Innovating Healthcare with Biomarker Discovery by Luminex

Connecting the xMAP Community to Innovative Applications & Resources

xMAP Insights ÂŽ

2017 Issue 2

Innovating Healthcare with Biomarker Discovery

xMAP® Insights A Magazine for the Multiplexing Community Greetings, Welcome to xMAP® Insights. We’ve set out to create a quarterly magazine that connects you to the multiplexing community by sharing innovative applications of xMAP Technology and providing valuable resources to catalyze your next experiment. Hilary Graham, MA Senior Manager, Scientific Marketing, Luminex Corporation

This issue focuses on high-throughput biomarker discovery and validation in support of drug discovery and diagnostic development. You’ll also find new product features from Luminex Licensed Technologies Partners (Bio-Rad, Invitrogen, MilliporeSigma, and R&D Systems). We are excited to announce that we are planning two user group meetings, called xMAP Connect, in Boston and Amsterdam for customers around the globe. We look forward to seeing you there! Next quarter, xMAP Insights will focus on vaccine development. If you have suggestions for future articles, please contact me directly at hgraham@luminexcorp.com. I’m grateful to have you as a reader and look forward to hearing from you. If you ever find yourself in Austin, please reach out, as we’d be happy to host you on a tour of our campus.

Warm Regards,

Hilary Graham, MA Senior Manager, Scientific Marketing Luminex Corporation

Luminex xMAP Insights | 2017 Issue 2

Contents 6

Putting an End to Insights | 2017 Issue 2 InflammatoryxMAP速 Disease "The automation2 reduces Letter from the Editor hands-on time and enables us to run plates continuously during the day and overnight, increasing throughput."

Collaboration

Taking Biomarkers from the Bench to the Bedside

"The resulting data clearly mandated us to amend all of our patents, and we believe that they are much more defensible now."

Connections

Meet Woei Tan: Master Multiplex Immunoassay Developer

"Plex level effect is a critical issue that is carefully addressed from the start."

Community

22 26

Attend xMAP速 Connect 2017

Delivering on the Promise of Personalized Medicine

5 Tips for Multiplex Kit Users and Assay Developers from Woei Tan

"I am conducting research in conjunction with three clinical trials to identify oncology response biomarkers."

Become a Partner

Discover New Products 26 MilliporeSigma 27 Bio-Rad 28 R&D Systems 30 Invitrogen 31 xMAP速 MultiFLEX速 BioAssays

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

Connections

Collaboration

Luminex xMAP Insights | 2017 Issue 2 | Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures. 4

Connections

Luminex xMAP Insights | 2017 Issue 2 | Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures. 5

Collaboration

Galapagos is Putting an End to Inflammatory Disease Automated assay workflow supports high-throughput target validation

Jan Stallen

Anais Legent Jan Stallen and Anais Legent are target discovery scientists at Galapagos, based in The Netherlands. Galapagos was founded in 1999 and has since built a pipeline that currently consists of 16 clinical, two pre-clinical, and 20 discovery programs that are advancing small molecule drug candidates against inflammatory diseases that were developed in-house. Stallen and Legent recently implemented a 4-plex cytokine assay in support of their inflammatory bowel disease program with an automated FLEXMAP 3DÂŽ workflow. 6

Luminex xMAP Insights | 2017 Issue 2

Tell us about your work at Galapagos. Jan Stallen (JS): I’ve been at Galapagos for seven years; I currently manage the target discovery and validation team, but in the past, I managed the high throughput screening (HTS) group. I am also involved in the development and implementation of new technologies within the company and manage bioinformatics. Anais Legent (AL): I started at Galapagos nearly three years ago and develop complex protein and cell-based assays that are used for target discovery screening.

What differentiates Galapagos from other clinical-stage biotechnology companies?

The consistency and reliability of the assay and instrument have enabled us to compare data across the entire screen with confidence.

How does the Luminex FLEXMAP 3D® fit into your drug discovery program?

The automation reduces hands-on time and enables us to run plates continuously during the day and overnight, increasing throughput.

JS: We aim to develop diseasemodifying drugs that address the root cause of the disease rather than just treating the symptoms. To accomplish this, we start screening in human primary cells and stay as close to the human as possible throughout the drug discovery process.

Galapagos relies on a patented HTS target discovery platform that uses an adenoviral shRNA library to target and silence the human drugable genome that results in the identification of disease-modifying targets.

Tell us about the co-culture assay workflow you developed. JS: We developed a co-culture (gut epithelia + immune cells) assay to further screen new targets for immune modulation of Inflammatory Bowel Disease (IBD). AL: I developed and optimized a 4-plex assay to further characterize hits from the functional screen that inhibited cytokine secretions, which elicited a high immune response. We decided to start with a ready-made kit from a Luminex partner to save time and to minimize batch to batch variation. To increase throughput, I ported the assay from the commercially available 96-well format to a 384-well format and optimized incubation time, bead concentration, and wash conditions – because no kits for our cytokine targets were available in the 384 format at that time.

JS: The FLEXMAP 3D meets our throughput needs and can take multiple analyte measurements at the same time. That’s why we adopted it for this project. AL: Luminex instruments have intuitive interfaces that are user-friendly. We were up and running in a couple of weeks with no prior experience.

I understand your workflow is automated?

AL: Yes. We have a PAA KinEDx robotic arm that feeds the FLEXMAP 3D, and a custom made waste system. The automation reduces hands-on time and enables us to run plates continuously during the day and overnight, increasing throughput. For this study, we ran approximately 40 multiplex assay plates that were generated from the supernatant of 160 96-well plates. We would not have been able to meet our timeline with the staff on hand if we would have run traditional ELISA assays.

