肠道病毒71抗原ELISA检测方法构建及其应用

目的 建立肠道病毒71型(EV71)抗原的ELISA检测方法.用于EV71的抗原定量、TCID50试验及EV71免疫原性与免疫保护性的关系分析.方法 以高亲和力的EV71中和单抗K8G2和Y8H2-HRP构建检测EV71抗原的双抗体夹心ELISA方法.比较本方法与显微镜观察法检测的EV71 TCID50.分析EV71抗原的特异比活与免疫血清中和抗体水平的关系.结果 本试剂定量检测抗原的线性范围为0.125 ～4.0 U/ml,R2 =0.9911,试剂不与EV71之外的受试物反应,试剂的回收率为0.89 ～ 1.16,变异系数小于15％,37℃9 d的热回收率大于85％.本法与显微镜观察法检测的EV71 TCID50的相关系数r=0.990.21株EV71抗原的特异比活与免疫血清中和抗体水平的相关系数r=0.930.结论 构建了EV71抗原ELISA检测试剂,可用于EV71的抗原含量检测、TCID50分析,为特异比活与免疫血清中和性关系研究提供了一些有价值的信息.

The development and application of a quantitative ELISA detection method for enterovirus 71 antigen

Objective To develop an ELISA method for quantitative determination of enterovirus 71 (EV71) antigen.The method can be applied to detect EV71 antigen contents and analyze the correlation between immunogenicity and immunoprotection.It also can be used for tissue culture infective dose( TCID50 ) assay.Methods A double antibody sandwich ELISA method was developed for quantitative determination of EV71 antigen on the basis of the high-affinity neutralizing monoclonal antibodies ( K8G2 and Y8H2-HRP).This method was compared with microscopic observation for the detection of EV71 TCID50.The correlation was analyzed between the specific activity of EV71 antigen and the EV71 neutralizing antibody titer in immune serum.Results The linear range of this method was 0.125-4.0 U/ml and the R2 value was 0.9911.The reagent did not react with other antigens except EV71 antigen.The recovery ratio of this method was 0.89-1.16.The coefficient of variation was less than 15％.The heat recovery rate was above 85％ when the reagent was in 37℃ for 9 days.There was a good correlation in TCID50 of EV71 between this method and microscopic observation,r=0.990.The specific activity of EV71 antigen had positive correlation with the neutralization titer of immune serum in 21 EV71 strains,r=0.930.Conclusion The quantitative ELISA method for EV71 antigen was developed,which could be used to detect EV71 antigen contents and analyze TCID50.The specific activity of EV71 antigen detected by the method could be used to evaluate the immunoprotection of the vaccine potency test.