百日咳毒素 丝状血凝素和百日咳黏附素血清抗体检测方法的建立及验证

Development and Validation of ELISA Assays for Determination of Serum Antibody against Pertussis Toxin, Filamentous Hemagglutinin, Pertactin of Bordetella Pertussis

目的建立定量检测抗百日咳毒素抗体(Antibody to Pertussis Toxin,Anti-PT)、抗丝状血凝素抗体(Antibody to Filamentous Haemagglutinin,Anti-FHA)和抗百日咳黏附素抗体(Antibody to Pertactin,Anti-Prn)的酶联免疫吸附试验(Enzyme-linked Immunosorbent Assay,ELISA)方法。方法分别应用纯化的百日咳毒素(PT)、丝状血凝素(FHA)和百日咳黏附素(Prn)作为包被抗原,百日咳血清抗体国际参考品为标准品,建立定量检测Anti-PT、Anti-FHA、Anti-Prn的ELISA方法。结果建立了三种定量检测ELISA方法,并进行方法学验证。结果显示,定量检测Anti-PT的ELISA方法的最低检测限度为1.31IU/ml(国际单位/毫升),批内和批间变异系数(Coefficient of Variability,CV)分别为9.99%和11.55%,回收率为97.19%;检测Anti-FHA方法的最低检测限度为1.02IU/ml,批内和批间CV分别为11.01%和12.76%,回收率为101.20%;检测Anti-Prn方法的最低检测限度为0.51IU/ml,批内和批间CV分别为9.00%和11.18%,回收率为107.83%。用建立的ELISA方法与国外同类方法对30份血清样本进行分析,检测结果差异无统计学意义。应用上述建立的三种ELISA方法,检测分析了三组份无细胞百日咳疫苗(Acellular Pertussis Vaccine,aP)基础免疫后免疫原性。结论所建立的百日咳血清抗体检测ELISA方法,准确度高和重复性好,无需特殊仪器,适合于一般实验室开展,可用于接种aP后血清学效果观察和百日咳流行病学研究。

Objective To develop three Enzyme-linked Immunosorbent Assay（ELISA）methods for quantitative determination of IgG antibody against pertussis toxin（PT）, filamentous hemagglutinin （FHA）, pertactin（Prn）in human serum, respectively. Method Purified PT, FHA and Prn and international reference pertussis serum were used as coating antigen and standard for development of three ELISA assays. Results Three ELISA assays had been established for determining anti-PT, FHA and Prn antibody, respectively. The limit of quantification of the assays had been demonstrated for anti-PT antibody with 1.31IU/ml, for anti-FHA antibody with 1.02IU/ml, for anti-Prn antibody with 0.51IU/ml. The average intra-assay coefficient of variation of three assays was 9.99%、11.01% and 9.00%. The average interassay coefficient of variation of three assays was 11.55%、12.76% and 11.18%, respectively. The average recovery rates of three assays were 97.19%、101.20% and 107.83%, respectively. Three antibody levels in 30 serum sample were detected by ELISA methods developed in this study, which were not difference significantly in comparison with that of similar method from other country. These methods were also applied for immunogenicity analysis of three-component acellular pertussis vaccine in the clinical trial. Conclusion Three quantitative ELISA methods had been demonstrated to be simple, accurate and reproducible. They were used for evaluation ofimmunogenicy of acellular pertussis vaccine and investigation of pertussis epidemiology.