柯萨奇病毒A组16型中和抗体假病毒荧光定量检测方法的建立及初步应用  

Establishment and preliminary application of a pseudovirus luciferase assay for quantitative detection of coxsackievirus A16 neutralizing antibody

作　　者：郝晓甜 陈盼[1] 毛群颖[1] 邵杰[1] 林惠娟[1] 罗震 吴星[1] 梁争论[1] HAO Xiao-tian CHEN Pan MAO Qun-ying SHAO Jie LIN Hui-juan LUO Zhen WU Xing LIANG Zheng-lun(Division of Hepatitis Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control, Beijing 100050, China)

机构地区：[1]中国食品药品检定研究院肝炎病毒疫苗室,北京100050 [2]华兰生物工程股份有限公司研发部,河南新乡453003

出　　处：《中国病毒病杂志》2016年第1期6-11,共6页Chinese Journal of Viral Diseases

基　　金：国家科技重大专项(2012ZX10004701)

摘　　要：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 pseudovirus luciferase assay（PVLA）for the detection of Coxsackievirus A16（CV-A16）neutralizing antibodies. Methods CV-A16 pseudoviruses was obtained by sequential transfection of CV-A16 capsid protein expresser plasmid and enterovirus 71 subgenomic RNA bearing luciferase reporter gene.PVLA method was developed based on in vitro neutralization of CV-A16 pseudoviruses and neutralizing antibody titers were quantified by measuring luciferase activity.CVA16 national neutralizing antibody standard was used to optimize PVLA assay.Fifteen mouse serum samples collected from mice immunized with CV-A16 virus candidates were used to compare PVLA method with traditional cytopathic effect（CPE）assay. Results A single-round CV-A16 pseudovirus was successfully established and a safe,convenient and quantitative method（PVLA）was developed to measure the CV-A16 neutralizing antibodies in serum.Neutralizing antibody titers of 15 mouse serum samples were measured by both PVLA and CPE methods.Pearson correlation analysis（r2=0.91）and Bland-Altman analysis revealed that these two assays were highly consistent. Conclusions The CV-A16 PVLA method established in this study can replace traditional CPE method for the rapid detection of CV-A16 neutralizing antibody.

关 键 词：柯萨奇病毒A16型 假病毒 中和抗体 荧光定量 

分 类 号：R392[医药卫生—免疫学]