COVID-19 and Characterizing the Immune Response: A Method for Isotyping Using xMAP® Technology

In a recent white paper, authors describe an assay modification to measure the COVID-19 antibody response in serum samples

While early measurements of COVID-19 patients’ antibodies against SARS-CoV-2 focused on the IgG isotype, IgA and IgM responses are also important for fully understanding the body’s immune response to COVID-19.

“IgA titers have been found to be significantly higher in severe COVID-19 patients, and remained higher for a longer time than what has been seen in milder cases,” the authors pointed out. “Serological IgM and IgA responses may also be predictive of survival, with anti-spike IgM and IgA enriched in surviving donors and nucleocapsid-specific IgM and IgA at higher levels in individuals who died.”

To help investigators better characterize an individual’s immune status, the Luminex Team designed a new method to expand our xMAP® SARS-CoV-2 Multi-Antigen IgG Assay for serological isotype profiling in a recent white paper.

The study began with the xMAP SARS-CoV-2 Multi-Antigen IgG Assay—a serological assay that uses multiplex beads to detect antibodies reactive to multiple SARS-CoV-2 antigens in human serum and plasma. The assay kit detects IgG antibodies in serum and plasma that bind to the viral nucleocapsid antigen, the spike 1 domain of the spike antigen, and the receptor binding domain portion of the spike protein. In the white paper, the assay was further modified to evaluate IgM and IgA responses.

For validation, the authors demonstrated the chronological evolution of the three isotypes in donor samples. They analyzed 20 samples collected prior to December 2019 and 66 samples collected from 40 unique donors who were SARS-CoV-2-positive. Nine donors were sampled at multiple time points, ranging from 1 to 116 days post-symptom onset. As part of this work, the team also tested multiple assay conditions and detailed the technical procedure and results.

The modified assay demonstrated the expected earlier onset of IgM and IgA, followed by a faster return to pre-infection levels compared to IgG (which is more prevalent). This was seen both in single-timepoint and longitudinal samples taken from individual donors post-onset of symptomatic illness.

Further, the authors investigated how the IgA, IgG, and IgM titers for all three antigens (nucleocapsid, RBD, and S1) changed following the onset of symptoms, and noted that serological “IgA and IgM collectively make up around 20% of total circulating antibody in the blood, and reflect different stages of the immune response during COVID-19 progression.”

This update to the xMAP assay enables the rapid and high-throughput quantification of the IgM and IgA responses to multiple viral antigens, supporting improved characterization of the human immune response to SARS-CoV-2. As the pandemic evolves, being able to determine a patient’s immune status will be a critical part of understanding COVID-19 progression.

Read more about the study here.

Read the White Paper on the Study Here.

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

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Five xMAP® Technology Tips from Luminex Tech Support

Whether you’re a new or experienced xMAP® Technology user, our Technical Support Team can help you! They’ve compiled five tips based on questions that users frequently ask.

Tip #1: Frustrated by verification failure?

Some MAGPIX® verification failures are the result of debris on the chamber or clogs in the sample probe and in the sample lines. As the composition of the debris is generally unknown, running the Enhanced Startup routine twice—once with 0.1N NaOH for the Sanitize and Clean commands, and again with 20% bleach⁠—will generally clear up minor debris and clogs. Still not fixed? The degree of clog can be determined by reviewing the Calibration/Verification Report and Support Utility.

Tip #2: Getting an error code?

The most common instrument code is 800C9580. This is a system code, not an error code, and will accompany both MAGPIX® and LXR (Luminex® 200™ and FLEXMAP 3D®) error codes. MAGPIX error codes resemble 2091[XXXX] and 2XXX; LXR codes will begin with “0x-“. If you receive this code, check the System Log or contact Luminex Customer Support with the support utility file to determine the actual error.

Tip #3: Looking for proteins to develop your own assay?

In addition to the xMAP® Kit Finder and the xMAP Cookbook, Linscott’s Directory is a great resource, with over 2,100,000 unique antibody listings and 1,000,000 other immunological and biological reagents.

Tip #4: Does your quantitative protein assay lack sensitivity?

Try increasing the primary incubation. Primary incubation times to test are one hour, two hours, and overnight. One and two hour incubations can be performed at room temp while shaking. Overnight incubations should be performed while refrigerated and shaking.

