrier protein for vitamin A in serum. Human retinol-binding protein circulating in blood consists of 183 amino acid residues. Several truncated isoforms of RBP4 lacking 1, 2, 4 or 6 of the very C-terminal residues were also described in literature (7). In blood RBP4 carries retinol (vitamin A) which is bound to RBP4 in equimolar ratio. Besides, a major part of circulating RBP4 forms complex with prealbumin (transthyretin) and according to Jaconi et al. only a small fraction of free RBP4 can be found in serum. (7) RBP4 has been studied since the 1960s, mainly as a transporter of retinol. However, recent data suggests that RBP4 may contribute to pathogenesis of type 2 diabetes. Yang et al. demonstrated that serum RBP4 levels are elevated in patients with obesity and type 2 diabetes. Studies in mice showed that serum RBP4 may cause insulin resistance (1).

Therefore, while on the one hand there is a growing body of evidence demonstrating that RBP4 is a promising marker of the risk of type 2 diabetes, on the other hand there is a conflicting situation in the literature regarding RBP4 clinical utility in terms

of predicting insulin resistance and type 2 diabetes (3). Some authors show a strict correlation between circulating RBP4 and magnitude of insulin resistance in subjects with obesity and type 2 diabetes and non-obese subjects with a family history of type 2 diabetes (2). On the contrary, others (4, 5) had not found any correlation between those variables. This confusing situation could at least partially be explained by the heterogeneity of the RBP4 in serum and by methodological shortcomings in determining level of circulating RBP4 (6). If epitope of diagnostic antibody is influenced by RBP4 truncation or by complex formation with retinol or prealbumin, then the level of RBP4 determined by the assay, utilizing such an antibody, would be different from the results of measurements by the assays with antibodies that are not susceptible to such modifications.

HyTest offers a set of mouse monoclonal anti-human RBP4 antibodies that are suitable for the development of sandwich immunoassays for the quantitative detection of circulating RBP4 in human plasma as well as for the immunodetection of RBP4 in direct ELISA, Western blotting or that can be used for the immunoprecipitation of the antigen.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

99

1. Purified endogenous RBP4

HyTest offers two types of purified native RBP4 antigen: free and complexed with prealbumin. Both forms of endogenous RBP4 (free and complexed) were purified from normal human serum in mild conditions using several chromatographic steps (Fig. 107).

Both native free and native complexed RBP4 antigens are unaffected by multiple (at least 5 - 7) freezethaw cycles (Fig. 108).

1200000

1000000

800000

CPS

Native RBP4 represents the most natural form of RBP4 and is therefore the antigen of choice for assay calibration. It is known that in serum RBP4 exist mostly as a 1:1 complex with prealbumin (transthyretin) and only a small part of RBP4 in the blood is presented as a free form. (7)

600000

native free RBP4 native complexed RBP4

400000

200000

95 kDa

0

72 kDa

0

1

2

3

4

5

6

7

55 kDa

Figure 108. Immunoreactivity of native free and native complexed RBP4 after several freeze-thaw cycles measured with the assay RB48 - RB42.

Capture antibody: RB48 (1 μg/well) Detection antibody: RB42 labeled with stable Eu3+ chelate (0.2 μg/well) Antigen: Native isolated RBP4.

36 kDa 28 kDa

RBP4

17 kDa

prealbumin (transthvretin)

Figure 107. RBP4 isolated from normal human serum (coomassiestained gel after SDS-electrophoresis in reduced conditions).

Lanes: 1: free native RBP4, 3 μg per track 2: native RBP4 complexed with prealbumin, 2 μg of total protein per track Molecular weight marker positions are marked by arrows.

Ordering information: Product

Cat.#

Purity

Source

Retinol-binding protein 4 (RBP4) from human plasma, complexed with prealbumin

8RP7

>70%

Human plasma

Retinol-binding protein 4 (RBP4) from human plasma, free

8RF9

>95%

Human plasma

100

8

freeze-thaw cycles

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

2. Anti-human RBP4 monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human recombinant retinol-binding protein Protein A affinity chromatography MAbs solution in PBS with 0.1% sodium azide ELISA, RBP4 sandwich immunoassay, Western blotting

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen the cells of Balb/c mice immunized with human recombinant RBP4.

2.1. Applications 2.1.1. Sandwich immunoassay for RBP4 detection in human plasma Anti-human RBP4 MAbs were obtained after mice immunization with human recombinant RBP4. All MAbs were tested in direct ELISA with human recombinant and native (endogenous, purified from human blood) RBP4. The best MAbs were further tested in sandwich immunoassay and several twosite combinations demonstrating the highest sensitivity for both recombinant and endogenous proteins were selected (Fig. 109) and recommended by our specialists for the development of RBP4 sandwich immunoassays.

HyTest offers MAbs RB42, RB48, RB45, RB49, RB51 and RB55, that are suitable for the immunodetection of native RBP4 in direct ELISA and sandwich immunoassay. Recommended combinations of antibodies for the development of sandwich immunoassay are (capture -detection): RB48 - RB42 RB48 - RB49 RB48 - RB51 RB55 - RB45 Selected assays recognize endogenous antigen in highly diluted human plasma (Fig. 110)

1000000

RB48-RB51

1000000

100000

RB48-RB51

CPS

CPS

100000

10000 10000 0.01

0.1

1

10

100

RBP4 concentration, (ng/ml)

1000 1

Figure 109. Calibration curve of RBP4 sandwich immunoassay.

