鸭源鸡杆菌抗体间接ELISA检测方法的建立及应用

利用鸭源鸡杆菌YU-PDS-RZ-1-SLG分离株制备超声波裂解抗原,建立了可以检测鸭源鸡杆菌多个血清型抗体的间接ELISA方法。包被抗原质量浓度为10mg/L,包被条件为37℃2h,再4℃过夜;封闭液为1%明胶,封闭条件为37℃1h;阴、阳性血清最佳稀释度为1∶100,酶标二抗工作滴度为1∶1 000;底物显色时间为15min。经交叉性试验、阻断试验和重复性试验证实建立的ELISA方法重复性好,特异性强。板内变异系数为2.01%～5.75%,板间变异系数为2.43%～6.20%。间接ELISA方法的灵敏度是微量凝集试验的25～100倍。利用所建立的ELISA方法检测了人工感染鸭源鸡杆菌的4日龄SPF鸡在感染后不同阶段感染组、同居组和空白对照组的血清抗体,并根据感染后不同阶段所测D450值绘制抗体消长曲线,其抗体水平在感染后32～47d开始上升,60d时达到高峰,但维持时间较短,2周后迅速下降。建立的间接ELISA方法可以用于临床病例的血清学快速检测,为进行鸭源鸡杆菌的血清流行病学调查提供了手段。

Development and primary application of indirect ELISA for detecting antibody against G. anatis

Indirect enzyme-linked immunosorbent assay(ELISA) was developed for detection of antibodies against all serotype Gallibacterium anatis(G.anatis) in chickens.The coating antigen was prepared by ultrasonic method with G.anatis strain YU-PDS-RZ-1-SLG.The optimum G.anatis antigen concentration was 10 mg/L,and coating time was 2 hours at 37℃ and then over night at 4℃.The blocking material was 1% gelatin and blocking time was 1 hour at 37℃.The dilution of the positive and negative sera was 1∶100,and the optimum dilution of rabbit-anti-chicken IgG labeled with horseradish peroxidase was 1∶1 000.The optimum reaction time of the enzyme-substrate was 15 min.The indirect ELISA was confirmed reproducible and specific by cross-assay,blocking-assay and reproductive assay.The coefficient of variation within plate was 2.01%-5.75%,and the coefficient of variation between plates was 2.43%-6.20%.The sensitivity of indirect ELISA was 25 to 100 times than that of micro-agglutination test.Twenty 4-day-old SPF chickens were used and divided into 3 groups,one group was inoculated with 1.4×106 colony-forming units of G.anatis strain,the other two groups were used as cohabitating group and negative control group,separately.The indirect ELISA was used for testing the antibody against G.anatis from these chickens in different time after infection.The response curve was established when the D450 nm values varied along with the time.The antibody level reached the peak at the 60th day post-infection,and then dropped rapidly.The results showed that the indirect ELISA could be used for detecting the serum antibody of chickens in the epidemiology survey of G.anatis.