xMAP Insights Magazine: The COVID-19 Issue Fall 2020 by Luminex

xMAP INSIGHTS 速

The COVID-19 Issue

An xMAP速-Based Multiplex Test for COVID-19 Antibodies That Works on Dried Blood Spots Read more on Page 4 > Rush University Medical Center's Dr. Imad Tarhoni (left), David Gerard (center), and Cristina Fheid (right).

How Multiplexing Can Improve COVID-19 Antibody Detection Read more on Page 6 >

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Mount Sinai Scientists Design COVID-19 Antibody Test Using xMAP速 Technology

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New xMAP速 Cookbook Addendum: How to Develop Multiplex Serology Assays

xMAP INSIGHTS ®

A Magazine for the Multiplexing Community Greetings, My name is Lisa Kessler, and I am the new editor of xMAP® Insights, your source for the latest innovations and applications of xMAP Technology from your colleagues in the multiplexing community. When we were trying to determine what this issue should focus on, it seemed obvious that we needed to spread the word about the amazing work members of our community have been doing to fight the COVID-19 pandemic. We hope you will find the articles we’ve featured informative, engaging, and inspirational. Our users have done tremendous things this year, and we are excited to share some of their highlights with you. This issue of xMAP Insights focuses on how xMAP users have used custom multiplex assays to detect COVID-19 in several different settings. Here are just a few of the stories from our Multiplexing Community: • RUSH University Medical Center created an xMAP®-Based Multiplex Test for COVID-19 Antibodies that works on dried blood spots • Scientists at Rochester University show “Why Multiplexing Matters for COVID-19 Antibody Detection” • Mount Sinai Scientists Design COVID-19 Antibody Test Using xMAP® Technology Although we are not meeting in person right now, I am looking forward to the opportunity to meet face-to-face in the future. In the meantime, please feel free to contact me directly at lkessler@luminexcorp.com if you have any story ideas for the next edition. Thank you for being a member of our community. Wishing you good health. All the Best,

Lisa Kessler, MBA Scientific Marketing Licensed Technologies Group Luminex Corporation

CONTENTS 2

Letter from the Editor

An xMAP®-Based Multiplex Test for COVID-19 Antibodies That Works on Dried Blood Spots

How Multiplexing Can Improve COVID-19 Antibody Detection

Mount Sinai Scientists Design COVID-19 Antibody Test Using xMAP® Technology

New xMAP® Cookbook Addendum: How to Develop Multiplex Serology Assays

An xMAP®-Based Multiplex Test for COVID-19 Antibodies That Works on Dried Blood Spots

Sample type requirements pose one of the biggest challenges to COVID-19 testing strategies, for both molecular and antibody tests. For molecular tests, saliva would be the ideal sample type, but tests using more invasive nasopharyngeal swabs tend to perform better. Antibody tests historically have to be based on blood samples, but trying to collect millions of blood draws during a pandemic puts too many people and their healthcare providers at risk of infection. In Chicago, a team of scientists set out to determine if high-quality results could be generated from something as easily accessible as a dried blood spot. In a recent LabRoots webinar, Dr. Imad Tarhoni at Rush University Medical Center in Chicago offered a look at how his team developed a multiplex serological assay that they believed could be the cornerstone to a large-scale national COVID-19 testing strategy. They built their SARS-CoV-2 serology assay using xMAP® Technology to get the most from its multiplexing capabilities. In his presentation, Dr. Tarhoni noted that large-scale community testing will be essential for controlling the pandemic, particularly because so many infected individuals never exhibit symptoms. Nucleic acid testing for viral RNA, while effective, is only useful for a short period after infection. That’s why Dr. Tarhoni believes that serological assays, which can detect antibodies to the SARS-CoV-2 virus long after infection, will be so important for population-scale testing. Unfortunately, many of the existing serology tests for COVID-19 antibodies have had performance issues. Sensitivity and specificity metrics have not been high enough to avoid false negative and false positive results. This is where xMAP Technology came in. Dr. Tarhoni and his team incorporated four viral antigens for testing, ensuring highly accurate results. Whether a person has developed antibodies to the virus’s spike, nucleocapsid, membrane, and/or envelope protein, this test can detect a true positive result.

