检测鸡传染性鼻气管炎病毒抗体的间接ELISA方法的研究

Development of an Indirect ELISA for Detection Antibody of Avian Rhinotracheitis Virus

分别用绿猴肾细胞和鸡胚成纤维细胞增殖鸡传染性鼻气管炎病毒NL 7784株 ,提纯抗原建立了检测鸡传染性鼻气管炎病毒抗体的间接酶联免疫吸附试验 (ELISA)。试验的最佳反应条件为 :抗原最佳稀释度为 1∶16 0 ,酶标抗体为 1∶10 0 0稀释 ,待检血清为 1∶16 0稀释 ,包被液为pH9.6的碳酸盐缓冲液 ,6 %犊牛血清 (CS)作封闭液 ,稀释液用含 10 %CS的Tween_2 0磷酸盐缓冲液 ,洗涤液用含 0 .5 %Tween_2 0的磷酸盐缓冲液 ,抗原与抗体的最佳反应时间为 30分钟 ,底物的反应时间为 15分钟。与中和试验进行了比较 ,证明所建立的方法具有良好的特异性、稳定性、可重复性和较高的敏感性。

Avian rhinotracheitis virus was grown in monolayer of chick embryo fibroblasts(CEF),Vero cells and tracheal_ring organ cultures(TROC).An indirect enzyme_linked immunosorbent assay(ELISA)for detecting ARTV antibody had been devloped by coating plate with ARTV NL 7784 strain propagated in Vero cells and purified with concentration.The test conditions were optimized by reagent titration utilizing known positive and negative sera.A positive/negative(P/N)value larger than 2.1 was determined as comprehensive positive standard for the ELISA The performance of the ELISA was assessed for reproducibility and specificity.The results showed that the ELISA had good reliability.