xMAP Insights Magazine Fall 2023: Possibilities in Biomarker Discovery

FALL 2023

xMAP INSIGHTS ®

P OSSIB IL IT IE S IN B IOM AR K ER DIS COVERY

xMAP INSIGHTS ®

A Magazine for the Multiplexing Community Endless Possibilities Welcome to the latest edition of Insights magazine! We are thrilled to present you with more captivating stories that exemplify the incredible strides being made in the world of science, all thanks to xMAP® Technology.

As you are well aware, xMAP Technology revolutionizes multiplexing, providing unparalleled capabilities not achievable with any other method. In this issue of Insights, we delve into the realm of biomarker discovery—an essential domain where the ability to analyze a vast array of analytes within each sample is paramount. Without the bead-based xMAP multiplexing platform, biomarker discovery would prove significantly more daunting, hindering progress in this vital field.

Across laboratories worldwide, researchers are harnessing the power of xMAP Technology to uncover novel biomarkers, validate their findings, and leverage this invaluable knowledge to bolster drug discovery and development endeavors.

We invite you to indulge in this edition of Insights magazine, filled with inspiring tales of scientific advancement. As always, we encourage you to reach out with any inquiries about xMAP Technology or to share your own remarkable success stories.

Together, let's continue pushing the boundaries of science and innovation.

Lauren Whitman Scientific Content Sr. Marketing Manager, Global LTG Luminex Corporation A DiaSorin Company

CONTENTS 2

Editor's Letter

4

Biologics Keep Getting Better and Better for Immune-Related Diseases

7

Grant Writing for the US NIH R01: Focus on Key Components to Make Your Application Shine

10

5 Reasons to Own Your Assay

12

Apolipoproteins as Biomarkers in Heart Disease and Beyond

16

Accelerating Biomarker Discovery with Luminex® Technology

20

Go Beyond Protein Quantitation and Discover Accurate and Precise RNA Quantitation with the Same Luminex Instrument

22

Selecting Your Immunoassay Platform

Biologics Keep Getting Better and Better for Immune-Related Diseases Dominic Andrada, MS Scientific Applications Senior Marketing Manager, LTG Biologic therapeutics keep getting better and better for inflammation-related diseases For patients with immune-related diseases, biologic therapeutics have been a welcome improvement compared to conventional medications.

These biologics include first generation monoclonal antibodies (mAbs) and subsequent iterations with improved efficacy for treating inflammation-related pathology. Anti-inflammatories work by suppressing the general immune system, decreasing inflammation, reducing pain, and mitigating damage to joints and organs. Steroids, analgesics, and non-steroidal anti-inflammation drugs (NSAIDS) are routinely used for acute and longer treatments. However, their longterm use often leads to significant dose-dependent toxicity, reducing the medication’s efficacy and causing potential harm.1 The first biologic therapeutics to treat rheumatoid arthritis (RA) were a welcomed choice for chronic inflammation, offering the same if not better inflammation control with less severe or toxic side effects.

However, this first generation did not always provide adequate relief and created other side effects unique to biologic therapeutics.2 A greater understanding of the body’s immune response and improved development of biologics has led to newer generations of therapeutics

that have greater success targeting a variety of specific cytokines, leading to better treatment of specific inflammation-related diseases with reduced immunogenic side-reactions.

Older Generations of Biologics Improved Our Understanding of Cytokine Activity in Inflammation The first generation of biologics for RA worked by targeting tumor necrosis factor (TNF), a proinflammatory cytokine. TNF antagonists act by inhibiting the binding of TNF-α to its receptor and prevents the body from producing too much TNF and reduces joint inflammation and damage. Etanercept (Enbrel®) treats autoimmune diseases by interfering with TNF, and was first to gain US Food and Drug Administration (FDA) approval in 1998.4 Other notable TNF antagonists include infliximab (Remicade®),5 and adalimumab (Humira®).6 A few of these TNF antagonists were analyzed using xMAP® Technology to assess mechanisms and other cytokines involved in the underlying pathology.3 Some of them worked by modulating different cytokines and inflammation pathways by other mechanisms downstream from TNF, including B-cell inhibition and depletion, T-cell inhibition, Interleukin (IL)-1 antagonist, IL-6R, IL-16 & IL-17 antagonists, and as selective co-stimulation modulators.7

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New Generations of Biologic Therapeutics Improve Upon the Old Newer biologics such as sarilumab (Kevzara®) utilize an alternative mechanism which improves upon early generation TNF-α blockers by treating rheumatoid arthritis that does not respond adequately or is intolerant to other disease-modifying antirheumatic drugs.8 Kevzara works downstream of TNF by attaching to the IL-6 receptor instead of directly with the IL-6 cytokine, preventing the proinflammatory biomarker from activating cytokines further downstream in the inflammation pathway. To continue development of ever-improving biologics, it’s necessary to identify which biomarkers and network of cytokines are involved in the cellular responses responsible for overactivation of inflammation that causes joint and organ damage.9 Another improvement with subsequent generations of biologics includes reduced immunogenicity or reduced neutralization of therapeutic mAbs and their effects by the body’s immune system.

Multiple formats and iterations of monoclonal antibodies are developed typically by either phage display or non-human expression systems. Humanization of fusion proteins, antibody fragments, and mAbs by replacing non-human portions with human sequences, has reduced the amount of immunogenic material within these biologics.10

Newer drug development practices have led to less and less residual material in these biologics resulting in improved therapeutic profiles such as longer drug half-lives with reduced side effects.2

Biologics and Multiplexing Multiplexed immunoassays utilizing Luminex's xMAP® Technology have greatly contributed to understanding chronic inflammation and aberrant stimulation of T-cells and B-cells associated with immunerelated diseases. xMAP® assays are routinely used for in-depth characterization and development of biologics, as well as uncovering potential anti-drug immune responses. (For more information, see the recent paper, Design and Evaluation of a Multiplexed Assay to Assess Human Immunogenicity Against Humira.)6 Multiple combinations, or panels of various cytokines, are available for looking at specific biomarkers involved in immune cell activation, cellular responses, phosphoprotein cell-signaling, and cytokine cascades.5,11 Many of these are available as off-the-shelf kits from several of our assay Partners, which can be found on our xMAP® Kit Finder page.

