一种检测乙型脑炎病毒抗体阻断ELISA的建立及其初步应用

doi:10.11843/j.issn.0366-6964.2018.09.017

摘要/Abstract

摘要：

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。

Abstract:

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 EDⅢ protein 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.

中图分类号:

S852.659.6

江雅, 牛季琛, 倪慧, 韦冬妹, 周国栋, 曹瑞兵. 一种检测乙型脑炎病毒抗体阻断ELISA的建立及其初步应用[J]. 畜牧兽医学报, 2018, 49(9): 1961-1968.

JIANG Ya, NIU Ji-chen, NI Hui, WEI Dong-mei, ZHOU Guo-dong, CAO Rui-bing. Development and Preliminary Application of a Blocking ELISA for Detection of Japanese Encephalitis Virus Antibody[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(9): 1961-1968.

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