What’s does the future hold for your group at Galapagos? JS: We are developing more complex screens that provide us a more detailed understanding of disease modifying signaling pathways while also enabling us to select better hits. We will be expanding the adoption of the Luminex platform as it proved useful for the IBD project and empowers us to ask and answer more complex and biologically relevant questions.

Want more tips on how to automate your assay workflow? Download the Automation of xMAP® TechnologyBased Multiplex Assays white paper to learn more. All photographs in this article are used with permission from Galapagos NV.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

Collaboration

Protagen is Taking Biomarkers from the Bench to the Bedside The FLEXMAP 3D® supports high-throughput discovery of autoantibody biomarker candidates

Peter Schulz-Knappe is the Chief Scientific Officer at Protagen AG, based in Germany. Protagen is using Luminex’s FLEXMAP 3D® as part of its SeroTag® Peter Schulz-Knappe biomarker identification and development platform to create assays to support the development of drugs and companion diagnostics.

Luminex xMAP Insights | 2017 Issue 2

When developing your protein arrays, what challenges did you face? Protagen had developed its own protein arrays for biomarker discovery, which allowed us to look at up to several thousand proteins at any one time. However, we felt that the data quality and reproducibility just weren’t high enough for the standard of research we wanted so we had to look for other technologies.

How did Luminex attract your attention? Luminex had developed a new multiplexing platform, the

FLEXMAP 3D, which allowed researchers to look at 500 different analytes simultaneously. We carried out experiments that compared our techniques to the Luminex technology, and we found it gave us data of much higher quality, higher throughput and speed, and improved the reproducibility of results. The dynamic range was also much better, and the results were quantitative as well as qualitative. Also, at Protagen, we used typical planar microarrays in batches of 100, which meant that a study of a thousand patients needed ten different batches. As the FLEXMAP 3D system could process up to 2,000 patient samples per batch,

with up to 500 data points per patient sample, we could eliminate batch-to-batch variability, meaning that we could be much more confident in our results.

Were there any challenges in the switch? We decided to shift completely to the Luminex system. It was a nerve-racking move, as everyone had to adapt to the new techniques and it left us wondering about all of our existing data and patents, which were based on the old technology. But we took the step, and bravely decided to re-run all of our existing patient samples. The resulting data clearly mandated

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

Collaboration us to amend all of our patents, and we believe that they are much more defensible now. Our research requires up to 8,000 data points per patient sample so, with the FLEXMAP 3D system, we still have to run a number of assays per patient, but because data quality and reproducibility are so high, we continue to see a huge benefit.

What difference did the shift to FLEXMAP 3D make? Precision medicine demands biomarkers that can move from the bench to the bedside by transforming biomarkers into in vitro diagnostics. Luminex technologies are available worldwide, and they are already used in a wide range of FDA cleared, diagnostic tests. This means that developers of diagnostic assays can stay with the same platform for discovery, validation, clinical testing, and commercialization while maintaining their line of evidence. It also makes it easier for third parties to be able to reproduce the results, for example, during clinical trials.

The resulting data clearly mandated us to amend all of our patents, and we believe that they are much more defensible now.

How have you applied FLEXMAP 3D to your research? Our SeroTag biomarker identification and development platform uses Luminex’s xMAP® Technology for high-throughput discovery of biomarker candidates by measuring the levels of autoantibodies in serum samples from thousands of patients. We screened samples from people with autoimmune disorders and used the outcomes to create NavigAID SLE, a multiplex assay that uses biomarkers to separate and define

Luminex xMAP Insights | 2017 Issue 2

subgroups of patients with systemic lupus erythematosus (SLE). NavigAID SLE can help drug companies develop more effective therapeutics for patients with SLE through precise disease characterization, patient stratification, and response prediction. Last year, we also launched our first in vitro diagnostics based on the SeroTag platform. Compared with the ten-plus years that it usually takes to move a biomarker assay from concept to the market, these projects have only taken around three and a half years to this point. Our Multilisa® SLE multiplex assay will incorporate up to 10 standard markers and up to five autoantigens for SLE and will act as the proof-of-concept for our approach. We are also developing NavigAID SSc for Systemic Sclerosis, NavigAID SjS for Sjögren Syndrome, and Multilisa RA for rheumatoid arthritis. Many markers are in assay development, with consecutive launches expected this year. These products will validate both our approach and Luminex’s technology.

We are also looking at other disease areas, including cancer. Patients who are treated with immuno-oncology drugs, such as checkpoint inhibitors and cancer vaccines, can develop immune-related problems which move them into our field of expertise. Here we have a substantial collaboration with

the National Cancer Institute to identify biomarkers that can be used to predict therapy responsiveness, monitor patients receiving immunotherapies, and detect adverse events early. These will be useful both in drug development and as companion diagnostics.

Watch this video to learn more about the FLEXMAP 3D.

Protagen Facility

All photographs in this article are used with permission from Protagen AG. SeroTag and Multilisa are registered trademarks of Protagen AG. Interview excerpted from Nature Inside View Advertorial published 1 June 2017.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

Collaboration

Delivering on the Promise of Personalized Medicine Yoshihiko Fujita is discovering biomarkers to inform cancer treatment Yoshihiko Fujita is on the Faculty of Medicine in the Department of Genome Biology at Kindai University. Kindai University is the fourth largest comprehensive Photo Caption university in Japan. Yoshihiko Fujita Dr. Fujita works in the laboratory of Kazuto Nishio, Chair of the Department of Genome Biology, who is renowned for his genomic analysis work of cancer that supports the development of predictive clinical biomarkers and personalized medicines. Dr. Fujita hopes to discover biomarkers that inform treatment selection and improve patient survival outcomes.