The scientists conjugated all four antigens onto xMAP beads and then optimized the assay for characteristics like linearity, detection limits, precision, cross-reactivity, and more. Next, they performed validation studies to ensure that the assay delivered reliable results, using dried blood spot samples, since they would be the most economical sample type for a nationwide testing strategy. In addition to confirming the utility of dried blood spots, they also demonstrated strong results from 384-well microtiter plates, which will be needed for any highthroughput approach. By reducing the antibody capture incubation time, they brought the turnaround time down to less than four hours with no loss in assay performance. For clinical validation, the team launched a number of studies, encompassing more than 1,000 samples from control populations and COVID-19-positive populations. The final review, including 1,178 cases collected at least six days after infection, demonstrated an assay sensitivity of 100%, specificity of 99.6%, and accuracy of 99.6%. “That is outstanding performance compared to other tests,” Tarhoni said. He noted that the ability to test dried blood spot samples—which could be collected at home and mailed to labs for analysis—provides a convenient, cost-effective means of testing large cohorts of the population. Information gathered from such testing could guide policy decisions about when to stay home and when it’s safe to return to work. It would also be important for evaluating plasma in blood banks for use in convalescent plasma therapy, monitoring vaccine efficacy, and epidemiological surveillance. View the whole webinar here.

xMAP® INSIGHTS | November 2020 Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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How Multiplexing Can Improve COVID-19 Antibody Detection As the COVID-19 pandemic continues to spread, xMAP® users around the world are turning to our bead-based multiplexing technology to develop new detection assays for antibodies to the SARS-CoV-2 virus. With the power of xMAP multiplexing technology, a single sample can be plexed to measure up to 500 antigens per well. If each of those targets had to be measured in a single reaction, and each sample were tested against all of the analytes, the number of reactions needed—not to mention the volume of reagents and sample used— would be impractical. Multiplexing offers significant advantages over single-plex analysis, which is especially important when running a large number of samples. Dr. Nicole Pecora, Assistant Professor of Pathology and Laboratory Medicine at the University of Rochester Medical Center, developed a serological antibody detection assay based on xMAP Technology and shared valuable insights into the process of building this assay in a recent LabRoots webinar. She was joined by Dr. Steve Angeloni, Senior Field Applications Scientist for Luminex, who offered a detailed look at the steps involved in developing and validating a multiplex immunoassay. Dr. Pecora and her team developed a five-plex assay based on xMAP Technology to measure SARS-CoV-2 antibody titers. The assay was designed to detect antibodies directed at the virus’s spike protein, its receptor-binding domain, and its nucleocapsid protein. The team also added internal controls for quality assurance.

Dr. Pecora and her team developed a five-plex assay based on xMAP Technology to measure SARS-CoV-2 antibody titers.

To develop the assay, the team confirmed coupling, optimized bead reactions, and tested several IgG detection reagents to determine titer levels. The assay was optimized to ensure high sensitivity with minimal serum input. The resulting test has a 45-minute workflow and is read using the FLEXMAP 3D® System. For assay validation, Dr. Pecora’s team established background metrics by testing sera collected prior to the emergence of SARS-CoV-2 and set thresholds to define a positive call. Since they

serve an area where the virus was not prevalent at the time of their research, specificity was the team’s biggest concern, so they focused on making sure the assay would not cross-react with other common respiratory infections. The assay was also validated with known positive samples for SARS-CoV-2, and results were compared to another test that had recently received Emergency Use Authorization from the FDA. They demonstrated that the xMAP assay compared favorably and can be easily adapted to the clinical environment. Sensitivity and specificity were excellent, and results were reproducible across runs and operators. Additionally, the assay’s large dynamic range makes it possible to generate quantitative results that can be used to qualify plasma donors, monitor plasma recipients, and eventually, develop vaccines. Before the emergence of SARS-CoV-2, scientists had already developed a multiplex serological assay for six coronaviruses based on xMAP Technology. The xMAP platform enables the addition of viral targets or other relevant pathogens, as new strains become important to monitor and study with custom assays. Although there are several types of COVID-19 tests currently available, serological assays provide a unique avenue for monitoring patients’ immune status following infection, as well as determining the success of vaccine candidates as they progress through clinical studies and begin to become commercially available. If you’d like to learn more about how this antibody detection assay was developed, you can view the entire webinar here.