Capture antibody: RB48 (1 μg/well) Detection antibody: RB51 labeled with stable Eu3+ chelate (0.2 μg/well) Antigen: Purified endogenous RBP4.

10

100

1000

1000

100000

1000000

Serum dilution factor

Figure 110. Titration curve of human plasma sample.

Capture antibody: RB48 (1 μg/well) 3+ Detection antibody: RB51 labeled with stable Eu chelate (0.2 μg/well) Antigen: Normal human serum diluted with PBS containing 0.1% Tween-20.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

101

Immunoreactivity of native RBP4, being measured by recommended MAb combinations, is unchanged in the presence of EDTA in the tested sample (Fig. 111).

2.1.3. Western blotting HyTest MAbs RB42, RB45, RB48, RB51 could be used for RBP immunodetection in Western blotting (Fig. 112).

2000000

CPS

1500000

1000000

+ EDTA - EDTA 500000

0 1

10

100

1000

100000

RBP4 native, ng/well

Figure 111. Immunodetection of purified endogenous RBP4 in sandwich immunoassay by RB48-RB42 MAb assay in presence of 5 mM EDTA (blue line) or in absence of EDTA (pink line).

Capture antibody: RB48 (1 μg/well) Detection antibody: RB42 labeled with stable Eu3+ chelate (0.2 μg/well) Antigen: Native isolated RBP4

All HyTest anti-RBP4 MAbs recognize both free RBP4 and RBP4 complexed with prealbumin.

2.1.2. Immunoprecipitation HyTest anti-RBP4 MAbs being immobilized onto BrCN-activated Sepharose could be used as an affinity matrix for the immunoprecipitation of RBP4.

1

2 3

4

Figure 112. Immunodetection of RBP4 in Western blotting after SDSelectrophoresis in reducing conditions by MAb RB42 (lane 1), RB45 (lane 2), RB48 (lane 3) and RB51 (lane 4).

1 μg per track of purified endogenous RBP4 was loaded onto gel.

Ordering information: MAb

Cat.#

Specificity

Subclass

Application

RB42

4RB2

Retinol-binding protein 4 (RBP4)

IgG1

EIA, WB

RB45

4RB2

Retinol-binding protein 4 (RBP4)

IgG1

EIA, WB

RB48

4RB2

Retinol-binding protein 4 (RBP4)

IgG1

EIA, WB

RB49

4RB2

Retinol-binding protein 4 (RBP4)

IgG1

EIA, WB

RB51

4RB2

Retinol-binding protein 4 (RBP4)

IgG1

EIA, WB

RB55

4RB2

Retinol-binding protein 4 (RBP4)

IgG1

EIA, WB

References: 1. Yang, Q., et al., Serum retinol binding protein 4 contributes to insulin resistance inobesity and type 2 diabetes. Nature, 2005, 436, 356-362.

2. Graham, T., et. al., Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects. New Engl. J. Med., 2006, 354, 24, 2552-2563.

3. Qi, Q., et al., Elevated retinol-binding protein 4 levels are associated with metabolic syndrome in Chinese people. J. Clin. Endocrinol. Metab. 2007, 92, 48274834.

4. Lewis, J., et al., Plasma retinol-binding protein is unlikely to be a useful marker of insulin resistance. Diabetes Res. Clin. Pract. 2008, 80, 13-15.

102

5. Promintzer, M., et al., Insulin resistance is unrelated to circulating retinol binding protein and protein C inhibitor. J. Clin. Endocrinol. Metab. 2007, 92, 4306-4312.

6. Graham, T., et al., Shortcomings in methodology complicate measurements of serum retinol binding protein (RBP4) in insulin resistant human subjects. Diabetologia, 2007, 50, 814-824.

7. Jaconi, S., Characterization of two post-translationally processed forms of human serum retinol-binding protein: altered ratios in chronic renal failure. J. Lip. Res., 1995, 36, 1247-1253.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

XXII Human Adiponectin Adiponectin is an abundant protein hormone that belongs to a family of so-called adipokines. Adiponectin is expressed mostly by adipocytes and is an important regulator of lipid and glucose metabolism.

It is established that adiponectin is an insulin-sensitizing hormone with anti-diabetic, anti-inflammatory and anti-atherogenic properties (1). Potential diagnostic usage of adiponectin was a subject of increasing interest in recent years. It was shown that decreased serum adiponectin concentration indicates insulin resistance and type 2 diabetes (2). Besides, hypoadiponectinemia was shown to be associated with coronary artery disease (3). Several authors point out that a high level of circulating adiponectin reduces the risk of coronary heart disease among type 2 diabetes patients (4) and is associated with a reduced risk of myocardial infarction in apparently healthy men (5). Consequently, there is growing interest among medical professionals in using adiponectin for insulin resistance diagnosis and the predicting of cardiovascular complications in subjects with type 2 diabetes.

Human adiponectin consists of 244 amino acid residues and has a distinct domain structure: it contains both collagen-like and globular C1q-like domains.

