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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Webinar: A Multiplex Serology Test for Detecting COVID-19 Antibodies in Dried Blood Spots

Rush University Medical Center develops a highly accurate test that can be used with dried blood spot samples.

In a recent LabRoots webinar, Dr. Imad Tarhoni from Rush University Medical Center in Chicago offered a look at how his team developed a multiplex serological assay that they believe could be the cornerstone to a large-scale national COVID-19 testing strategy. They built the assay using xMAP® Technology to take advantage of its strong performance and multiplexing capabilities.

In his presentation, 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 of time after infection. That’s why Tarhoni believes that serological assays, which can detect antibodies to the SARS-CoV-2 virus after infection, will be so important for population-scale testing.

This kind of testing has been limited thus far because until recently, existing COVID-19 serology tests weren’t sensitive or specific enough to avoid false negative or false positive results.

To address this problem, Tarhoni and his team used xMAP Technology to develop a new serology assay for COVID-19—one that would take advantage of its multiplexing capabilities by incorporating four viral antigens, ensuring the most accurate results possible. Whether a person has developed antibodies to the virus’s spike, nucleocapsid, membrane, or envelope protein, this test can detect a true positive result.

The scientists conjugated all four antigens onto xMAP beads and optimized the assay for linearity, detection limits, precision, cross-reactivity, and more. Next, they dove into validation studies to ensure that the assay delivered reliable results using dried blood spot samples, which would be the most useful 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 high-throughput approach. Importantly, by reducing the antibody capture incubation step, 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 groups of people. 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.

For more information about how their test was developed, view the entire presentation here.

To learn how their test was developed, view the full webinar.

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Coming Soon: A New Multiplex Serology Test for SARS-CoV-2

Luminex Partner Tetracore designed a serology assay for SARS-CoV-2 using xMAP® Technology.

Coming Soon: A New Multiplex Serology Test for SARS-CoV-2

A new multiplex serology test to determine whether someone has been exposed to the SARS-CoV-2 virus is coming soon, thanks to assay developer Tetracore. The company—one of Luminex’s research partners—developed this test based on our xMAP Technology. We spoke with Tetracore Senior Scientist Neeraja Venkateswaran to learn more about the test and how it can help fight the COVID-19 pandemic.

Q: Many SARS-CoV-2 serology tests in development focus on one antibody. Why was it important to your team to take a multiplex approach?

A: It’s based on our experience with infectious disease testing. When Zika happened, we saw that an ELISA-based serology test had high cross-reactivity with dengue fever. This is always an issue when looking at only one antigen at a time. At the time, we used the Luminex platform to develop a very complex panel with several different antigens to detect whether a patient was positive for dengue or for Zika.

Q: What did you learn from the multiplex Zika test?

A: When the dust settled from that outbreak, we realized that there was a problem with the single ELISA assay. All of these RNA viruses have many conserved domains. Since dengue and Zika looked similar to the immune system, we saw that patients who had been previously infected with dengue and were exposed with Zika began producing the old dengue antibodies at first, because that’s the body’s response when it recognizes some of those conserved domains.

Q: How did that inform your approach to testing for SARS-CoV-2?

A: The moment that SARS-CoV-2 hit, we thought that people who had been exposed to the original SARS virus and now got the new virus would start making antibodies for SARS first, because their immune system would remember that. We knew that a serology test would have to be able to distinguish between SARS, MERS, and SARS-CoV-2—this is why a multiplex test is so important. We immediately began screening antigens available for SARS, and now we have created a test that successfully discriminates between SARS, MERS, and SARS-CoV-2.

Q: With so many molecular assays for SARS-CoV-2 now available, what’s the role of a serology test?

A: Serology tests tell us who’s been exposed, including people who were asymptomatic or who had symptoms but missed the window for molecular testing. Detecting a robust IgG response could identify people who are safe to go back to work. Serology is also very important for understanding the epidemiology of disease, determining whether we have developed herd immunity, and conducting routine surveillance. Once there are vaccines ready for testing, we will need to see the human immune response to them—serology can be applied for that, too.