xMAP® INSIGHTS | November 2020 Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

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Mount Sinai Scientists Design COVID-19 Antibody Test Using xMAP® Technology Susan ZollaPazner, PhD. Professor of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai

This xMAP-based assay was also compared to ELISA tests, revealing that the multiplex assay had significantly higher sensitivity—in some cases five to ten times higher—compared to the traditional immunoassay. The webinar also discussed additional advantages of the xMAP assay, including: • xMAP beads can be coated with antigen ahead of time and stored for at least a month, making preparation more convenient and efficient. • Luminex beads require 20-fold less antigen compared to ELISAs, resulting in significant cost savings.

New York City has been hit hard by the COVID-19 pandemic. As thousands of people test positive each day and hospitals struggle to serve all in need, the city still has a unique advantage: the presence of the world’s leading medical centers. Some of the earliest American studies of COVID-19 antibodies came from the Icahn School of Medicine at Mount Sinai in New York City, so it’s no surprise that scientists there were among the first to develop an antibody test. Using xMAP Technology to design this test allowed them to make the most of its multiplexing capabilities, low sample input requirements, and fast results. Unlike molecular tests—which detect active SARS-CoV-2 infections— antibody tests can be used to identify people who have been exposed to the virus and mounted an immune response. Such tests will be important for population-scale analysis to establish a broader epidemiological understanding of the pandemic, and essential for evaluating the effectiveness of new vaccines.

• The xMAP assay delivers results for both spike and RBD antibodies in less than two hours—a significant improvement compared to ELISAs, where plates can only detect one antibody at a time and the complete workflow from prep to results takes two days. In addition to the data she presented, Dr. Zolla-Pazner also shared results from additional studies that were enabled by the multiplex assay. For example, she showed that it was possible to track rising antibody levels over time by testing longitudinal specimens from the same patient, which will be extremely useful for characterizing the long-term immune response to COVID-19. Further, using the same method from her team’s original xMAP assay, she analyzed isotyping data to investigate differences in the IgG and IgM immune response. This information could inform donor selection for convalescent plasma therapy by helping clinicians identify the best donors and tracking antibody levels in those donors over time.

The development of this multiplex antibody test was the focus of a LabRoots webinar presented by Dr. Susan Zolla-Pazner, a Professor of Medicine at Mount Sinai’s Infectious Disease Division. Her team’s antibody test targets two SARS-CoV-2 antigens—the full spike protein and its receptor-binding domain (RBD)—and uses two bead sets, each designed to bind to antibodies associated with one of these antigens. Validation studies of the multiplex test using confirmed SARS-CoV-2 positive and negative samples demonstrated robust signals for both antigens from human plasma and serum.

xMAP® INSIGHTS | November 2020 Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

Watch the entire webinar! View Now >

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SELF-REGISTER @ ACADEMY.BIO-TECHNE.COM Academy.Bio-Techne.com 1. CLICK ‘REGISTER’ AT THE BOTTOM OF THE LOGIN SCREEN. 2. CREATE YOUR PROFILE WITH YOUR EMAIL AS YOUR USERNAME. 3. CHOOSE THE MAGPIX® LUMINEX® LEARNING PLAN.