Tip #5: Want stellar protein coupling?

Before coupling proteins (including antibodies) to beads, verify the protein concentration via NanoDrop™, bicinchoninic acid (BCA) assay, or Bradford assay. While we don’t suggest a specific concentration, it’s always best to verify the protein concentration that’s on the label or in the certificate of analysis, as they aren’t always correct.

Take a look at our Customer Center, where you’ll find contact information, product documents, target values and Certificates of Quality, training resources, and more.

Have a question and don’t know where to start? View this flyer to see how we can help.

Interested in our Luminex Learning Sessions?

Multiplex Technology Optimizes Traditional Immunogenicity Testing

Scientists combine anti-drug screening and isotyping into a single assay using xMAP® Technology in a recent study

Assessing immunogenicity, or the potential of a biologic to elicit an immune response, is a critical step in drug discovery and development. In vaccine development, a strong immune response indicates that protection against an infectious disease has been achieved. However, in biotherapeutic development, signs of immunogenicity suggest that anti-drug antibody (ADA) production may lead to adverse events or limit the drug’s efficacy.

Traditional immunogenicity characterization methods are deployed in a tiered strategy, requiring multiple steps to determine the presence and titer of ADAs, antibody isotypes, and the neutralizing capacity of a drug. This process tends to be time-consuming and expensive, especially since it involves repeated freeze/thaw cycles and frequent sample handling, increasing the likelihood of unreliable results. Additionally, in some cases, sample volume may be limited.

However, multiplex immunoassays can combine immunogenicity tests to potentially overcome these obstacles—saving time and costs, preserving sample volume, and delivering high-confidence results.

Multiplexing delivers benefits by consolidating assays while delivering reliable results

In a recent study, researchers used xMAP® Technology to design a multiplex assay from two separate immunogenicity assays to simultaneously identify and isotype ADAs in humans who received Humira®, a TNF inhibitor with a well-documented immunogenicity profile that is commonly used to treat rheumatoid arthritis and other inflammatory disorders.

To evaluate the multiplex assay, scientists screened and isotyped 50 serum samples from healthy individuals and 20 rheumatoid arthritis patients that had never received Humira®. For screening, a bead set was coupled to Humira® and was then used to capture ADAs from the samples. For isotyping, human ADA isotypes were evaluated by coupling mouse isotype-specific capture antibodies to a unique bead set. The coupling confirmation test showed a high level of Humira® on the bead surface—with a median fluorescence intensity (MFI) of 26,923—and high MFI values for the isotype-specific bead sets and their respective human control antibodies as well.

Ultimately, this study demonstrates how multiplex technology can help streamline the traditional tiered approach for immunogenicity studies while reducing hands-on time and preserving both sample volume and integrity. The authors offer a detailed discussion on how their multiplex assay design could be customized, expanded, and optimized, and indicate that xMAP Technology could be valuable for immunogenicity assessment in clinical settings.

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Bead-Based Multiplex Assay Shines in Performance Analysis

xMAP® Technology offers significant advantages in recent comparison to electrochemiluminescence platform.

It’s clear why multiplex immunoassays play a prominent role in many modern laboratories. In addition to saving time, labor, and resources, multiplexing provides simultaneous quantification of multiple targets within small sample volumes.

Different multiplex platforms have individual strengths, and in a recent study, scientists at the University of Tübingen in Germany compared two commonly used technologies: an xMAP® Technology bead-based assay and an electrochemiluminescence (ECL) planar assay. Their goal was to compare the analytical performance characteristics of both multiplex systems when measuring 16 cytokines in identical human plasma samples. They assessed each platform based on quantification limits, dynamic range, detection rate, and hands-on time.

Clear benefits found with the bead-based assay

While both systems were found to have strong reproducibility and a similar dynamic range, there were key differences in the reported results. For example, the xMAP assay detected many cytokines that the ECL assay missed entirely.

“While comparative standard and quality control (QC) sample data suggested more sensitive cytokine detection with [ECL] vs [xMAP], these findings did not translate to results obtained with actual human plasma samples,” the scientists report in the study. “[xMAP] markedly outperformed [ECL] when assessing actual human plasma samples.”

