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Updated: Dec 7, 2025
Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells
Published on: November 22, 2017
A Sensitive and High-Throughput Flow Cytometry-Based Assay for Measuring Antibody Neutralization of Human Adenovirus
Zhenwei Liu1, Xingui Tian1, Wenkuan Liu1
1State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, 510000, China.
Abstract:
The assessment of neutralization activity is an important step in the evaluation of neutralizing antibodies (NAbs). The traditional methods for measuring the antibody neutralization of human adenovirus type 3 (HAdV-3) are the microneutralization (MN) assay, which has insufficient sensitivity, and the plaque reduction neutralization test (PRNT), which is not suitable for high-throughput screening. Herein, we describe the development of a flow cytometry-based neutralization (FCN) assay for measuring the neutralization of sera, cell culture supernatants, and chimeric antibodies against HAdV-3 on the basis of a recombinant HAdV-3 (rHAdV-3) construct expressing the enhanced green fluorescent protein (EGFP). For flow cytometry-based assays, the optimal cell confluence was determined as 90%, and the virus was titrated using the assay. The established FCN assay follows the percentage law and an optimal MOI of not less than 5 × 10-4 was determined by using a purified chimeric antibody. In addition, comparison of the anti-HAdV-3 NAb titers of 72 human serum samples by the MN and FCN assays, showed that both assays correlated strongly with each other. Our FCN assay was an improvement over the MN assay because the observation period was reduced from 3 to 1 days and data analysis could be performed objectively and robotically. Importantly, the newly established FCN assay allows measurement of the neutralization activity of chimeric antibodies expressed in cell culture supernatants. Thus, this sensitive and high-throughput FCN assay is a useful alternative to the MN assay for measuring the antibody neutralization of HAdV-3 and for screening anti-HAdV-3 NAbs in cell culture supernatants.
Insights
A new flow cytometry-based neutralization (FCN) assay effectively measures neutralizing antibodies against human adenovirus type 3 (HAdV-3). This high-throughput method improves upon traditional assays, offering increased sensitivity and objectivity for HAdV-3 NAb screening.
Area of Science:
- Virology
- Immunology
- Biotechnology
Background:
- Neutralizing antibodies (NAbs) are crucial for evaluating viral immunity.
- Traditional methods like microneutralization (MN) and plaque reduction neutralization tests (PRNT) for human adenovirus type 3 (HAdV-3) have limitations in sensitivity and throughput.
- There is a need for a more efficient and sensitive assay for HAdV-3 NAb assessment.
Purpose of the Study:
- To develop and validate a flow cytometry-based neutralization (FCN) assay for quantifying HAdV-3 NAbs.
- To assess the assay's performance using sera, cell culture supernatants, and chimeric antibodies.
- To establish an optimized protocol for the FCN assay.
Main Methods:
- Development of a recombinant HAdV-3 (rHAdV-3) expressing EGFP for detection.
- Optimization of cell confluence (90%) and multiplicity of infection (MOI ≥ 5 × 10⁻⁴) for the FCN assay.
- Comparison of HAdV-3 NAb titers in 72 human serum samples using both MN and FCN assays.
Main Results:
- The FCN assay demonstrated strong correlation with the traditional MN assay for HAdV-3 NAb titers.
- The FCN assay reduced the observation period from 3 days to 1 day compared to the MN assay.
- The assay allows for objective, robotic data analysis and can measure neutralization activity in cell culture supernatants.
Conclusions:
- The developed FCN assay is a sensitive, high-throughput, and objective alternative to traditional methods for HAdV-3 NAb assessment.
- This assay facilitates the screening of NAbs in cell culture supernatants, including those from chimeric antibodies.
- The FCN assay provides a valuable tool for research and diagnostics involving HAdV-3 neutralization.
