间接竞争性ELISA法快速诊断TGEV的研究

利用基因工程技术制备的猪传染性胃肠炎病毒（TGEV）N基因原核表达产物重组N蛋白作为抗原诊断试剂，抗重组N蛋白的单克隆抗体作为抗体诊断试剂建立了间接竞争性ELISA检测细胞培养物中的TGEV病原的方法，并确定了间接竞争性ELISA操作流程中的最佳反应条件。在间接竞争性ELISA中，最佳抗原包被量为0．25卢g／孔；竞争抗原与单克隆抗体作用的最适时间为1h，反应温度为37℃；选择1h作为酶标抗体作用的最佳时间；底物液最佳作用时间为10min；选择样品的抑制率50％为其临界值；所用封闭液0．5％的聚乙烯醇PBS溶液在4℃冰箱中可密封保存6个月，封闭时间为120min；特异性试验表明与猪轮状病毒、猪流行性腹泻病毒等肠道腹泻性病毒均无交叉反应。本试验建立的间接竞争性ELIsA诊断方法具有良好的敏感性和特异性，为TGEV的疫情监测、及时而准确的诊断奠定了基础。

Detection of TGEV with indirect competitive ELISA

Three methods were established for rapid detection to TGEV using TGEV recombinant nucleoprotein（rNP）and MeAb against rNP. In indirect competitive ELISA the optimal amount of coated antigen was 0.25 μg per well; the optimal time in which MeAb reacted with competitive antigen was 1 h and the reaction temperature was 37℃ ; the reaction time to HRP was 1 h; the re- action time to substrate was 10 rain; the inhibited rate of 50% was the critical value. The optimal blocking solution was 0. 5G polyvinyl alcohol （PVA） of PBS, the blocking time was 120 min; blocking solution can be kept for 6 monthes at 4℃. The special test indicates TGEV had no cross- ing reaction with PRV and PEDV. The method for detection was rapid, sensitive, accurate and sim- ple. The result would be gained in 2-3 hours. The method for rapid detection to TGEV was estab- lished firstly by using rNp and MeAb against rNP in domestic, and founded a material base for TGE detection accurately and rapidly.