Development and verification of specific serum bactericidal assay in vitro for dysentery serum antibody
10.13200/j.cnki.cjb.004340
- VernacularTitle:痢疾血清抗体特异性体外血清杀菌试验方法的建立及验证
- Author:
HU Xiaohua
- Publication Type:Journal Article
- Keywords:
Shigella dysenteriae;
Serum bactericidal assay(SBA);
Working bacteria stocks;
Complements
- From:
Chinese Journal of Biologicals
2024;37(11):1354-1360+1366
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop and verify a specific serum bactericidal assay(SBA) in vitro for dysentery serum antibody,in order to evaluate the immune efficacy of dysentery bivalent conjugate vaccine.Methods A standardized SBA was developed by identification of strains, preparation of working bacteria stocks, determination of the percentage recovery rates of working bacteria stocks and complement screening according to the reference method published by WHO reference laboratory in US University of Alabama at Birmingham(UAB), and verified for the specificity, linearity, precision as well as robustness. The titers of 20 pairs of serum samples were determined using the developed SBA method.Results The strains were confirmed to be Shigella flexneri 2a(S.flexneri 2a) and Shigella sonnei(S.sonnei), and their working bacteria stocks were constructed respectively. The percentage recovery rates of working bacteria stocks of the two types were 92% and 94% after cryopreservation for more than 2 h, and 91% and 96% after cryopreservation for 6 months, respectively. The both working dilutions were determined to be 20 000, and the numbers of surviving bacteria were 115 and 109 CFU/spot respectively at this working dilution. Two batches(07634EL and 011834EL) of complements could be used as working complements. The CV of reproducibility of the method was less than 30%, and the inter-assay CV was less than 50%, indicating that the reproducibility and intermediate precision of the method were good. The bactericidal titers of the two serotypes were completely inhibited when the concentrations of homologous dysentery polysaccharide were 50 and 40 μg/mL, respectively, while the inhibition was still lower than 20% even when the concentration of heterologous dysentery polysaccharide increased to 200 μg/mL, which indicated that the method had a good specificity. The logarithmic value of bactericidal titer and the logarithmic value of initial dilution multiple of both two serotypes were significantly negatively correlated, with R~2 of 0.997 4 and 0.996 2, respectively.The antibacterial titers of S.flexneri 2a and S.sonnei antibodies increased significantly compared with those before immunization.Conclusion A standardized SBA in vitro for dysentery specific antibody was successfully developed, which can be used to assess the immune efficacy of dysentery bivalent vaccine.
