禽白血病病毒ELISA检测方法的建立与初步应用

本研究以在毕赤酵母系统中表达并纯化的p27重组蛋白为包被抗原,HRP标记的兔抗鸡IgY为酶标二抗,建立了一种快速有效的禽蛋白血病病毒ELISA检测方法,并对其反应条件进行优化。结果显示,该方法具有良好的特异性和重复性;与市售商品试剂盒相比,符合率为96.92%,且更为敏感。应用建立的ELISA方法检测来自吉林省内鸡场314份疑似样品,阳性检出率为75.15%。本研究为禽白血病病毒的检测与诊断提供了一种简便、有效的检测方法。

DEVELOPMENT AND PRELIMINARY APPLICATION OF ELISA METHOD FOR DETECTION OF AVIAN LEUKOSIS VIRUS

In the present study,the p27 gene of avian leukemia was cloned into the Pichia pastoris vector pPIC9 K and the expressed protein was used for development of indirect ELISA method for detecting Avian leucosis virus.In this ELISA method,the purified protein was used as the coating antigen and horseradish peroxidase-labeled rabbit anti-chicken IgY as the secondary antibody.Then the ELISA method was optimized for the best performance.The optimal parameters included the coating antigen concentration at 2.5μg/mL,serum dilution at 1:200,blocking time for 1.5 h with 5%non-fat dry milk,HRP-labled rabbit anti-chicken dilution at 1:5000,incubation at temperature at 37℃for 1 h and development time for 12 min.Under these conditions,the cut value was determined as 0.1979.The ELISA method was applied to test 314 suspected samples from chicken farms in Jilin Province.The positive rate was 75.15%in general.This ELISA method developed here provided a simple and effective detection for avian leukemia virus.