For developing potentially new biologics or looking at novel immune pathways, specifically-configured panels of cytokines and custom developed assays are available from several assay-development services, including the LuminexPLORE Lab.

REFERENCES 1. Rice JB, White AG, et al. "Long-term systemic corticosteroid exposure: a systematic literature review." Clinical therapeutics 39.11 (2017): 2216-2229. https://doi.org/10.1016/j.clinthera.2017.09.011 Curtis JR and Jasvinder A Singh. “Use of biologics in rheumatoid arthritis: current and emerging paradigms of care.” Clinical therapeutics vol. 33,6 (2011): 679-707. doi:10.1016/j.clinthera.2011.05.044 Rose S, Eren M, et al. “A novel mouse model that develops spontaneous arthritis and is predisposed towards atherosclerosis.” Annals of the rheumatic diseases vol. 72,1 (2013): 89-95. doi:10.1136/ annrheumdis-2012-201431. 2. https://www.enbrelpro.com/resources-and-support 3. Engström J, Lönnkvist M, et al. “Comparison of fecal calprotectin and serum C-reactive protein in early prediction of outcome to infliximab induction therapy.” Scandinavian journal of gastroenterology vol. 54,9 (2019): 1081-1088. doi:10.1080/00365521.2019.1660402. 4. Alleyn, Matthew et al. “Design and Evaluation of a Multiplexed Assay to Assess Human Immunogenicity Against Humira®.” The AAPS journal vol. 22,5 104. 3 Aug. 2020, doi:10.1208/s12248-020-00487-4. 5. Xiaochen L, et all. The global landscape of approved antibody therapies, Antibody Therapeutics, Volume 5, Issue 4, October 2022, Pages 233–257, https://doi.org/10.1093/abt/tbac021 6. https://www.kevzarahcp.com 7. Khan IH, Krishnan VV, et al. “A comparison of multiplex suspension array large-panel kits for profiling cytokines and chemokines in rheumatoid arthritis patients.” Cytometry. Part B, Clinical cytometry vol. 76,3 (2009): 159-68. doi:10.1002/cyto.b.20452. 8. Harding R, Sticker M, et al. “The immunogenicity of humanized and fully human antibodies.” mAbs 2:3, 256-265; May/June 2010. DOI: 10.4161/mabs.2.3.11641 9. Rheumatoid Arthritis information. WebMD (Internet). Citied 2023 Aug. Available from: https://www.webmd.com/rheumatoid-arthritis/biologics#:~:text=The%20FDA%20has%20approved%20these%20medications%20to%20treat,every%20week%20or%20by%20IV%20once%20 a%20month. 10. Solberg SM, Sandvik LF, et al. “Serum cytokine measurements and biological therapy of psoriasis - Prospects for personalized treatment?.” Scandinavian journal of immunology vol. 88,6 (2018): e12725. doi:10.1111/sji.12725.

xMAP® INSIGHTS | Fall 2023 ® xMAP INSIGHTS | Spring 2022

Luminex supports life science research with our Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures. 5 5Luminex supports life science research with our Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

xTEND YOUR

RESEARCH NOT YOUR WORKDAY

xMAP® Technology helps deliver more data from less sample, giving you some of your time back in an increasingly busy world. Additional benefits include: • A broad dynamic range, helping you avoid repeat testing due to out-of-range values • A compact footprint, with an embedded PC and an easy-to-use touchscreen interface • NEW! Flexible, secure, easy to use xMAP INTELLIFLEX® Software 2.0 enables integration to an automated workflow and 21 CFR Part 11 records compliance • An xTENSIVE menu of commercially available kits • Custom assay support, including assay development services through the LuminexPLORE Lab

Whatever your needs are, xMAP Technology offers ways to save. Learn more about the many benefits of multiplexing with xMAP INTELLIFLEX® on our website: luminexcorp.com/INTELLIFLEX.

For Research Use Only. Not for use in diagnostic procedures.

6 2023 Luminex Corporation. A DiaSorin Company. All Rights reserved. Luminex , xMAP INTELLIFLEX, © and xMAP are trademarks of Luminex Corporation, registered in the US and other countries.

xMAP® INSIGHTS | Fall 2023 Luminex supports life science research with our Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

Grant Writing for the US NIH R01: Focus on Key Components to Make Your Application Shine Dominic Andrada, MS Scientific Applications Senior Marketing Manager, LTG So, your xMAP® data looks great for your project and now you want to write a grant proposal? This is exciting news! Now that you have your experiments planned out, biomarkers of interest identified, and some ideas on how your project can help you solve the complexities of biology and cure disease, you will need funding to take your vision to the next level. Therefore, as you continue to refine your vision of what your research will look like, you should think about how you will plan and write your proposal for grant funding.

Applying for a Research Grant The Research Program Grant (R01) is the US National Institutes of Health (NIH) oldest and most commonly used program for funding work in health-related research and development. R01s can be investigator-initiated or can be solicited via a Request for Applications.

The NIH website enables access to the investigator-initiated R01 application, which means there are no specific program requirements to fulfill in order to apply. However, the R01 research plan proposed by the applicant must be related to the stated program interests of one or more of the NIH Institutes and Centers, based on their missions.

Specific information about the mission of each NIH Institute and Center may be found on the List of NIH Institutes, Centers, and Offices website.

Before You Apply…

To maximize your chance of receiving funding, you should plan properly for your application prior to submission. This involves developing clear goals and methods in your written grant proposal. Talk to NIH program directors in your field and colleagues with R01 grants for advice and support; they can help you determine whether your idea fits the priorities of the funding agency you are targeting as well as help you identify additional funding opportunities. Continue to hone your research proposal based on feedback and further review of the relevant research literature.

