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EQUINE VETERINARY JOURNAL Equine vet. J. (2007) 39 (5) 414-416 doi: 10.2746/042516407X204573
Antibodies to elastin peptides in sera of Warmblood horses at different ages L.
VAN
BRANTEGEM*, H. E. V.
DE
COCK, V. K. AFFOLTER†, L. DUCHATEAU‡, J. GOVAERE§, G. L. FERRARO# and R. DUCATELLE¶
Laboratory of Pathology, Department of Veterinary Medicine, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium; †Department of Pathology, Microbiology and Immunology, #Centre for Equine Health, School of Veterinary Medicine, University of California, Davis, USA; and ‡Department of Physiology and Biometrics, §Department of Obstetrics, Reproduction and Herd Health, ¶Laboratory of Pathology, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. Keywords: horse; anti-elastin antibodies; ELISA; elastin
Summary Reasons for performing study: Early diagnosis and monitoring progression of chronic diseases in elastin-rich tissues, such as chronic progressive lymphoedema in draught horses and chronic obstructive pulmonary disease (COPD) is still a real challenge in the horse. Use of an enzyme-linked immunosorbent assay (ELISA) to detect anti-elastin antibody (AEAb) levels might be useful to assess the status of such diseases. Baseline levels, representing physiological breakdown of elastin in normal horses, are not available at present. Hypothesis: Levels of AEAb in healthy horses are generally low and follow the same age-related pattern as found in man. Therefore, elevation of AEAb levels in serum can be used to evaluate pathological elastin breakdown in elastinrich tissues. Methods: Sera of 84 clinically healthy Warmblood horses were evaluated for the presence of AEAbs by means of a modified version of an ELISA technique used in man. The horses were divided in 5 age groups: A) <4 months; B) 4–23 months; C) 2–3 years; D) 4–10 years; and E) >11 years. Results: Antibodies to elastin were found in all equine serum samples tested. Their levels were lowest in Group A, low in Groups B and E and highest in animals age 2–10 years. Conclusions: Measuring AEAbs in serum of horses by an ELISA technique proved to be possible and levels were stable during well-defined life stages. Potential relevance: Changes in AEAb levels are expected to be useful for early diagnosis and for monitoring progression of diseases that affect elastin-rich tissues, such as chronic progressive lymphoedema and COPD. Introduction The elastic properties of many tissues, such as lung, dermis and large blood vessels, are due to the presence of elastic fibres in the extracellular space (Rosenbloom et al. 1993). Under normal *Author to whom correspondence should be addressed. [Paper received for publication 11.10.06; Accepted 30.01.07]
circumstances elastin is synthesised in high quantity only by embryonic and rapidly growing tissues (Rosenbloom et al. 1993). In healthy human subjects, elastin degradation is extremely slow due to extensive cross-linking, with a half-life of about 70 years (Petersen et al. 2002). There is, however, a small physiological turnover resulting in some soluble elastin derived peptides (EPs), such as α-elastin being released, reaching the circulation and inducing the production of antielastin antibodies (AEAbs). Increased degradation of elastin in man has been shown to occur in several pathological conditions, such as lung emphysema (Kucich et al. 1985; Frette et al. 1997), arteriosclerosis (Fülöp et al. 1989, 1990), abdominal aortic aneurysms (Satta et al. 1998; Petersen et al. 2002; Shinohara et al. 2003), diabetic retinopathy (Nicoloff et al. 2000a,b) and a variety of skin diseases (Colburn et al. 1992, 2003; Daskalova et al. 1997). In these conditions, the released EPs induce the production of AEAbs (Bako et al. 1987; Baydanoff et al. 1987; Fülöp et al. 1989; Colburn et al. 1992, 2003). Serum levels of AEAbs have been studied extensively to detect and monitor progression of diseases in elastin-rich tissues such as skin, lung and blood vessels in man (Kucich et al. 1985; Fülöp et al. 1989, 1990; Colburn et al. 1992, 2003; Nicoloff et al. 2000b). Diseases that affect elastin-rich tissues in horses include chronic obstructive pulmonary disease (COPD) (Beech 1991; Robinson et al. 1996) and the recently recognised chronic progressive lymphoedema (CPL) in draught horses (De Cock et al. 2003; Ferraro 2003). Identifying tissue damage at an early stage and monitoring progression is still a challenge in horses with these conditions (Couëtil et al. 2001; De Cock et al. 2003). One possible approach is the detection of AEAbs induced by EPs, derived from elastin degradation. As a first step in exploring this possibility the usefulness of an enzyme-linked immunosorbent assay (ELISA) to detect AEAbs in sera of healthy horses was evaluated in the authors’ laboratory. In man, an age related change in the quantity of AEAbs has been found (Baydanoff et al. 1987, 1991). Therefore, in this study sera of 84 horses, ranging from newborn to age 31 years, were evaluated for the presence of AEAbs.
