狂犬病病毒糖蛋白抗原含量双抗体夹心ELISA检测方法的建立及验证

目的建立可快速检测狂犬病病毒(rabies virus,RabV)糖蛋白抗原含量的双抗体夹心ELISA方法,并进行验证及初步应用。方法用抗RabV糖蛋白基因工程抗体建立双抗体夹心ELISA法,检测RabV糖蛋白抗原含量。对建立的方法进行特异性、线性、准确性、板内与板间精密性、灵敏度验证,对不同厂家(毒株分别为aG、PV、CTN、PM)生产的狂犬病疫苗与不同工艺阶段的狂犬病疫苗样品进行适用性检测。结果捕获抗体与酶标抗体最佳稀释度分别为1∶2 000和1∶4 000,最佳封闭剂为1. 5%BSA。该方法与其他人用疫苗和辅料成分无交叉反应;线性范围在0. 003 2~0. 103 1 IU/mL之间,R2> 0. 98;准确性验证的回收率在97. 0%~110. 1%之间;精密性验证的板内变异系数<8. 6%,板间变异系数<13. 9%;检测不同厂家的狂犬病疫苗和不同工艺阶段的疫苗样品呈良好的剂量依赖效应。结论建立了适用于人用狂犬病疫苗糖蛋白抗原含量检测的双抗体夹心ELISA方法,符合定量检测的需求,可用于aG、PV、CTN、PM等不同毒株生产的狂犬病疫苗检测;也可用于病毒收获液、浓缩液、灭活液及原液等不同工艺阶段样品检测。

Development and validation of double antibody sandwich ELISA for determination of rabies virus glycoprotein antigen content

Objective To develop,validate and preliminarily apply a double antibody sandwich ELISA for rapid determination of rabies virus(RabV)glycoprotein antigen content. Methods The double antibody sandwich ELISA was developed with recombinant antibodies against RabV glycoprotein and used for the determination of RabV glycoprotein content. The developed method was validated for specificity,linearity,accuracy,inplate and interplate precisions and sensitivity. The rabies vaccines produced by different manufacturers from different virus strains(aG,PV,CTN and PM)as well as the vaccine samples at different steps of production procedure were determined by the method to evaluate the suitability. Results The optimal dilutions of capture antibody was 1 ∶ 2 000 while that of enzyme-labeled antibody was1 ∶ 4 000,and the optimal blocking agent was 1. 5% BSA. There were no cross reactions with other vaccines or subsidiary materials. The linear range of the developed method was 0. 003 2 ~ 0. 103 1 IU/mL,with a R2 value of more than 0. 98. The recovery rate in validation for accuracy was 97. 0% ~ 110. 1%. The CVs in validation for inplate and interpolate precisions were less than 8. 6% and less than 13. 9% respectively. Dose-dependent effects were observed by the developed method in determination of rabies vaccines from different manufacturers and samples at different steps of production procedure. Conclusion A double antibody sandwich ELISA suitable for RabV glycoprotein antigen content was developed,which met the requirements for quantitative determination,and might be used for the rabies vaccines prepared from different virus strains such as aG,PV,CTN and PM as well as the samples at different steps of production procedure,such as virus harvest,concentrate,inactivation liquid and bulk.