What technology does your lab use most often? Our department operates the Genome Center at the Life Science Research Institute of Kindai University, which performs in-house clinical sequencing for hospital patients using next generation sequencing (NGS) and digital polymerase 12

Luminex xMAP Insights | 2017 Issue 2

chain reaction (PCR). We use NGS to identify gene mutations, such as copy number variation, gain of function, and fusion proteins. Once we identify aberrantly expressed genes, we then move to our Bio-Plex® 200 system for further analysis. We also have a Bio-Plex Pro™ Wash Station which increases throughput and improves the reliability of our data. For these studies, we rely exclusively on xMAP®-based kits to quantitatively measure growth factors and angiogenesis-associated factors from pre-treatment patient plasma.

Tell me about your work. Currently, I am conducting research in conjunction with three clinical trials to identify oncology response biomarkers. Two studies seek to discover predictive biomarkers for multikinase inhibitors (sorafenib and sunitinib) and one study is investigating opioid treatment in palliative care. • We examined 40 factors from hepatocellular carcinoma patients before dosing with sorafenib to assess treatment response, specifically progression-free survival, overall survival, and response rate. Using a combination of the BioPlex Pro™ Human Cancer Biomarker Panel 1, 2, and TGF-β

panels, we found low expression levels of osteopontin and sVEGFR1 were linked with extended overall survival. • We evaluated 15 factors from metastatic renal cell carcinoma patients being treated with sunitinib to monitor therapeutic efficacy, specifically progression-free survival, overall survival, and adverse events. Using the MILLIPLEX® MAP Human Soluble Cytokine Receptor Panel, we found that high expression levels of sEGFR were correlated with significantly extended progression-free survival time. • We tested the levels of 40 chemokines before morphine administration, post one day, and post eight days to establish a prognostic indicator between the concentration of each chemokine and pain control, morphine dose requirement, and adverse effects. We are using the Bio-Plex Pro™ Human Chemokine Panel for this ongoing study.

I am conducting research in conjunction with three clinical trials to identify oncology response biomarkers.

site (CUP). CUP is a heterogeneous group of cancers that are currently diagnosed by immunohistochemical testing. There is no standard treatment regimen, and prognosis outcomes are poor. Preliminary microarray and immunostaining data indicate that programmed death-ligand 1 (PD-L1) and indoleamine-2,3-dioxygenase (IDO) expression is doubled in CUP versus normal tissue. We are using xMAP Technology to develop a quantitative method that will be more sensitive than immunostaining to detect CUP associated prognostic factors. This xMAP-based method will be leveraged in future studies where we plan to introduce nivolumab, the antibody-based immunotherapy against programmed death receptor-1 (PD-1). • The majority of our projects are focused on cancer, but we are also interested in identifying biomarkers relevant to disease progression in sepsis. While some patients readily recover from sepsis, many others become severely ill and often die. Early intervention is critical to positive patient outcomes, so we hope to discover predictive biomarkers. We collected samples from mildly and seriously ill patients at day 1 and day 3 and are using the Bio-Plex Pro™ Human Chemokine 40-plex kit for analysis.

What about upcoming projects?

Explore the xMAP® Kit Finder.

I am currently working on a couple of exploratory projects:

You can browse more than 1,400 commercially available multiplex immunoassays from Luminex Licensed Technology Partners.

• We are interested in better understanding the role of immune checkpoint proteins in cancers of unknown primary

All photographs in this article are used with permission from Yoshihiko Fujita. Bio-Plex Pro is a trademark of Bio-Rad Laboratories, Inc. Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

Connections

Luminex xMAP Insights | 2017 Issue 2 | Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures. 14

Connections

Luminex xMAP Insights | 2017 Issue 2 | Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures. 15

Connections

Meet Woei Tan:

Master Multiplex Immunoassay Developer Tan has developed more than 400 Bio-Plex® multiplex immunoassays Who are the people behind your multiplex immunoassay kits? Dr. Woei Tan is a Senior Staff Scientist at Bio-Rad Laboratories in the San Francisco Bay Area. He has developed more than 400 Bio-Plex® multiplex immunoassays over the past 15 years. When Woei isn’t at work, you’ll find him traveling or taking close-up photographs of nature.

Plex level effect is a critical issue that is carefully addressed from the start.

What do you do at Bio-Rad? I lead assay development for the Bio-Plex product line. I manage the development of multiplex sandwich immunoassays and then work closely with the manufacturing and quality control teams to bring new kits to market. I also interface with product marketing managers to share technical assay development knowledge with customers through seminars, workshops, and conferences.

