Development of a coxsackievirus A16 neutralization test based on the enzyme-linked immunospot assay

Wangheng Hou; Lisheng Yang; Delei He; Jun Zheng; Longfa Xu; Jian Liu; Yajing Liu; Huan Zhao; Xiangzhong Ye; Tong Cheng; Ningshao Xia

doi:10.1016/j.jviromet.2015.02.010

Development of a coxsackievirus A16 neutralization test based on the enzyme-linked immunospot assay

J Virol Methods. 2015 Apr:215-216:56-60. doi: 10.1016/j.jviromet.2015.02.010. Epub 2015 Feb 19.

Authors

Wangheng Hou¹, Lisheng Yang¹, Delei He¹, Jun Zheng¹, Longfa Xu², Jian Liu¹, Yajing Liu¹, Huan Zhao², Xiangzhong Ye³, Tong Cheng⁴, Ningshao Xia⁵

Affiliations

¹ State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, 361102, China.
² National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen 361102, China.
³ Beijing Wantai Biological Pharmacy Enterprise Co., Ltd., Beijing, 102206, China.
⁴ State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen 361102, China. Electronic address: tcheng@xmu.edu.cn.
⁵ State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen 361102, China.

PMID: 25704596
DOI: 10.1016/j.jviromet.2015.02.010

Abstract

Coxsackievirus A16 (CA16) is one of the major pathogens responsible for hand, foot and mouth disease (HFMD). The assessment of the humoral immunity response is indispensable in the development of vaccines against enteroviruses. The neutralization test based on the inhibition of cytopathic effects (Nt-CPE) is a common method for measuring neutralizing antibodies against CA16. However, an efficient neutralization test needs to be developed for seroepidemiological surveys and clinical trials of CA16 vaccines because Nt-CPE is time-consuming and labor-intensive. In this study, a high-throughput neutralization test for CA16 based on the enzyme-linked immunospot assay (Nt-ELISPOT) was developed. The monoclonal antibody 7D10, which reacted with the viral protein VP1, was used to detect the cells infected with CA16. The neutralizing titers of sera were proven to be unchanged over an infectious dose range from 10 to 10,000TCID50 per well. The Nt-ELISPOT results correlated well with the Nt-CPE results (R(2) = 0.9250), and the detection period was shortened from five days to approximately 30h. Overall, the Nt-ELISPOT is a reliable and efficient method for measuring neutralizing antibodies against CA16.

Keywords: Antibody; Coxsackievirus A16; ELISPOT; Enterovirus; Neutralization.

Publication types

Evaluation Study
Research Support, Non-U.S. Gov't

MeSH terms

Antibodies, Neutralizing / blood*
Antibodies, Viral / blood*
Enterovirus / immunology*
Enzyme-Linked Immunospot Assay / methods*
High-Throughput Screening Assays*
Humans
Neutralization Tests / methods*
Time Factors

Substances

Antibodies, Neutralizing
Antibodies, Viral