Aside from investigator and institution eligibility requirements, focus on refining key components of your grant application to make your application shine with reviewers.

• Administrative Components: • Biographies and letters of collaboration • Resources and Facilities • Budget Allowable costs by the R01 grant are listed on the NIH website and can include Luminex equipment and supplies.7 A successfully written R01 grant proposal will highlight your distinct vision for how your research could be improved or advanced with specific items. For example, projects requiring multiplex immunoassays could include Luminex instruments running multiplexed proteomic and genomic assays.

Show Enthusiasm for the Specific Aims of your Research Reviewers will want to see your enthusiasm—make sure your passion for your research shines through in your application. If you are not excited about the proposal, you cannot expect the reviewers to be excited, either. Regarding the overall impact/benefits of your specific aims, offer insight into your research question(s) and why your work is important within the field of health science. Provide a rationale for your hypotheses and hook the reviewer to want to champion your application. The specific aims section should contain the key elements of your proposal, such as specific goals and long-term goals of your research project. What gaps in your field of study will you address? What alternative possibilities could be tested, and why are your choices superior to those possibilities? Provide testable and measurable indicators of your proposed research and methodologies.

An example Specific Aims by the National Cancer Institute provides the following:8 • Introductory paragraph: Hook the reader, define the topic and critical need • Second paragraph: Include the goals of the intended research, central/working hypotheses • Specific research paragraphs • Summary: Highlight innovation, broad base to support your entire proposal

Components of the R01 application include:

Develop an Abstract to summarize your proposal for reviewers and the public:

• Scientific Components: • Specific aims • Abstract • Research plan

• Introduce the problems you are addressing • State the overall hypotheses you are testing • Illustrate the main approaches your research will take, as well as an experimental plan you will implement, if funded

xMAP® INSIGHTS | Fall 2023 Luminex supports life science research with our Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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A Crucial Part of Your Proposal: Research Plan The Research Plan Component is a significant section and can comprise the bulk of your application. To score high in this component:7 Write a condensed story of your project with information about the topic and scope of the problem • Provide a well-grounded basis for your study through a critical review of the relevant literature and prior research • Defend why your study needs to be done, why it is relevant and necessary, and its implications • Approach leading to proposed studies: Rigor of prior research, preliminary data and methodologies, feasibility of the approach • Summarize your previously published relevant data to establish that you have the right expertise for executing the experimental plan and interpreting the results Include technical innovations, such as novel biomarkers or customdeveloped xMAP® assays, that you plan to implement.

Enhance Your Grant Proposal with Facilities and Personnel Information While information on your facilities and personal bibliography do not influence the priority score rating for your application, the following elements need to be included in the grant application:8 • Facilities & Other Resources that are directly applicable to the proposed work • Bibliography of any references cited in the Research Plan

An example from the National Cancer Institute provides the following:7 • Bibliography: • Personal statement describing why you are well suited to lead the project • Cite up to four publications or research products that highlight your experience and qualifications • Describe up to five of your most significant contributions to science. List your ongoing or completed relevant research to provide evidence that you can manage a grant award • Curriculum Vitae of all key personnel and collaborators who will contribute substantive effort • Facilities & Other Resources • Provide sufficient details of your scientific work environment and available resources that will contribute to the probability of success • Include description of appropriate resources available to you through collaborators • Acquire letters of support from your department and collaborators attesting to the resources and expertise available to you and describing the institutional investment in your success as an investigator • Budget • Provide updated price quotations and specifications for relevant instruments and supplies needed for your Research Plan • Obtain pricing information on Luminex instruments, training, and reagents

Conclusion

Click here to learn more about xMAP Technology and the portfolio of Luminex instruments.

While the R01 grant is ubiquitous in many life science research laboratories, writing a proposal for it can be hard work. It requires you to closely refine your vision and consider how your research and approach will effectively respond to a gap or address a problem. Often, R01 applicants are rejected, so continue to reapply.

Therefore, award of the R01 grant is often celebrated among new academic investigators and is considered to be a welcome renewal in established labs. For labs that are new to xMAP® Technology or are experts in biomarkers, the R01 grant has been helpful to advance their multiplexing needs.

REFERENCES 1. Rice JB, White AG, et al. "Long-term systemic corticosteroid exposure: a systematic literature review." Clinical therapeutics 39.11 (2017): 2216-2229. https://doi.org/10.1016/j. clinthera.2017.09.011 2. Curtis JR and Jasvinder A Singh. “Use of biologics in rheumatoid arthritis: current and emerging paradigms of care.” Clinical therapeutics vol. 33,6 (2011): 679-707. doi:10.1016/j.clinthera.2011.05.044 3. Rose S, Eren M, et al. “A novel mouse model that develops spontaneous arthritis and is predisposed towards atherosclerosis.” Annals of the rheumatic diseases vol. 72,1 (2013): 89-95. doi:10.1136/annrheumdis-2012-201431. 4. https://www.enbrelpro.com/resources-and-support 5. Engström J, Lönnkvist M, et al.

“Comparison of fecal calprotectin and serum C-reactive protein in early prediction of outcome to infliximab induction therapy.” Scandinavian journal of gastroenterology vol. 54,9 (2019): 1081-1088. doi:10.1080/00365521.2019.1660402. 6. Alleyn, Matthew et al. “Design and Evaluation of a Multiplexed Assay to Assess Human Immunogenicity Against Humira®.” The AAPS journal vol. 22,5 104. 3 Aug. 2020, doi:10.1208/s12248-020-00487-4. 7. Xiaochen L, et all. The global landscape of approved antibody therapies, Antibody Therapeutics, Volume 5, Issue 4, October 2022, Pages 233–257, https://doi.org/10.1093/abt/tbac021 8. https://www.kevzarahcp.com 9. Khan IH, Krishnan VV, et al. “A comparison of multiplex suspension array large-panel kits for profiling cytokines and chemokines in rheumatoid arthritis patients.” Cytometry. Part B, Clinical cytometry vol. 76,3 (2009): 159-68. doi:10.1002/cyto.b.20452.

