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Updated: Aug 16, 2025
Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection
Published on: June 5, 2021
A highly sensitive bead-based flow cytometric competitive binding assay to detect SARS-CoV-2 neutralizing antibody
Xiangyu Yao1, Zhichao Zhang2, Qingmin Mei3
1The Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi Provincial Key Laboratory of Medical Molecular Cell Biology, Institute of Biomedical Sciences, Shanxi University, Taiyuan, China.
A new bead-based flow cytometric assay accurately detects SARS-CoV-2 neutralizing antibodies (nAb). The iACE2/RBD-Fc model demonstrates superior sensitivity and specificity for assessing immunity post-infection or vaccination.
Area of Science:
- Immunology
- Virology
- Biotechnology
Background:
- Accurate detection of SARS-CoV-2 neutralizing antibodies (nAb) is crucial for evaluating immunity after infection or vaccination.
- Competitive binding (CB) assays offer a fast, cost-effective alternative to viral neutralization tests.
- CB assays quantify nAb by measuring inhibition of spike (S) protein binding to the ACE2 receptor.
Purpose of the Study:
- To establish and evaluate a bead-based flow cytometric CB assay for SARS-CoV-2 nAb detection.
- To compare the performance of six different assay configurations.
- To identify the most sensitive and specific CB assay model.
Main Methods:
- Development of a bead-based flow cytometric competitive binding (CB) assay.
- Testing six combinations of immobilized ACE2/S1/RBD and soluble ACE2/S1/RBD protein variants.
- Validation using monoclonal antibodies, convalescent sera, and vaccinated subject sera.
Main Results:
- The iACE2/RBD-Fc, iACE2/S1-Fc, and iS1/ACE2-His models specifically detected SARS-CoV-2 nAb.
- The iACE2/RBD-Fc model exhibited the highest sensitivity, outperforming a commercial surrogate virus neutralization test (sVNT) ELISA kit.
- Assay performance was influenced by ACE2 tag, spike protein type, and binding measurement methods.
Conclusions:
- The iACE2/RBD-Fc CB assay is a highly sensitive and specific method for detecting SARS-CoV-2 nAb.
- This assay effectively tracks nAb kinetics against different SARS-CoV-2 variants after vaccination.
- The iACE2/RBD-Fc model shows strong correlation with live and pseudovirus neutralization tests, indicating its clinical potential.
