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Updated: Aug 10, 2026
A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System
Published on: April 6, 2021
Single-reaction multi-antigen serological test for comprehensive evaluation of SARS-CoV-2 patients by flow cytometry
Yaiza Cáceres-Martell1, Daniel Fernández-Soto1, Carmen Campos-Silva1
1Department of Immunology and Oncology, National Centre for Biotechnology, CNB-CSIC, Madrid, Spain.
Insights
A new multiplex serological test detects multiple antibodies against SARS-CoV-2 antigens, offering a clearer picture of immune response than single-antigen tests. This advanced assay accurately distinguishes COVID-19 patients from healthy individuals.
Area of Science:
- Immunology
- Virology
- Medical Diagnostics
Background:
- Most current serological tests for SARS-CoV-2 detect antibodies to only one viral antigen.
- Some laboratory-confirmed COVID-19 patients show no detectable SARS-CoV-2-specific antibodies with single-antigen assays.
- A comprehensive understanding of immune responses to SARS-CoV-2 is crucial for interpreting diverse clinical presentations.
Purpose of the Study:
- To develop and validate a novel, highly multiplexed serological assay for SARS-CoV-2.
- To compare the diagnostic performance of multiplexed antigen detection with single-antigen assays.
- To investigate the breadth and specificity of antibody responses to multiple SARS-CoV-2 antigens.
Main Methods:
- Utilized flow cytometry to detect multiple immunoglobulin (Ig) isotypes binding to four key SARS-CoV-2 antigens simultaneously: Spike glycoprotein, Receptor Binding Domain (RBD), nucleocapsid protein, and main protease.
- Developed a single reaction assay for high-throughput analysis.
- Compared antibody profiles between COVID-19 patients and healthy controls.
Main Results:
- The multiplex assay provides a more complete picture of seroconversion compared to single-antigen assays.
- Demonstrated 100% confidence in discriminating between COVID-19 patients and healthy controls.
- Observed varied antibody responses, with some patients showing bias towards antibodies against surface proteins and others against internal viral proteins.
Conclusions:
- The developed multiplex serological test is a simple and effective tool for diagnosing SARS-CoV-2 infection.
- Analyzing responses to multiple antigens and Ig isotypes may correlate with disease severity and provide insights into COVID-19 pathogenesis.
- This assay enhances the understanding of patient-specific immune responses to SARS-CoV-2.
Abstract:
Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.
