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目的建立一种简单、快速、高通量的检测柯萨奇病毒A组16型(CV-A16)中和抗体假病毒荧光定量检测方法 (pseudovirus luciferase assay,PVLA)。方法首先构建CV-A16核衣壳蛋白表达子及插入萤火虫荧光素酶报告基因的肠道病毒71型(EV-A71)复制子,再顺次转染293T细胞,互补包装得到CVA16假病毒。通过条件优化并使用CV-A16国家中和抗体标准品,建立CV-A16中和抗体定量检测方法(PVLA)。用该方法检测15份CV-A16病毒免疫小鼠血清,并与传统CPE法的检测结果进行统计学分析,比较两种方法检测结果的一致性。结果得到CV-A16假病毒,建立了CV-A16假病毒中和抗体检测方法,该方法检测CV-A16中和抗体滴度的结果与传统CPE法的检测结果高度一致(相关系数r2为0.91)。结论CV-A16假病毒中和抗体检测方法可作为CPE法的替代方法,用于CV-A16中和抗体的快速检测。
Objective To establish a simple, rapid and high-throughput method for the detection of Coxsackievirus A virus type 16 (CV-A16) neutralizing antibody pseudovirus (PVLA). Methods CV-A16 nucleocapsid protein expression vector and enterovirus 71 (EV-A71) replicon with firefly luciferase reporter gene were constructed and transfected into 293T cells. CV-A16 neutralizing antibody quantitation assay (PVLA) was established by conditional optimization and using CV-A16 national neutralizing antibody standards. Fifteen CV-A16 virus-immunized mice were tested for serum by this method, and the results were statistically analyzed with the traditional CPE method. The consistency between the two methods was compared. Results The CV-A16 pseudovirus was obtained and the neutralizing antibody against CV-A16 was established. The titer of CV-A16 neutralizing antibody was highly consistent with that of the traditional CPE assay (r2 was 0.91) . Conclusion The detection method of neutralizing antibody of CV-A16 pseudovirus can be used as an alternative method of CPE method for the rapid detection of neutralizing antibodies of CV-A16.