检测百日咳黏着素的酶联免疫吸附法的建立及初步应用

 目的  建立定量检测白喉-破伤风-无细胞百日咳疫苗生产过程中黏着素（pertactin， Prn）的方法。 方法   纯化的Prn免疫家兔以制备高效价血清抗Prn抗体。辛酸-硫酸铵沉淀法纯化抗Prn多克隆抗体并进行辣根过氧化物酶标记，建立双抗体夹心ELISA法。 结果   建立的方法与丝状血凝素和百日咳毒素无交叉反应，特异性较好。该ELISA法在1.25~80 .00 μg/L Prn测量区间有最佳线性，相关系数＞0.99。实验内和实验间检测64、32、16 μg/L Prn，变异系数为2.6%~7.7%，回收率为84.9%~95.5%，精密度和准确度均符合常规质控要求，因此该法的定量限度为16 μg/L。 结论   建立的ELISA法可有效检测百日咳疫苗纯化过程中的Prn含量，为组分百日咳疫苗质量控制奠定了重要基础。

Establishment and preliminary application of an ELISA method for determination of pertactin

Objective  To establish an ELISA method for quantitative determination of pertactin (Prn) during production of combined diphtheria, tetanus and acellular pertussis vaccine.  Methods   Rabbits were immuned by purified Prn to prepare High-titer serum antibodies against Prn. The polyclonal antibodies against Prn were purified by octanoic acid-ammonium sulfate precipitation method and labeled with horseradish peroxidase to establish a double antibody sandwich ELISA method.   Results  The ELISA method had good specificity for Prn and no cross reaction with filamentous hemagglutinin and pertussis toxin. The best linearity of the ELISA method was formed in a range of 1.25-80.00 mg/L (r＞0.99). The coefficient of variation and recover rates of intra- and inter-assay were 2.6%-7.7% and 84.9%-95.5% respectively when 64, 32 and 16 mg/L standard Prn were detected, and the precision and accuracy both met requirements of quality control. The quantitative limit was 16 mg/L.  Conclusion   The established ELISA method can be applied to detecting Prn during purification of pertussis vaccine and lay the foundation for quality control of Prn.