The authors further clarify that the ECL assay classified most cytokine levels at less than half of what was found with the xMAP assay, suggesting that this contributed to the lower detection rates for the ECL assay.

In addition, the xMAP bead-based assay reduced hands-on time by more than a third compared to the ECL assay. It also offers greater multiplexing capacity. The use of beads makes it possible to query up to 500 analytes per well, while ECL assays are typically limited to just 10 analytes per well. Three different ECL 10-plex assay kits were used in this study to evaluate the same 16 cytokines, resulting in increased hands-on time.

“The potential cost, labor, and time savings associated with a larger multiplex set might be advantageous for certain applications, particularly with rare, expensive, or limited volume samples,” the scientists conclude.

To read the full study comparing these multiplex platforms, download our free white paper, or view the original publication in Frontiers in Immunology.

xMAP-based immunoassay kits offer significant benefits to researchers who need to get more data from less sample, as noted by the results of this study. For a catalog of over 1,300 available off-the-shelf kits, take a look at the xMAP Kit Finder. Over 1,300 kits are available from Luminex Partners. The Kit Finder allows you to filter by analyte, species, and vendor. Or, you are interested in exploring our custom assay development services, connect with our LuminexPLORE Lab—and see how our experts can help your lab reach its goals.

Download our free white paper here.

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How xMAP® Technology Advances Veterinary and Agricultural Research

New white paper reviews a host of recent studies based on xMAP® Technology

When Luminex scientists first created xMAP® Technology back in the 1990s, they couldn’t possibly have imagined the vast array of applications for which it would eventually be utilized. We are continually impressed with our users’ creativity and scientific vision as they bring multiplexing technology to numerous new research areas.

In this recent white paper, Multiplex Methods in Veterinary Medicine and Agriculture, our team explores several ways xMAP-based assays are used in veterinary medicine and agricultural science. There are a wide range of applications in this research area alone—including livestock and crop production, food and water safety, translational research, companion animal medicine, zoonotic disease, wildlife and ecosystem health, and more.

Adaptable, efficient multiplex assays

While this is a broad landscape representing enormous diversity, there is a common thread across these applications: they are all characterized by a need for highly accurate measurement of biochemical processes. xMAP Technology powers multiplex assays that can detect up to 500 analytes in a single, low-volume sample, making them more time and cost-efficient than traditional immunoassay methods. Specifically, in veterinary research, the ability to use small input volumes makes it possible to study birds and other small animal species.

The flexibility of xMAP Technology is one of the reasons it’s well-suited to so many different research uses. Along with our partners, we offer an extensive menu of commercially available assays, as well as the option to design custom assays for novel research applications.

Below are just a few examples of how xMAP is being used across veterinary and agricultural research:

  • Studying host immune proteins and infectious agents in veterinary research
  • Detecting foodborne pathogens, hormones, antibiotics, and other drugs in food safety testing
  • Measuring mycotoxins in feed
  • Differentiating viruses in plants
  • Evaluating water quality and other environmental substrates

Multiple studies with reliable results

The studies described in the white paper show how widely adaptable xMAP-based assays are, supporting research across multiple fields.

These studies also reveal how xMAP-based assays can bridge the gap between veterinary medicine and human health. Using xMAP Technology, investigators are determining appropriate animal models for human disease.

xMAP Technology is open-architecture—reducing time, labor, and costs compared to traditional methods—delivering reliable answers to complex questions. It offers the versatility to perform a wide range of multiplexing assays, from proteins to nucleic acids, on a single platform.

Download our free white paper to read more about how scientists are using xMAP-based assays to enhance their research in veterinary medicine and agricultural science.

Free White Paper: New Veterinary and Agricultural Applications with xMAP Technology

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The Year in Review—How COVID-19 Transformed Our World

Reflecting on a challenging year in global health

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As 2021 inches closer, many of us have begun our annual reflection, reviewing the events of the past year to gain perspective and shape the months ahead. Ordinarily, one might identify several themes that characterized the year—but in 2020, everything was filtered through the lens of the COVID-19 pandemic.

With tens of millions of cases and more than one million global deaths, the dramatic loss of life and complete societal and economic disruptions have created lasting changes in all of our lives. The pandemic changed how we work, interact with each other and our communities, teach our children, and measure our days.