Q: Why did you choose the xMAP® platform for your multiplex tests?

A: I have been working with the xMAP platform for years. Once you use xMAP Technology, you don’t ever want to do an ELISA again. We don’t test for a single target when we do nucleic acid testing, and I believe we should look for more than one antigen for any pathogen. Luminex gives me the ability to do that. It also lets me put an internal control into every single reaction, which can’t be done with ELISA testing.

Interested in learning more about Luminex’s SARS-CoV-2 testing solutions? Check out our website.

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The Versatility of Multiplex Antibody Titer Assays for COVID-19 and Beyond

The COVID-19 outbreak that began in Wuhan, China in December 2019 was marked by an increase in the number of cases showing pneumonia-like symptoms.1,2

It was not until about a month after the outbreak began that genomic analysis identified a novel coronavirus as the causative agent. With the emergence of new pathogen strains, genomic assays are not always able to detect new variants, and after an infection, may not be able to identify which pathogens were involved. However, with serological assays that pick up polyclonal antibody responses, the conservation of epitopes—even with new variants—make it possible to identify the different pathogens circulating in an epidemic.

While genomic tests can be modified to distinguish between different pathogen strains once they are identified, the identification of causative agents during and post- epidemics can be better determined with serological assays. This would be especially useful when screening patients for COVID-19, which presents with flu- or pneumonia-like symptoms—similar to infections caused by other respiratory pathogens.3-5 An assay that can determine which pathogens are eliciting immune responses in patient populations would provide significant value for triaging patients into appropriate treatment categories. Post-pandemic, this type of serological assay could not only more precisely identify the causative pathogens that have passed through, or are still passing through a population, it could also monitor the pathogens involved in second waves of infection.

With Luminex’s xMAP® bead-based platforms, multiplex serological assays can be developed for this type of immune surveillance of different pathogens, as well as for vaccine testing and developing immune therapies.6-10 The process of coupling antigens to Luminex beads is simple and can be completed in a few hours. By multiplexing, an assay that could distinguish antibody titers of a number of different pathogens in a single reaction with very small sample volumes would enable clinicians to determine the causative pathogen(s) of a patient’s illness within a few hours.

These types of multiplex immune surveillance assays would be beneficial for other diseases with similar clinical symptoms as well. Examples include hemorrhagic fevers that are caused by viruses from several viral families, as well as chronic fatigue syndromes, which can be caused by a number of different viral, bacterial, and microbial pathogens—to name a few.11,12

With its multiplexing ability to measure antibody titers to multiple antigens in one reaction, Luminex xMAP® Technology provides the ideal platform for building these types of multiplex serological assays. For more information on Luminex assay development, download the xMAP® Cookbook. Luminex also offers custom assay development services through LuminexPLORE Lab.

For genomic assay screening, Luminex has several platforms that can screen for multiple pathogens involved in both respiratory and gastrointestinal infections, in addition to food safety applications. Information about Luminex diagnostic assays can be found on our webpage.

