Related Experiment Video
Updated: Jul 19, 2025
Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection
Published on: June 5, 2021
Development of a Cell-Based SARS-CoV-2 Pseudovirus Neutralization Assay Using Imaging and Flow Cytometry Analysis
Jerilyn R Izac1, Edward J Kwee1, Linhua Tian1
1Biosystems and Biomaterials Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD 20899, USA.
Abstract:
COVID-19 is an ongoing, global pandemic caused by the novel, highly infectious SARS-CoV-2 virus. Efforts to mitigate the effects of SARS-CoV-2, such as mass vaccination and development of monoclonal therapeutics, require precise measurements of correlative, functional neutralizing antibodies that block virus infection. The development of rapid, safe, and easy-to-use neutralization assays is essential for faster diagnosis and treatment. Here, we developed a vesicular stomatitis virus (VSV)-based neutralization assay with two readout methods, imaging and flow cytometry, that were capable of quantifying varying degrees of neutralization in patient serum samples. We tested two different spike-pseudoviruses and conducted a time-course assay at multiple multiplicities of infection (MOIs) to optimize the assay workflow. The results of this assay correlate with the results of previously developed serology and surrogate neutralization assays. The two pseudovirus readout methods produced similar values of 50% neutralization titer values. Harvest-free in situ readouts for live-cell imaging and high-throughput analysis results for flow cytometry can provide unique capabilities for fast evaluation of neutralization, which is critical for the mitigation of future pandemics.
Insights
A new vesicular stomatitis virus (VSV)-based assay rapidly measures neutralizing antibodies against SARS-CoV-2. This method aids in tracking immune response and developing effective COVID-19 treatments.
Area of Science:
- Virology
- Immunology
- Infectious Diseases
Background:
- The COVID-19 pandemic necessitates accurate measurement of neutralizing antibodies to SARS-CoV-2.
- Existing methods for assessing antibody neutralization can be slow or complex.
- Rapid and reliable assays are crucial for vaccine efficacy assessment and therapeutic development.
Purpose of the Study:
- To develop and validate a rapid, user-friendly neutralization assay for SARS-CoV-2.
- To quantify functional neutralizing antibodies in patient serum samples.
- To provide a tool for faster diagnosis and treatment evaluation during pandemics.
Main Methods:
- Development of a vesicular stomatitis virus (VSV)-based pseudovirus neutralization assay.
- Utilized two distinct readout methods: live-cell imaging and flow cytometry.
- Optimized assay parameters including pseudovirus type and multiplicity of infection (MOI).
Main Results:
- The VSV-based assay successfully quantified varying levels of SARS-CoV-2 neutralization in patient sera.
- Assay results demonstrated strong correlation with established serological and surrogate neutralization assays.
- Both imaging and flow cytometry readouts yielded comparable 50% neutralization titer (NT50) values.
Conclusions:
- The developed VSV-based assay offers a rapid and efficient method for evaluating SARS-CoV-2 neutralizing antibody responses.
- In situ and high-throughput capabilities enable swift assessment critical for pandemic mitigation.
- This assay serves as a valuable tool for monitoring vaccine effectiveness and guiding therapeutic strategies.