Dedicating all available resources to address challenges

The pandemic also brought a beautiful side of humanity into focus: how we came together to fight for better health outcomes. At Luminex, this was a very personal lesson for us. Each day, we develop technologies and assays to improve clinical laboratory testing capabilities, and COVID-19 had a major impact on how we work together to quickly address time-sensitive challenges. In 2020, every member of the Luminex family worked diligently to develop, validate, manufacture, and support a host of new assays to facilitate SARS-CoV-2 detection and COVID-19 research.

Although developing these new solutions was an intense process, we knew our team was experienced, knowledgeable, and up to the challenge. We immediately recognized that building one assay wouldn’t overcome the enormous testing obstacles faced by labs around the world, so our goal was to develop a COVID-19 assay for every clinical testing platform in our portfolio. We wanted all Luminex customers to get the support they needed.

Creating a united front against COVID-19

By early March, our internal efforts were well underway. Later that same month, we received our first Emergency Use Authorization (EUA) from the US Food and Drug Administration (FDA) for a SARS-CoV-2 assay as part of our ® SARS-CoV-2 Assay” href=”https://investor.luminexcorp.com/news-releases/news-release-details/luminex-receives-fda-emergency-use-authorization-ariesr-sars-cov”>ARIES® SARS-CoV-2 Assay, and then, we received authorization for our ® Technology Users Battle COVID-19 with New Serology Assays” href=”/blog/xmap-technology-users-battle-covid-19-with-new-serology-assays/”>xMAP® community mobilized quickly against the pandemic, deploying research partners and colleagues in other organizations as they engaged in COVID-19 research. We were also honored to support webinars featuring scientists from the Icahn School of Medicine at Mount Sinai and Rush University Medical Center to help others learn from their developments.

We won’t be sorry to leave 2020 in the past—and there are still challenges ahead as we move into the new year while continuing to fight the pandemic. However, even in the most difficult moments of this past year, we were struck by the dedication and tenacity of both our global workforce, as well as the clinical laboratory community at large, and we are honored to be part of the network of assay and technology developers supporting their unprecedented efforts.

We remain dedicated to supporting our customers and partners fighting the pandemic, which is why we have developed multiple flexible solutions for automating and facilitating SARS-CoV-2 testing and COVID-19 research needs. Click here to learn more about these solutions and stay informed on the most up-to-date COVID-19 testing information.

Learn more about Luminex's SARS-CoV-2 testing solutions.

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Training Tomorrow’s Custom Assay Development Experts: A Summer Internship Program Hosted by the LuminexPLORE Lab

UT Austin students share their experience working in our xMAP® Custom Assay Development lab this past summer

Internships in our industry aren’t particularly common, and they’re even harder to find in places like Austin, which isn’t really considered a biotech hub. However, it’s important to offer these types of programs so that students interested in a job in the life sciences field have an opportunity to explore career options that aren’t limited to academia. This is why we were so proud to offer an internship program in the LuminexPLORE Custom Assay Development Lab this past summer.

Atharwa Mankame and Chad Papenfuhs, two students at the University of Texas at Austin, spent several months working in the Custom Assay Development Lab where Development and Applications Scientist Jackie Surls, PhD, mentored them during the program. As they prepared to wrap up their internships, we asked for their feedback about their experience. They told us that they both enjoyed the program and were grateful to have had the opportunity to work on real projects while they were here.

“What’s unique about our internship opportunity is that we get to take on these projects,” Papenfuhs said, noting that he was able to work directly with customers. In one project, he helped to determine whether Luminex assays could be used to detect guide RNAs for a company running CRISPR gene editing workflows. “It’s been fun to have real responsibility in the biotech world,” he adds.

For his role, Mankame relied on his operations management focus at school to analyze process flows, identify bottlenecks, and make recommendations about how to streamline resource allocation. For example, he suggested that exploring options to help standardize the time it takes to review project contracts could allow the team to better predict when staff resources would be needed for new custom assay programs. He also mentioned that he appreciated the opportunity to present this and other projects he worked on while here to the Luminex team.

