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.

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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

Multiplexing Provides Richer Information for Veterinary Testing

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

Luminex Licensed Technologies Partner Spotlight: EpiCypher

As one of Luminex’s Licensed Technologies Partners, EpiCypher is one of many companies that uses 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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For COVID-19 Detection and Epidemiology, xMAP® Technology Offers Unique Advantages

Multiplex serology tests have yielded impressive results, showing promise for use in the COVID-19 pandemic.

For COVID-19 Detection and Epidemiology, xMAP<sup>®</sup> Technology Offers Unique Advantages” width=”592″ height=”395″ /></div> <p>xMAP-based serology tests have been widely implemented for the detection of pathogenic bacteria, viruses, and parasites. In the COVID-19 era, xMAP Technology has helped build several new multiplex serology tests designed to detect antibodies to different SARS-CoV-2 antigens.</p> <p>To demonstrate how these tests may make a difference, we’ve summarized how some xMAP-based serology tests have been used for clinical lab applications, as well as for epidemiology and surveillance.</p> <h3><em>Providing multiplex results you can rely on </em></h3> <p>One of the earliest applications of a multiplexed xMAP serological assay involved the screening of HIV antibodies in dried blood spot specimens for early detection of HIV exposure in neonates. Developed by <a title=Bellisario et al., this assay was able to simultaneously measure antibodies to three different purified recombinant HIV-1 antigens (p24, gp120, and gp160). The extra specificity of this assay meant it could distinguish 92 previously tested newborn specimens as either HIV-negative or HIV-positive, and was also able to clearly differentiate anti-HIV-1 blood spot controls (zero, low, and high) from the CDC.

In another application, Wu et al. used the xMAP platform to develop a sensitive, rapid, high-throughput multiplexed serological assay for identifying specific IgG antibodies to nine hemorrhagic fever viruses. Performance data with clinical serum samples demonstrated sensitivities of 90.7–98.04% and specificities of >90% for all but two of the viruses.

Waterboer et al. developed a multiplexed serological immunoassay that could simultaneously detect antibodies against 100 in situ affinity–purified recombinant human papillomavirus (HPV) proteins. Their assay could measure antibodies at serum dilutions of >1:1,000,000 and demonstrated high reproducibility and excellent concordance with single-plex ELISAs.

Respond to outbreaks with trusted technology

xMAP bead-based serological assays have also been implemented in outbreak situations. Ayouba et al. developed a multiplexed serological assay for the Zaire Ebola virus using nine recombinant proteins. The performance of the assay was evaluated with 94 positive and 108 negative samples, and revealed high sensitivity and specificity. Additionally, this high-throughput xMAP assay was extremely cost-effective when compared to the single-plex ELISA ($4 vs. $54 for each sample), and could therefore be easily and economically implemented to screen large sample volumes in an outbreak situation.

In addition to pathogen identification, detection of serological markers can provide additional information to estimate recent and past exposure to the pathogen. xMAP-based multiplex serological assays have been well-established in epidemiology for surveillance purposes.

Customized immunoassays for serosurveillance

To monitor vaccine-preventable diseases, Caboré et al. developed a pentaplex immunoassay for the simultaneous detection of IgG antibodies against diphtheria, tetanus, pertussis toxins, and other pertussis antigens. Their assay demonstrated good correlation with ELISA, a low intra- and inter-assay variability score, and was successfully implemented for performing large serosurveillance/seroprevalence studies.

O’Hearn et al. developed a similar multiplexed assay to detect IgG antibodies against Lassa, Ebola, Marburg, Rift Valley fever, and Crimean-Congo hemorrhagic fever viruses, as well as pan-assays for flaviviruses and alphaviruses. Their study highlights the importance of surveillance, as the results revealed that in addition to Lassa virus (which is endemic in West Africa), other strains of viruses are also responsible for hemorrhagic fever, but often go undetected, and ultimately, untreated.

Applying xMAP Technology to the COVID-19 pandemic

To aid in the detection of COVID-19, Luminex recently received Emergency Use Authorization for an xMAP-based antibody assay to help identify individuals who were exposed and developed an immune response to SARS-CoV-2. The luminexcorp.com/?wpdmdl=42265).

Free White Paper: xMAP Technology: A Benchmark for Serological Testing

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How to Reduce Coupling Variability with Luminex Bead Assays

Top 10 tips for a successful coupling process

How to Reduce Coupling Variability with Luminex Bead Assays

If you’re designing a new multiplex bead-based assay or adding new targets to an existing xMAP Cookbook recipes can make all the difference.

If you’ve never performed coupling before, or if you’ve done it in the past and noticed problems, there are a few common issues that can be easily worked through.

Variability is the number one issue to avoid. Let’s say you’re performing a coupling confirmation assay to verify coupling efficiency. If there’s a really low signal, this could indicate an issue with the coupling process. As a general guideline, we recommend 5 micrograms per million beads, but always encourage users to do a titration to check the optimal concentration to put on the bead.

