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目的建立检测伤寒沙门菌(Salmonella typhi)抗体的间接ELISA方法,并进行验证。方法对常规间接ELISA方法进行优化,确定抗原最适包被质量浓度及被检血清最佳稀释度、最适包被温度及时间、包被抗原与血清的最适温度和时间、血清与酶标二抗及封闭的最适温度和时间、酶标二抗最适工作质量浓度,并验证该方法的灵敏度、精密性、特异性及耐用性;用伤寒Vi多糖结合物原液和伤寒Vi多糖衍生物经小鼠大腿内侧皮下免疫,分别于免疫后第14、21、28、35天,经眼眶采全血,分离血清,应用建立的间接ELISA方法检测抗体水平,计算抗体几何平均滴度(GMT)。结果确定了间接ELISA方法最适工作条件:伤寒Vi荚膜多糖抗原最适包被质量浓度为5μg/ml,血清最佳稀释倍数为1∶40;最适包被温度及时间为4℃12 h;包被抗原与血清最适反应温度和时间为37℃2 h;血清与酶标二抗最适反应温度和时间为37℃1 h;封闭液最适封闭温度和时间为室温1 h;HRP标记的羊抗鼠IgG最佳反应浓度为1∶8 000。该方法灵敏度为1∶1 600;孔间和板间CV平均值分别为2.8%和5.8%;与其他血清无交叉反应;pH值、包被时间、包被温度及酶结合时间调整前后,同一样品检测的A450值差异无统计学意义(P>0.05)。伤寒Vi多糖与蛋白载体偶联后,免疫原性明显增强,且白喉类毒素(diphtheria toxin DT)和重组铜绿假单胞菌外毒素A(recombinantPseudomonasaeruginosaexotoxinA,rEPA)两种不同载体偶联得到的结合物免疫原性无明显差异。结论成功建立了一种检测伤寒沙门菌抗体的间接ELISA方法,该方法具有较强的特异性、灵敏度及精密性。
Objective To establish an indirect ELISA method for detecting Salmonella typhi antibody and verify the results. Methods The conventional indirect ELISA method was optimized to determine the optimal concentration of antigen and the optimal dilution of serum to be tested, the optimum temperature and time of coating, the optimum temperature and time of coating antigen and serum, The optimal temperature and time of secondary antibody and blocking, the optimal working concentration of enzyme-labeled secondary antibody, and verify the sensitivity, precision, specificity and durability of the method; with the combination of tyrosinase Vi polysaccharide conjugate and tyrosinase Vi polysaccharide derivatives The mice were immunized subcutaneously on the medial thighs on the 14th, 21st, 28th and 35th day after immunization respectively. The whole blood was collected through the orbit and the serum was separated. The indirect ELISA was used to detect the antibody level. The geometric mean antibody titer (GMT) . The results showed that the optimum conditions of indirect ELISA were as follows: the optimum concentration of coating for Vi-capsular polysaccharide of typhoid Vi was 5μg / ml, the optimal dilution of serum was 1:40; the optimum temperature and time of incubation were 12 ℃ for 4h ; The optimum reaction temperature and time of coating antigen and serum were 37 ℃ for 2 h; the optimum reaction temperature and time of serum and enzyme labeled secondary antibody was 37 ℃ for 1 h; the optimal closing temperature and time of blocking solution was 1 h at room temperature; HRP The optimal reaction concentration of labeled goat anti-mouse IgG was 1: 8000. The sensitivity of this method was 1: 1 600; the average CV between wells and plates was 2.8% and 5.8% respectively; no cross-reaction with other serums; pH, coating time, coating temperature and enzyme binding time were the same There was no significant difference in A450 values between samples (P> 0.05). After conjugation of tyrosinase Vi with protein carrier, the immunogenicity was significantly enhanced and the conjugate obtained by diphtheria toxin DT conjugate with recombinant Pseudomonas aeruginosa exotoxin A (rEPA) Immunogenicity no significant difference. Conclusion An indirect ELISA method for the detection of Salmonella typhi was successfully established. The method has strong specificity, sensitivity and precision.