基于FAdV-4中国流行株Fiber2蛋白的间接ELISA抗体检测方法的建立

Development of an indirect ELISA for antibody detection based a Chinese fowl adenovirus serotype 4 fiber2 protein

利用在大肠杆菌中表达的FAdV-4中国流行株Fiber2蛋白作为包被抗原，建立了检测抗禽腺病毒血清4型（FAdV-4）中国流行株抗体的间接ELISA方法。所建立方法的最佳条件为：抗原最适包被量为0．25μg／μL，包被时间为37℃1h，然后4℃过夜；被检血清的最佳稀释度为1：800，作用时间为2h；二抗选用HRP标记的兔抗鸡IgY，稀释度为1：4000，作用时间为1．5h；显色液TMB作用条件为室温5min。所建立ELISA的判定标准为：样品的S／P值大于或等于0．0553判为阳性，小于0．0418判为阴性。用建立的ELISA方法对抗鸡新城疫病毒、禽流感病毒、传染性法氏囊病病毒、传染性支气管炎病毒、减蛋综合征病毒阳性血清进行了检测，均无交叉反应，表明该方法有较好的特异性，组内和组间重复的最大变异系数为5．6％和5．7％，所建立的ELISA方法重复性良好。用所建立的ELISA方法对50份来自于疑似患肝炎-心包积液综合征病鸡的血清样品进行检测，阳性捡出率为98％，与FAdV-4病原学检测结果符合率为100%。结果表明：本试验所建立的ELISA方法可用于抗FAdV-4中国流行株抗体的检测，为肝炎-心包积液综合征病监测和流行病学调查提供了方法。

To establish a method for fowl adenovirus serotype 4（FAdV-4） epidemiological surveillance,an indirect ELISA was developed using the purified recombinant ChineseFAdV-4 fiber2 protein as coating antigen. The optimal reaction conditions were as follow.amount of coating antigen was 0.25 μg/well,and coating condition was at 37℃ for 1 h then 4℃ overnight ; dilution of serum sample was 1 ： 800,and incubated for 2 h;working dilution of second antibody was 1 ： 4 000,incubation time was 1.5 h;TMB incubation condition was 5 min at room temperature. Serum was determined as positive when its S/P≥O. 055 3 and negative when its S/P〈0. 041 8,respectively. There was no cross-reactivity between FAdV-4 antigen and anti-NDV,-H9/H5 subtype AIV,-IB- DV,-IBV and-EDSV serum,indicating that the method had a good specificity. The maximal intra- and inter-assay coefficient of variation was 5.6 % and 5.7 %,indicating that the method had a good reproducibility. Fifty serum samples from clinical suspected FAdV-4-infected chicken were detected for FAdV-4-specific antibody,the results showed the coincidence with those of FAdV-4 antigen detection was 100%. This indirect ELISA can be used for surveillance of FAdV-4 infection in chicken flocks.