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

References

  1. Dong E, Du H, and Gardner L. An interactive web-based dashboard to track COVID-19 in real time. The Lancet Correspondence. February 2020.
  2. Ding Q, Lu P, Fan Y, et al. The clinical characteristics of pneumonia patients coinfected with 2019 novel coronavirus and influenza virus in Wuhan, China. J Med Virol. 2020 Mar. doi: 10.1002/jmv.25781.
  3. Centers for Disease Control and Prevention. Causes of Pneumonia. March 2020. Accessed March 2020. Available from: https://www.cdc.gov/pneumonia/causes.html.
  4. The World Health Organization. Pneumonia Fact Sheet. August 2019. Accessed March 2020. Available from: https://www.who.int/news-room/fact-sheets/detail/pneumonia.
  5. The Mayo Clinic. Pneumonia. March 2018. Accessed March 2020. Available from: https://www.mayoclinic.org/diseases-conditions/pneumonia/symptoms-causes/syc-20354204.
  6. Khaliq A, Ravindran R, Hussainy S, et al. Field evaluation of a blood based test for active tuberculosis in endemic settings. Plos One. https://doi.org/10.1371/journal.pone.0173359.
  7. Trivedi S, Miao C, Sanchez J, et al. Development and Evaluation of a Multiplexed Immunoassay for Simultaneous Detection of Serum IgG Antibodies to Six Human Coronaviruses. Scientific Reports. February 2019.
  8. Elberse K, de Greef S, Wattimena N, et al. Seroprevalence of IgG antibodies against 13 vaccine Streptococcus pneumoniae serotypes in the Netherlands. Vaccine Volume 29, Issue 5, 29 January 2011, Pages 1029-1035. https://doi.org/10.1016/j.vaccine.2010.11.054.
  9. Opalka D, Lachman C, MacMullen S, et al. Simultaneous Quantitation of Antibodies to Neutralizing Epitopes on Virus-Like Particles for Human Papillomavirus Types 6, 11, 16, and 18 by a Multiplexed Luminex Assay. Clin Diagn Lab Immunol. 2003 Jan; 10(1): 108–115. doi: 10.1128/CDLI.10.1.108-115.2003.
  10. Berglund J, Vink P, Tavares Da Silva F, et al. Safety, immunogenicity, and antibody persistence following an investigational Streptococcus pneumoniae and Haemophilus influenzae triple-protein vaccine in a phase 1 randomized controlled study in healthy adults. lin Vaccine Immunol. 2014 Jan;21(1):56-65. doi: 10.1128/CVI.00430-13.
  11. Centers for Disease Control and Prevention. Viral hemorrhagic fevers (VHFs). January 2014. Accessed March 2020. Available from: https://www.cdc.gov/vhf/index.html.
  12. Centers for Disease Control and Prevention. Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. July 2018. Accessed March 2020. Available from: https://www.cdc.gov/me-cfs/about/possible-causes.html.

Learn more about our xMAP COVID-19 offering here.

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xMAP® Connect Virtual Conference – Inspiring Multiplexing Innovation

xMAP<sup>®</sup> Connect Virtual Conference – Inspiring Multiplexing Innovation” width=”592″ height=”395″ /></div> <p> </p> <p>Luminex is proud to announce the <a title=® Connect Virtual Conference” href=”https://www.labroots.com/ms/virtual-event/xmap-connect?campaign=LRblog” target=”_blank” rel=”noopener noreferrer”>xMAP® Connect Virtual Conference, hosted online this year by LabRoots. The goal of this conference is to connect fellow xMAP enthusiasts and provide best practices for multiplex assays, virtually from anywhere in the world.

Luminex’s

During this virtual program, you will hear from 20+ multiplex experts about how they’ve developed, validated, and optimized their xMAP-based methods. You can also explore the virtual poster and exhibit hall to gain insights from industry experts, or visit Luminex Partner booths to learn more about xMAP-based multiplexing solutions.

The xMAP Connect Virtual Conference is free to attend and will remain open through April 2021. For more information about the event, speakers, poster hall, and exhibit hall, or to register, click

Download the xMAP Connect Virtual Conference here.

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Questions to Ask When Choosing a Multiplex Assay Kit

With the COVID‑19 pandemic in full swing, many researchers are looking to multiplex assays to evaluate what they call the “cytokine storm.”

The “cytokine storm” refers to the increase in pro-inflammatory cytokines like IL-6 in patients with COVID‑19, which is believed to predict disease severity.1 Many of Luminex’s Partners already have multiplex assays available for research use. But because there are many kits available for all types of research, we put together a list of questions to consider when determining which assay would work best for you.

Q: Will my samples fall within the dynamic range of the assay?

A: The dynamic range of the assay should include values that you expect your samples to fall in. If you are analyzing endogenous samples, you may need a range that covers lower concentrations. When looking at induced or disease state models, a range that includes higher concentrations would be more suitable. When evaluating both, an assay with a large dynamic range is best.

Q: What is the sensitivity of the assay?

A: Because endogenous levels of analytes can be very low, it is important that the sensitivity of the assay is high enough that it can detect low levels of analytes in your samples if needed.

Q: What is the accuracy of the kit?