Collagen-like parts of three adiponectin molecules can interact, forming a triple coiled coil structure that is very similar to that in collagen (6). C1q-like domains form a “head” of adiponectin globula (Fig. 113) and share a considerable degree of structural similarity to complement component C1q. Several oligomeric forms of native adiponectin circulating in the blood are described in literature: trimers (low-molecular weight form, LMW), hexamers (medium molecular weight form, MMW) and higher order multimers (high molecular weight form, HMW). Three monomers of adiponectin form a trimer. Trimers linked by disulfide bond form a hexamer. The exact structure

of the HMW form of adiponectin is not yet known. It is most likely that several combined hexamers and/ or trimers constitute a high-molecular weight form of adiponectin. It is generally believed that disulfide bonds, as well as some bonds with participation of modified amino acid residues in the collagen domain of adiponectin, take part in holding subunits of the HMW form of adiponectin together (Fig. 113). It is also believed that those oligomeric forms exist in the bloodstream as separate moieties and do not convert into one another. (7) It has been shown recently that adiponectin oligomers are capable of binding Ca2+ ions, which are thought to participate in the maintenance of conformational stability of adiponectin (10). The concentration of total adiponectin in the blood is approximately 3-30 μg/ml, whereas the concentration of the closest structural homolog of adiponectin, C1q, is approximately 80-200 μg/ml. It is therefore of utmost importance that antiadiponectin antibodies have no cross-reactivity with human C1q. (8) Some authors describe significant gender differences in adiponectin level in healthy adults and these differences are believed to contribute to discrepancies in adiponectin concentration reported by various authors. It was shown, that the biologic activity of adiponectin is mediated by highmolecular weight form and, unsurprisingly it has been recently suggested that the concentration of the HMW form of adiponectin or ratio HMW/total adiponectin (sum of three types of oligomers) in serum correlates more strongly than the total adiponectin with insulin resistance and other measures of type 2 diabetes (9).

HyTest offers a new generation of anti-human adiponectin monoclonal antibodies suitable for both research purposes (Western blotting, direct ELISA) and for the development of adiponectin-specific sandwich immunoassays.

Figure 113. Schematic representation of adiponectin oligomeric forms.

S–S

trimer (LMW form of adiponectin)

hexamer (MMW form of adiponectin)

HMW form of adiponectin

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

103

1. Native purified adiponectin antigen NEW! Native adiponectin purified from normal human plasma is the best calibrator for immunoassays. Native adiponectin was isolated from normal human plasma using a combination of chromatographic methods. Its purity is approximately 92%, as calculated by densitometry of protein bands stained with Coomassie Brilliant Blue R-250 following SDS-electrophoresis in reduced conditions (Fig. 114).

Native purified adiponectin fully recovers its immunoreactivity after lyophilization and reconstitution by the addition of deionized water (Fig. 115).

10000000

1000000

100000

CPS

before freeze-drying after freeze-drying 10000

72 kDa 1000

Adn dimer 55 kDa

100 1

10

100

1000

100000

10000

native purified Adn, ng/ml

Figure 115. Lyophilization does not affect immunological activity of native purified adiponectin measured by assay Adn94-Adn63.

36 kDa

Adn monomer 28 kDa

Purified native adiponectin contains all three oligomeric forms of Adn (Fig. 116) and can therefore serve as a calibrator for all types of Adn assays: total Adn, HMW- or LMW-specific.

Figure 114. SDS-electrophoresis in reducing conditions and Western blotting of native purified adiponectin from human plasma.

Lane 1 – 2 μg of purified adiponectin loaded onto track, stained with Coomassie Brilliant Blue R-250.

Lane 2 – 200 ng of purified adiponectin loaded onto track, stained with Adn23 MAb in Western blotting.

HMW

MMW

0.4

Adn94-Adn63

350000 300000

0.2

250000 200000

– 0.2

150000

LMW

– 0.4

100000

– 0.6

50000

– 0.8

669 kDa

440 kDa 232 kDa

158 kDa

CPS

A 280

0.0

0 –50000

100

150

200

Elution volume (ml)

Figure 116. Native purified adiponectin contains all oligomeric forms.

3μg of adiponectin was applied onto gel-filtration column and immunoreactivity in fractions was measured with HyTest assay Adn94-Adn63.

Molecular weight markers are depicted by arrows, black curve represents optical density measured at 280 nm.

2UGHULQJ LQIRUPDWLRQ 3URGXFW

Cat.#

3XULW\

Source

Adiponectin, human, native

8AN7

>95%

Pooled human plasma

104

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

2. Anti-human adiponectin monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human adiponectin Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide Adiponectin sandwich immunoassay, adiponectin immunodetection in Western blotting (Adn20, Adn23, Adn63, Adn214, Adn222 and Adn243)

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice that are immunized with either human recombinant adiponectin or native human adiponectin.

All antibodies were tested in direct ELISA for crossreaction with C1q, which is the most abundant adiponectin homolog in blood. None of the selected MAbs showed any cross-reaction with human C1q.