Photo Caption

Woei Tan

How does Bio-Rad develop new products? Product marketing managers, R&D scientists, and data scientists closely monitor trends in publications, conferences,

Configuring the assay

Stages of

Multiplex

Assay Development

1 Screening raw materials and selecting antibodies

Luminex xMAP Insights | 2017 Issue 2

and clinical trials to understand the complex networks of antibodies coexist. We became acutely aware of this biomarkers across many different disease states. We work interference in suspension-based immunoassays when we collaboratively with our transitioned to MagPlexÂŽ beads around 2009. I remember Eschscholzia customers to hone these reading a 2014 article by Juncker that addressed the issue of californica non-specific signal in serological assays. target lists into biologically relevant panels. When we are configuring multiplex assays, the plex level Once the target effect is a critical issue that is carefully addressed from the analytes for the new start. The larger the panel, the more severe the effect panel are set, a team Daucus carota L. can be on the assay. Our largest panel is currently a of scientists gets to 40-plex, so great care was taken in development work on assay developto effectively control and minimize the plex ment, which requires effect. Being able to provide customers with tremendous effort and deep top quality products that perform reliably assay development expertise. The first is rewarding. step is the most important and timeconsuming; we work to identify the best How do you spend your time antibody pairs available because when away from work? you start with something clean, the I travel around the world meeting new downstream workflows become easier. people and enjoying their cultures. Most The six stages of assay development are: recently, Iâ&#x20AC;&#x2122;ve been to Southeast Asia and 1. Screening raw materials and India. In future trips, I hope to experience selecting antibodies more remote parts of the world like Greenland. 2. Configuring the assay As part of my traveling experience, I take close-up photos 3. Refining buffer formulation of plants and insects, which is also known as macro or 4. Minimizing assay cross-talk microphotography. 5. Testing samples 6. Optimizing the assay

Want reliable multiplex assay results?

Tell me about plex level effect. Plex level effect is the accumulated effect of non-specific binding that occurs when a large number of different

Woei Tan shares 5 tips for kit users and assay developers.

Minimizing assay cross-talk

Optimizing the assay

Refining buffer formulation

Testing samples All photographs in this article are used with permission from Woei Tan. Bio-Plex is a trademark of Bio-Rad Laboratories, Inc.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

Connections

Woei Tan Has 5 Tips for Multiplex Kit Users and Assay Developers

Tip #1 There is a difference between plasma and serum

tips for multiplex assay users

Tip #3 Always follow the assay protocol

Plasma is processed from blood in the presence of an anticoagulant. Serum is processed from blood without an anticoagulant. The sample matrices are different for plasma and serum so conduct your study with one sample type to ensure data continuity.

Be consistent with the method of sample collection and storage as these factors can impact Tip #2 the consistency in sample measurement. For cytokine Consistency in analysis, be sure to avoid sample preparation multiple freeze-thaw cycles and storage is critical as this will decrease the for precision stability of certain cytokines in the sample. Instead, prepare and freeze multiple aliquots of the same sample for single use.

Bio-Plex assays are specially optimized for the specific volume of the assay as written, not just the proportion of the reagents. If you must deviate from the default instructions, be sure you run a validation study. While diluting reagents or splitting kits might be tempting, it will compromise assay performance and data reliability, negating any small cost savings you may think you are gaining.

Bio-Plex is a trademark of Bio-Rad Laboratories, Inc.

Luminex xMAP Insights | 2017 Issue 2

Woei Tan, PhD, is a Senior Staff Scientist at Bio-Rad Laboratories in the San Francisco Bay Area. Tan shares a few best practices that he’s learned while developing more than 400 Bio-Plex multiplex immunoassays over the past 15 years.

Tip #1 Key development steps of multiplex sandwich immunoassays are:

5. Optimizing assays to reduce cross-talk

2. Sourcing of antibodies from trusted vendors and screening of antibody pairs

1. Selecting targets

3. Refining of buffer formulation

tips for multiplex assay developers

4. Constructing and calibrating curves

Don't rely solely on validation data from western blot or ELISA, as antibodies validated on these platforms don’t always perform as expected in the multiplex assay environment. For example, in ELISA, antibodies are attached via passive adsorption versus covalent coupling in Bio-Plex, which may result in altered antibody behavior. It’s essential to validate extensively and explicitly in a multiplex assay environment.

Tip #2 Antibodies used in development of multiplexing assays require platformspecific validation

Want more assay development tips? Check out this BioRadiations article.

Download the xMAP® Cookbook.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

Connections

Community

Luminex xMAP Insights | 2017 Issue 2 | Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures. 20

Connections

Luminex xMAP Insights | 2017 Issue 2 | Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures. 21

Community

Let’s Connect! Whether you’re an expert eager to share your work or just getting started – xMAP® Connect is the premier multiplexing user group meeting. Analyze both protein and gene expression from a

Boston September 19th

single sample Protein and gene expression data can be generated from the same sample, giving researchers more information about that sample. After a cell culture is centrifuged, the supernatant is analyzed for multiple cytokines using the Invitrogen™ ProcartaPlex™ assay. The cell pellet is then lysed and run using the Invitrogen QuantiGene™ Plex assay to obtain gene expression data.

Centrifuged sample

rtaPlex assay Proca

Protein expression data

Supernatant cell pellet Qua ntiGen e Plex assay

Gene expression data

Amsterdam November 8th and 9th

Protein and gene expression data from a single sample.

Attend xMAP Connect to: • Foster collaborations with multiplexing enthusiasts • Learn about new applications and assays • Share assay development best practices • Optimize your xMAP experiments with help from Luminex Application Scientists • Discover multiplexing kits and services available from Luminex Partners: Find out more at thermofisher.com/luminex

Luminex xMAP Insights | 2017 Issue 2

Interested in attending? Sign up to receive more information about xMAP Connect 2017. Boston

Amsterdam

Watch this video to hear from xMAP Connect 2016 attendees about the why they participated and what they most enjoyed.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

Community

Become a Partner Are you interested in commercializing a multiplexed assay on a platform that is efficient, flexible, and widely-adopted? xMAP® might be the right technology platform to grow your business. There are currently more than 70 Luminex Licensed Technologies Partners that represent a diversity of:

• Market segments: life science research, drug and vaccine development, clinical diagnostics, agriculture, and biodefense

• Company sizes: from a handful of employees to multinational corporations

• Geographies: Belgium, Canada, China, Estonia, France, Germany, Greece, Japan, Korea, Norway, South Korea, Spain, Taiwan, The Netherlands, and the United States

• Offerings: IVD and RUO kits, custom assay development, and testing service providers

You won’t go it alone when you partner with Luminex. Over the past 20 years, Luminex has developed a robust toolkit of resources to support partners in the development and commercialization of xMAP-based assays.

