牛D型产气荚膜梭菌肠毒血症间接ELISA诊断方法的建立

Development of an indirect ELISA for diagnosis of bovine enterotoxemia resulted from Clostridium perfringens type D

根据菌体蛋白可以刺激机体产生相应抗体的原理,建立了以产气荚膜梭菌粗提菌体蛋白为抗原的检测牛D型产气荚膜梭菌抗体的间接ELISA。结果显示,D型产气荚膜梭菌菌体裂解抗原最适包被浓度为50μg/mL,最适包被条件为37℃1 h;被检血清的最适稀释度为1∶100,酶标二抗的最适工作浓度为1∶1 000,血清与酶标二抗的反应时间均为37℃1 h;最适封闭时间为37℃1 h;PBST稀释液中脱脂乳的浓度为30 g/L;底物最适反应条件为室温25 min;阴阳性临界值为0.032。对采集于吉林省延边州的100份牛血清样品采用建立的间接ELISA进行了检测;结果,阳性率为11.0%。经特异性试验、重复性试验和与琼脂扩散抗体检测法比较,采用裂解菌体蛋白为抗原建立的ELISA方法具有良好的特异性、敏感性和重复性。

Based on the principle that tropina make animal body produce antibody,an indirect ELISA for detection of the antibody against bovine Clostridium perfringens type D was developed with the tropina from C. perfringens. In the indirect ELISA, the optimal concentration of the antigen from C. perfringens was 50 μg/mL,the optimal coating condition was for 1 h at 37 ℃ ,the dilution rate of serum sample was 1 ： 100,the optimal working concentration of the HRP-labeled rabbit anti-bovine IgG was 1 ： 1 000,sera and rabbit anti-bovine IgG should be incubated for 1 h at 37 ℃ ,the optimal confining liquid was 30 g/L skimmed milk in PBST,substrate should be incubated for 25 min at room temperature,and the threshold of the negative to the positive was 0. 032. The 100 bovine serum samples collected from Yanbian Prefecture of Jilin Province were detected by the indirect ELISA,and the positive rate of the samples was 11.0%. The specificity experiment,reproducibility experiment and comparison experiment with agar diffusion test indi- cated that the indirect ELISA possessed satisfactory specificity, sensibility and reproducibility.