HAV抗原检测ELISA试剂盒的验证及应用

Verification and application of the HAV antigen detection ELISA kit

目的验证甲型肝炎灭活疫苗(人二倍体细胞)(甲肝疫苗)生产场地变更后原生产场地制备的ELISA试剂盒对HAV抗原检测的一致性.方法用原生产场地制备的抗HAV多克隆抗体包被的酶标板、辣根过氧化物酶标记的抗HAV抗体制备ELISA试剂盒,并对该试剂盒进行线性、精密度、准确度、专属性和稳定性验证,同时用该试剂盒检测甲肝疫苗中间品的HAV抗原浓度,考察其适用性.结果在新生产场地中ELISA试剂盒的检测线性范围为0.2~6.4 IU/ml,线性决定系数大于0.99.该试剂盒的精密度良好,检测结果的变异系数均小于15%.不同HAV抗原浓度样品的回收率为84%~104%.该试剂盒可特异性检测HAV,不与非HAV及HAV培养基质发生反应.包被抗体的酶标板于37℃放置7 d对检测结果没有影响.该试剂盒检测结果显示,HAV收获液、HAV纯化液和甲肝疫苗半成品的平均HAV抗原浓度分别为213.25、146.70和48.91 IU/ml,符合在生产过程中HAV抗原含量下降的规律.结论原生产场地制备的ELISA试剂盒可用于检测新生产场地生产的甲肝疫苗.

Objective To verify the consistency of the ELISA kit prepared in original manufacturing site for the HAV antigen detection after manufacturing site change of inactivated hepatitis A vaccine (human diploid cell). Methods The ELISA kit was prepared with the anti-HAV polyclonal antibody-coated plate and the horseradish peroxidase-labeled anti-HAV antibody prepared in original manufacturing site, and its linearity, accuracy, precision, specificity and stability were verified. Meanwhile, ELISA kit was used to detect HAV antigen concentrations in the intermediate products of inactivated hepatitis A vaccine. Results In new manufacturing site, the linear range of the ELISA kit was 0.2-6.4 IU/ml, and the linear determination coefficient was >0.99.The precision of the ELISA kit was good, and the coefficients of variation were all <15%. The recovery rates of samples with different HAV antigen concentrations were 84%-104%. The ELISA kit specifically detected HAV antigen, and did not react with non-HAV and cell matrix for HAV culture. There was no effect on the detection results when the antibody-coated plate was placed at 37 ℃ for 7 d. The detection results showed that the average antigen contents of HAV harvests, HAV purified liquid and final bulk of vaccine were 213.25, 146.70 and 48.91 IU/ml, respectively, meeting with decline pattern of the HAV antigen content in the process. Conclusion The ELISA kit prepared in original manufacturing site can be used to detect the inactivated hepatitis A vaccine (human diploid cell) prepared in new manufacturing site.