Ⅰ群禽腺病毒间接100K-ELISA检测方法的建立

Establishment of an indirect 100K-ELISA method for detection of antibodies against fowl adenovirus group Ⅰ

为了建立一种Ⅰ群禽腺病毒(FAVⅠ)感染的抗体间接ELISA检测方法,试验以FAVⅠ100K重组蛋白作为包被抗原,优化间接ELISA反应条件,建立了FAVⅠ感染产生抗体的间接100K-ELISA检测方法,检测FAVⅠ活病毒感染血清和灭活苗免疫血清各50份。结果表明:经优化筛选的最佳反应条件如下,包被抗原浓度为5.4μg/mL,包被条件为37℃作用1 h加4℃过夜,5%脱脂奶封闭1 h,血清稀释度为1∶100,作用时间为60 min,酶标二抗的最佳浓度为1∶3 000,作用时间为45 min。该方法的阴性、阳性临界值为0.345。96%感染动物血清检测结果为阳性,而98%灭活苗免疫动物血清检测结果为阴性。说明研究建立的100K-ELISA方法特异性强、灵敏度高、重复性好。

To establish an indirect EL1SA method for detection of infected antibody against fowl adenovirus group I （ FAV I ） , the 100K re- combinant FAV I protein was used as the coated antigen, and the ELISA reaction conditions were optimized, and then an indirect 100K - ELISA method was established for detecting antibody against FAV I in infected serum. The method was used to detect fifty infected and fifty in- activated immune serum samples,respectively. The results that the optimized reaction conditions were as follows： tile antigrn -coating concen- tration of 5.4 μg/mL, the coating condition at 37 ℃ for I h and 4 ℃ overnight, 1h blocking with 5 % skimmed milk, 1 ： 100 dilution of serum with the reaction time of 60 rain, and 1：3 000 HRP -labeled goat anti -chicken IgG with the reaction time of 45 min. The cut -off value of the indirect 100K -ELISA was 0. 345 for positive or negative results. The 96% of serum samples from the infected animals were positive, whereas the 98% of serum samples from the inactivated vaccinated animals were negativel It indicates that the established indirect 100K - ELISA meth- od has strong specificity, high sensitivity and good repeatability.