人肠道病毒71型中和抗体检测方法的实验室评价

肠道病毒71型（Enterovirus 71，EV71）属于小

RNA 病毒科肠道病毒属，为危害程度仅次于脊髓灰质炎病毒的嗜神经性肠道病毒，可引起5岁以下儿童手足口病（Hand -foot -mouth disease ，HFMD ），少数

重症患者可并发严重的中枢神经系统疾病和肺部感

染，重症患儿病程进展迅速，预后差［1-4］

。目前，HFMD 尚缺乏有效的预防措施和治疗方法，研制疫苗是控制HFMD 流行的重要手段。

中和效价是评价疫苗免疫原性的关键指标之一［5-8］，检测结果的准确性对疫苗研发和应用均具有重要意义。目前，EV71中和抗体检测方法多采用微量细胞病变法，该方法影响因素较多，且国内各实验室的应用时间尚短，为探讨和规范该测定方法，准确评价EV71疫苗毒株的免疫原性和保护能力，本实验对实验室内、实验室间以及不同毒株对EV71抗

基金项目：国家科技支撑项目（2008BAI69B01）资助项目.

作者单位：1中国药品生物制品检定所（北京100050）；2华兰生

物工程股份有限公司研发部（河南新乡453003）；3北京生物制品研究所（北京100024）；4北京科兴生物制品有限公司研发部（北京100085

）；5中国医学科学院医学生物学研究所免疫室（昆明650118）.通讯作者：毛群颖，E -mail:maoqunying@

*：

共享第一作者中国图书分类号R373.2R392-33文献标识码A 文章编号1004-5503（2010）08-0885-04

【实验技术】

人肠道病毒71型中和抗体检测方法的实验室评价

毛群颖1*何鹏1*于祥2李楠2郝春生3高强4董承红5梁争论1李凤翔1沈心亮3王军志1

【摘要】目的

对人肠道病毒71型（Enterovirus71，EV71）中和抗体检测方法进行实验室评价，为该方法的标准化提供

依据。方法5个实验室按照统一制定的EV71中和抗体检测操作细则（SOP ），应用A 、B 和C 基因型的10株EV71病毒株，分别测定10份人免疫球蛋白、20份健康成人血浆和15份EV71动物高效价免疫血清样品中EV71中和效价，检测实验室内、实验室间重复性及不同EV71病毒株对中和抗体检测的影响。结果相同实验室应用各病毒株重复测定中和效价的最大值与最小值（Max -Min ）倍数差均在4倍以内，3次重复试验结果间差异无统计学意义（P 值均＞0.05）；不同实验室应用相同病毒株检测结果的Max -Min 倍数差在4倍以内；

不同实验室应用不同病毒株检测结果为：A 型株与其他基因型病毒株中和效价的Max -Min 差在192倍以内，B3与C4亚型的病毒株中和效价的Max -Min 倍数差在64倍以内，C4亚型不同病毒株之间中和效价的Max -Min 倍数差在16倍以内。结论按统一SOP 操作后,EV71中和抗体检测方法应用相同毒株在实验室内和实验室间具有较好的重复性，而不同病毒株对中和效价的测定结果有较大影响。【关键词】肠道病毒A 型，人；手足口病；中和抗体；基因型

Laboratory Evaluation of Method for Determination of Neutralizing Antibody against Human Enterovirus 71

MAO Qun -ying △,HE Peng,YU Xiang,LI Nan,HAO Chun -sheng,GAO Qiang,DONG Cheng -hong,LIANG Zheng -lun,LI Feng -xiang,SHEN Xin -liang,WANG Jun -zhi （△National Institute for the Control of Pharmaceutical

and Biological Products,Beijing 100050,China

）【Abstract 】

Objective

To evaluate the method for determination of neutralizing antibody against human enterovirus 71

（EV71）in laboratory and provide a basis for standardization of the method.Methods

The neutralizing antibody titers against EV71

in 10human immunoglobulin,20healthy adult plasma and 15high titer animal immune serum specimens were determined with 10EV71strains of genotypes A,B and C by 5laboratories according to the SOP for EV71neutralizing antibody,based on which the in －tra -and inter -reproducibility of the method as well as effect of various EV71strains on determination of neutralizing antibody were analyzed.Results

The fold differences in maximum and minimum （Max -Min ）of determination results of neutralizing antibody titers

with various EV71strains by the same laboratory were less than 4,and the results of 3repeat tests showed no significant difference （P >0.05）.The fold differences in determination results with the same EV71strain by various laboratories were also less than 4.The determination results with various EV71strains by various laboratories were as follows:the fold differences of the Max -Min of neu －tralizing antibody titers of EV71strain genotype A with other genotypes were not more than 192,while those of subtypes B3with C4were not more than 64,and those of subtype C4with other strains were not more than 16.Conclusion By operation according to the unitary SOP,the method for determination of neutralizing antibody against EV71showed satisfactory intra -and inter -reproducibility.However,various EV71strains showed significant effect on the determination result.

【Key words 】Enterovirus A,human;Hand -foot -mouth disease;Neutralizing antibody;Genotype