Download the Accelerate Your Commercial Success white paper. Learn about three current partners' businesses and their experience working with Luminex: • Beijing Unionluck Biological Technology Company • Myriad RBM • NMI TT Pharma Services

Luminex xMAP Insights | 2017 Issue 2

8 Benefits of Becoming a Luminex Partner Unparalleled Adoption

Unmatched Expertise

Proven Protocols

Support from Luminexâ&#x20AC;&#x2122;s Business Managers worldwide, who are in the field talking to your representatives and potential customers every day

More than 14,000 xMAP instruments sold globally

Manufacturing consultation from the undisputed multiplex assay marketing leader with more than 20 years of experience, and a history of continuous quality improvement

Take the guesswork out of getting started with the xMAP Cookbook, a collection of proven protocols and reagent recommendations

Global Presence

Every Luminex Partner Full menu of microspheres, an antibody coupling kit, and ancillary instrument control reagents

Proven Reagents

We provide assistance in your lab. Field Application Scientists are available to aid in the development and optimization of your assays

Bench-side Assistance

In-person and online classes to enhance your knowledge of Luminex technology and systems

Technical Training

Enjoys Our Success Tool Kit Marketing Support

Opportunities include access to our digital presence, display space in our trade show booth and collateral, and participation in webinars and xMAP-related scientific symposia

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

MILLIPLEX® MAP Mouse High Sensitivity T Cell Panel Low levels of inflammation are involved in many clinical and subclinical disease states. Measuring picogram levels of cytokines is critical for understanding the pathogenesis of these diseases, particularly in model organisms like mice. Cytokines and chemokines function as biomarkers for various diseases such as: • Autoimmune disease • Cardiovascular disease • Cancer

• Diabetes • Neurological disorders

Design your multiplex kit by choosing available analytes within the MILLIPLEX® MAP Mouse High Sensitivity T Cell Panel: GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-13, IL-17A, KC/CXCL1, LIX, MCP-1, MIP-2 and TNF-α. • MILLIPLEX MAP Mouse High Sensitivity T Cell Panel: MHSTCMAG-70K • MILLIPLEX MAP Mouse High Sensitivity T Cell Premixed Panel: MHSTCMAG-70KPMX • MILLIPLEX MAP Mouse High Sensitivity T Cell Panel- Premix Bulk (Space Saver) Packaging: MHSTCMAG-70KPXBK Plasma samples from an LPS-challenge in vivo mouse time course experiment showed a spike in analyte concentrations over the course of the study in multiple analytes (M-CSF, IL-1β, IL-2, and IL-5 are shown below).

Milliplex and the MilliporeSigma logo are trademarks of Merck KGaA.

Luminex xMAP Insights | 2017 Issue 2

Assay Notes

Bio-Plex™ Pro Human Apolipoprotein Panel

Apolipoproteins are important proteins that serve many functions. They bind to lipids and transport them through the lymphatic and circulatory systems. Apolipoproteins also serve as enzyme cofactors, receptor ligands, and lipid transfer carriers that regulate lipoprotein metabolism and their uptake in tissues. Apolipoproteins also function as biomarkers for various conditions such as: • • • • • •

Cardiovascular diseases Diabetes Alzheimer’s Disease Cancer Liver Disease Sepsis

Detecting changes in biomarker levels or the presence of biomarkers such as apolipoproteins can be difficult with traditional ELISA techniques. Multiplex solutions offer higher sensitivity and conserve precious sample by testing for multiple analytes in a single well. While there are several multiplex options available, the new Bio-Plex Pro™ Human Apolipoprotein Panel allows you to rapidly and efficiently detect nine key apolipoproteins plus C-Reactive Protein (CRP) from 10µl of sample in a single well in less than 4 hours. The Human Apolipoprotein Panel enables the detection and quantification of the following ten analytes in human biological samples: Apo A1, Apo A2, Apo B, Apo C1, Apo C3, Apo D, Apo E, Apo H, Apo J, and CRP (C-Reactive Protein). Bio-Plex Pro™ Human Apolipoprotein Assay Panel, 10-plex 12003081 Go to www.bio-rad.com/NewApo to learn more.

Bio-Plex and the Bio-Rad logo are trademarks of Bio-Rad Laboratories, Inc.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

RnDSy-lu-2945

Luminex速 High Performance Assays 101: Luminex High Performance Assays 101: The Devil inDiluents the Diluents The Devil is inisthe 速

the simultaneous detection of multiple protein analytes presents many challenges. This includes assay ctivity, and antibody interference. R&D Systems recognizes these unique difficulties. The diluents provided with Performance Assays have been tested and specifically formulated to overcome the challenges listed above that ssays.

