产气荚膜梭菌β2毒素单克隆抗体间接ELISA检测方法的建立

Establishment of indirect ELISA assay method for monoclonal antibody against Clostridium perfringensβ2 toxin

以产气荚膜梭菌β2毒素重组蛋白为包被抗原,筛选出4株具有ELISA检测功能的单克隆抗体,并选取其中1株(9/1E23)作为一抗,以HRP-羊抗鼠IgG为二抗,建立并优化产气荚膜梭菌β2毒素抗体间接ELISA检测方法。最终确定抗原的最佳包被浓度为0.5μg/mL,抗体的稀释比为1∶2^(10)(0.029μg/μL),二抗稀释比为1∶2000,封闭条件为5%的脱脂牛奶37℃封闭2 h,试验结果表明,该方法可特异性的识别β2毒素抗体,敏感性可达2^(14)且具有良好的批间和批内稳定性。利用所建立的间接ELISA检测方法,对临床收集的201份正常牛血清、2份阳性血清和1份阴性血清样品进行ELISA检测,阴阳性符合率均为100%。研究建立的ELISA检测方法能够用于检测动物血清中产气荚膜梭菌β2毒素抗体的水平,为检测动物的产气荚膜梭菌感染情况提供有效的检测手段,在临床血清流行病学调查和疫苗免疫效果评价方面具有广泛应用前景。

The expressed and purified Clostridium perfringensβ2 toxin recombinant protein was used as the coating antigen,and four monoclonal antibodies(McAbs)with ELISA detection function from 21 strains of Clostridium perfringensβ2 toxin monoclonal antibodies were selected.Then using the recombinantβ2 toxin protein as the coating antigen,an indirect ELISA method for the detection of Clostridium perfringensβ2 toxin antibody was established by optimizing the reaction conditions.And it was finally determined that the optimal coating concentration of the antigen was 0.5μg/mL,the dilution ratio of the antibody was 1∶2^(10)(0.029μg/μL),the dilution ratio of the secondary antibody was 1∶2000,and the blocking condition was 5%.The skimmed milk was blocked at 37℃for two hours.The test results showed that the method had good specificity,sensitivity and stability.Using the established indirect ELISA detection method,201 normal bovine sera,two positive sera,and one negative serum were tested by ELISA.Both the negative coincidence rate and the positive coincidence rate were 100%.The ELISA detection method established in this study can be used to detect the level of Clostridium perfringensβ2 toxin antibody in animal serum,which provides an effective detection method for understanding animal cPB2 toxin infection and antibody level,and has broad application prospect in clinical seroepide miological investigations and vaccine immunity evaluation.