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New xMAP® Cookbook Addendum: How to Develop Multiplex Serology Assays

Since SARS-CoV-2 emerged in late 2019, single-target serological assays have played a critical role in detecting viral antibodies induced by prior infection. However, single-target assays have some limitations, especially if there are varying levels of

It’s clear why demand is surging for these tests—many xMAP® assays have already been developed and adapted to detect several antibodies associated with the SARS-CoV-2 virus in labs across the world.

antibodies to different parts of the virus in recovered patients. Bead-based multiplex technology is ideal for developing serological assays, especially because it can detect antibodies to more than one viral antigen at a time. It’s clear why demand is surging for these tests—many xMAP® assays have already been developed and adapted to detect several antibodies associated with the SARS-CoV-2 virus in labs across the world. Given the current state of the global pandemic, assay developers are eager to contribute to the clinical research community. To help familiarize scientists with the process of developing bead-based assays, especially as they relate to the SARS-CoV-2 virus, the Luminex Team has developed an addendum to the latest version of the xMAP Cookbook, titled “Planning SARS-CoV-2 Serological Assay Development.” This addition can help guide users in key considerations and decisions that are critical to optimal assay performance. Highlights of the addendum include:

Combined with the complete xMAP Cookbook and the support of Luminex’s Field Applications Team, this new addendum will help assay developers of all experience levels get started quickly and build novel, rapid-result xMAP serological assays to help manage the evolving COVID-19 outbreak.

• A review of relevant structural proteins • Protein acquisition guidance • Considerations for coupling antigens to xMAP® beads • Supply lists specific to SARS-CoV-2 serological assay development • A step-by-step development guide

xMAP® INSIGHTS | November 2020 Luminex supports life science research with its Research Use Only (RUO) product portfolio. Not for use in diagnostic procedures.

Check out the latest version of the xMAP Cookbook today! Download Here >

LOOK CLOSER Immunoassay resources for viral research

MILLIPLEX® panels to assist your research efforts for critical vaccine development, epidemiology, and population research studies, including: • MILLIPLEX® Human SARS-CoV-2 Antigen Panel 1 for IgM (Cat. No. HC19SERM1-85K), IgG (Cat. No. HC19SERG185K), and IgA (Cat. No. HC19SERA1-85K) - Each kit detects the immunoglobulin type antibodies which recognize four viral antigens: SARS-CoV-2 spike protein subunits (S1 and S2), receptor binding domain (RBD), and nucleocapsid protein (N) • MILLIPLEX® Multiplex Immunoassays for Cytokine Storm Research - Cytokine panels, such as our 48-plex Human Cytokine/ Chemokine/Growth Factor Panel A (Cat. No. HCYTA-60K) and Non-Human Cytokine/Chemokine Panel I (Cat. No. PRCYTOMAG-40K)

For more kit listings, visit SigmaAldrich.com/milliplex-covid-19 SigmaAldrich.com/milliplex-covid-19

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HAVE CONFIDENCE IN YOUR COVID-19 ANTIBODY TEST.

In uncertain times, laboratories need to have confidence in their COVID-19 antibody test results—which is why Luminex developed the comprehensive xMAP® SARS-CoV-2 Multi-Antigen IgG Assay. The xMAP® SARS-CoV-2 antibody assay delivers rapid and reliable detection of SARS-CoV-2 antibodies in human serum and plasma samples. By detecting three different SARS-CoV-2 antigens, the xMAP SARS-CoV-2 antibody assay may provide earlier, more sensitive results. Key Features: • Broad Coverage: Detects IgG antibodies against 3 SARS-CoV-2 antigens: • S1 subunit of the spike protein • Receptor binding domain (RBD) of the spike protein • Nucleocapsid protein • Flexible Throughput: Test up to 96 samples in less than 3 hours • Versatile System Options: Validated on MAGPIX®, Luminex® 200™, and FLEXMAP 3D® Systems • Common Sample Types: Designed for use with human serum and plasma samples

luminexcorp.com ©2020 Luminex Corporation. All rights reserved. The xMAP® SARS-CoV-2 Multi-Antigen IgG Assay has not been FDA Cleared or Approved. This test has been authorized by the FDA under an EUA for use by authorized laboratories. This test has been authorized only for the detection of antibodies against SARS-CoV-2, not for any other viruses or pathogens. This test is only authorized for the duration of the declaration that circumstances exist justifying the authorization of emergency use of in vitro diagnostic tests for detection and/or diagnosis of COVID-19 under Section 564(b)(1) of the Act, 21 U.S.C. §360bb-3(b)(1), unless the authorization is terminated or revoked sooner.

For more information, please visit: luminexcorp.com/xmap-SARSCoV2

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