百日咳丝状血凝素酶联免疫检测法的建立

Establishment of ELISA in determination of filamentous hemagglutinin in DTaP

目的建立百日咳组分疫苗丝状血凝素(FHA)抗原含量监控的ELISA检测法。方法制备的多克隆抗血清,经辛酸硫酸铵法纯化抗体,用过碘酸钠氧化法辣根过氧化物酶标记,以棋盘滴定法确定最佳包被抗体及酶标抗体的浓度配伍,建立了双抗体夹心ELISA检测法。结果对双抗体夹心ELISA法的特异性、最佳线性范围、检测限度、精密度、准确度、测定限量、适用性的一系列验证试验表明,该方法与百日咳组分疫苗中PT和Prn无明显交叉反应,特异性较好。在0至20 ng/mL测量区间有最佳线性,相关系数大于0.99;经实验内12次及不同试验间3次测定16、8、4 ng/mL中的FHA含量,变异系数在0.2%～11.4%间,回收率在96.9%～114.5%间,精密度及准确度验证均符合常规质控要求,因此测定限量为4 ng/mL。结论该方法能有效检测出百日咳杆菌培养上清中的FHA含量,可用于百日咳组分疫苗生产过程的中间品质量控制。

Objective To develop an ELISA in quantitative determination of filamentous hemagglutinin （ FHA） in DTaP （diphtheria,tetanus and acellular pertussis combined vaccine, adsorbed ）. Methods The high potency antisera for FHA were obtained by using FHA immunized rabbit, the polyclonal antibody was purified by capric acid and ammonium sulphate precipitation, the antibody was coupled to horseradish peroxidase （ HRP ） by periodate oxidase method in order to develop a double antibody sandwich ELISA in quantitative determination of filamentous hemagglutinin （ FHA） in DTaP. Results This method showed an excellent specificity in reaction with FHA, there were no cross reactions with PT or Prn. The best linearity of dose-response curve was found in a range of 0-20 ng/mL （ r〉0.99 ） , the good precision was obtained with the coefficient of variation as 0.2% roll. 4% and the good accuracy with the recovery of 96.9%-114.5% by both the intra-and inter-assays while in test for standard FHA, such as 16 ng/mL, 8 ng/mL, 4 ng/mL, the quantitative limitation was de-termined as 4 ng/mL, which was in accordance with requirements for conventional quality control measures. Conclusion This method could be applied in detection content of FHA in quality control for the intermediate preparations in the process of production of DTaP.