2.1. Applications New data!

All MAbs were tested in two-site combinations as capture or detection antibodies in sandwich ELISA with native adiponectin. Seven two-site combinations were selected for the development of sandwich immunoassays on the basis of sensitivity and specificity to different oligomeric forms of adiponectin: Adn20 Adn36 Adn94 Adn279 Adn214 Adn222 Adn305

-

Adn23 Adn27 Adn63 Adn94 Adn27 Adn94 Adn279

10000000

1000000

CPS

2.1.1. Adiponectin sandwich immunoassay

100000

Adn279-Adn94

10000

1000 0.1

1

10

100

1000

10000

native purified human Adn, ng/ml

A representative curve demonstrating detection of purified native adiponectin by the assay Adn279Adn94 is shown on Fig. 117.

Figure 117. Calibration curve for sandwich adiponectin immunoassay.

MAb Adn279 was used as a coating (1 μg/well), MAb Adn94 was labeled with stable Eu3+ chelate and was used as a detection (0.2 μg/well) antibody. Native adiponectin purified from human plasma was used as a calibrator.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

105

10000000

Adn94-Adn63

1000000

CPS

All assays were tested with serial dilutions of normal human serum to evaluate the interaction of MAbs with native adiponectin in a complex environment. All assays demonstrated a steady decrease of signal correlating with the degree of serum dilution. The representative titration curve for the assay Adn94-Adn63 (capture antibody-detection antibody, respectively) is shown in Fig. 118.

100000

Figure 118. Normal human serum titration curve in sandwich immunofluorescent assay. Adn94 MAb was used as a coating antibody (1 μg/well), MAb Adn63 was used as a detection antibody (0.2 μg/well). Normal human serum, serially diluted with phosphate-buffered saline (10 mM K-phosphate, pH 7.4, 150 mM NaCl, 0.1% Tween-20) was used as an antigen.

10000 0.1

10

100

1000

10000

100000

serum dilution factor

10000000

plasma serum

1000000

CPS

Assays Adn36-Adn27 and Adn20-Adn23 react differently with adiponectin in serum and citrate plasma (Fig. 119). Other MAbs two-site combinations (Adn94-Adn63, Adn279-Adn94, Adn214-Adn27, Adn222-Adn94, Adn305-Adn279) react identically with antigen in serum and plasma identically.

100000

10000

Figure 119. Normal human serum or citrate plasma titration curves for MAb assay Adn36-Adn27. Normal human pooled serum or citrate plasma, serially diluted with phosphate-buffered saline (10 mM K-phosphate, pH 7.4, 150 mM NaCl, 0.1% Tween-20) was used as an antigen.

1000 1

10

100

1000

10000

serum/plasma dilution factor

Figure 120. Serum titration curve for the assay Adn20-Adn23. Pooled normal human serum was serially diluted with phosphate-buffered saline with EGTA or w/o EGTA (10 mM K-phosphate, pH 7.4, 150 mM NaCl, 0.1% Tween-20, 10 mM EGTA).

106

10000000

1000000

serum serum+EGTA

CPS

Recognition of adiponectin by assays Adn20Adn23 and Adn36-Adn27 in serum is Ca 2+-sensitive (Fig. 120). Chelating of Ca2+ ions by EGTA leads to the rearrangements in adiponectin structure and changes in the interaction of one of the antibodies with the antigen. Other assays do not demonstrate Ca2+-dependence in the antigen recognition and react identically with adiponectin in serum or citrate plasma.

100000

10000

1000 1

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

10

100

serum dilution factor

1000

10000

2.1.2. Assays detecting total, HMW or LMW forms of human adiponectin New data!

HMW

A

1600

40000

1400 1200

30000

LMW

1000

A 280

35000

800

MMW

25000 20000

600

15000

400

CPS

To establish an oligomer specificity of HyTest assays, serum proteins were separated according to their molecular masses by means of size-exclusion chromatography and immunoreactivity in fractions was measured. The assay Adn20-Adn23 detects two oligomeric forms of adiponectin: mostly HMW and to a lesser extent, the MMW form (Fig. 121A). The assay Adn94-Adn63 recognizes all three Adn oligomeric forms - total adiponectin (Fig. 121B) and the assay Adn214-Adn27 reacts primarily with the LMW form of adiponectin (Fig. 121C).

Adn20-Adn23*

10000

200

5000

0

0

-200 100

150

200

Elution volume (ml)

669 kDa

B

1600

158 kDa Adn94-Adn63*

40000 35000

1200

30000

1000

25000

800

20000

600

15000

400

10000

200

5000 0

0 -200 100

CPS

1400

A 280

2.1.3. Western blotting All MAbs were tested on their ability to recognize adiponectin in Western blotting. Only six the of tested antibodies – MAbs Adn20, Adn23, Adn63, Adn214, Adn222, and Adn243 – reacted with adiponectin transferred onto nitrocellulose membrane after SDSPAGE in reducing conditions (Fig. 122).

440 kDa 232 kDa

-100000 150

200

Elution volume (ml)

C 72 kDa

1600

Adn214-Adn27

80000

1400 1200

55 kDa

60000

1000

A 280

800

36 kDa

40000

CPS

Adn dimer

600 400

20000

200

Adn monomer

28 kDa

0 -200 100

0 150

200

Elution volume (ml)

1

2

3

4

5

Figure 121. Immunoreactivity in protein fractions after size-exclusion chromatography, measured by assay Adn20-Adn23 (A) and by assays Adn94-Adn63 (B), Adn214-Adn27 (C) in sandwich ELISA. 1 ml of normal human serum was applied onto column. Positions of oligomeric forms of adiponectin and molecular weight markers are depicted on the picture. Black line is optical density detected at 280 nm.

