RÉSUMÉ
Objective@#To establish a functional antibody detection method for acellular pertussis vaccines in order to conveniently and effectively evaluate the production consistency and potency of acellular pertussis vaccine bulks and final products.@*Methods@#Chinese hamster ovary (CHO) cell clustering assay was optimized and used to measure titers of neutralizing antibodies against pertussis toxin in mouse immune serum samples.@*Results@#Vaccine samples were determined to be immunized intraperitoneally with 1/5 the human dose to ten female NIH mice (20-24 g, 5-week-old). Four weeks after immunization, blood samples were collected to isolate serum. Serially diluted serum samples were used to neutralize 0.1 IU/ml of pertussis toxin national reference product for 2 hours. Results of clustering were determined after 48 hours of incubation in pre-cultured CHO cell wells. The geometric mean of the serum dilution of the final unclustered wells was the neutralizing antibody titer of vaccine sample. There were significant differences in the titers of neutralizing antibodies elicited by acellular pertussis vaccines prepared with different manufacturing processes. Vaccine samples succeed or failed the modified intracerebral challenge assay (MICA) were easily distinguishable by neutralizing antibodies.@*Conclusion@#The method of detecting neutralizing antibodies to pertussis toxin greatly reduces the amount of animals used in research. CHO cell clustering assay that has better repeatability and precision can be used for monitoring and initial evaluation of the consistency and potency of the bulks and final products of pertussis vaccines prepared with different manufacturing processes.
RÉSUMÉ
Objective To understand serotype and fimbriae-genotype of B. pertussis vaccine strains and isolates from different periods in China. Methods Serotype of eighty isolates and three vaccine strains were determined using anti-fim2 and fim3 monoclonal antibodies compared with polyclonal antisera. Fim2 and fim 3 genes were amplified by PCR and the amplified products were sequenced and analyzed . Results The serotype of three vaccine strains and all isolates but only one tested by the slide agglutination and micro-plate assay of anti-fim2 and fim3 monoclonal antibodies were the same in comparison with that of the slide agglutination of polyconal antisera. In this study, seventeen isolates and vaccine strains CS and P3S10 were fim2&3 serotype, and forty-eight isolates were tim2 serotype while fifteen isolates and vaccine strain 18530 were fim3 serotype. The predominant serotypes were fim2 and fim2&3 before Expanded Program on Immuni-zation in 1978, while the find became the most popular serotype after nation-wide pertussis vaccination in China. The fim2-1 and fim3-A genotype was the most common type, which was identified in 92.5% and 95.0% of the isolates, respectively. The genotype of vaccine strain 18530 was fim2-2 and fim3-A while oth-er vaccine strains were fim2-1 and fim3-A. The isolates contained fim3-B and fim3-D subtypes were found since 2000. These data indicated that the serotype and fimbriae genotype of B. pertussis isolates have been changed for immune environment of national-wide pertussis vaccination in China. Conclusion The validity and specificity of anti-fim2 and fim3 monoclonal antibodies have been validated for serotyping of B. pertussis strains. The information of serotype and fimbirae genotype of B. pertussis vaccine strains and isolates from dif-ferent time periods have been obtained. These data can facilitate the studies on quality control of vaccine strain, epidemiology and the evolution of B. pertussis in China.
RÉSUMÉ
Objective To establish a rapid,accurate,specific quantitative assay for detecting B.pertussis,and apply to clinical diagnosis.Methods According to the specific sequence of B.pertussis IS481 gene,the primers and the fluorescence probe were designed and synthesized.Then a fluorescence quantitative PCR for detecting B.pertussis was developed.The specificity,sensitivity and reproducibility of the method were evaluated.255 specimens including 225 nasopharyngeal swabs from suspected pertussis patients and 30 normal nasopharyngeal swabs were detected by fluorescence quantitative PCR.Results A rapid specific quantitative method for detecting B.pertussis was established.The standard curve of the method indicated that there was a good linear relationship between the CT value and the template concentration with the correlation coefficient being 0.998.The linear range of the system was from 102 to 108 copies/μl and the minimum was 102 copies.It had a high sensitivity and good specificity.The intra.and inter-assay coefficients of variation were 5.78%-16.7% and 8.25%-14.9% respectively.The fluorescence quantitative PCR identified 41 positive results for specimens from suspected pertussis patients and results of 30 normal specimens were all negative.Conclusions The method can quantitatively detect the B.pertussis rapidly with high sensitivity and specificity,it can be applied to clinical diagnosis.