Both interns credit Jackie’s management style with fostering an interesting and challenging environment. “Jackie’s been great. She told me to take the initiative and go for it when I suggested projects,” Mankame says. “That allowed me to explore areas I wasn’t familiar with.”

“What’s unique about our internship opportunity is that we get to take on [customer] projects. It’s been fun to have real responsibility in the biotech world.”

— Chad Papenfuhs, UT Austin Student & LuminexPLORE Lab Intern

Looking to the future

When asked about their plans for their own careers, Mankame told us he intends to go to medical school and hopes to be able to consult on technology development once he’s a physician. Papenfuhs, who plans to complete a PhD in microbiology and then work in the biotech industry, is hopeful that the connections he made here will come in handy down the road. We’re not looking forward to saying goodbye to our interns, but we are so proud of everything they’ve accomplished during their time at Luminex.

As Mankame and Papenfuhs head back to full-time classes, they offered some advice to future interns: summer break is a great time for a training program, because there’s less competition between school coursework and the internship. This approach also lets interns spend bigger blocks of time in the lab, which Mankame notes is important to be able to see an assay all the way through.

We’re thankful to our interns for their valuable contributions during their time with us, and to our team members for helping make this important program such a success!

Although next year’s program is still a ways away, if you’re interested in learning more about the xMAP Technology our interns employed during their time here, we have a variety of in-depth resources to learn more about custom assay development. Download the xMAP® Cookbook, a collection of methods and protocols for developing multiplex assays, or click here to learn more about LuminexPLORE Lab, a custom services program designed to accelerate your research.

Interested in developing your own assays? Get started with the xMAP Cookbook

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Multiplex Bead Mixes Made Easy with an Excel-Based Bead Calculator

Whether you’re developing a multiplex assay for 500 different markers in one reaction or developing lower-plex assays, knowing how to make a multiplex bead mix from different types of bead stocks is critical for proper assay performance.

Unconjugated beads can be purchased as individual stocks at different concentrations of 12.5 or 2.5 million beads per mL. After coupling with different capture molecules for proteomic or genomic applications, we recommend resuspending coupled beads to a concentration of about 20 to 50 million beads/mL (<20,000 to 50,000 beads/µL), or less for proteomic vs. genomic applications respectively; see the xMAP® Cookbook for coupling protocols.

Choosing Your Beads

Whether coupling your own beads, or using MagPlex®-Avidin or MagPlex®-TAG beads, we recommend that the multiplex mix contains about 2,500 beads of each region per reaction in a user-defined volume. The Cookbook recommends that each bead region used for making a multiplex mix be at 50 or 75 beads/µL (50,000 or 75,000/mL) for different applications. As the plex of an assay increases and the volume of bead mix needed per reaction varies based on assay design, using the standard 50 or 75 beads/µL leads to errors with making a multiplex mix.

Determining Bead Stock Concentrations

When making multiplex assays, it is more efficient to have all the stocks at the same concentration and at concentrations other than the 50 or 75 beads/µL recommended. For example, in a 40-plex assay, if all the beads were at 1 million beads/mL, the same volume of each bead stock can be used to make a multiplex mix, rather than having to change the pipette volume 40 times to dispense the right amount of beads into the mix for each bead region.

No matter how capture molecules are coupled or bound to Luminex beads, the first step after attaching capture molecules is to confirm bead recovery and determine the bead concentration. The next step is to dilute all the bead preparations to the same bead concentration. While each bead preparation has a different concentration, getting them all to the same concentration can be easily calculated with the appropriate bead stock dilution calculator template.

Download the Coupled Bead Stock Dilution Calculator Template

Using the Multiplex Bead Mix Calculator

Once all of the bead stocks are adjusted to the same concentration, the bead mix calculator can be used to determine how to make a multiplex bead mix. The bead mix calculator eliminates potential calculation errors that can result from the unique needs of each experiment, including a varying number of bead regions per reaction (plex), final bead concentration of all the coupled bead stocks, and the specific volume of bead mix needed for each reaction. When using these variable parameters above certain plexes, there are different points where a concentration step will be needed to correctly make a multiplex mix. The bead mix calculator can indicate if concentrating is needed or not, and can let you know if you have enough beads to make a multiplex mix.