6

Figure 122. Immunodetection of native adiponectin with anti-Adn MAbs in Western blotting after SDS-electrophoresis in reducing conditions.

40 ng of native purified adiponectin was loaded onto each track, nitrocellulose membrane was stained with 5 μg/ml of various anti-adiponectin MAbs in phosphate-buffered saline, containing 5% dry milk and 0.1% Tween-20.

Lane 1 – Adn20 Lane 2 – Adn23 Lane 3 – Adn63 Lane 4 – Adn214 Lane 5 – Adn222 Lane 6 – Adn243 Molecular weight markers are marked by arrows.

MARKERS OF CARDIOVASCULAR DISEASES AND METABOLIC SYNDROME

107

2UGHULQJ LQIRUPDWLRQ MAb

Cat.#

6SHFL多FLW\

Subclass

Application

Adn20

2AN6

Human adiponectin

IgG2a

WB

Adn23

2AN6

Human adiponectin

IgG2a

WB

Adn27

2AN6

Human adiponectin

IgG2a

EIA

Adn36

2AN6

Human adiponectin

IgG2a

EIA

Adn63

2AN6

Human adiponectin

IgG1

EIA, WB

Adn94

2AN6

Human adiponectin

IgG1

EIA

Adn97

2AN6

Human adiponectin

IgG1

EIA

Adn130

2AN6

Human adiponectin

IgG2a

EIA

Adn214

2AN6

Human adiponectin

IgG1

EIA, WB

Adn222

2AN6

Human adiponectin

IgG1

EIA, WB

Adn243

2AN6

Human adiponectin

IgG2a

EIA, WB

Adn279

2AN6

Human adiponectin

IgG1

EIA

Adn305

2AN6

Human adiponectin

IgG1

EIA

References: 1. Wang, Y., et al., Adiponectin inhibits cell proliferation by interacting with several growth factors in an oligomerization-dependent manner. J.B.C., 2005, 280, 18, 18341-18347.

2. Ryo, M., et al., Adiponectin as a biomarker of the metabolic syndrome. Circ.

J., 2004, 68, 975-981.

3. Kumada, M., et al., Association of hypoadiponectinemia with coronary artery disease in men. Arterioscler. Thromb. Vasc. Biol., 2003, 23, 85-89.

4. Schulze, M., et al., Adiponectin and future coronary heart disease events among men with type 2 diabetes. Diabetes, 2005, 54, 534-539.

Pischon, T., et. al., Plasma adiponectin levels and risk of myocardial infarction in men. JAMA, 2004, 291, 14, 1730-1737.

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5. Pajvani, U., et al., Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin. J.B.C., 2003, 278, 11, 9073-9085.

6. Wang, y., et al., Hydroxylation and Glycosylation of the Four Conserved Lysine Residues in the Collagenous Domain of Adiponectin. J.B.C., 2002, 277, 22, 19521-19529.

7. Wouters, D, Evaluation of classical complement pathway activation in rheumatoid arthritis. Arhtritis & Rheumatism, 2006, 54, 1143-1150.

8. Lara-Castro et al., Adiponectin multimeric complexes and the metabolic syndrome trait cluster. Diabetes, 2006, 55, 249-259.

9. Schraw T, Wang ZV, Halberg N, Hawkins M, Scherer PE. Plasma adiponectin complexes have distinct biochemical characteristics. Endocrinology, 2008, 149(5), 2270-82.

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XXIII Leptin One of the most frequently encountered risk factors for cardiovascular disorders in developed countries is that of obesity (1). The link between obesity and cardiovascular disease is very complex. Obesity is associated with high insulin and leptin levels, with abnormalities in leptin homeostasis being suggested as increasing the propensity to obesity. Meanwhile, recent studies have demonstrated that high serum levels of leptin represented an independent risk factor for such cardiovascular thrombotic events as heart attacks and strokes (2).

Leptin is expressed and secreted by adipose tissue cells and the level of circulating leptin is directly proportional to the total amount of fat in the body. Leptin exerts an influence in many physiological processes, including food intake, thermoregulation, fertility, thyroid function, adrenal function, sympathetic nerve activation, renal function, blood vessel tone and blood pressure.

Amino acid sequence of human leptin is 85% identical to mouse protein and 84% identical to rat leptin.

Mouse and rat leptin demonstrate 96% identity with one another (3, 4). Human leptin is a 146 amino acid residue, 16 kDa, non-glycosylated protein.

1. Anti-leptin monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human recombinant leptin Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide Leptin immunoassay

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with human recombinant leptin. Different combinations of monoclonal antibodies could be used for immunoassay development.

