牛传染性鼻气管炎病毒重组gE蛋白间接ELISA诊断方法的建立

Indirect Enzyme-linked Immunosorbent Assay with Recombinant Glycoprotein E of Infectious Bovine Rhinotracheitis Virus

以重组牛传染性鼻气管炎病毒gE蛋白,经纯化后作为诊断抗原建立了检测牛血清特异性gE抗体的间接酶联免疫吸附试验,确定其最佳包被量为每孔1.075μg。样品稀释度为1:40,兔抗牛IgG辣根过氧化物酶标记抗体稀释度为1∶5000。判定标准为样品OD值大于0.582为阳性,小于0.472为阴性,在0.472与0.582之间为可疑。经抗特异性试验和重复性试验证明该方法特异性高、重复性好。在403份血清样品中,进口试剂盒检出234份阳性样品,该诊断方法检出248阳性样品,与进口试剂盒的符合率为94.3%,但该诊断方法检出率更高。在43份血清样品中,中和试验检出24份阳性样品,该诊断方法检出28份阳性样品,与中和试验的符合率为85.7%。应用该诊断方法调查了我国部分地区IBRV的感染率,发现这些地区的IBRV感染率为68.7%。

An indirect enzyme-linked immunosorbent assay （iELISA） was developed for detection of antibodies against infectious bovine rhinotracheitis virus（IBRV） using the purified recombinant gE protein. The optimum conditions for iELISA were determined. The concentration of the recombinant gE protein was 1. 075μg for coating ELISA plates ; the dilution folds of the rabbit anti-bovine IgG conjugated with perosidase and the sera were 1：5 000 and 1 ： 40 respectively. OD value of the sample higher than 0. 582 was determined as positive standard for the ELISA and lower than 0.472 as negative standard. The ELISA was confirmed to be specific and reproducible. In all 403 serum samples,234 were confirmed positive by the imported IBR ELISA kit. But 248 were confirmed positive by this method, 94.35% agreement was obtained by comparing to imported IBR ELISA kit and 85.71% by comparing to virus neutralization. In all 43 serum samples, 24 were confirmed positive by neutralization while 28 were confirmed positive by this method. The infection with IBRV in some districts of China was investigated by this iELISA. The result showed that the infection rate with IBRV in these districts was 68.74%.