定量检测百日咳黏附素的ELISA法建立及初步应用

Establishment and preliminary application of ELISA method for the quantitative determination of pertussis pertactin

目的:建立定量检测吸附无细胞百白破(组分)联合疫苗(DTaP)生产过程中黏附素(pertactin,PRN)含量的方法。方法:以高效价兔抗PRN多克隆抗体为包被抗体,羊抗PRN多抗为检测抗体,采用辣根过氧化物酶标记的兔抗羊抗体,建立双抗体夹心间接酶联免疫吸附试验(ELISA)法测定PRN抗原的含量并进行方法学验证。结果:建立的ELISA法在3.906 25~500 ng·mL^(-1)PRN浓度区间有最佳线性,相关系数r> 0.95;该法对PRN检测有良好的特异性;实验内和实验间检测250,60,15 ng·mL^(-1)浓度下PRN含量,变异系数为1.987%~9.785%、回收率为93.1%~103.5%。该法精密度和准确度较好,该法的定量限度为15 ng·mL^(-1)。同时探讨了建立的双抗体夹心ELISA法的初步应用,测定了PRN纯化步骤的回收率以及联合疫苗中PRN组分的含量和吸附率。结论:建立的ELISA法可有效检测百日咳疫苗纯化过程中PRN的含量,为DTaP生产过程的质量控制奠定了重要基础。

Objective: To establish an ELISA method for the quantitative determination of pertactin(PRN) during production of Diphtheria, Tetanus and Acellular Pertussis( component) Combined Vaccine( DTaP).Methods: The rabbit anti-PRN polyantibody with high potency was taken as the capture antibody, while sheep anti-PRN polyantibody was taken as the detection antibody. The content of PRN antigen was detected by using horseradish peroxidase-labeled rabbit anti-sheep antibody, and the double-antibody sandwich indirect enzyme-linked immunosorbent assay(ELISA) method was established and validated.Results: The best linearity of the ELISA method is in the range of 3. 906 25 ~ 500 ng·mL^(-1)(r> 0. 95). The ELISA method has good specificity for PRN. The coefficient of variation and the recovery rates of intra-and inter-assay are 1. 987% ~9. 785% and 93. 1% ~103. 5%, respectively,under the concentrations of standard PRN as 250, 60, and 15 ng·mL^(-1). The precision and accuracy of this method are good. The quantitative limit was 15 ng·mL^(-1). The preliminary application of this method was discussed, also the recovery rate of the purification step of PRN, the content and adsorption rate of PRN component in the combined vaccine were determined.Conclusion: The established ELISA method effectively detected the content of PRN in the purification process of pertussis vaccine, which build an important foundation for the quality control of the production process of DTaP combined vaccine.