Ⅰ群禽腺病毒间接33K-ELISA检测方法的建立

Establishment of indirect ELISA for detection of antibodies against fowl adenovirus group Ⅰ(FAVI)

【目的】建立一种鉴别I群禽腺病毒(FAVI)灭活苗免疫和野毒感染的检测方法,为FAVI的防控与净化提供技术支撑。【方法】以FAVI33K重组蛋白为包被抗原,筛选和优化间接ELISA的条件,建立FAVI抗体的间接33K-ELISA检测方法,检验其特异性、敏感性和重复性,并鉴别检测FAVI活病毒感染血清和灭活苗免疫血清各50份。【结果】33K重组抗原最佳包被浓度6.0μg/mL,包被条件为37℃孵育1h后4℃过夜,最佳封闭液为5%脱脂奶,封闭时间1.0h;待检血清稀释度1:100,抗原抗体最佳作用时间1.0h;酶标二抗最佳稀释度1:3000,作用时间45min。优化后的间接33K-ELISA的阴、阳性临界值为0.316。其特异性、敏感性、重复性试验及鉴别检测结果表明,优化后的间接33K-ELISA具有特异性强、灵敏度高、重复性好等优点,且能成功鉴别FAVI感染与灭活疫苗接种。【结论】间接33K-ELISA操作简便、特异性强、灵敏度高、重复性好、适于批量检测,可有效鉴别FAVI感染和灭活苗免疫接种,利于挑选出FAVI感染动物,为FAVI的防控与净化提供了新思路和新方法。

[Objective]The aim of the present experiment was to establish a detection method to differentiate infected animals from inactive-and-vaccinated animals in the fowl adenovirus group I（FAVI） in order to provide technical support for controlling and purifying FAVI.[Method]Using nonstructural protein 33K recombinant protein as coating antigen,indirect 33K-ELISA was constructed through screening and optimizing reaction conditions;its specificity,sensitivity,and repeatability were tested and used to detect fifty infected and fifty inactive-and-vaccinated sera,respectively.[Result]The optimal concentration of coating antigen was 6.0 μg/ml,and the optimal coating condition was incubating at 37℃ for 1h and then coating overnight at 4℃.The optimal blocking buffer was 5% skimmed milk with 1.0 h blocking time.The optimal dilution of serum vs.enzyme labeled antibody were 1：100 and 1：3000,respectively.The optimal reaction time for coating antigen with serum and enzyme labeled antibody were 60 min and 45 min,respectively.With critical value of 0.316,the optimized 33K-ELISA was proved to be of favorable specificity,high sensibility,and excellent stability;it could also successfully distinguish the infected animals from inactive-and-vaccinated animals of FAVI.[Conclusion]Indirect 33K-ELISA,which can differentiate infected ones from inactive-and-vaccinated animals,is straightforward,specific,sensitive,stable,and suitable for batch detection.Due to its multiple positive traits,indirect 33K-ELISA introduced new viewpoints and methods for FAVI control and purification.