e, Non-specific Binding, Cross-reactivity Assay diluents matter becauseand the simultaneous detection of multiple protein analytes presents many challenges. This includes assay

non-specific binding, cross-reactivity, and antibody interference. R&D Systems recognizes these unique difficulties. The diluents provided with

diluents matter simultaneous detection 速速 because ems LuminexAssay Assays are carefully and to tested and specifically formulated to overcome the challenges listed above that R&D Systems Luminex Highdesigned Performance Assaystested have been of multiple protein analytes presents many challenges. Detection Antibody are unavoidable in multiplex assays. nd ensure an optimally functional sandwich immunoassay This includes assay non-specific binding, cross-reactivity, Target Analyte erence occurs when a substance within the assay prevents the and antibody interference. R&D Systems recognizes Defining Interference, Non-specific Binding, and Cross-reactivity Off-target t analyte. It can occur due to non-specific antibody these unique difficulties. The diluents providedbinding, with Interfering Substance Reagents provided in R&D Systems Luminex Assays are carefully designed and testedAntibody to tibody interference. In addition, sampleAssays matrices, R&D Systems' Luminexcomplex High have such Detection Antibody minimize assay interference and Performance ensure an optimally functional sandwich immunoassay Capture AntibodyTarget Analyte been tested and specifically formulated overcome en contain interfering factors. Interfering factors may also bewithin the assay prevents the (Figure 1). Immunoassay interference occurs to when a substance Off-target accurate of the target analyte. It can occur due non-specific antibody binding, the challenges listed above that are unavoidable in es via the reagents ordetection equipment used. Interference, noto matter Interfering Substance Antibody antibody cross-reactivity, or antibody interference. In addition, complex sample matrices, such assays.signal relative to the actual ither reducedmultiplex elevated Capture Antibody Figure Optimally Functional Sandwich Immunoassay asor serum and plasma, can often contain interfering factors. Interfering factors may1.also be introduced to biological samples via the reagents or equipment used. Interference, no matter . This meansDefining untrustworthy data. Read below to learn how we Interference, Non-specific Binding, and the mechanism, can result in either reduced or elevated signal relative to the actual Figure 1. Optimally Functional Sandwich Immunoassay igh Performance Assay diluents to overcome these challenges, Cross-reactivity concentration of target analyte. This means untrustworthy data. Read below to learn how we ust the data you generate. carefully design our Luminex High Performance Assay diluents to overcome these challenges, Reagents provided in R&D Systems' Luminex Assays are ensuring that you can always trust the data you generate. carefully designed and tested to minimize assay interference and ensure an optimally functional sandwich immunoassay (Figure 1). Immunoassay interference occurs when a substance within the assay prevents the accurate detection of the target analyte. It can occur due to non-specific antibody binding, antibody cross-reactivity, or antibody interference. In addition, complex sample matrices, such as serum and plasma, can often contain interfering factors.

Interfering factors may also be introduced to biological samples via the reagents or equipment used. Interference, no matter the mechanism, can result in either reduced or elevated signal relative to the actual concentration of target analyte. This means untrustworthy data. Read below to learn how we carefully design our Luminex High Performance Assay diluents to overcome these challenges, ensuring that you can always trust the data you generate.

Find Your High Performance Assay | rndsystems.com/diluents

R&D Systems, the R&D Systems logo, and Quantikine are trademarks of Bio-Techne Corporation.

28 Luminex xMAP Insights | 2017 Issue 2 ind Your High Performance Assay | rndsystems.com/diluents

d (Figure 2). Non-specific binding to microparticles, microplates, or other assay surfaces can be reduced by blocking. contributes to background (Figure 2). Non-specific binding to mic Capture Antibodyblocking reagents to ensure low Capture Antibody non-specific binding. igh Performance AssaysHigh contain assay-optimized ection Antibody Luminex Figure 2. Examples of Non-Specific &D Systems Performance Assays contain assay-optimized blocking reagents to ensure low Binding in an Immunoassay. Diluents provided with R&D Systems Luminex High Performance Target Analyte non-specific binding. Detection Antibody Figure 2. Examples of Non-Specific Binding in an ImmunoDetection assay. Antibody Off-target