All leptin specific antibodies were tested in sandwich immunoassay as capture and detection antibodies. All MAbs recognize antigen in human blood with high sensitivity. The best pairs recommended for sandwich immunoassay are: 3G7 4F12 3G7 4F12

– – – –

4F12 3G7 9C10 10H1

Ordering information: MAb

Cat. #

Specificity

Subclass

Application

3G7 4F12 5F10 9C10 10H1 4F8

2LE1 2LE1 2LE1 2LE1 2LE1 2LE1

Leptin Leptin Leptin Leptin Leptin Leptin

IgG1 IgG1 IgG1 IgG1 IgG1 IgG1

EIA, Sandwich immunoassay (capture, detection) EIA, Sandwich immunoassay (capture, detection) EIA, Sandwich immunoassay EIA, Sandwich immunoassay (detection) EIA, Sandwich immunoassay (detection) EIA, Sandwich immunoassay

References: 1. El-Atat F et al. (2003) Obesity and hypertension. Endocrinol Metab Clin North Am. 32(4):823-854.

2. Rahmouni K, Haynes WG. (2004) Leptin and the cardiovascular system. Recent Prog Horm Res. 59:225-244.

3. Zhang Y. et al. (1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372: 425-432.

4. Ogawa Y. et al. (1995) Molecular cloning of rat obese cDNA and augmented gene expression in genetically obese Zucker fatty (fa/fa) rats. J Clin Invest 96: 1647-1652.

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XXIV Proinsulin, Insulin and C-peptide Diabetes mellitus (or simply diabetes) is one of the diseases of metabolic syndrome. It usually arises due to a combination of hereditary and environmental causes, resulting in abnormally high blood sugar levels (hyperglycemia). Diabetes develops in the case of type 1 diabetes due to a diminished production of insulin, while in type 2 diabetes it develops because of resistance to the insulin hormone. Insulin is a hormone that regulates carbohydrate metabolism and has intensive effects on both the metabolism and several other body systems (e.g. vascular compliance).

Insulin causes most of the body’s cells to take up glucose from the blood (including liver, muscle, and fat tissue cells) storing it as glycogen in the liver and muscle, as well as stopping the use of fat as an energy source. As its level is a central metabolic control mechanism, insulin status is also used as a control signal to other body systems (such as amino acid uptake by body cells) and has several other anabolic effects throughout the body.

In mammals, insulin is synthesized in the pancreas within B-cells of the islets of Langerhans from the proinsulin precursor molecule by the action of proteolytic enzymes, known as prohormone convertases (PC1 and PC2), as well as the exoprotease carboxypeptidase E. These modifications of proinsulin remove the middle portion of the molecule, known as C-peptide, from the C- and N- terminal ends of proinsulin. The remaining polypeptides are B- and Achains, which are bound together by disulfide bonds/ disulfide bonds forming insulin.

Insulin has a highly conservative sequence over mammals, reptiles, birds and fish. Meanwhile, C-peptide (abbreviated from Connecting peptide) instead demonstrates considerable interspecies variability.

It is a polypeptide molecule that comprises 31 amino acid residues with a molecular mass of approximately 3.0 kDa. The precise function of C-peptide remains unclear but its positive functions in the repair

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of the muscular layer of the arteries and therapeutic effects on some complications associated with diabetes mellitus, such as diabetic neuropathy, have been recently demonstrated. Proinsulin as a precursor of both insulin and C-peptide, demonstrates interspecies variability and for the majority of species (including human) only one form of proinsulin is described. Unlike others, rats and mice produce two proinsulin isoforms – I and II, which differ from one another in two (rat) or three (mouse) amino acid residues of the C-peptide part of proinsulin.

Analysis of proinsulin synthesis and processing, as well as insulin and C-peptide clearance, are very important for the improved understanding of carbohydrate metabolism abnormalities. Assays for insulin, proinsulin and C-peptide are widely used in the monitoring of hypoglycemia, pathogenesis and treatment of diabetes mellitus. It has been demonstrated that C-peptide measurements in blood or urine display several advantages over the direct insulin quantification. Therefore, C-peptide measurements perhaps present the only method of determining insulin production in cases of diabetes treatment where endogenous insulin is mixed in blood with the exogenous molecule. Being released into the bloodstream, insulin is very quickly utilized by the liver. Quick excretion and quick elimination leads to considerable fluctuations of insulin concentrations in the blood. Cpeptide is eliminated and degraded mainly by kidneys and this process is not as impetuous as insulin elimination. In addition insulin in blood is less stable than C-peptide. Consequently the half life of insulin in blood is significantly shorter than that of C-peptide (4 and 33 minutes respectively). Finally, hemolysis is known to significantly reduce measured insulin concentration. Therefore, C-peptide would appear to be the more reliable indicator of insulin production than insulin on its own.

HyTest delivers a whole set of monoclonal antibodies for the monitoring of proinsulin metabolism of both human and rat origin. Carefully chosen epitopes and

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original approaches for selecting specific monoclonal anti-C-peptide (rat I, rat II and human) antibodies have enabled us to develop highly sensitive and specific antibodies that make C-peptide detection possible without cross-reactivity with native proinsulin or some forms of partially processed proinsulin. We are also able to offer pairs of antibodies that are capable of detecting either both isoforms of rat C-peptide

(C-peptides I and II) or one of two isoforms (C-peptide I or II). Finally, we have generated monoclonal antibodies that specifically detect intact and partially processed proinsulin and do not interact with free Cpeptide. Furthermore a set of anti-human insulin and anti-proinsulin (both human and rat) MAbs of different specificity are available.

