基于鸡白痢沙门菌LPS的D群沙门菌抗体间接ELISA检测方法的建立

Establishment of indirect ELISA method for detecting antibody of group D Salmonella based on the LPS antigen of Salmonella pullorum

通过热酚水法提取并纯化鸡白痢沙门菌的脂多糖(lipopolysaccharide,LPS),以LPS作为包被抗原,建立D群沙门菌抗体间接ELISA检测方法。结果显示,通过方阵滴定法等确定抗原包被质量浓度为2.233 mg/L;血清最佳稀释度为1∶200,最佳作用时间为60 min;最适封闭条件为5%脱脂乳封闭60 min;酶标二抗最适工作质量浓度为1∶8000,作用60 min;底物显色时间为15 min;阴阳性判定标准为S/P值≥0.5。特异性、敏感性和重复性验证表明,该方法可特异性检测鸡白痢沙门菌等D群沙门菌的阳性血清,与鼠伤寒沙门菌、大肠杆菌等家禽病原菌的阳性血清无交叉反应,其检测灵敏度是鸡白痢平板凝集的400倍,批间重复和批内重复变异系数均小于10%。对临床血清的检测结果表明,该方法与Biochek公司的D群抗体检测试剂盒的阳性符合率为94.67%,阴性符合率为98.11%,总符合率为98.59%。结果表明,该检测方法可用于临床鸡白痢沙门菌等D群沙门菌感染的抗体检测。

The lipopolysaccharide(LPS)of Salmonella pullorum was extracted and purified by hot phenol water method.An indirect ELISA method for detecting the antibody against group D salmonella infection was established using LPS as the coating antigen.The coating concentration of LPS was determined to be 2.233 mg/L by checkerboard titration experiment;the optimal dilution of serum to be tested was 1:200,and the optimal action time was 60 min;the optimum sealing condition was 5%skim milk for 60 min;the optimum working mass concentration of HRP-labeled rabbit anti-chicken IgG antibody was 1:8000,and the action time was 60 min;the color development time of substrate was 15 min;the criterion between negative and positive samples was S/P value≥0.5.Results from the specificity,sensitivity and repeatability tests showed that this method could specifically detect the positive serum of group D Salmonella,such as Salmonella pullorum,and had no cross-reaction with the positive serum of other poultry pathogens,such as Salmonella typhimurium and Escherichia coli.The detection sensitivity was 400 times than that of the plate agglutination test for pullorum disease,and the variation coefficients of inter-and intra-batch repeats were lower than 10%.The detection results of clinical serum showed that the positive coincidence rate between our method and BioChek group D antibody detection kit was determined as94.67%,the negative coincidence rate was 98.11%,and the total coincidence rate was 98.59%.This study showed that our detection method could be used for detection of antibodies of Salmonella pullorum,and other group D Salmonella infections.