10. Harding R, Sticker M, et al. “The immunogenicity of humanized and fully human antibodies.” mAbs 2:3, 256-265; May/June 2010. DOI: 10.4161/mabs.2.3.11641 11. Rheumatoid Arthritis information. WebMD (Internet). Citied 2023 Aug. Available from: https://www.webmd.com/rheumatoid-arthritis/biologics#:~:text=The%20FDA%20has%20approved%20 these%20medications%20to%20treat,every%20week%20or%20by%20IV%20once%20a%20month. 12. Solberg SM, Sandvik LF, et al. “Serum cytokine measurements and biological therapy of psoriasis - Prospects for personalized treatment?.” Scandinavian journal of immunology vol. 88,6 (2018): e12725. doi:10.1111/sji.12725.

xMAP® INSIGHTS | Fall 2023 Luminex supports life science research with our Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Join us at

Atlanta, GA | November 2nd Emory Conference Center Hotel 1615 Clifton Rd Atlanta, GA 30329

Registration: 9:00 a.m. - 9:30 a.m.

Sessions and Lunch: 9:30 a.m. - 4:30 p.m.

Social Hour: 4:30 p.m. - 6:00 p.m.

Whether you’re a multiplexing expert or just getting started, xMAP® Connect is the premier event for connecting with fellow xMAP enthusiasts and learning best practices for multiplex assays. During this program, you’ll hear about the latest research innovations and clinical applications powered by xMAP Technology—and be sure to visit our Luminex partner booths to further explore the possibilities of xMAP multiplexing solutions.

Register now! info.luminexcorp.com/research/xmap-connect-2023-atlanta For Research Use Only. Not for use in diagnostic procedures. ©2023 Luminex Corporation. A DiaSorin Company. All rights reserved.

FL627375.0823

5 Reasons to Own Your Assay Carrie Wittmer, M.S. Manager, Scientist III, LuminexPLORE Lab

With services from the LuminexPLORE Lab, you can rest easy knowing that your assay is your own

3. Variation Visibility

There are plenty of situations where it makes sense to use a commercially available kit or assay—but then there are times when it’s better to build your own. There are many advantages to developing your own assay, but what if you need help along the way?

The LuminexPLORE Lab was launched for this exact reason.

We knew that many xMAP® Technology customers were eager for their own assay but didn’t have the time or resources to perform the full development process themselves. Whether it’s performing a discrete step such as bead coupling or it’s complete development and validation of the assay, our service team can handle the work—and hand off a carefully documented assay that is fully yours.

To learn more about why it’s so important to own your assay, we consulted with Carrie Wittmer, Scientist III Manager in the LuminexPLORE Lab. With more than 20 years of experience at the bench, Carrie has extensive knowledge of molecular assays and life science technologies. Here’s what she had to say.

1. Intuitive Troubleshooting Knowing the ins and outs of your assay allows you to troubleshoot potential causes when it does not perform as expected. Does equipment need to be calibrated? Were wash cycles performed as often as needed? When you have full control over your assay, you’ll know every element that factors into its performance—and what to suspect if there’s a problem.

2. Sample Type Optimization If you buy an off-the-shelf kit and it doesn’t generate great results for your sample type, there may not be much you can do to improve the situation. But with your own assay, you have full control over assay optimization. You can fine-tune the assay to produce excellent results for plasma, cell culture, or any other matrix of interest—or even adapt from one sample type to another when the need arises.

With a commercial assay, you may find yourself at the mercy of the vendor for ensuring reliable reagents. So when you start to see variation in assay results, it’s very difficult to pinpoint a source. Was there a difference in manufacturing from one batch of reagents to the next? When you own your assay, on the other hand, you have full transparency into every component of the workflow. If there’s lot-to-lot variation in raw materials or reagents, you’ll spot it—and be able to adjust for it to continue to get great results.

4. Stability Monitoring The more you run your assay, the better you get to know it.

Insights that can’t be rushed include things like how stable the assay remains over time, and how materials needed for the assay are affected by repeated freeze/thaw cycles. You can make these same observations with commercial assays, but when the assay is yours, you’re in a better position to monitor over time and understand exactly which variables might be affecting stability and material performance.

5. Supply Chain Management One of the many lessons we learned from the COVID-19 pandemic is that the supply chain cannot be taken for granted.

If an assay is mission-critical for your operations, then you need a detailed plan to ensure that you can continue to produce results even if certain supplies or reagents become unavailable. This is virtually impossible with a commercial assay, but with your own assay, you’re in charge. You can test raw materials from a broad range of vendors and review results to see which ones perform best. These immediately become your go-to backup plan if your primary supply source gives out.

If you’re interested in learning more about custom assay development services to help you own your assay, please reach out to your Luminex representative or visit luminexcorp.com/luminexplore.

xMAP® INSIGHTS | Fall 2023 10

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THE FINAL FRONTIER IN

ASSAY DEVELOPMENT Like the vast expanse of outer space, medical science is largely uncharted territory. And its navigators need the right vessel.

That vessel? LuminexPLORE Lab. With our custom, best-in-class xMAP® multiplexing assay development and testing services, we ensure fast, affordable, reliable results, so you can continue exploring the true final frontier…

The health of humankind.

Make first contact: luminexcorp.com/luminexplore

xMAP® INSIGHTS | Fall 2021 xMAP® INSIGHTS | Spring 2022 Forportfolio.

Research Not for use in diagnostic procedures.

Luminex supports life science research with our Research Use Only (RUO) product Not forUse use in Only. diagnostic procedures.

© 2023 Luminex Corporation. A DiaSorin Company. All Rights reserved. Luminex , xMAP INTELLIFLEX, and xMAP are trademarks of Luminex Corporation, registered in the US and other countries.

