Development of an efficient neutralization assay for Coxsackievirus A10
Development of an efficient neutralization assay for Coxsackievirus A10
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柯萨奇病毒 A10 有效中和试验的开发
DOI：
10.1007/s00253-018-09598-7
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发表时间：
2019-02-01
影响因子：
5
通讯作者：
Xia, Ningshao
中科院分区：
文献类型：
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作者：
Liu, Dongxiao;Xu, Longfa;Xia, Ningshao
柯萨奇病毒A10（Coxsackievirus A10，CVA 10）是近年来世界范围内引起儿童手足口病（Handfoot，and Mouth Disease，HFMD）的主要病原体之一，但目前尚无有效的疫苗或治疗方法。对CVA 10的群体免疫进行血清学评价将促进疫苗的发展。传统的基于细胞病变效应抑制的中和试验（Nt-CPE）是测定抗CVA 10中和抗体效价的常用方法，费时费力。在这项研究中，开发了一种基于抗CVA 10单克隆抗体（mAb）3D 1的有效中和试验，称为Elispot中和试验（Nt-Elispot）。在Nt-Elispot中，以辣根过氧化物酶（HRP）标记的单抗3D 1检测1：4000稀释度的CVA 10感染的RD细胞，CVA 10的最佳感染剂量为105 TCID 50/孔，孵育时间固定为14 h。与Nt-CPE法相比，Nt-Elispot法能有效缩短检测周期，且相关性好，用该方法检测了123份健康儿童血清中的CVA 10中和抗体，总阳性率为49.3%（54/123），几何平均滴度（GMT）为574.2。进一步通过Nt-Elispot筛选获得2株抗CVA 10中和mAb。该方法可作为一种高效、高通量的检测CVA 10免疫力和筛选中和抗体的方法。
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 105TCID50/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.