联合疫苗中白喉类毒素抗原质量控制方法的研究

Research in quality control method of diphtheria toxoid antigen in combined vaccine

目的建立白喉类毒素含量测定方法,用于百白破为基础的联合疫苗的质量控制。方法采用棋盘滴定法确定抗白喉类毒素包被抗体及酶标抗体浓度,并对方法进行全面验证。结果棋盘滴定法确定包被抗体浓度为5μg/ml,酶标记抗体稀释2000倍为宜。验证双抗体夹心ELISA线性检测范围为0.0027 lf/ml^0.0429 lf/ml(相关系数r>0.99),检测方法特异性强、精密度及准确度均符合要求(精密度验证CV<10%,准确度验证回收率在85%~115%)。利用该法测定多批联合疫苗中DT的吸附率,分别使用该法和絮状单位法测定了17批类毒素原液中DT的含量,比较2种检测方法呈显著相关(r=0.594)。结论建立了白喉类毒素抗原含量检测方法,为白喉疫苗生产过程及联合疫苗中DT吸附率的评价提供有效质控方法。

Objective To develop a sandwich ELISA for the quantification of diphtheria toxoid(DT).Methods Checkerboard titration was used to determine the concentration of anti-diphtheria toxoid coated antibody and enzyme-labeled antibody,and the method was fully verified.Results By checkerboard method,the optimal concentration of coating antibody was 5μg/ml,and the working dilution of HRP-labeled antibody was 1∶2000.By validation,the linear range of the assay was within 0.0027 lf/ml-0.0429 lf/ml(r>0.99).The detection method has strong specificity,precision and accuracy that meet the requirements(CV<10%,the accuracy verification recovery rate is within 85%-115%).This method was used to determine the adsorption rate of DT in multiple batches of combined vaccines.The content of DT in 17 batches of toxoid stock solution was determined using this method and the flocculent unit method,and the two detection methods were compared,showing a significant correlation(r=0.594).Conclusion A sandwich ELISA for quantification of DT was developed,which can be applied as an effective quality control method for the quantification of DT in DT vaccine and DTP-based combined vaccines during manufacture process.