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 Health, Xiamen University, Xiamen, 361102, People's Republic of China.
Xiamen Center for Disease Control and Prevention, Xiamen, 361012, People's Republic of China.
Appl Microbiol Biotechnol. 2019 Feb;103(4):1931-1938. doi: 10.1007/s00253-018-09598-7. Epub 2019 Jan 8.
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 10 TCID/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.
柯萨奇病毒 A10（CVA10）最近已成为全球儿童手足口病（HFMD）的主要病原体之一，但目前尚无针对 CVA10 的治疗方法或疫苗。对 CVA10 群体免疫的血清学评估将促进疫苗的开发。基于细胞病变抑制（Nt-CPE）的传统中和测定是测量针对 CVA10 的中和抗体滴度的常用方法，但该方法既耗时又费力。在这项研究中，开发了一种基于针对 CVA10 的单克隆抗体（mAb）3D1 的高效中和试验，称为基于酶联免疫斑点的中和试验（Nt-Elispot）。在 Nt-Elispot 中，用辣根过氧化物酶（HRP）标记的 mAb 3D1 用于检测 RD 细胞中感染的 CVA10，当与固定孵育时间为 14 h 结合时，将 CVA10 的最佳感染剂量设置为 10 TCID/孔。与 Nt-CPE 相比，Nt-Elispot 方法有效地缩短了检测周期，并且与 Nt-CPE 具有良好的相关性。使用 Nt-Elispot，对 123 份来自健康儿童的血清进行了针对 CVA10 的中和抗体检测，结果表明总体血清阳性率为 49.3%（54/123），几何平均滴度（GMT）已计算为 574.2。此外，通过 Nt-Elispot 筛选获得了 2 种抗 CVA10 的中和 mAb。总体而言，建立的 Nt-Elispot 可用于评估针对 CVA10 的免疫和筛选中和抗体的高效高通量方法。