Low signal can also be a sign that there is a reagent quality issue. If you’re using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) that has degraded, you may experience poor coupling efficiency. This is a common problem. Take care of those EDC vials! Be sure to store EDC in the freezer with a desiccant.

Over-vortexing can also cause problems. We recommend that xMAP users vortex and sonicate to make sure the beads are in homogenous suspension, but over-vortexing can lead to clumping, which will affect coupling.

To help you streamline the bead coupling process, here are our top 10 tips for reducing variability:

  1. Microspheres are light-sensitive. There’s no need to couple in the dark, but cover tubes in foil during incubation steps. If exposed to light for too long, the beads will photobleach.
  2. When resuspending the uncoupled beads in the coupling tube, make sure you have vortexed and sonicated the beads. Bead loss is often due to not mixing the beads well.
  3. There is such a thing as too much vortexing and sonicating. Stick to the recommended recipe to avoid bead clumping.
  4. Make sure you are using the tubes specified in the xMAP Cookbook, as tube type can have an impact on coupling efficiency. We recommend:
    1. USA Scientific® (Catalog # 1415-2500), or
    2. Eppendorf™ Protein LoBind (Catalog # 022431081)
  5. Make sure you have an appropriate magnet for the tube type containing your beads. The right kind will pull the beads to one side, not split them between both sides, or leave beads floating in the tube.
  6. Magnets can lose magnetism if not taken care of properly (e.g., do not store magnets on top of each other).
  7. Embrace single-use 10 mg EDC vials. Bulk EDC is hygroscopic, so it readily absorbs moisture from the air. Over time, the EDC degrades, which greatly diminishes coupling efficiency.
  8. When performing wash steps, make sure you place the beads on the magnet for at least 30 to 60 seconds for typical couplings. For large-scale coupling, visually check to ensure beads have been pulled. If they haven’t, wait an additional 30 to 60 seconds.
  9. When washing and aspirating beads, be careful not to disturb the beads to avoid having them stick to the transfer pipette.
  10. If using automation, check bead recovery before and after coupling. Ensure automation is optimally set up to avoid significant bead loss.

Please feel free to reach out with any questions. We’re confident that you can master the science of bead coupling!

Learn how LuminexPLORE can help your lab!

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Webinar: Mount Sinai Scientists Design Multiplex COVID-19 Antibody Test

Implementing xMAP® Technology allows for faster, more accurate results

Webinar: Mount Sinai Scientists Design Multiplex COVID-19 Antibody Test

When the global outbreak hit the US, the Icahn School of Medicine at Mount Sinai in New York City published some of the earliest COVID-19 studies in the US. It’s no surprise then that scientists at Mount Sinai were among the first to develop an antibody test. Using webinar presented by Dr. Susan Zolla-Pazner, a Professor of Medicine at Mount Sinai. Developed by Dr. Zolla-Pazner’s team, the assay 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. In the webinar, they presented the results from their validation study, which demonstrated that their test could provide robust signals for both antigens from plasma and serum using positive and negative COVID-19 samples.

Compared to ELISAs, this xMAP-based antibody test had significantly higher sensitivity—in some cases five to ten times higher. ELISA-based testing also led to more false negative results than the multiplex assay, according to Dr. Zolla-Pazner.

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.
  • The xMAP assay delivers results for both spike and RBD antibodies in less than two hours, while ELISA plates can only detect one antibody at a time, and the complete workflow from prep to results for ELISAs takes two days.

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. 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. These results could inform donor selection for convalescent plasma therapy.

Watch the entire webinar here, or view Luminex’s COVID-19 testing resources here.

Watch the entire presentation with Dr. Zolla-Pazner.

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xMAP® Technology Users Battle COVID-19 with New Serology Assays

Designed and built by researchers around the world, xMAP® assays shine light on the antibody response to SARS-CoV-2.

xMAP<sup>®</sup> Technology Users Battle COVID-19 with New Serology Assays” width=”592″ height=”395″ /></div> <p>Our <a title=® Technology” href=”https://www.luminexcorp.com/xmap-technology/”>xMAP® Technology empowers researchers to build their own assays quickly, creatively, and effectively. Designed and built by researchers around the world, xMAP® assays shine light on the antibody response to SARS-CoV-2. If you haven’t seen the studies, they are well worth your time. Here, we provide a quick overview of some of the latest studies using xMAP to address the pandemic.

Identifying SARS-CoV-2 patients who have seroconverted.

Weiss et al. developed an xMAP serological assay and used it to assess the presence of antibodies for two SARS-CoV-2 proteins in sera from COVID-19 infected and uninfected individuals. This assay is critical because it can identify patients who have been infected with SARS-CoV-2 and have seroconverted, which will help characterize the immune response to the disease.