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To correct for day-to-day variations, results were expressed as corrected optical densities (COD) obtained as follows:
Materials and methods Animals and sera In total, 84 privately owned, Warmblood horses were used. All were in general good health and body condition at the time of serum sampling. The animals were divided into 5 groups according to their age: Group A) age <4 months; Group B) 4–23 months; Group C) 2–3 years; Group D) 4–10 years; and Group E) 11–31 years. Normal, negative control sera were taken from 8 foals immediately after birth, before colostrum was administered. Positive control serum was obtained from a pony with severe chronic COPD. All serum samples were immediately frozen after collection and stored at -20°C until analysed. Enzyme-linked immunosorbent assay The performed ELISA was a modified version of the method described by Colburn et al. (2003). The antigen used was a commercial α-elastin1 prepared from bovine neck ligament by the neutral extraction method of Partridge et al. (1955). For the ELISA, wells of polystyrene microtitre plates (MaxiSorb)2 were coated with 120 µl antigen (α-elastin) dissolved in a 0.05 mol/l carbonate buffer (pH 9.6) at a concentration of 40 µg/ml. The plates were incubated for 24 h at 4°C. After washing the plates in an automated plate-washer (Wellwash 4 Mk 2)3 4 times with 400 µl PBS-Tween (PBST: PBS-Tween 20 0.05%)4, the plates were dried and stored at 4°C until used. At the time of use, unspecific binding was blocked with 300 µl of a 1% solution of bovine serum albumin (BSA) in PBST for 2 h at a temperature of 25 ± 3°C. Next, the plates were washed 4 times with 400 µl PBST and incubated with 100 µl of test serum, diluted at 1:50 with PBST containing 1% BSA. The plates were then incubated for 1 h at 37°C. After washing 4 times with 400 µl PBST, 100 µl of alkaline phosphatase labelled goat-anti equine Ig G (H+L)5 diluted 1:1000 in PBST containing 1% BSA, was added. After an incubation of 1 h at 37°C, the plates were washed 4 times with 400 µl PBST. Subsequently, 100 µl of p-nitrophenyl phosphate (Sigmafast)6, made up in 0.2 mol/l Tris buffer at a concentration of 1 mg/ml, was added to the ELISA plates. After 30 min at 25°C +/- 3 °C in a dark chamber, the reaction was stopped by adding 25 µl of 3 mol/l NaOH. The optical densities (OD) were measured spectrophotometrically at 405 nm using an automated plate reader (Multiskan)3. Optimisation of the ELISA The optimal concentrations of antigen, sera and conjugate were defined in preliminary checkerboard assays. Reproducibility of the test procedure was determined by intra- (CV1) and interassay (CV2) coefficients of variance (CV = s.d./mean) by measuring 4 replicates of the positive and negative controls in a single plate and subsequently in 4 separate assays (Jacobson 1998).
COD =
ODsample - ODnegative control ODpositive control - ODnegative control
In preliminary studies, several dilution series of all serum samples were carried out. The obtained optical density values were placed in a serial dilution curve. The 1:50 dilution of each serum sample represented a point on the linear portion of the serial dilution curve. The values measured at a 1:50 dilution of the sera were, therefore, used for statistical evaluation. Statistical analysis Analysis of variance was used to compare the log-transformed COD between the age and sex groups at a global significance level of 5%. The log-transformed COD values were normally distributed (Shapiro-Wilks test, P = 0.27). Multiple comparisons between the age groups were done, based on Tukey’s adjustment technique for multiples comparisons. Results The absolute OD values of the positive and negative control sera were 0.529 (range 0.449–0.613) and 0.088 (range 0.080–0.101), respectively. The CV1 was 5.36% and 4.84% for the positive and negative controls, respectively, while the CV2 was 9.91% and 5.34%. The mean COD and s.e. of mean of the AEAb levels for the different age groups are summarised in Table 1. The young mature horses (Group C) had the highest mean AEAb levels. In the youngest (Group A) and oldest animals (Group E), mean AEAb levels were substantially lower. When the different age groups were statistically evaluated, a significant difference in AEAb levels was found (P = 0.0094). A statistically significant difference in AEAb levels was found when the youngest horses (Group A) were compared pairwise to Group C (P = 0.0038) and to Group D (P = 0.0088). Mean ± s.e. COD for the mares, stallion and geldings were 0.40 ± 0.03, 0.28 ± 0.05 and 0.29 ± 0.03, respectively. These differences in AEAb levels were not significantly different (P>0.05). Discussion In this study, an ELISA technique was used to detect anti-elastin antibody (AEAb) levels in sera of Warmblood horses of different age groups. In human medicine, the technique has been optimised and used extensively to detect and monitor progression of several pathological conditions (Kucich et al. 1985; Fülöp et al. 1989, 1990; Colburn et al. 1992, 2003; Nicoloff et al. 2000b). AEAbs were found in all serum samples tested except in the precolostral sera. This proves that, as in man, there is a constant physiological production of antibodies against EPs during a horse’s life.