11 PRTA563707

Apolipoproteins as Biomarkers in Heart Disease and Beyond While the role of apolipoproteins and low-density lipoproteins (LDL) in cardiovascular disease (CVD) has been understood for some time, emerging research is revealing that these proteins may prove to be valuable biomarkers beyond CVD: they are now linked to cancer and other chronic diseases.

What are Apolipoproteins?

Along with other proteins, apolipoproteins bind lipids to form lipoproteins such as high-density lipoprotein (HDL), lowdensity lipoprotein (LDL), and very low-density lipoprotein (VLDL). These lipoproteins are responsible for transporting lipids throughout the lymphatic and circulatory systems. There are eight classes of apolipoproteins, each including numerous subclasses, and each apolipoprotein has a unique function in maintaining health or driving disease (Table 1).

Table 1. An Overview of Apolipoprotein Analyte

Gene ID

UniProt ID Description

Apo A1

335

P02647

Major protein component of HDL particles in plasma.

Apo A2

336

P02652

Found in HDL cholesterol in plasma.

Apo B

338

P04114

Primary organizing protein component of the shell enclosing fat molecules, and is required for the formation of LDL, VLDL, and intermediate-density lipoprotein (IDL).

Apo C1

341

P02654

Expressed primarily in the liver and activated when monocytes differentiate into macrophages.

Apo C3

345

P02656

A component of VLDL that inhibits lipoprotein lipase and hepatic lipase. It is thought to inhibit hepatic uptake of triglyceride-rich particles.

Apo D

347

P05090

A component of HDL that has no marked similarity to other apolipoprotein sequences. It has a high degree of homology to plasma retinol-binding protein, also known as lipocalins. Apo D is also closely associated with the enzymes involved in lipoprotein metabolism.

P02649

Expressed by the liver and macrophages, it is primarily found in IDL that are essential for the normal catabolism of triglyceride-rich lipoprotein constituents in peripheral tissues. In the central nervous system, Apo E is mainly produced by astrocytes and transports cholesterol to neurons via Apo E receptors. Apo E is the principal cholesterol carrier in the brain.

Apo E

348

Apo H

350

P02749

One of its functions is to bind cardiolipin. When bound, the structure of cardiolipin and Apo H both undergo large changes in structure that allow for involvement in phospholipid binding. Apo H is complexly involved in the agglutination of platelets. Normally, Apo H assumes an anti-coagulation activity in serum; however, changes in blood factors can result in a reversal of that activity.

Apo J

1191

P10909

Also known as clusterin, Apo J is associated with the clearance of cellular debris and apoptosis. In humans, it is a molecular chaperone responsible for aiding protein folding of secreted proteins. Its three isoforms have been differentially implicated in pro- or anti-apoptotic processes.

xMAP® INSIGHTS | Fall 2023 12

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

A large body of work has linked various apolipoproteins to neurodegenerative and cardiovascular diseases.

However, there are many studies indicate that apolipoproteins are critical biomarkers in many other diseases, from autoimmunity to cancer (Table 2).

Table 2. The Percentage of Scientific Literature on Apolipoproteins as Biomarkers in Various Diseases from 1972–2017 APO A1

APO A2

APO B

APO C1

APO C3

Alzheimer's Disease

1%

1%

Arthritis

8%

Autoimmune

8%

2%

3%

2%

2%

Cancer

5%

4%

6%

1%

2%

Cholesterol

21%

3%

16%

3%

8%

Diabetes

9%

2%

10%

2%

Inflammation

5%

2%

1%

Lupus

1%

1%

Neurodegenerative Disease

4%

Sepsis

8%

Statin Pathway

10%

APO D

APO E

1%

89%

6%

13%

6%

2%

1%

1%

16%

40%

5%

2%

18%

1%

23%

1%

39%

1%

1%

9%

33%

1%

2%

1%

11%

1%

2%

1%

7%

54%

1%

2%

2%

APO J

15%

4%

2% 3%

APO H

4%

78%

10%

10%

2%

31%

2%

Bone Healing

50%

Apolipoproteins Serve as Biomarkers for Assessing Risk or Protection in Cardiovascular Disease Specific apolipoproteins have been identified as key markers of cardiovascular risk. The apolipoprotein B (Apo B): apolipoprotein A1 (Apo A1) ratio was shown to be superior to HDL and LDL cholesterol ratios for estimating the risk of acute myocardial infarction.7 The correlation between Apo B and cardiovascular disease (CVD) has been demonstrated across all ethnic groups and sexes.7 This therefore serves as a measure for initiating lipid-lowering therapy in these patients.10 In contrast, low levels of apolipoprotein C3 (Apo C3) are associated with cardiovascular protection.5 While cholesterol levels are still used to measure cardiovascular risk, apolipoproteins have emerged as relevant biomarkers for assessing the risk of various CVD and initiating prophylactic and therapeutic strategies.

xMAP® INSIGHTS | Fall 2023 Luminex supports life science research with our Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Apolipoprotein Involvement in the Development of Type-II Diabetes The incidence of type II diabetes (T2D) has been increasing globally, which has led to major efforts to identify markers that determine risk. Similar to CVD, the Apo B to Apo A1 ratio is associated with the development of T2D and has been proposed as a new biomarker for predicting disease.6 High concentrations of Apo A1 were found to be predictive of T2D and demonstrated a significant linear association that was superior to other lipid and lipoprotein variables.4