Ordering information: MAb

Cat. #

Specificity

Subclass

Remarks

3A1 1G4 1D6 1D3 7D6 CCI-10 CCI-17 CCI-3 7F5 C7C9 D4B8 7F8 3A6 8E2 D6C4

2P9 2P9 2P9 2P9 2P9 2PR8 2PR8 2PR8 2I1 2I1 2I1 2I1 2I1 2I1 2IP10

IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG2b IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1

Kd = 1.1 x 10-8 M, EIA, IHC Kd = 2.6 x 10-7 M, EIA, IHC Kd = 3.0 x 10-8 M, EIA EIA Kd = 5.0 x 10-8 M, EIA EIA (capture) EIA (capture) EIA (capture) Kass = 1.0 x 109 M-1 C-terminal Pentapeptide of B-chain, Kass = 1.0 x 109 M-1 Kass = 1.5 x 108 M-1, EIA (detection), IHC Kass = 7.5 x 109 M-1, EIA (capture) Kass = 8.0 x 108 M-1, EIA (capture) Kass = 6.0 x 108 M-1, EIA (detection) Kd = 8.1 x 10-8 M, EIA (capture), IHC

D3E7

2IP10

IgG1

Kd = 6.3 x 10-8 M, EIA (detection), IHC

1H8 2B7 4H8 5B8 7E10 2A11 6H1 CC18 CC24 CC27 CC34 CI-0 CII-106 CII-11 CII-138 CII-29 CII-55 CII-97

2I2 2I2 2I2 2I2 2I2 2I2 2I3 2I3 2I3 2I3 2I3 2I3 2I3 2I3 2I3 2I3 2I3 2I3

Proinsulin, Human Proinsulin, Human Proinsulin, Human Proinsulin, Human Proinsulin, Human Proinsulin, Rat Proinsulin, Rat Proinsulin, Rat Insulin, Human Insulin, Human Insulin, Human Insulin, Human Insulin, Human Insulin, Human Insulin/Proinsulin, Rat-mouse Insulin/Proinsulin, Rat-mouse C-peptide, Human C-peptide, Human C-peptide, Human C-peptide, Human C-peptide, Human C-peptide, Human C-peptide, Rat C-peptide, Rat C-peptide, Rat C-peptide, Rat C-peptide, Rat C-peptide, Rat C-peptide, Rat C-peptide, Rat C-peptide, Rat C-peptide, Rat C-peptide, Rat C-peptide, Rat

IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1

N/cr with Human, Bovine, Porcine, Mouse/rat Insulin, EIA N/cr with Human, Bovine, Porcine, Mouse/rat Insulin, EIA N/cr with Human, Bovine, Porcine, Mouse/rat Insulin, EIA N/cr with Human, Bovine, Porcine, Mouse/rat Insulin, EIA N/cr with Human, Bovine, Porcine, Mouse/rat Insulin, EIA N/cr with Human, Bovine, Porcine, Mouse/rat Insulin, EIA EIA EIA EIA EIA EIA EIA EIA EIA EIA EIA EIA EIA

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XXV Brain S-100 Protein S-100 protein derived from brain tissue is an acidic calcium-binding protein with a molecular weight of approximately 21 kDa. It is predominantly synthesized in the brain by astroglial cells and mainly presented by two isoforms; alpha-beta heterodimer (S-100a) and beta-beta homodimer (S-100b).

Due to its predominant location in astroglial cells, S-100 protein can be used as a sensitive and reliable marker for central nervous system damage. Structural damage of glial cells causes leakage of S-100 protein into the extracellular matrix and cerebrospinal fluid, further releasing into the bloodstream. S-100 protein appears to be a promising marker for the severity of brain injury and neuronal damage. There is a good correlation between S-100 concentration

in patients’ serum samples and outcome following traumatic and ischemic brain injury. Measurements of S-100 protein could prove to be very useful in the diagnosis and prognosis of clinical outcome in acute stroke and the estimation of the ischemic brain damage during cardiac surgery. Elevated serum levels of S-100 correlate with duration of circulatory arrest.

S-100 protein is purified from human brain tissue by several chromatographic methods including gel-filtration and ion-exchange chromatography. After native gel electrophoresis by Ornstein-Davis the protein is presented by two bands that correspond to alpha-beta and beta-beta forms (Fig. 123).

Figure 123. Native gel electrophoresis of S-100 protein (by Ornstein – Davis).

Antigen loaded: Lane 1: 2 μg Lane 2: 5 μg Gel staining: Coomassie brilliant blue R-250

AB BB

2 μg

5 μg

1. Human brain S-100 protein Source: Purity: Presentation: Application: Storage:

Human brain. Blood sample from the tissue donors was tested and found negative for HBsAg, HIV-1 and HIV-2 antibodies, HCV and syphilis >95% Lyophilized Immunogen for antibody production, immunological and mass S-100 standard, S-100 biochemical and immunochemical studies -20°C

Ordering information: Product

Cat. #

Purity

Source

Human S-100 Human S-100, beta-beta homodimer

8S9h 8S9-2h

>95% >95%

Human brain Human brain

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2. Anti-S-100 monoclonal antibodies Host animal: Cell line used for fusion: Antigen: Purification method: Presentation: Application:

Mice Balb/c Sp2/0 Human brain S-100 protein Protein A affinity chromatography MAb solution in PBS with 0.1% sodium azide S-100 immunoassay, S-100 immunoaffinity purification, S-100 immunodetection in Western blotting

Hybridoma clones have been derived from the hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with purified human brain

100000

S-100 protein. The best combinations of MAbs for sandwich immunoassay: 8B10 – 6G1 (Fig. 124) 3B10 – 6G1

Figure 124. S-100 calibration curve. One step assay in streptavidin coated plates.