TABLE 1: Mean ± s.e. corrected optical density (COD) and median of anti-elastin antibodies (AEAbs) in horses of 5 age groups
Number of horses Mean ± s.e. COD Median
Group A <4 months
Group B 4–23 months
Group C 2–3 years
Group D 4–10 years
Group E >11 years
5 0.16 ± 0.06a 0.11
4 0.35 ± 0.10ab 0.35
18 0.40 ± 0.04b 0.40
41 0.38 ± 0.03b 0.36
16 0.35 ± 0.06ab 0.28
a, b: different letters indicate significant differences
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The optical density (OD) values for AEAbs in this study were lower than the reported values in man. Antibodies produced against EPs, such as α-elastin, generally map to 2 immunodominant epitopes, which are exposed when elastin is degraded (Barnard et al. 1982; Wrenn and Mecham 1987). These antigenic determinants define 2 antibody classes. Class I AEAbs show broad interspecies cross-reactivity binding to a cross-linking region and reveal highly conserved animo acid sequences across species. Class II AEAbs show little or no interspecies cross-reactivity (Barnard et al. 1982; Wrenn and Mecham 1987). The detected AEAbs in horses were therefore of the Class I type, as they specifically recognised bovine α-elastin. This might explain the lower OD values compared to the human studies, where both Classes I and II antibodies were detected (Baydanoff et al. 1987, 1991). In man, there is an age-related change in the physiological production of AEAbs (Baydanoff et al. 1987, 1991). Levels increase during youth, are highest at age 18–20 years, then stabilise and remain constant until age 60 years when the AEAb levels start to decrease significantly (Baydanoff et al. 1987, 1991). In horses, a similar pattern was found. AEAbs increase during the first years and reach their highest level at age 2–3 years. This is the age at which horses reach their sexual maturity and approach their mature size, comparable to the life stage of man of age 18–20 years. As in man, AEAbs in horses stabilise later in life and finally decrease with ageing. The significantly low AEAb levels in horses age <4 months was noteworthy. The reason for this is not known, but it might be related to the immature immune system of neonates. No comparison can be made with human subjects for this age group as the youngest human age group studied was children age 1–7 years (Baydanoff et al. 1987, 1991). Finally, no significant difference in AEAb levels was found between the sexes. This adds to the value and usability of the test, as one reference value for both sexes can be used. There was no literature found on AEAb levels in the different sexes in man. In conclusion, the results of this study show that AEAbs can be measured in equine sera and that the evolution of the physiological AEAb levels during a horse’s life is comparable with that of man. Further research is warranted to evaluate the usefulness of measuring pathological AEAbs in sera of horses with a variety of medical conditions associated with tissue damage in elastin rich tissues such as lung, skin, arteries and lymphatics.
Baydanoff, S., Nicoloff, G. and Alexiev, C. (1987) Age-related changes in anti-elastin antibodies in serum from normal and atherosclerotic subjects. Atherosclerosis 63, 267-271.
Acknowledgements
Partridge, S.M., Davis, H.F. and Adair, S.T. (1955) The chemistry of connective tissues: 2. soluble proteins derived from partial hydrolysis of elastin. Biochem. J. 61, 11-21.
The authors thankfully acknowledge the excellent technical assistance of Karolien Hermy, Marleen Foubert and Nathalie van Rysselberghe. We also acknowledge all veterinarians who collected the blood samples. We are grateful to the Center for Equine Health, School of Veterinary Medicine, University of California, Davis, for providing us with financial support. Manufacturers’ addresses 1Elastin Products Company Inc., Owensville, Missouri, USA. 2Nunc, Roskilde, Denmark. 3Thermo Electron Corporation, Waltham, Massachussets, USA. 4Merck-Schuchardt, Darmstadt, Germany. 5Southern Biotech Ass. Inc., Birmingham, Alabama, USA. 6Sigma-Aldrich, St Louis, Missouri, USA.
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Author contributions Initiation, conception and planning were by L.V.B., H.D.C., V.K.A. and R.D. Pathology was by L.V.B., J.G. and G.L.F. Execution was by L.V.B. and H.D.C., and statistics by H.D.C. and L.D. All authors contributed to the writing.