基于E^rns蛋白的猪非典型瘟病毒间接ELISA检测方法的建立

猪非典型瘟病毒(APPV)是新近发现的仔猪先天震颤的病原体,其血清学检测方法亟待建立.E^rns蛋白是APPV诱导机体产生中和抗体的重要保守性抗原,是血清学检测的特异性靶标之一.本研究制备了猪非典型瘟病毒原核表达的Erns蛋白,以其作为包被抗原,建立了间接ELISA检测方法.结果表明,最佳抗原包被浓度为8μg/mL,最佳血清稀释度为1:10,阴阳性临界值D450=0.318,灵敏度达到1:64,特异性好,与猪瘟、猪伪狂犬、猪繁殖与呼吸障碍综合征、猪流感及圆环病毒病的阳性血清均不发生交叉反应.该检测方法重复性好,批内变异系数最大值为7.84%,批间变异系数最大值为8.57%.利用建立的间接ELISA方法对50份猪临床血清样本进行了检测,其阳性率为10%.基于Erns蛋白间接ELISA检测方法的建立为APPV的流行病学调查和临床诊断提供了有效的工具.

DEVELOPMENT OF AN INDIRECT ELISA METHOD OF DETECTION OF ANTIBODIES TO E^rns PROTEIN OF ATYPICAL PORCINE PESTIVIRUS

Atypical porcine pestivirus(APPV)is a newly discovered virus causing congenital tremor in piglets and a serological method is urgently needed for diagnosis.E^rns protein of APPV is an important conservative antigen that induces neutralizing antibodies and makes it to be one of the specific targets for serological detection.In this study,the recombinant E^rns protein obtained from prokaryotic expression was used as the coating antigen for development of an indirect ELISA method.The optimal coating antigen concentration was determined to be 8μg/mL and serum dilution was 1/10.The positive and negative critical ratio was D450=0.318 and the sensitivity of the indirect ELISA was 1/64.This ELISA method had no cross reactions with classical swine fever,porcine pseudorabies,porcine reproductive and respiratory syndrome,swine influenza and porcine circovirus disease,indicating that it had a good specificity.The coefficient of maximum variation was 7.84% and 8.57% in intra-and inter-assays,respectively,showing that the detection method was low variability.Clinical porcine serum samples were detected using this indirect ELISA method and the positive rate was 10%(5/50).The development of the indirect ELISA method based on Erns protein provided an effective tool for epidemiological investigation and clinical diagnosis of congenital tremor in piglets.