Capture MAb: 8B10 (biotinylated), 200 ng/well Detection MAb: 6G1 (Eu-labeled), 200 ng/well Antigen: S-100 protein from human brain Incubation time: 20 minutes Temperature: 20°C

CPS

10000

1000

100 0.01

0.1

1

10

100

S-100 concentration (ng/ml)

The MAbs also work in Western blotting (Fig. 125).

AB

BB 1

2

3

4

Figure 125. Interaction of monoclonal antibodies with S100 protein from human brain in Western blotting (after native gel electrophoresis by Ornstein – Davis).

Antigen loaded: 1μg Lane 1: MAb 4B3 Lane 2: MAb 8B10 Lane 3: MAb 6G1 Lane 4: MAb 3B10

Ordering information: MAb

Cat. #

Specificity in WB

Subclass

Application

3B10 4B3 8B10 6G1

4S37 4S37 4S37 4S37

AB AB and BB AB and BB AB and BB

IgG2a IgG2a IgG1 IgG1

EIA, Sandwich immunoassay (capture), WB WB EIA, Sandwich immunoassay (capture), WB EIA, Sandwich immunoassay (detection), WB

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XXVII Special Products 1. Immunosorbents Immunosorbents based on monoclonal antibodies are widely used for antigen puriďŹ cation or antigen extraction from crude protein mixtures. At the request of the customer, our specialists will prepare immunosorbents with antibodies attached to different types of matrixes. As an example, we have good level of experience in working with agarose-based immunosorbents.

2. Labeled Antibodies Different types of antibody conjugates are used for research and production purpose. At the request of the customer, all antibodies presented in the catalogue can be labeled with FITC, Biotin, HRP and Texas Red.

3. Fab and F(ab)2 Fragments of Monoclonal Antibodies Fab and F(ab)2 fragments of any MAb can be prepared at the request of the customer.

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XXVIII Abbreviations Used in the Catalog a.a.r.

A/C AACC AMI BNP C/r CHF CK-MB Capture CPS CRP Detection dPAPP-A EIA FABP Fg FITC GFR GPBB, GPMM, GPLL HF HRP HSA hsCRP htPAPP-A IVD LOX-1 MAb ME MI MPO MW N/A N/cr NT-proBNP NYHA PAPP-A PBS PCT proBNP proMBP RBP4 RIA SAA sCD40L SDS-PAGE sLOX-1 TAFI TnC TnI, cTnI, skTnI TnT, cTnT, skTnT U-II WB

Amino acid residues Affinity chromatography American Association of Clinical Chemistry Acute myocardial infarction Brain natriuretic peptide Cross-reactivity, cross-reaction Congestive heart failure Creatine kinase isoenzyme MB Antibody that can be used for capture Counts per second C-reactive protein Antibody that can be used for labeling Dimeric form of pregnancy associated plasma protein Enzyme immunoassay Fatty acid binding protein Fibrinogen Fluoresceine isothiocyanate Glomerular filtration rate Glycogen phosphorylase isoenzymes BB, MM and LL Heart failure Horseradish peroxidase Human serum albumin High sensitive C-reactive protein Heterotetrameric PAPP-A/proMBP complex In vitro diagnostic activities Lectin – like oxidized LDL receptor Monoclonal antibody Mercaptoethanol Myocardial infarction Myeloperoxidase Molecular weight Not applicable No cross-reaction N-terminal fragment of brain natriuretic peptide proform New-York Heart Association Pregnancy associated plasma protein A Phosphate buffered saline Procalcitonin Brain natriuretic peptide proform Proform of major basic protein Retinol binding protein 4 Radioimmunoassay Serum amyloid A Soluble CD40 ligand Electrophoresis in polyacrylamide gel in presence of sodium dodecyl sulfate Soluble form of LOX-1 Thrombin activatable fibrinolysis inhibitor Troponin C Troponin I, cardiac and skeletal Troponin T, cardiac and skeletal Urotensin II Western blotting

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General Product Catalog 2010-2011

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Markers of Cardiovascular Diseases and Metabolic Syndrome Troponin ProBNP, BNP and NT-proBNP Myoglobin Pregnancy Associated Plasma Protein A (PAPP-A) Myeloperoxidase (MPO) Soluble CD40 Ligand (sCD40L)

Zeeland Turku Oy/Finepress Oy 06/2010

Cystatin C D-Dimer and HMW Fibrin Degradation Products Fatty Acid Binding Protein (FABP) High Sensitivity C-Reactive Protein (hsCRP) Retinol-Binding Protein 4 (RBP4) Adiponectin, Leptin Proinsulin, Insulin, C-peptide