Download the Multiplex Bead Mix Calculator

Instructions on how to use these workbooks can be found in this video:

Tools for Calculating Bead Concentrations and Making Multiplex Bead Mixes

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Precision Coupling with Molar Amounts of Protein [Blog]
Getting Started with xMAP® Technology [Video]
Browse 1,200+ Partner Kits with xMAP® Kit Finder [Online Tool]
xMAP® Cookbook to Design Your Own Assays [Download]
View the xMAP Multiplexing Blog [Blog]

Multiplexing Provides Richer Information for Veterinary Testing

Cornell scientists use Luminex® technology to skip long ELISAs and advance diagnostic test interpretation

At Cornell University’s Animal Health Diagnostic Center (AHDC), scientists provide high-quality testing for a variety of animals while developing valuable new veterinary tests.

Dr. Bettina Wagner, a veterinarian and immunology expert serving as the AHDC’s Director of Serology, has built several important assays using xMAP® Technology. When she first joined the ADHC in 2006, she acquired a new Luminex® 100™ System, which proved so effective that she upgraded to the Luminex 200™. She now has four of these units—three in the AHDC and another in her research lab, where she performs much of her initial work on developing new assays.

Early in Dr. Wagner’s tenure at the AHDC, she established an xMAP-based test to replace western blot and ELISA tests for Borrelia burgdorferi, the pathogen responsible for most cases of Lyme disease.

“When we introduced the test in 2011, it was the first multiplex serological assay for veterinary species,” Wagner recalls. The bead-based test could be used across multiple species and, unlike its predecessor assays, offered more than just a ‘yes or no’ answer.

“This test gives more information than the previous testing for Lyme,” she adds. “It can quantify antibodies to three specific antigenic proteins of the pathogen with broad linear range. Differences in antibody pattern against the three proteins can also tell us whether an infection is early or chronic, and it can evaluate treatment success as antibodies decline,” she says. The test also includes a marker to examine vaccine success and antibody titers.

Taking advantage of the many applications of xMAP Technology

Around the same time she created the test for B. burgdorferi, Dr. Wagner began a USDA-funded project to develop monoclonal antibodies for equine immunological molecules. She designed a multiplex cytokine assay—the first available for horses—using xMAP Technology. “We have run this assay for many years for the equine research community,” she adds.

Following the success of these initial projects, Dr. Wagner and her team continue to design additional multiplex tests. Most recently, Wagner and her team developed a test to detect antibodies associated with COVID-19 in felines, which they tested on cat samples collected from veterinarians in New York City.

“Luminex technology is great—we use it every day,” Dr. Wagner says. “Multiplexing is very important for us. We can offer advanced interpretation by using multiple targets, so it is an experiment life-saver for many samples where we simply don’t have enough volume to run several separate assays.”

Find detailed instructions on developing your own assays.

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Luminex Licensed Technologies Partner Spotlight: EpiCypher

EpiCypher delivers high-quality reagents for epigenetics and chromatin research

As one of Luminex’s Licensed Technologies Partners, EpiCypher is one of many companies that uses xMAP® Technology to deliver important solutions for improving research and molecular diagnostics.

Based out of the Research Triangle Park in North Carolina, EpiCypher is a biotechnology company that helps scientists push the boundaries of research in chromatin regulation, chromatin mapping, and epigenetics. They do this through the development and production of modified recombinant nucleosomes, and have leveraged this technology for a wide range of quantitative chromatin assays.

To find out more about how xMAP Technology supports EpiCypher’s developments, we spoke with Leslie Shannon, Marketing Communications Manager, Keli Rodriguez, Lab Manager, and Carolina Lin, Research Associate.

What does EpiCypher do?

Founded in 2012, EpiCypher aims to address the growing need for high-quality epigenetic reagents. They pioneered the production of recombinant designer nucleosomes (dNucs™), which contain defined post-translational modifications (PTMs) and recapitulate the natural nucleosome structure observed in vivo, making them ideal substrates for a variety of chromatin assays.

One application of dNucs is SNAP-ChIP™ Spike-in technology, which uses panels of DNA-barcoded dNucs as spike-in controls to examine antibody specificity (on- and off-target binding) and efficiency (percent pulldown relative to input) during a ChIP-seq workflow. Spike-ins can also be used for data normalization, highlighting the utility of these reagents for chromatin profiling.