Apolipoproteins Involvement in Tumor Development and Metastasis; Role as Diagnostic Biomarkers Lipid metabolism has been implicated as a major metabolic pathway in cancer.1 Accordingly, several recent studies have investigated the association between proteins involved in lipid metabolism, such as lipids and apolipoproteins, and cancer risk and progression. While Apo A1 and Apo B are firmly established in CVD, they also play a role in cancer development. It is suggested that Apo A1:Apo B100 might provide superior risk prediction for certain cancers than standard lipid markers.3 The Malmo Diet and Cancer Study of over 28,098 individuals demonstrated a positive correlation between the Apo B:Apo A1 ratio as well as Apo B and overall cancer incidence.2 The association between Apo A1 and cancer risk has been further demonstrated in lung, pancreatic, colon, and ovarian cancer as well as metastatic nasopharyngeal carcinoma.2 Based on these results, Apo A1, along with transthyretin, transferrin, HDL-associated proteins, β2-microglobulin, prealbumin, and CA125, is currently part of a US Food and Drug Administration approved plasma test to identify patients with early-stage ovarian cancer.11 The global incidence of chronic diseases has been increasing, which has led to major efforts to reduce risk and develop novel therapies. Identifying useful biomarkers is critical for developing better treatment, and apolipoproteins have been shown to be useful biomarkers in many diseases. To support scientists in their effort to understand apolipoproteins in health and disease, Bio-Rad has developed the Bio-Plex Pro™ Human Apolipoprotein Assay Panel. This panel allows sensitive detection and quantification of ten apolipoprotein analytes including Apo A1 and Apo C3 which are mentioned in this article.

Learn more about this apolipoprotein magnetic bead-based multiplex assay.

Adapted from Bio-Rad's Bioradiations Multiplex Immunoassays article. View the full article here: bioradiations.com/apolipoproteins-as-biomarkers-inheart-disease-and-beyond/

REFERENCES 1. Baenke F et al. (2013). Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development. Dis Model Mech 6, 1353–1363. 2. Borgquist S et al. (2016).

Apolipoproteins, lipids and risk of cancer. Int J Cancer 138, 2648–2656. 3. Chandler PD et al. (2016). Lipid biomarkers and long-term risk of cancer in the Women’s Health Study. Am J Clin Nutr 103, 1397–1407. 4. Jian ZH et al. (2013). The association between the apolipoprotein A1/ high density lipoprotein-cholesterol and diabetes in Taiwan— a cross-sectional study. BMC Endocr Disord 13, 42. 5. Maeda N et al. (1994). Targeted disruption of the apolipoprotein C-III gene in mice results in hypotriglyceridemia and protection from postprandial hypertriglyceridemia. J Biol Chem 269, 23,610–23,616. 6. Mao Y et al. (2017). The nonlinear association between apolipoprotein B to apolipoprotein A1 ratio and type 2 diabetes.

Medicine (Baltimore) 96, e5834. 7. McQueen MJ et al. (2008). Lipids, lipoproteins, and apolipoproteins as risk markers of myocardial infection in 52 countries (the INTERHEART study): a case-control study. Lancet 372, 224–233. 8. Onat A et al. (2010). The paradox of high apolipoprotein A-I levels independently predicting incident type-2 diabetes among Turks.

Int J Cardiol 142, 72–79. 9. Ooi et al. (2008). Apolipoprotein C-III: understanding an emerging cardiovascular risk factor. Clin Sci 114, 611–624. 10. Sniderman AD et al. (2016). Age and cardiovascular risk attributable to apolipoprotein B, low-density lipoprotein cholesterol or non-high-density lipoprotein cholesterol. J Am Heart Assoc 5, e003665. 11. ZamanianDaryoush M and DiDonato JA (2015). Apolipoprotein A-I and cancer. Front Pharmacol 6, 265. 12. Zheng S et al. (2017). Associations of apolipoprotein B/apolipoprotein A-I ratio with pre-diabetes and diabetes risks: A cross-sectional study in Chinese adults. BMJ Open 7, e014038. 13. The Bio-Plex Suspension Array System includes fluorescently labeled microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc. by the Luminex Corporation.

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DISCOVER CELL SORTING MADE SIMPLE. CONNECTIONS Now can focus Could you apolipoproteins be on the results, not procedures. biomarkers you are missing?

Discover the Bio-Plex Pro Human Apolipoprotein Multiplex Immunoassay Panel If you are studying cancer, neurological and cardiovascular disease, sepsis, or a host of other disease states, apolipoproteins may be the key to a more complete understanding. Visit our Apolipoprotein Resource Page to learn more about how these proteins are connected to disease.

Visit bio-rad.com/apo to see what you may be missing.

Accelerating Biomarker Discovery with Luminex® xMAP® Technology Biomarkers continue to rise in importance due to their broad spectrum of uses in helping to improve diagnostic accuracy, predict disease progression, and evaluate therapeutic response. Biomarkers for cancer are a good example.

As cancer progresses, changes in protein profiles and differences in protein distribution (both in tissues and bodily fluids) can be examined through the quantitative profiling of numerous proteins.

Studies that profile protein expression patterns in bodily fluids present new opportunities for the development of novel, highly sensitive diagnostic tools for early cancer detection. One specific focus of this research has been the detection of bladder cancer. Although it is not one of the most common types of cancer, bladder cancer has one of the highest rates of recurrence. To date, there are no diagnostics capable of detecting bladder cancer prior to clinical presentation.

Because of this severe limitation, nearly 30% of patients initially present with stage 2 and higher bladder cancer. Stage 2 bladder cancer has a 5-year survival rate of 50%.

In recent years, new proteomic-based technologies have enabled the identification of potential biomarkers that can be used to better detect cancers and tumor progression. One such technology, Oncuria®, is a first-of-its-kind bladder cancer test from Nonagen Bioscience. Developed using Luminex xMAP® Technology and high-quality reagents from R&D Systems™, this custom multiplex immunoassay measures the concentrations of 10 urinary analytes simultaneously. Validated in more than 4,300 patients, Oncuria was shown to have 93% sensitivity and 95% specificity for detecting bladder cancer. Additionally, Oncuria has demonstrated consistent sensitivity and specificity across bladder cancer tumor grades and stages.

