日本脑炎病毒NS1蛋白双抗体夹心ELISA检测方法的建立  

Establishment of Double Antibody Sandwich ELISA for Detection of Japanese encephalitis virus NS1 Protein

作　　者：杨克露 裴超[1] 刘朝霞 周登元 曹胜波[1] 陈焕春[1] 宋云峰[1] YANG Ke-Lu;PEI Chao;LIU Zhao-Xia;ZHOU Deng-Yuan;CAO Sheng-Bo;CHEN Huan-Chun;SONG Yun-Feng(College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]华中农业大学动物医学院,武汉430070

出　　处：《农业生物技术学报》2020年第7期1322-1330,共9页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2018YFD0501004);国家自然科学基金(31772711);湖北省重大科技创新计划(2018ABA107)

摘　　要：日本脑炎病毒(Japanese encephalitis virus,JEV)是危害人民健康的一种重要人畜共患病原,仍然缺乏敏感性高的抗原检测方法。NS1蛋白是JEV表达的一种分泌性蛋白,是该病毒诊断的理想靶标。本研究用构建的非结构蛋白1(non-structural protein 1,NS1)单克隆抗体,建立了可检测NS1蛋白的双抗体夹心酶联免疫吸附试验(double antibody sandwich ELISA,DAS-ELISA)方法。用针对JEV NS1蛋白不同位点的单克隆抗体杂交瘤细胞株(5H2和2F6)制备腹水,经Protein G纯化后,以5H2包被酶标板作为捕获抗体,HRP标记的2F6为检测抗体。ELISA条件优化的结果显示,捕获抗体和酶标抗体最佳浓度分别为25和2.296μg/mL。用该方法检测系列稀释的NS1蛋白,蛋白浓度范围在78.125~625.000 ng/mL时,检测OD450值和蛋白浓度之间具有良好的线性关系,说明该方法可用于NS1蛋白的定量检测。用该方法检测猪伪狂犬病病毒、猪繁殖与呼吸综合征病毒、猪细小病毒等均显示阴性,说明特异性良好。重复性试验结果显示,该方法批内重复性为0.7%~2.6%,批间重复性为0.6%~2.1%。用该方法检测了JEV感染的小鼠(Mus musculus)脑组织匀浆,在小鼠发病期可明显检测到NS1蛋白。本研究建立了检测JEV NS1蛋白的ELISA方法,为该病毒的诊断提供了一种工具。Japanese encephalitis virus(JEV)is an important zoonotic pathogen theating human health,and still lacks sensitive antigen detection method currently.NS1 protein is a secreted protein expressed by JEV and is an ideal target for diagnosis of the virus.In this study,a double antibody sandwich ELISA(DAS-ELISA)method for detecting non-structural protein 1(NS1)was established using the constructed NS1 protein monoclonal antibodies.Monoclonal antibody hybridoma cell lines(5H2 and 2F6)against different sites of the JEV NS1 protein were used to prepare ascites.After purification by Protein G,the ELISA plate was coated with 5H2 as the capture antibody,and 2F6 labeled with HRP as detection antibody.The optimized ELISA conditions showed that the optimal concentrations of capture antibody and enzyme-labeled antibody were 25 and 2.296μg/mL,respectively.Using this method to detect serially diluted NS1 protein in concentration of 78.125~625.000 ng/mL,there was a good linear relationship between the OD450 value and the protein concentration,indicating that this method can be used for quantitative detection of NS1 protein.Detection of porcine pseudorabies virus,porcine reproductive and respiratory syndrome virus,and porcine parvovirus by this method showed negative results,indicating good specificity.The reproducibility test results showed that the repeatability within the batch was 0.7%~2.6%and the repeatability between batches was 0.6%~2.1%.Using this method,brain tissue homogenates of JEV-infected mice were detected,and NS1 protein was clearly detected during the onset of mice(Mus musculus).This study established an ELISA method for detecting JEV NS1 protein,which provides a tool for the diagnosis of the virus.

关 键 词：日本脑炎病毒 NS1蛋白 单克隆抗体 双抗体夹心ELISA 

分 类 号：S855.3[农业科学—临床兽医学]