Related Experiment Video
Updated: Nov 10, 2025
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
Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19
Linhua Tian1, Elzafir B Elsheikh1, Paul N Patrone2
1Biosystems and Biomaterials Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD 20899, USA.
Insights
New quantitative serology assays using flow cytometry can measure antibodies against SARS-CoV-2 spike proteins. These assays are specific and correlate with neutralizing antibody levels, aiding COVID-19 research.
Area of Science:
- Immunology
- Virology
- Biotechnology
Background:
- Quantitative serology assays are crucial for COVID-19 diagnostics, surveillance, and vaccine development.
- Robust and reliable antibody measurements are needed to understand immune responses to SARS-CoV-2.
Purpose of the Study:
- To develop and validate quantitative, multiplexed flow cytometry-based serological and neutralization assays for SARS-CoV-2.
- To assess the specificity and accuracy of these novel assays against various coronaviruses and reference standards.
Main Methods:
- Development of flow cytometry assays to detect IgG and IgM antibodies against full-length spike and receptor binding domain (RBD) antigens.
- Quantification of anti-SARS-CoV-2 RBD antibody titers using a reference standard.
- Testing for cross-reactivity with spike proteins from MERS, SARS1, OC43, and HKU1 viruses.
- Correlation analysis between anti-RBD antibody titers and neutralizing antibody titers.
Main Results:
- The developed assays accurately quantified anti-SARS-CoV-2 RBD antibody titers within a defined range (37.6 µg/mL to 31.0 ng/mL).
- The assays demonstrated high specificity, showing no cross-reactivity with other common coronavirus spike proteins.
- A good correlation was observed between quantified anti-RBD antibody titers and neutralizing antibody titers.
Conclusions:
- This proof-of-concept demonstrates a quantitative, multiplexed flow cytometry approach for serological and neutralization assays.
- These assays enhance measurement confidence and provide foundational tools for COVID-19 clinical and epidemiological studies.
- The developed assays are specific, quantitative, and correlate with functional neutralization, supporting their utility in global health initiatives.
Abstract:
Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.