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Antibody molecule other than the targeted Non-specific binding occurs in an immunoassay when the antibody pair interacts withand the sample container or otherour assay surfaces and Detection Antibody sample is often challenging. R&D Systems carefully selects exhaustively tests Cross-reactivity, the interaction of the antibody pair with a contributes to background (Figure 2). Non-specific binding to microparticles, microplates, or other assay surfaces can be reduced by blocking. e detection antibody and often in-house developed antibody pairs used in our Luminex High Performance Assays to ensure Target Analyte Cross-reactivity, the interaction oftargeted the antibody pair aAssays molecule other than the targeted molecule other than the targeted analyte, be with caused by either Detection Diluents provided with R&D Systems Luminex Highcan Performance contain assay-optimized blocking reagents to ensure low Antibody pair with a molecule other than the Detection Antibody Antibody interference occurs when endogenous antibod toanalyte, the analyte of interest (Figure 3). can be caused by either the capture antibody or the detection antibody and often analyte specificity. non-specific binding. Off-target the capture antibody or the detection antibody and often occurs tibody or the detection antibody and often Target Analyte tar Interfering sample (Figure E) and prevent proper Target Analytewithin the action ofwhen the antibody pair with a molecule other than the targeted bstances within the biological Substance the interaction of 4; theD,antibody pair3.with a molec Figure Cross-Reactiv Detection Antibody occurs proteins sample are structurally similar toanalyte the analyteCross-reactivity, of interest (Figure 3). when inin the sample are structurally similar to the Antibody rally similar to proteins the analyte ofthe interest (Figure 3). Off-target interfering substances in biological samples include Off-target yand either theDetection capture antibody the detection antibody and often within exhaustively tests analyte, can be caused by either the capture antibody orSubstance the hum det Interfering Antibody Detection Antibody Optimizing antibodies soour toor avoid cross-reactivity with substances the biological TargetInterfering Analyte interest (Figure 3).as Optimizing antibodies so as to avoid crossCapture Antibody ivity withofsubstances within the biological Substance Antibody Antibody (Figure 3). occurs when any substance present in exceptionally high concentratio he sample are structurally similar to the analyte of interest are structurally similar to the hsample Performance Assays to ensure Target Analyte Target Analyte is often challenging. R&D Systems carefullysample selectsis and tests ourproteins in the sample reactivity with substances within the biological oftenexhaustively fully selects and exhaustively tests our Off-target Antibody Capture Antibody Antibody interference occurs when endogenous antibodies withinprevent samples cross-link with assay (Figure Interfering antibody interference from antibodies HAMACapture and RF in biol as to avoid cross-reactivity with substances within the biological Optimizing antibodies so as to avoid cross-reactivity with substan Substance Off-target Off-target in-house developed antibody pairs used selects in our Luminex High Performance Assays Antibody to ensure Luminex High Performance Assays to ensure challenging. R&D Systems carefully and Interfering exhaustively tests Substance Interfering Substance Figure 3. Cross-Reactivity in an Immunoassay. Antibody Antibody within the sample (Figure 4;pairs D, E) and prevent proper target-analyte binding to both R&D the capture detection ng. R&Dour Systems selects and exhaustively our sample is often challenging. Systems and carefully selectsantib and analyte specificity. in-housecarefully developed antibody used in ourtests Luminex High Capture Antibody Capture Antibody Capture Antibody A. B. Figure 3. Cross-Reactivity inhuman an Immunoassay. Figure 3. Cross-Reactivity inLuminex an Immunoassay. in-house developed antibody pairs used in our High Per( interfering substances in biological samples include anti-mouse antibodies (HAMA) and rheumatoid factor ody pairs used in our Luminex High Performance Assays to ensure Performance Assays to ensure analyte specificity. analyte specificity. any substance present in exceptionally high concentrations. Diluents provided with our Luminex High Performance within samples cross-link with assay antibodies (Figure 4; A–C) or substances

Figure 2. Examples of Non-Specific Binding in an Immunoassay. Figure 3. Cross-Reactivity in an Immunoassay. Antibody occurs when endogenous antibodies Detection s-analyte antibodies withininterference samples cross-link assay antibodies (Figure 4; Common A–C) or substances prevent antibody interference from HAMA and RF in biological samples. Antibody interference occurs whenwith endogenous antibodies within samples cross-link with assay antibodies (Figure 4; A–C) or substance binding to both the capture and detection antibodies. antibody Antibody Interfering within samples cross-link with assay antibodies (Figure 4; A-C) or roper target-analyte binding to both the capture and detection antibodies. Common antibody Antibody within the sample (Figure 4; D, E) and prevent proper target-analyte binding to both the capture and detection antibodies. Common antib n anti-mouse antibodies (HAMA) and rheumatoid factor (RF) antibodies as well as substances withinantibodies the sample (Figureand 4; D, E) and prevent proper lude human anti-mouse (HAMA) rheumatoid factor (RF) antibodies as well as Antibody interference occurs when endogenous antibodies curs when endogenous antibodies within samples with assay antibodies (Figureand 4; A–C) or substances substances in biological samples includecross-link human anti-mouse antibodies rheumatoid factor (RF) aswith wel A. provided B. are C. antibodies . interfering Diluents with our Luminex High Performance Assays designed to (HAMA) Cross-reactivity, the interaction of thethe antibody pair withdetection a molecule other than the targetedantibody Off-target target-analyte binding towith both capture and antibodies. Common interfering substances in biological Detection Antibody ncentrations. Diluents provided our Luminex High Performance Assays are designed to within the sample (Figure 4; D, E) and prevent proper target-ana Detection ecal 4; D, E) and prevent proper target-analyte binding to both the capture and detection antibodies. Common antibody any substance present in exceptionally high concentrations. Diluents provided with our Luminex High Performance Assays are designed t Substance analyte, can include be caused by either the capture antibody or the detection antibody and often samples. samples factor (RF) antibodies, as well as any Targetsubstance Analyte Capture RF in biological samples. human anti-mouse antibodies (HAMA) and rheumatoid Antibody interfering substances in biological samples include human anti prevent antibody interference fromare HAMA and RF in biological samples. biological samples include anti-mouse antibodies (HAMA) and rheumatoid antibodies as well as occurs when proteins in the human sample structurally similar to the analyte of interest (Figure 3). factor (RF) Antibody Off-target present in exceptionally high concentrations. Diluents provided with our Luminex High Performance Assays are designed Detection Interfering Optimizing antibodies so as to avoid cross-reactivity with substances within the biological any substance present in exceptionally high Substance exceptionally high concentrations. Diluents provided with our Luminex High Performance Assays are designed to concentrations. Dilu Interfering Antibody C. Antibody B. C.selects to prevent antibody interference from HAMA and RF inexhaustively biological tests samples. Interfering sample is often challenging. R&D Systems carefully and our prevent antibody interference from HAMA A. from HAMA C. Antibody Capture Antibody and RF in biological sa ence and RF in biological Antibody samples. B. Detection Detection

