Development, verification and preliminary application of indirect ELISA for antibody detection of pertussis toxin, filamentous hemagglutinin and pertussis adhesin
10.13200/j.cnki.cjb.004318
- VernacularTitle: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
- Author:
SUN Bo
- Publication Type:Journal Article
- Keywords:
Indirect enzyme-linked immunosorbent assay(ELISA);
Pertussis toxin(PT) antibody;
Filamentous hemagglutinin(FHA) antibody;
Pertussis adhesin(PRN) antibody;
Quantitative detection
- From:
Chinese Journal of Biologicals
2024;37(10):1218-1224
- CountryChina
- Language:Chinese
-
Abstract:
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 Enzyme-labeled 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. 005 3-0. 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. 022 3-1. 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. 009 4-0. 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.
