马动脉炎病毒双抗体夹心ELISA检测方法的建立

Development of a double antibody sandwich ELISA for detection of equine arteritis virus

为建立马动脉炎病毒(EAV)的快速检测方法,本实验以EAV N蛋白单克隆抗体(MAb)2B9为捕获抗体,辣根过氧化物酶(HRP)标记的EAV N蛋白MAb 2B3(HRP-2B3)为检测抗体,建立EAV双抗体夹心ELISA检测方法。结果显示,该方法的最佳工作条件为:MAb 2B9的包被浓度为2μg/mL,HRP-2B3的工作浓度为2μg/mL,以OD450nm≥0.124为阳性判定标准。该方法对马传染性贫血病毒、马疱疹病毒I型和IV型、马流感病毒等均无交叉反应,特异性好;最低检出限为病毒标准品200倍稀释(103.2TCID50),敏感性高。3次重复试验建立的标准曲线的相关系数(R2)均大于0.997。本实验建立的EAV双抗体夹心ELISA方法具有特异性好、敏感性高等优点,适于EAV的快速检测。

To develop a method for EAV detection, a double-antibody sandwich ELISA （DAS-ELISA） was developed using MAb 2B9 against EAV nucleoprotein as capture antibody and MAb 2B3 conjugated to HRP （HRP-2B3） as detecting antibody under the optimized reaction conditions of coating with MAb 2B9 at 2 μg/well, detecting with HRP-2B3 at 2 μg/well with a cutoff value of 0.124 （OD450nm）. The specificity test shown the DAS-ELISA was specifically detected EAV with a detection limit of 103.2 TCIDt0, but no crossing-reaction to equine herpes virus I, equine herpes virus IV, equine influenza virus and equine infectious anemia virus. In addition, the standard curve of EAV quantification by the DAS-ELISA had linear correlation with three times repeated tests and the R2 were above 0.997. These results demonstrated that the DAS-ELISA method was specific and sensitive, which had a potential to be applicable for the EAV detection in horse.