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

Development and verification of specific serum bactericidal assay in vitro for dysentery serum antibody

目的为评价痢疾双价结合疫苗的免疫效果,建立痢疾血清抗体特异性体外血清杀菌试验(serum bactericidal assay,SBA)方法,并进行验证。方法参考美国阿拉巴马大学研究的痢疾特异性SBA方法,进行菌种鉴定及工作菌库的构建、冻存工作菌复活率测定、补体筛选,建立本实验室痢疾血清抗体特异性SBA方法,并对方法的精密性、特异性、线性、耐用性进行验证。采用建立的方法对20对痢疾临床血清样本进行SBA检测。结果确定菌株分别为福氏2a志贺菌(S.flexneri 2a)和宋内志贺菌(S.sonnei),成功构建了工作菌库,2个血清型工作菌冻存2 h以上冻存复活率分别为92%和94%,冻存6个月冻存复活率分别为91%和96%;确定工作稀释度均为20000,在该稀释度下,存活的菌数分别为115和109 CFU/spot。2批(07634EL和011834EL)补体均可作为工作补体。该方法重复性变异系数(CV)<30%,试验间CV<50%,表明方法的重复性和中间精密性较好;两种血清型的杀菌滴度在同源痢疾多糖浓度分别为50和40μg/mL时达到完全抑制,而异源痢疾多糖浓度即使增加至200μg/mL,抑制均仍低于20%,表明方法的特异性较好;两种血清型杀菌滴度的对数值与起始稀释倍数的对数值均呈显著负相关,R^(2)分别为0.9974、0.9962。福氏2a志贺菌、宋内志贺菌抗体杀菌滴度与免疫前相比均明显增加。结论成功建立了标准化的痢疾特异性抗体体外SBA方法,此方法可用于评价痢疾双价苗的免疫效果。

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.