Application Note: Multiplexed SARS-CoV-2 Serological Assay Using Advanced Flow Cytometry

The coronavirus disease 2019 (COVID-19) pandemic has had a profound impact on human life. The causative agent of COVID-19 is the betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which continues to be a global public health threat.

Upon infection, SARS-CoV-2 initiates entry into target cells through the interaction of the receptor binding domain (RBD) on the S1 subunit of its envelope Spike glycoprotein with the angiotensin-converting enzyme 2 (ACE2) on human cells. This interaction then induces a conformational change in the S2 subunit of the Spike glycoprotein that results in virus-host cell membrane fusion and viral entry.

Antibodies directed against the RBD of the SARS-CoV-2 Spike protein can effectively neutralize the virus by blocking virus interaction and entry into host cells. Production of these neutralizing anti-Spike RBD antibodies is also an important factor in the protective immunity induced by current COVID-19 vaccines. Several isotypes of neutralizing anti-Spike RBD antibodies (IgG, IgM and IgA), have been identified in both COVID19 patients and in vaccine recipients.


In this study, use of the iQue® SARS-CoV-2 Kit allowed for rapid, simultaneous detection of all three major isotypes (IgG, IgM and IgA) of antibodies specific for SARS-CoV-2 Spike RBD, and further revealed the heterogeneity of antibody responses induced in both patients and vaccine recipients. In addition, measurement of multiple anti-Spike RBD antibody isotypes was shown to be required for accurate detection of seroconversion. This highly sensitive and specific multiplexed assay provided reliable quantification of all three anti-Spike RBD antibody isotypes in a single 10 μL sample of serum or plasma diluted at least 100-fold, with antibody detection levels in the pg/mL range.

Overall, the combined detection of all three antibody isotypes (IgG, IgM and IgA) in a single well of a 96- or 384-well plate not only saves time and precious patient samples, but also provides a dynamic, comprehensive profile of the humoral response over time following natural infection with SARS CoV-2 or immunization with COVID-19 vaccines. This assay thus provides a powerful tool in the continuing fight against SARS-CoV-2.

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Key Takeaways

This application note highlights the following topics:

  1. The iQue® SARS-CoV-2 (IgG, IgM and IgA) Kit is a Highly Sensitive and Specific Serological Assay
  2. Analysis of anti-SARS-CoV-2 Spike RBD Antibody Profiles Produced During Infection with SARS-CoV-2
  3. Production of anti-SARS-CoV-2 Spike RBD IgG, IgM and IgA Antibodies Induced in Response to Vaccination with COVID-19 mRNA Vaccines

Page count: 12
Read time: 10 minutes
Authors: Julie Lovchik, Ariel Munitz, Motti Gerlic and Mark Carter

Target audience:

Immunologists, virology researchers, drug and vaccine discovery researchers, in biotechnology, pharmaceuticals, biopharma and academia.   

Applications covered:

Flow cytometry

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