百日咳毒素、丝状血凝素、百日咳黏附素抗体间接ELISA检测方法的建立、验证及初步应用

Development,verification and preliminary application of indirect ELISA for antibody detection of pertussis toxin,filamentous hemagglutinin and pertussis adhesin

目的建立小鼠血清中百日咳毒素(pertussis toxin,PT)、丝状血凝素(filamentous hemagglutinin,FHA)及百日咳黏附素(pertussis adhesin,PRN)抗体间接ELISA定量检测方法,并进行验证及初步应用,为百日咳疫苗生产工艺的优化提供参考。方法分别用PT、FHA及PRN抗原包被酶标板,加入百日咳抗小鼠血清标准品及待测小鼠血清,以HRP标记的山羊抗鼠IgG作为酶标二抗,通过TMB显色的指示程度定量检测小鼠血清中PT、FHA、PRN抗体含量。对建立的间接ELISA检测方法进行专属性、线性范围、准确性、精密性进行验证。采用建立的方法对百日咳疫苗及效价合格的内部参比品免疫小鼠血清进行检测。结果采用PT、FHA、PRN抗体ELISA方法检测百日咳抗小鼠血清标准品及待测样品的结果为阳性,其余样本检测结果均为阴性。PT抗体ELISA检测方法的线性范围为0.0053~0.17 U/mL,回收率为90.91%~104.77%,重复性验证RSD为6.31%,人员间RSD为6.40%;FHA抗体ELISA检测方法的线性范围为0.0223~1.43 U/mL,回收率为95.84%~102.18%,重复性验证RSD为9.02%,人员间RSD为5.79%;PRN抗体ELISA检测方法的线性范围为0.0094~0.3 U/mL,回收率为86.27%~100.22%,重复性验证RSD为6.94%,人员间RSD为8.90%。采用建立的方法检测百日咳疫苗样品及效价合格的内部参比品免疫小鼠血清,6组样品PT抗体水平明显高于内部参比品,FHA、PRN抗体水平与内部参比品相当。结论建立的各抗体ELISA检测方法专属性强,线性相关性良好(R2>0.99),准确性高,精密性良好,可用于小鼠血清中百日咳(PT、FHA、PRN)抗体的定量检测,为百日咳疫苗生产工艺的优化提供了参考。

Objective To develop an indirect ELISA method for quantitative detection of antibodies against pertussis toxin(PT),filamentous hemagglutinin(FHA)and pertussis adhesin(PRN)in serum of mice,and verify and preliminarily apply the method in order to provide reference for the optimization of production process of pertussis vaccine.Methods Enzymelabeled plates were coated with PT,FHA and PRN antigens respectively,and the pertussis anti-mouse serum standard and mouse serum samples were added.Using goat anti-mouse IgG labeled with HRP as the enzyme-labeled secondary antibody,the contents of PT,FHA and PRN antibodies in serum of mice were quantitatively determined by the indication degree of TMB color development.The specificity,linear range,accuracy and precision of the developed indirect ELISA detection method were verified.The serum of mice immunized with pertussis vaccine and qualified internal reference products was detected by the developed method.Results The results of the pertussis anti-mouse serum standard and mouse serum samples detected by PT,FHA and PRN antibody ELISA were positive,while the other samples were negative.The linear range of ELISA for PT antibody was 0.0053-0.17 U/mL,the recovery rate was 90.91%-104.77%,the RSD of reproducibility verification was 6.31%,and the RSD among personnel was 6.40%.The ELISA for FHA antibody had the linear range of 0.0223-1.43 U/mL,the recovery of 95.84%-102.18%,the RSD of reproducibility verification of 9.02%,and the RSD among personnel of 5.79%.The ELISA for PRN antibody showed the linear range of 0.0094-0.3 U/mL,the recovery rate of 86.27%-100.22%,the RSD of reproducibility verification of 6.94%,and the RSD among personnel of 8.90%.The detection results of mouse serum immunized with pertussis vaccine and qualified internal reference products by the developed method showed that,the PT antibody levels of six groups of samples were significantly higher than those of the internal reference products,while the FHA and PRN antibody levels were equivalent to those of the internal reference products.Conclusion The developed ELISA methods have strong specificity,good linear correlation(R2>0.99),high accuracy and good precision,and can be used for quantitative detection of pertussis(PT,FHA,PRN)antibodies in mouse serum,providing a reference for the optimization of pertussis vaccine production process.