A: When researching kits, evaluate the vendor-provided accuracy data. This should be provided with all kits. A range of 80–120% when performing a spike-and-recovery experiment is the industry standard.

Q: What is the intra- and inter-assay precision?

A: It is important that your results are precise and accurate, whether all samples are contained on one plate or are on multiple plates—this is vital when evaluating different treatment groups. You want any differences seen between groups to be solely due to treatment effects, not a variation in your assay.

Q: What is the lot-to-lot variability of the kit and how is it evaluated?

A: When you are expecting to evaluate samples over a long period of time, it is important to determine the lot-to-lot variability of the assay to ensure reliable, accurate, and consistent results. This is especially important in pre-clinical work, when evaluating several compounds in an animal model over months or years, or when processing a large number of samples whose evaluation will exceed a single lot of kits.

Q: What is the percent of cross-reactivity between analytes?

A: When multiple analytes are present in one assay, cross-reactivity between the capture or detection reagents can occur, leading to inaccurate results. This information should be provided by the vendor, and any kits with high cross-reactivity should be avoided.

Q: How does the kit account for matrix effects?

A: All matrices—including plasma, serum, cell culture media, and tissue homogenates—can affect the performance of the assay. Ensure that the kit either provides a buffer to prevent these effects or provides instruction on how they can be mitigated.

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

References:

  1. Puja M, et al. COVID-19: consider cytokine storm syndromes and immunosuppression. The Lancet, 2020 Mar 13. 395, 10229; 1033-1034. Available from: https://doi.org/10.1016/S0140-6736(20)30628-0.

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How Luminex Is Supporting Research Partners’ Pandemic Response

Licensed Technologies Group is finding new ways to boost partner programs

Here at Luminex, we are working on all fronts to help fight the coronavirus pandemic. The most visible activities have been the launch of new diagnostic tests—namely, the NxTAG® CoV Extended Panel and the ARIES® SARS-CoV-2 Assay—both of which received Emergency Use Authorization from the FDA and are being deployed in the field right now.

We’ve also been engaged in the coronavirus response in other ways. Our Licensed Technologies Group (LTG), which works closely with our many research partners to help develop assays and services based on xMAP® Technology, has pivoted to be more involved in coronavirus efforts. Employees in this area are used to traveling all the time to visit partners. Now, the whole team is working from home and finding new ways to help colleagues in other organizations who are involved in characterizing the COVID-19 immune response, studying disease progression, developing serology tests or vaccine candidates, and more.

“We are all scientists first and, with our research partners, we are throwing everything we have at this—from epitope masking and phage display technology to HLA analysis and cytokine storm profiling,” says Heather Darby, a Field Applications Scientist on the LTG team.

Luminex partner organizations are already making impressive strides in this work. An academic hospital in the Midwest is in the final stages of validating a four-plex serology assay that will allow testing of more than 16,000 patients per week. Other partners are using serology assays as the basis for developing antigen-based vaccines and COVID-HIG therapies.

The LTG team continues to support our partners, by:

  • Increasing the time and resources we allocate to partners who can help the broadest base of COVID-19 patients as quickly as possible, especially for designing serology tests to identify people who have recovered from the virus and may safely return to work
  • Driving initiatives to educate teams across Luminex on serological and bead-based applications relevant to the novel coronavirus
  • Using our extensive network to connect partners with the resources they need, such as positive sera or reagents
  • Reaching out more frequently to partners to check in
  • Shifting Scientific and Sales Support to online and video calls

“This feels like a very personal initiative,” says Joe Balsanek, Regional Business Manager in the LTG unit, who began his career as a certified laboratory scientist before moving to the business side. “These aren’t customers as much as they are friends, family, and colleagues. In addition to supporting their pandemic efforts, we simply want to know that they’re safe and healthy.”

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To kickstart your multiplex assay kit search, use our xMAP Fit Finder

Considerations Regarding RNA Extraction Kits for SARS-CoV-2 Detection

The novel coronavirus disease (COVID-19) pandemic continues to trigger rapid development of molecular diagnostic tests around the world.

