鸡传染性支气管炎病毒抗体间接ELISA检测方法的建立与初步应用

Establishment and preliminary application of an indirect ELISA for detection of antibodies against infectious bronchitis virus

建立了一种检测鸡传染性支气管炎病毒(infectious bronchitis virus,IBV)抗体终点滴度(end-point titer,ET)的间接ELISA方法,以应用于监测鸡免疫疫苗或者感染野毒后的抗体。以大肠杆菌表达的IBV核衣壳(N)蛋白作为包被抗原,以SPF鸡血清和感染IBV的SPF鸡血清分别作为阴性血清和阳性血清对照,优化确定出ELISA的各项技术参数。对66份SPF鸡血清进行测试,确定了阴、阳性S/P临界值为0.385。特异性试验表明包被抗原与其他常见病原的阳性血清均不发生交叉反应,批内和批间重复性试验的变异系数均小于10.00%。选取了50份临床血清样品,通过阳性-阴性阈值(positive negative threshold,PNT)基线确定ET值,建立了固定血清稀释倍数(1∶500)处S/P值与ET的线性回归方程,相关系数(R2)为0.9593(P<0.0001)。通过检测2个商品鸡群在1~49日龄不同时间点采集的461份血清样本,比较了建立的IBVN-ELISA方法与IDEXX公司提供的商品化试剂盒。本研究建立的IBVN-ELISA方法与该商品化试剂盒相比总体符合率为93.06%,抗体滴度的消涨趋势基本一致,ELISA的敏感性和特异性分别达到99.75%和100.00%(P<0.0001)。结果表明,本研究建立的ELISA方法是一种良好的检测方法。

An indirect ELISA method for the end-point titer(ET)detection of infectious bronchitis virus(IBV)antibodies was established to monitor the antibody in chicken vaccinated or infected by field virus.IBV nucleocapsid(N)protein expressed in E.coli was used as coating antigen,serum from SPF chickens and the SPF chickens infected by IBV were used as negative and positive control,respectively.Parameters of ELISA method were optimized.The cutoff value of negative and positive S/P was determined to be 0.385by testing 66SPF chicken sera.Specificity assay showed that there was no cross reaction between coating antigen and serum of other common avian pathogens,and the coefficients of variation in both intra-and inter-batch repeatability tests were less than 10.00%.Fifty serum samples were selected to determine ET of IBV-specific antibodies by positive-negative threshold(PNT)baseline,and the linear regression equation between S/P value and ET at fixed serum dilution ratio(1∶500)was established with a correlation coefficient(R2)of 0.9593(P<0.0001).Comparison of the established IBVN-ELISA method with commercial kits provided by IDEXX were performed to detect a total of 461serum samples collected from two commercial chicken flocks at different time points from 1to 49days of age.Compared with the commercial kit,IBVN-ELISA method had the overall coincidence rate of 93.06%with the trend of nearly consistent antibody titer variation.The sensitivity and specificityu12288 for the ELISA were 99.75%and 100.00%,respectively(P<0.0001).The above results showed that the ELISA method established in this study is a good detection method.