“We’re excited to be working with Bio-Techne and appreciate their passion and commitment to bringing innovative clinical diagnostic tools to the market. Bio-Techne has proven to be a reliable partner and quality supplier of its R&D Systems brand of Luminex® assays, which has helped accelerate our path to LDT approval for our Oncuria® diagnostic test for early detection, monitoring, and prediction of bladder cancer.”

— Hideki Furuya, Assistant Professor, Cedars-Sinai Improving Testing Sensitivity While advances in genomic testing have led to progress in developing tools for early diagnosis, many current tests lack the sensitivity required to detect cancers early enough to improve long-term survival. One drawback to geneexpression analysis is that tumors shed cell-free DNA in a stage-dependent manner. In the early stages, when tumor burden is low, there is not much cell-free DNA entering the bloodstream. Moreover, that small amount of cell-free DNA needs to be picked out of a higher baseline of other, non-tumor derived cell-free DNA.1 xMAP® INSIGHTS | Fall 2023 16

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

To move diagnostic tests closer toward clinical utility, research trials need sensitive and highly reproducible immunoassay systems that can simultaneously interrogate large numbers of targets per sample in a short time. This is where precision Luminex multiplex assays are delivering promising results. By adding proteomic signature data as part of a multiomics testing strategy, researchers can increase analytical specificity and sensitivity and improve the probability of earlier detection.

An important advantage of using a robust multiplexing platform like Luminex’s xMAP® Technology is that it allows researchers to explore more applications and examine a wide variety of markers simultaneously. Scientists can then use data on multiple biomarkers to evaluate therapeutic response or refine their drug candidates. Assessing multiple targets in a single assay also allows you to be judicious with precious samples, alleviating the need to prioritize assays based on limited sample volumes.

While identifying more accurate biomarkers is of the utmost importance, we know that there is no single biomarker that will be the key to early cancer detection. Instead, we will likely need to measure a dozen proteins and determine the algorithms that will give us insights through many clinical samples and trials. These algorithms are the proprietary secret sauce to detecting cancers early and accurately. This reality underscores the necessity of equipping yourself with the best tools and working with a trusted immunoassay partner.

The multiomics blood test demonstrated 90% sensitivity and 100% specificity for CRC, whereas plasma ctDNA achieved 47% sensitivity and 75% specificity.

CRC sensitivity ws higher for the multiomics blood test versus plasma ctDNA. The use of proteins along with nucleic acids is a strong tool for early detection. In this chart, we can see the advantages not just of proteomics but of multi-omics in increasing sensitivity.

xMAP® INSIGHTS | Fall 2023 Luminex supports life science research with our Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Human Immuno-Oncology Panel

by R&D Systems™ Multiplex Immuno-oncology targets with the new Human Immuno-Oncology Luminex® Panel. Confidently move your immune checkpoint and immune therapy research forward with this new, premixed panel.

Panel features include: • Customizable panel with 14 different immuno-oncology markers such as CLTA4, PD-L1, PD-L2, TIM-3, LAG-3 • Optimized assay diluents for accurate and reproducible results • Rigorous quality testing of each panel to provide confidence in your results • Validated for human serum, plasma, cell culture supernates, and cell lysates

Discover more | www.bio-techne.com/Human-IO-Panel

Bio-Techne® | R&D Systems™

Novus Biologicals™

Tocris Bioscience™

ProteinSimple™

ACD™

ExosomeDx™

Asuragen®

For research use or manufacturing purposes only. Trademarks and registered trademarks are the property of their respective owners.

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Immunology

Deeper insights with high-plex ProcartaPlex panels The largest panels for human and mouse immune response profiling Step into the next dimension of multiplexing— the largest panel just got better

Panel features include: •

True multiplexing—Run only one plate instead of multiple assays

•

Scalability—Achieve reproducible performance regardless of plex size

•

High throughput—Increase sample throughput with the 384-well plate format

•

Customizable—Easily modify the panel with our menu of 600+ analytes

Accelerate your immune response profiling and biomarker discovery with the largest ready-to-use panels on the market.

The new Invitrogen ProcartaPlex Human Immune Response ™

™

Panel 80-Plex and Invitrogen™ ProcartaPlex™ Mouse Immune Response Panel 64-Plex are meticulously designed to deliver reliable and precise results, helping ensure you can confidently make informed decisions based on your findings.

Learn more about our high-plex solutions at thermofisher.com/80plex

For Research Use Only. Not for use in diagnostic procedures. © 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are ®the property of Thermo Fisher Scientific and its subsidiaries unless xMAP INSIGHTS | Spring 2022 otherwise specified. COL28005 0923 Luminex supports life science research with our Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Go Beyond Protein Quantitation and Discover Accurate and Precise RNA Quantitation with the Same Luminex Instrument

Biomarker discovery is a complex process that involves identifying, validating, and verifying biomarkers that can provide insights into normal or pathological biological processes. To accelerate the discovery and development of clinically relevant biomarkers, it is important to meet certain requirements, including speed and high throughput.

RNA levels can be transient and may not correlate with protein levels, which are likely to remain stable for longer periods of time to maintain cellular functions. Due to differences between timing and expression levels, one assay may detect changes that the other does not.

Therefore, analyzing both data sets can provide more detailed insights.

The ability to perform both RNA and protein measurements using unified, high-throughput workflows make Thermo Fisher Scientific’s Invitrogen ProcartaPlex and QuantiGene Plex assays ideal tools for large-scale screening studies that benefit from automated workflows.

A more robust and accurate picture of cell states can be obtained by examining variations in both RNA and protein expression.

A perfect example of this strategy was shown in a recent publication in the journal Viruses. Researchers from the University of Tennessee Health Science Center in Memphis, TN, leveraged Thermo Fisher Scientific’s multiplex assays for gene and protein analysis. Data generated by ProcartaPlex multiplex protein assays, and QuantiGene Plex multiplex gene expression assays were crucial in investigating the replication, innate immune response, and pathology of closely related, clinical variants circulating during the first wave of the pandemic at both the RNA and protein level.

xMAP® INSIGHTS | Fall 2023 20

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

In another recent publication in the Journal Neuropeptides, researchers from the University of New South Wales published data supporting a novel inflammatory role for the neuropeptide Hemokinin-1 in human colon to regulate the release of a broad spectrum of proinflammatory mediators. QuantiGene and ProcartaPlex multiplex data demonstrated that 4-hour incubation with Hemokinin-1 significantly stimulated transcript expression and release of MCP-1, MIP-1α and β, RANTES, TNF-α, IL-1β and IL-6 proteins from the mucosa.

