肠道病毒71型抗原定量检测方法的建立

Quantitative detection assay development for enterovirus 71 antigen

目的 建立肠道病毒71型（enterovirus 71,EV71）抗原的定量检测方法,用于EV71疫苗生产过程中EV71抗原含量的监测.方法 用EV71抗原免疫新西兰兔制备抗EV71多克隆抗体,纯化后用辣根过氧化物酶标记.采用双抗体夹心ELISA法建立抗原检测系统.以EV71国家抗原标准品为定量标准,建立剂量反应曲线,确定该方法的线性范围和定量限,并对该方法的特异性、精密度、准确性、稳定性及适用性等进行验证.结果 建立的ELISA法的线性范围为5～80 U/ml,定量限为5 U/ml,线性决定系数均大于0.990 0;不同浓度样品回收率为85.0％～110.0％,变异系数均小于15％;置于2～8℃及37℃3、6d的抗体包被板对不同浓度样品的检测值变异系数均小于15％;该方法可特异性检测EV71原液,与非EV71样品无交叉反应.结论 建立了EV71抗原定量检测方法,为EV71疫苗生产工艺过程的质量控制奠定了基础.

Objective To develop a quantitative detection assay of enterovirus 71 （EV71） antigen for monitoring virus antigen content during vaccine production.Methods Anti-EV71 polyclonal antibody was prepared by immunizing New Zealand rabbits with EV71 antigen.Purified antibody was labeled with horseradish peroxidase to develop a double-antibody sandwich ELISA.A dose-response curve was plotted using EV71 antigen national reference as quantitative standard.The linear range and quantitative detection limit of the ELISA method were determined.The specificity,precision,stability,and feasibility of the method were evaluated.Results The linear range was 5-80 U/ml and coefficient of determination was 〉 0.990 0.The quantitative detection limit was 5 U/ml.The recovery rate of internal reference was 85%-110% and the coefficient of variation（CV） in reference detection was 〈 15%.The CV for same sample detection between coated plates placed in 2-8 ℃ and 37 ℃ for 3,6 days was 〈 15 %.No cross reaction with non-EV71 samples was observed.Conclusion A quantitative detection assay for EV71 antigen is developed,which may provide a simple monitoring method for quality control during vaccine production.