Similarly, Randad et al. developed an xMAP-based ten-plex SARS-CoV-2 immunoassay for detecting salivary antibodies that demonstrated significant correlation with serum sample results. The assay had 100% sensitivity and specificity for detecting prior SARS-CoV-2 infection.

Dobaño et al. developed quantitative, xMAP-based multiplex assays for detecting IgG, IgM, and IgA against a panel of eight SARS-CoV-2 antigens. For the best-performing combination of Ig-isotypes and antigens, the assay demonstrated 100% specificity for SARS-CoV-2. Sensitivity was 94.94% for positive samples collected at ≥14 days following the onset of symptoms, and 96.08% for those collected at ≥21 days.

Additionally, the Wadsworth Center of the New York Department of Health developed an xMAP-based serological assay for the detection of total antibody (IgG, IgM, and IgA) to SARS-CoV-2 in human serum, which received Emergency Use Authorization from the Food and Drug Administration. The assay demonstrated a sensitivity of 88% and a specificity of 98.8%, and was implemented to detect reactive and non-reactive SARS-CoV-2 antibodies.

Flexible solutions to complex problems

If a ready-to-use solution works better for your workflow, Luminex was recently granted Emergency Use Authorization for our

Free White Paper: xMAP Technology: A Benchmark for Serological Testing

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Luminex Supports New Award for Scientific Contributions to Cytokine and Interferon Biology

Luminex Supports New Award for Scientific Contributions to Cytokine and Interferon Biology

At Luminex, we believe in the value of recognizing excellence, which is why we are excited to support a new award through the International Cytokine & Interferon Society. The ICIS-LUMINEX John R. Kettman Award for Excellence in Interferon & Cytokine Research will be given to a mid-career investigator who has made exceptional contributions to the field of interferon or cytokine biology.

The awardee will receive a cash prize of $5,000 to cover ICIS meeting registration, and travel support if needed, to present their research in an award seminar. The submission deadline is September 15, 2020, so if you know someone who would be a good fit for this award, please submit a nomination. Entrants will be considered based on their publications, discoveries, and collective contributions to interferon or cytokine biology.

The award is named after John Kettman—better known as Jack—one of Luminex’s co-founders. He is an accomplished immunologist who served as a faculty member at the University of Texas Southwestern Medical School for nearly 30 years, and continues his work there through a professor emeritus appointment. Dr. Kettman has published more than 100 papers in peer-reviewed scientific journals.

Through this award, we are pleased to honor the remarkable immunology career of one of our founders, by supporting the accomplishments of up-and-coming scientists in the field. Good luck to all of the candidates!

Learn more about past winners here!

Past Award Winners >>

Precision Coupling with Molar Amounts of Protein

Precision coupling is useful for multiplex antibody assays, screening recombinant antibodies, and more!

I’ve been getting a lot of requests lately to provide more information on the molar coupling method. If you’ve been wondering how it works, here’s a quick look at how to incorporate molar coupling into your assay development.

When coupling whole immunoglobulins or antibody fragments for sandwich capture assays, coupling μg amounts of protein per million beads is the best way to optimize the amount of antibody needed to develop the most sensitive assay. However, to develop a multiplex assay for antibody titer or antigen specificity determination, it is best to couple antigens with the same moles per million beads. This is especially true when unrelated antigens of different molecular weights show some degree of cross-reactivity. By coupling with moles of protein per million beads, this promotes coupling the same number of molecules to bead surfaces, regardless of the protein’s molecular weight. For example, when coupling 1 μg per million beads of a 20 kDa protein and 1 μg of a 40kDa protein, the smaller protein will have twice as many molecules on the beads’ surface. This makes the signal a function of the number of molecules on the bead surface rather than reflecting antibody specificity.

To measure the effect molar coupling can have on determining antibody specificity, six antigens ranging in molecular weight from 24 kDa to 36.25 kDa were coupled at three different μg per million beads or pmol per million beads coating levels. Differences in signal were determined with 50X diluted sera specific for antigen D (Figure 1).

Figure 1. Comparison of Signal for Antigen D-Specific Sera with μg vs. Molar-Coupled Beads.

Precision Coupling with Molar Amounts of Protein

Beads coupled with 5 μg, 2.5 μg, and 1.25 μg/million beads showed good signal for antigen D as expected, but also high titers for several of the other unrelated antigens. By coupling at 20 pmol, 10 pmol, and 1 pmol, the specific titer for antigen D was significantly higher than the non-specific signal, which was decreased in comparison.

This coupling approach is also useful for screening recombinant antibodies for specificity, as well as for some protein-protein interaction assays. Excel-based workbooks for calculating how to prepare your protein stocks for standard coupling to beads or binding to MagPlex®-Avidin beads are available from your Field Application Scientist (FAS). Reach out to your FAS about how to use these tools, or for more information about molar coupling using xMAP® Technology.

Stephen Angeloni, PhD, is a Senior Field Application Scientist at Luminex.

Learn more about xMAP Technology here.

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