中文摘要：

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。

英文摘要：

Objective To develop an indirect ELISA method for detection of Salmonella typhi antibody. Methods The condition for routine indirect ELISA,including antigen concentration for coating,dilution of serum to be tested,temperatures and times for coating,for reactions of coating antigen with serum and serum with enzyme-labeled secondary antibody and for blocking,as well as working mass concentration for enzyme-labeled secondary antibody,were optimized. The developed method was verified for sensitivity,precision,specificity and durability. Mice were immunized s.c. with bulk of typhoid Vi polysaccharide conjugate and derivative of typhoid Vi polysaccharide. Serum samples were collected from ten mice on days 14,21,28 and 35 after immunization respectively,and determined for antibody level by the developed indirect ELISA,based on which the GMTs of antibody were calculated. Results The optimal antigen concentration for coating,dilution of serum and temperature and time for coating were 5 μg / ml,1 ∶ 40,4 ℃ and 12 h respectively,while the optimal temperature and time for reaction of coating antigen with serum were 37 ℃ and 2 h,for reaction of serum with enzyme-labeled secondary antibody were 37 ℃ and 1 h,and for blocking were room temperature and 1 h,respectively.The optimal reaction concentration of HRP-labeled goat anti-mouse IgG was 1 ∶ 8 000. The sensitivity of the developed method was 1 ∶ 1 600. The mean CVs of determination results of test samples in various wells and on various plates were 2. 8% and 5. 8% respectively. No cross reactions with other sera were observed. The A450 values before and after the pH value, time for coating, temperature for coating and time for enzyme binding were tuned showed no significant differences（P 〉 0. 05）. The immunogenicity of typhoid Vi polysaccharide was enhanced after conjugation to protein carrier,while showed no significant difference after conjugation to diphtheria toxin（DT） and to recombinant Pseudomonas aeruginosa exotoxin A（rEPA）. Conclusion An indi