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