In addition to developing and commercializing reagents, EpiCypher scientists also contribute research. “Our goal is to not only bring our customers cutting-edge epigenomic technologies, but also serve as a research partner,” says Leslie. “We have a unique business model that allows us to work with other scientists, either through collaborations, services, or projects, to answer challenging research questions.”

Why are nucleosome controls important for epigenetic research?

“Nucleosome controls offer a physiologically relevant measurement of success to epigenetic research,” Leslie says. For instance, histone PTM antibodies for ChIP assays are commonly tested using modified histone peptide arrays, which lack the nucleosome context. “Compared to our SNAP-ChIP Spike-in Controls, the histone peptide arrays didn’t accurately predict antibody specificity on chromatin in ChIP experiments. We also found cross-reactivity to other PTMs in many commercial ‘ChIP-grade’ antibodies. Overall, we concluded that nucleosome context is essential for identifying antibodies with reliable on-target performance in ChIP-seq.”

This revelation led to an initiative by the EpiCypher science team to leverage SNAP-ChIP controls to screen for reliable histone PTM antibodies for ChIP-seq experiments. “The SNAP-ChIP Spike-in Controls identify next-generation antibodies that are truly ‘ChIP-grade,’” Leslie mentions.

How is EpiCypher using Luminex technology?

The EpiCypher team needed an efficient technique to rapidly screen and identify candidate antibodies for final testing against SNAP-ChIP Spike-ins in a ChIP experiment. “It was costly and time-consuming to test all antibodies in standard ChIP workflows against SNAP-ChIP Spike-ins. Luminex offered a cheaper and faster way of performing these tests,” Leslie says.

To effectively analyze these large sets of antibodies, they first conjugate nucleosome panels to Luminex xMAP beads so the beads denote PTM identities. Then, they add an antibody of interest. “We run the reaction on the Luminex FLEXMAP 3D® to detect what nucleosome or nucleosomes bind to the antibody,” Keli explains. If 90% of antibody-bound nucleosomes are attached to the expected PTM of interest, then it is approved for further testing in ChIP with SNAP-ChIP Spike-ins.

After screening hundreds of antibodies, EpiCypher identified a collection of the highest-performing ChIP antibodies—this data will be published by the end of this year, which will allow researchers to be able to more easily identify the best antibodies for future ChIP experiments.

Why does multiplexing matter for EpiCypher’s work?

Multiplexing was a key feature EpiCypher scientists were looking for as they evaluated options for screening antibodies. “Some of the other assays we ran prior to utilizing xMAP took a lot longer,” Keli explains. “The Luminex workflow gives a lot of information in a very short period of time.”

By multiplexing, EpiCypher can test an antibody against an entire panel of modified nucleosomes in one reaction. “It’s a great feature to be able to work with 500 different analytes in a single well, using extremely low volumes for each reaction,” Carolina says.

Another advantage: the system’s robustness. The team performs all the washes typically used in ChIP assays, thus replicating key experimental steps that are necessary to predict an antibody’s performance using SNAP-ChIP Spike-ins. “The beads are able to withstand all these different reagents and stringent wash conditions,” Carolina adds.

As a result, EpiCypher’s Luminex-based antibody screening method can be adapted for a range of buffer washes, and can be applied for antibody testing across different assays—this flexibility will be helpful when the EpiCypher team expands multiplex testing to their other genomic mapping assays.

“We are developing similar nucleosome controls for CUT&RUN and CUT&Tag experiments, and plan to use Luminex for those as well, so we can identify top-performing antibodies for these novel chromatin profiling assays,” Keli concludes.

Multiplexing offers significant advantages over traditional methods for delivering reliable answers to complex questions. Whether it’s saving time and resources for researchers like our partners at EpiCypher, or accelerating discovery for large-scale screening projects, there’s a multiplex solution that can meet the needs of your workflow.

Are you interested in developing, manufacturing, and commercializing your own multiplex assay on an efficient, flexible, and widely-adopted platform? Contact us here.

To learn more about EpiCypher’s latest technologies, check out their website at: epicypher.com

Are you interested in developing, manufacturing, and commercializing your own multiplex assay on an efficient, flexible, and widely-adopted platform?

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