Development of an efficient neutralization assay for Coxsackievirus A10

  • Dongxiao Liu
  • Longfa Xu
  • Rui Zhu
  • Zhichao Yin
  • Yu Lin
  • Wangheng Hou
  • Shuxuan Li
  • Shuizhen He
  • Tong ChengEmail author
  • Ningshao Xia
Methods and protocols


Coxsackievirus A10 (CVA10) recently has become one of the major pathogens of hand, foot, and mouth disease (HFMD) in children worldwide, but no cure or vaccine against CVA10 is available yet. Serological evaluation of herd immunity to CVA10 will promote the development of vaccine. The traditional neutralization assay based on inhibition of cytopathic effect (Nt-CPE) is a common method for measuring neutralizing antibody titer against CVA10, which is time-consuming and labor-intensive. In this study, an efficient neutralization test based on a monoclonal antibody (mAb) 3D1 against CVA10, called Elispot-based neutralization test (Nt-Elispot), was developed. In the Nt-Elispot, the mAb 3D1 labeled with horseradish peroxidase (HRP) was used to detect the CVA10-infected RD cells at a 1:4000 dilution and the optimal infectious dose of CVA10 was set at 105 TCID50/well when combined with a fixed incubation time of 14 h. Compared with the Nt-CPE, the Nt-Elispot method effectively shortened the detection period and presented a good correlativity with it. Using the Nt-Elispot, a total of 123 sera from healthy children were tested for neutralizing antibody against CVA10, demonstrating that the overall seroprevalence was 49.3% (54/123) and the geometric mean titer (GMT) had been calculated as 574.2. Furthermore, 2 anti-CVA10 neutralizing mAbs were obtained by screening via the Nt-Elispot. Overall, the established Nt-Elispot could be used as an efficient and high-throughput method for evaluating immunity to CVA10 and screening the neutralizing antibodies.


Coxsackievirus A10 Neutralizing antibody Elispot Serological epidemiology 



We thank the editors at NPG Language Editing who provided editing assistance to the authors during the preparation of this manuscript.


This work was supported by a grant from the National Science and Technology Major Project of Infectious Diseases (No.2017ZX10304402), the National Natural Science Foundation of China (Nos. 81801646, 31670933, and 81701999), and the National Science and Technology Major Projects for Major New Drugs Innovation and Development (No. 2018ZX09711003-005-003). The sponsors had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving animal experiments were approved by Xiamen University Laboratory Animal Center (XMULAC) and conducted in accordance with animal ethics guidelines and approved protocols. The Animal Ethics Committee approval number was XMULAC20160049. All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committees of the Xiamen City Center for Disease Control and Prevention and the Xiamen University.

Informed consent

Informed consent was obtained from all individual participants included in the study.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Dongxiao Liu
    • 1
  • Longfa Xu
    • 1
  • Rui Zhu
    • 1
  • Zhichao Yin
    • 1
  • Yu Lin
    • 1
  • Wangheng Hou
    • 1
  • Shuxuan Li
    • 1
  • Shuizhen He
    • 2
  • Tong Cheng
    • 1
    Email author
  • Ningshao Xia
    • 1
  1. 1.State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, School of Public HealthXiamen UniversityXiamenPeople’s Republic of China
  2. 2.Xiamen Center for Disease Control and PreventionXiamenPeople’s Republic of China

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