一种检测乙型脑炎病毒抗体阻断ELISA的建立及其初步应用

Development and Preliminary Application of a Blocking ELISA for Detection of Japanese Encephalitis Virus Antibody

流行性乙型脑炎是由日本脑炎病毒(JEV)引起的蚊媒传播人畜共患传染病,严重危害人类和猪、马等动物的健康。为建立检测多种动物血清中JEV抗体的ELISA,应用重组囊膜蛋白和辣根过氧化物酶标记的JEV单克隆抗体建立了检测JEV抗体的阻断ELISA。结果显示:通过对100份JEV抗体阴性猪血清的检测结果进行统计学分析,确定阻断ELISA的判定标准:阻断率(PI)≥34%时判定为阳性,PI≤25%时判定为阴性,25%<PI<34%时判定为可疑。该阻断ELISA与其他常见猪病毒病抗体阳性血清无交叉反应;批内和批间重复试验的变异系数均小于5%;与商品化ELISA试剂盒的对比检测表明,敏感性为98.5%、特异性为94.3%,两者的符合率为97.2%。应用该阻断ELISA对临床收集的猪、牛和羊共515份血清样品进行检测,JEV抗体阳性检出率分别为74.5%、13.3%和9.2%,抽样的血清中和试验结果与阻断ELISA检测结果的符合率为100%(9/9)。本研究成功建立了可适用于猪、牛和羊临床血清JEV抗体检测的阻断ELISA方法。

Japanese encephalitis is a mosquito transmitted zoonosis caused by Japanese encephalitis virus(JEV)that seriously harms the health of human and animals,such as pigs and horses.In order to establish a specific method for detection the JEV antibody in various animal serum,a blocking ELISA method was established using recombinant EDⅢprotein and horseradish peroxidase(HRP)conjugated JEV specific Monoclonal antibody 1B10.Results were as follows:The percentage of interruption rate(PI)of 100 JEV antibody negative serum samples were statistically analyzed,the cut-off of blocking ELISA was determined as the sample presenting a calculated PI≥34%were considered positive;while samples with a calculated PI≤25%were rated negative;and those presenting PI between 25%and 34%were considered inconclusive.No cross-reaction with antibody positive serum against PCV2,PRV,CSFV,FMDV and PRRSV was observed in the test.And the intra-and inter-assay variation were less than 5%.Compared with the commercialization JEV antibody detection ELISA kit,the agreement rates of the new established blocking ELISA was 97.2%.The specificity and sensitivity of the blocking ELISA were 98.5%and 94.3%,respectively.The blocking ELISA was used to test against 515 serum samples collected from swine,cattle and sheep,among which 74.5%,13.3%and 9.2%of samples were JEV specific antibody-positive.The detection results of randomly selected swine,cattle and sheep by the serum neutralization test were agree with blocking ELISA.As a result,a blocking ELISA was successfully established for detecting JEV antibody of swine,cattle and sheep in the present study.