Nucleic acid amplification-based tests (NAATs)—such as real-time PCR and isothermal amplification—are the gold standard in molecular diagnostic technologies that are being utilized to detect SARS-CoV-2, the COVID-19 disease-causing virus.1

There are two critical and sequential steps that must occur prior to running a SARS-CoV-2 NAAT. The first is proper sample collection per the CDC’s guidelines,2 because inadequate collection may lead to inaccurate or inconclusive diagnostic results. The second is effective viral RNA extraction. The CDC has qualified and validated several extraction systems for use with their 2019-nCoV real-time RT-PCR Diagnostic Panel,3 including systems from Qiagen, Roche, and Biomerieux, but the rapid increase in testing has led to a global shortage of commercially available extraction kits. Consequently, lab test sites need to explore alternative vendors for extraction kits to qualify and validate with their respective NAAT.

Most commercial RNA extraction kits utilize similar principles for RNA extraction, with guanidine thiocyanate being the most popular chaotropic agent to lyse the virus, denature RNases, and protect RNA from degradation. These kits often include additional protocol steps, some designated as optional to improve purity, yield, or analyte detection. When selecting an RNA extraction kit, remember that SARS-CoV-2 is an RNA virus, and thus requires protection from RNases. Understanding the kit reagents and protocol will help ensure quality RNA extraction and reproducible results.

Luminex’s ARIES® SARS-CoV-2 Assay uses a sample-to-answer PCR platform that performs nucleic acid extraction, target amplification, and detection. It is one of the commercially available NAATs on the market.

Jackie Surls, PhD, is a Development & Applications Scientist at Luminex Corporation.

References

  1. “Coronavirus Test Tracker: Commercially Available COVID-19 Diagnostic Tests.” Genome Web (Internet). Cited Mar 2020. Available from: https://www.360dx.com/coronavirus-test-tracker-launched-covid-19-tests.
  2. “Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons for Coronavirus Disease 2019 (COVID-19).” The Centers for Disease Control and Prevention (Internet). Cited Mar 2020. Available from: https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html.
  3. “CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel.” The Centers for Disease Control and Prevention (Internet). Cited Mar 2020. Available from: https://www.fda.gov/media/134922/download.

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

Webinar: Mouse Model Analysis Is Better with Multiplexing

Query hundreds of immunoassay targets, even with limited sample volume

To make the most of immunology-focused assays using the limited tissue and fluid samples available from mouse models, scientists should consider using xMAP® Technology from Luminex, according to Daniel Braunschweig, Proteomics Field Application Scientist Leader at Bio-Rad Laboratories.

That’s the takeaway from a 30-minute webinar hosted by Labroots, which is now available for on-demand viewing and offers continuing education credits. In the presentation, Braunschweig spoke about getting the most out of bead-based multiplexing, experimental considerations, and key attributes for multiplex assays.

Variability in Mouse Models

Mouse models represent a $2 billion industry, Braunschweig noted, in part because they’re a mainstay for pharmaceutical R&D and are accepted models even for cutting-edge treatments such as immunotherapies and cell or gene therapies. Knock-out strains and assay content are readily available. However, variety among mouse strains, experimental design, and interpretation processes can lead to inherent variability when dealing with this type of data, he added.

Generating Data with Minimal Biological Sample

Perhaps the biggest challenge in working with mouse models, though, is that tissues and fluids are precious because they’re available in such small volumes. Because of this, researchers require cost-effective platforms with high sensitivity and specificity, comprehensive results, and strong reproducibility, Braunschweig said. He noted that xMAP Technology meets all of those criteria, generating data from potentially hundreds of targets per sample, even when there’s minimal sample to use. Automated multiplexing also reduces hands-on time and minimizes variability across experiments.

The webinar also introduces viewers to key experimental considerations, such as how analyte profiles differ by mouse age or tissue type, and assay attributes, including a large dynamic range and the ability to profile entire biological pathways.

If you work with mouse models and need immunoassay data, be sure to carve out a little time for this informative webinar.

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Webinar: Making the Most of Your Mouse Model with Bead-based Multiplexing