乙型脑炎病毒抗体捕获ELISA检测方法的初步建立

摘要

目的 建立检测乙型脑炎病毒抗体的抗体捕获ELISA方法.方法 以乙型脑炎病毒E蛋白抗原表位E39特异的单克隆抗体包被ELISA板,吸附乙型脑炎减毒活疫苗株SA14-14-2病毒粒子,再以吸附的病毒粒子捕获乙型脑炎病毒抗体,建立抗体捕获ELISA方法:同时与以乙型脑炎病毒细胞培养上清包被的间接ELISA方法进行对比,并检测临床血清样品105份.结果 间接ELISA方法背景无法消除,1:10、1:100、1:1000各稀释度的阳性血清与阴性血清吸光度(A)值相近,A阳性血清/阴性血清分别为1.02、0.99、1.13,均<2.1.而抗体捕获ELISA方法1:10、1:100稀释度的A阳性血清/阴性血清分别为3.57、2.94,均>2.1;1:1000稀释度的A阳性血清/阴性血清为1.42,<2.1,表明血清稀释度为1:100时可以显著区别乙型脑炎病毒感染的阳性血清与阴性血清,明显消除间接ELISA方法中背景过高的问题.抗体捕获ELISA方法检测105份临床血清样品,A阳性血清/阴性血清为0.257～0.321(0.262±0.050),均<2.1,为乙型脑炎病毒抗体阴性血清.结论 初步建立了乙型脑炎病毒抗体捕获ELISA方法,该方法特异性较高,对于建立乙型脑炎病毒群的快速鉴别诊断方法具有重要意义.%Objective To set up an antibody-capture ELISA method to detect the Japanese encephalitis virus(JEV)antibody.Methods ELISA plate was coated with the monoclonal antibody which was specific to the envelope protein epitope E39 of JEV,JEV SA14-14-2 strain as the source of antigen was used to absorb the monoclonal antibody,the absorbed virus used to capture the JEV'S antibody.The antibody that captured ELISA was established.The indirect ELISA method using the virus particles from cell culture was compared with coating ELISA plate,105 clinical serum were checked.Results The background in indirect ELISA assay could not be abscised,positive and negative serum diluted in a ratio of 1:10,1:100,1:1000,the relative value of A posative/A negative were 1.02,0.99,1.13,all<2.1.But the antibody-captured ELISA method when the serum dilution was 1:10,1:100,the A posative/A negative were 3.57,2.94,all>2.1;when the dilution was 1:1000,the A posative/A negative was 1.42,<2.1,it meant the method could distinguish the positive and negative serum efficiently when the dilution Was 1:100,the background problem in indirect ELISA assay could be solved.Antibody-capture method was used to check 105 serum samples,the A posative/A negative over a range of 0.257～0.321(0.262±0.050),all<2.1,no positive sample found.Conclusion The antibody-capture ELISA method has been preliminary set up with a high specificity,capable of quickly identifying JEV from other virus.