Learn more about the benefits of performing proteomics and genomics assays on the same instrument with the Behind the Bench blog.

xMAP® INSIGHTS | Fall 2023 Luminex supports life science research with our Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Selecting Your Immunoassay Platform Researchers rely on commercial immunoassays to study biomarker expression in diverse research applications. With recent technological advances pushing the limits of protein quantification in high-sensitivity and multiplex analyte detection, MilliporeSigma is committed to delivering the best possible immunoassays for your research needs. Whether you are using industry-leading MILLIPLEX® multiplex panels to broadly survey multiple analytes, or performing femtogram/mL biomarker analysis using the Single Molecule Counting (SMC®) ultrasensitive immunoassay platform, you can confidently expect reliable measurements, a simplified user experience, and knowledgeable scientific partners to help drive your biomarker project from hypothesis to publication.

MILLIPLEX® Multiplex Immunoassays MILLIPLEX® multiplex assays contain all the necessary components and reagents required to simultaneously detect multiple analytes and offer the largest selection of kits, analytes, and species across many research areas. The benefits of these assays include saving time, cost, reagents, and sample.

With this broad portfolio, researchers can find assays for analytes they need, achieve greater consistency by purchasing assays from one vendor, and retain the flexibility to meet needs now and in the future. From kit development and verification to manufacturing and quality control, researchers can rely on the quality built into each MILLIPLEX® kit to produce consistent, reliable results. Protocols are written with assay performance and workflows in mind, making it easy to find relevant information. In addition to the assay specifications listed in the protocol, performance criteria during kit development and verification include cross-reactivity, dilutional linearity/ parallelism, kit stability, and sample behavior.

Luminex’s xMAP Technology combines advanced fluidics, optics, and digital signal processing with proprietary microsphere technology to deliver high-density and highthroughput multiplexed assay capabilities at the same time.

As a Luminex partner, we are a preferred distributor and offer the entire portfolio of Luminex instruments, including the new xMAP INTELLIFLEX® platform, covering the full range of multiplexing capabilities. ®

SMC® Immunoassays SMC® technology is an advanced ultrasensitive immunoassay technology that gives researchers the power to detect biomarkers down to the femtogram/mL level. This platform provides an indispensable tool to help move novel biology forward, fueling the discovery and development of new therapeutics. SMC® technology relies on a basic sandwich immunoassay format utilizing two antibodies specific to the analyte of interest: a capture antibody coated on a plate or magnetic bead and a detection antibody conjugated to a fluorescent protein.

Scientists familiar with immunoassays know that sensitivity, matrix effects, and dynamic range affect measurements.

Combining a traditional immunoassay workflow with patented SMC® technology enables the detection of low-abundant biomarkers with unparalleled sensitivity and accuracy.

Researchers can detect, and monitor changes in, extremely low levels of biomarkers.

Distinguishing SMC® assays from traditional immunoassays, an elution buffer breaks apart the immunoassay complex once constructed. The eluate containing the fluorescent reporter molecule is transferred to a 384-well plate, thus removing other assay components that contribute to high background fluorescent signal. The plate is loaded into the SMCxPRO® instrument where a laser excites the fluorescent-labeled detection antibody as it passes through a narrow interrogation window. Individual photons are captured by an avalanche photodiode and the signal is recorded, allowing for the digital quantification of individual molecules.

Belysa® Software Belysa® Immunoassay Curve Fitting Software provides a userfriendly tool to ensure your methods are reproducible over time at the single assay level, over multiple plates, and over multiple lots. With Belysa® analysis software you can quickly check replicate %CVs and standard point recovery, check that plates ran consistently, confirm lot-to-lot similarity, and analyze data from ELISA readers and Luminex or SMCxPRO® instruments.

SigmaAldrich.com/immunoassays

xMAP® INSIGHTS | Fall 2023 22

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Set Your Sights

on Cytokines

Multiplex with MILLIPLEX® Human Cytokine Panels A & B MILLIPLEX® Human Cytokine Panels A and B offer you: • The ability to analyze nearly 100 cytokines in 50 µL of sample • The flexibility to choose any combination of analytes from each panel of 48 analytes • Superior performance with rigorous assay development and quality control testing • Ready-to-use reagents with everything you need in one box

Discover more at SigmaAldrich.com/milliplex-cytokines © 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

MilliporeSigma, the vibrant M, Millipore, and MILLIPLEX are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.

45271 11/2022

For Research Use Only.

Not For Use In Diagnostic Procedures.

MilliporeSigma is the U.S. and Canada Life Science business of Merck KGaA, Darmstadt, Germany.

KEYNOTE:

Dr. Paul Rhyne,

Bill and Melinda Gates Foundation Meet xMAP INTELLIFLEX® in Amsterdam

xMAP® Connect Amsterdam 2023 8-9 November DeLaMar Theater Marnixstraat 402, 1017 PL Amsterdam, Netherlands Now featuring Dr. Paul Rhyne from the Bill and Melinda Gates Foundation!

Keynote: Opportunities and Challenges in the Fight Against Shigella Whether you’re a multiplexing expert or just getting started, xMAP Connect is the premier event for connecting with fellow xMAP enthusiasts and learning best practices for multiplex assays.

Meet our xMAP INTELLIFLEX platform, and bolster your research with multiplexing expertise.

We hope to see you in Amsterdam!

More details on our program and a registration form can be found here: info.luminexcorp.com/xmap-connect-emea-2023-in-amsterdam