以重组无乳链球菌GapC1-150作抗原的牛血清抗体间接ELISA检测方法建立

Establishment of an Indirect ELISA Method for Detection of Bovine Antibody in Seru Using Recombinant GapC1-150 of Streptococcus agalactiae as Antigen

为了检测奶牛乳腺炎无乳链球菌GapC1-150抗原相关疫苗免疫血清抗体,我们用重组蛋白GapC1-150作为包被抗原建立了间接ELISA牛血清抗体检测方法。通过方阵法确定包被抗原浓度和血清稀释度;通过对阴性牛血清和免疫牛血清的OD450值检测分析确定cut-off值;通过与多种抗原免疫血清和病原感染血清进行检测确定该方法具有良好的特异性;通过对阳性和阴性血清倍比稀释检测确定该检测方法灵敏性较好,并对临床收集的牛血清样本进行了检测。以上结果表明,所建立的间接ELISA检测方法可用于GapC1-150抗原相关疫苗免疫血清抗体检测。

In order to detect serum antibody in bovine immunized with vaccine candidate composed of recombinant GapC1-150 against bovine mastitis caused by Streptococcus agalactiae,an indirect ELISA for detection of serum antibody of the immunized bovine was established using recombinant protein GapC1-150 as the antigen.The concentration of coated antigen and serum dilution were deter-mined as 2.5 g·mL^-1 and 1∶1000.In addition,the indirect ELISA using gGapC1-150 as coating antigen was used for serological detection.Optimal conditions for the indirect ELISA detection were determined,including PBS(pH 7.2)as the best solution coating for 45 min under 37℃,1%BSA(PBST)as the best solution blocking for 1.5 h under 37℃,and the optimal incubation with serum for 30 min under 37℃.The cut-off value was determined according to mean OD450 values of negative serum and positive serum.The sera from the bovine immunized with vaccine candidate or antigens and sera collected from cow farms were tested were by the established indirect ELISA,and the serum from vaccine immunized bovine showed positive and others negative.These results indicated that the indirect ELISA was a sensitive,specific and suitable for the detection of antibody against the vaccine candidate.