in-house developed antibody pairs used in our Luminex High Performance Assays Detection to ensure D. E.Target A Antibody Antibody analyte specificity. Antibody Interfering Off-target B. Off-target Interfering A. C. Interfering Figure 3. Target Cross-Reactivity in an Immunoassay. B. Analyte Detection nterfering Interfering Antibody Antibody InterferingSubstance Substance Antibody Interfering Antibody Antibody Target Analyte Capture Detection Interfering Antibody Antibody Capture Target Analyte Detection Antibody Antibody Target Analyte Detection Antibody Antibody Detection Antibody Off-target Antibody interference occurs when endogenous antibodiesOff-target within samples cross-linkAntibody withAntibody assayDetection antibodies (Figure 4; A–C) or substances Target Analyte DetectionI Antibody Substance Target Analyte Off-target Antibody Interfer Substance binding to both the capture and Antibody within the sample (Figure 4; D,Off-target E) and prevent proper target-analyte detectionInterfering antibodies. Common antibody Interfering Off-target Target Analyte Interfering Substance Interfering Capture Antibody Target Analyte Antibo Substance Antibody Capture Substance interfering substances in biological samples include human anti-mouse antibodies (HAMA) and rheumatoid factor (RF) antibodies as well as Antibody apture Antibody Antibody CaptureCapture Antibody Capture Target Analyte Detection Capture substance present in exceptionally high concentrations. Diluents provided with our Luminex High Performance AssaysCapture are designed to ntibodyanyD. Antibody E. Antibody Antibody Antibody Antibody Off-target Antibody Off-target prevent antibody interference from HAMA and RF in biological samples. Off-target Target Analyte Substance Detection Interfering Substance Capture Substance Capture Antibody Capture E. Antibody Antibod Antibody Capture A. B. C. Antibody Target Analyte Detection Antibody Interference in an Immunoassay. D. E. FigureTarget 4. Examples of Antibody Detection Antibody Detection Off-target Detection Antibody Detection Analyte Interfering Interfering TargetSubstance Antibody Antibody Antibody Interfering Antibody Detection Antibody Interfering AntibodyAnalyte Interfering D. E. Antibody Target Analyte Interfering Target Target Antibody E. Antibody Off-target Antibody Target Analyte Detection Analyte Interfering Detection Analyte Off-target Substance Detection Interfering Antibody Off-target ering Antibody Antibody Off-target Substance Capture Off-target Capture Target Analyte Antibody Antibody Substance body Substance Target Analyte Substance Antibody Capture Capture Antibody Off-target Analyte Capture

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research use or manufacturing p Antibody Figure 4. Examples assay. Learn more and design your Luminex High-Performance assay ofatAntibody Interference in anForImmuno Off-target

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Figure 4. Examples of Antibody Interference in an Immunoassay.

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protein and gene expression from a e ne expression data can be generated from ple, giving researchers more information Analyze both protein and gene expression from a mple. After a cell culture is centrifuged, the analyzed for multiplesingle cytokinessample using the Protein and gene expression data can be generated from cartaPlex™ assay. The cell pellet is then lysed he Invitrogen QuantiGene™ Plex assay to giving researchers more information the same sample, pression data.

Did you know that you can analyze both protein and gene expression from about that sample. After a cell culture is centrifuged, the a single sample? supernatant is analyzed for multiple cytokines using the Invitrogen™ ProcartaPlex™ assay. The cell pellet is then lysed

Protein andProtein gene expression data can be generated from the same sample, giving researchers more information ssay expression a x and run using the Invitrogen QuantiGene™ Plexforassay e l P a t r a about that sample. After a cell culture is centrifuged, the supernatant is analyzed multipleto cytokines using the Proc data Invitrogen™ ProcartaPlex™ assay. The cell pellet is then lysed and run using the Invitrogen QuantiGene™ Plex assay obtain gene expression data. to obtain gene expression data. Qua ntiGen e Plex assay

Gene expression data

Centrifuged sample

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Protein expression data

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Qua ntiGen e Plex assay

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Contact LuminexFAS@thermofisher.com for personalized assistance and to get started.

Invitrogen and QuantiGene are trademarks of Affymetrix. ProcartaPlex is a trademark of ore at thermofisher.com/luminex Thermo Fisher Scientific.

Luminex xMAP Insights | 2017 Issue 2

Limited Time Offer: Have up to 10 samples tested free of charge! xMAP® MultiFLEX® BioAssays are configurable bead-based nucleic acid assays designed to detect select emerging biothreat and infectious disease agents. These multiplex panels enable the simultaneous evaluation of dozens of targets in a single well, saving time and preserving sample as compared to traditional qPCR methods. xMAP MultiFLEX BioAssays include: • Panels for Biothreat, Mega Febrile, Mega Mosquito, and Mega Tick • Ability to customize your panel by selecting and paying only for your targets of interest • Complete kits with primers, coupled microspheres, buffer, and SAPE for up to 100 reactions; plus external and internal controls to ensure reliable results Not sure which MultiFLEX analytes are the best fit for your work? Let the lab at GenArraytion, Inc.® test 10 of your samples and provide you target-specific data1; evaluate the assay for your distinct needs! Using these results, GenArraytion can build a customized one-reaction multiplex molecular assay from existing panels to detect your targets of interest. Please contact your Business Manager or servicesales@luminexcorp.com for more information. 1. Offer valid for new xMAP MultiFLEX customers only. Limit ten (10) extracted nucleic acid samples. The customer is responsible for shipment of samples to GenArraytion and selection of initial MultiFLEX panel of interest.

Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

www.luminexcorp.com Â© 2017 Luminex Corporation. All Rights Reserved. Luminex, xMAP, FLEXMAP 3D, and MagPlex are all trademarks of Luminex Corporation, registered in the U.S. and other countries. 200 is a trademark of Luminex Corporation. MultiFLEX and MultiFLEX BioAssays are trademarks of GenArraytion Inc.