A Sensitive and High-Throughput Flow Cytometry-Based Assay for Measuring Antibody Neutralization of Human Adenovirus Typ
- PDF / 543,348 Bytes
- 8 Pages / 595.276 x 790.866 pts Page_size
- 70 Downloads / 171 Views
www.virosin.org www.springer.com/12250
(0123456789().,-volV)(0123456789().,-volV)
RESEARCH ARTICLE
A Sensitive and High-Throughput Flow Cytometry-Based Assay for Measuring Antibody Neutralization of Human Adenovirus Type 3 Zhenwei Liu1 • Xingui Tian1 • Wenkuan Liu1 • Yuting Xian1 • Weilue Chen1 • Rong Zhou1 Received: 18 January 2020 / Accepted: 24 August 2020 Ó Wuhan Institute of Virology, CAS 2020
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 9 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 highthroughput 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. Keywords Sensitive High-throughput Flow cytometry Neutralization Adenovirus
Introduction Human adenovirus (HAdV), which belongs to the genus Mastadenovirus, was first isolated from adenoids in 1953 (Rowe et al. 1953). So far, 103 types of HAdV (http:// hadvwg.gmu.edu/), including candidates from HAdV-53 to HAdV-103, have been identified and classified into seven species (A to G) on the basis of phylogenomics, serology,
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12250-020-00295-2) contains supplementary material, which is available to authorized users. & Rong Zhou [email protected] 1
State 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
and whole-genome sequencing (Jones et
Data Loading...
