一种检测传染性喉气管炎病毒的夹心ELISA方法的建立  

Development of a Sandwich ELISA Method for Defection Infectious Laryngotracheitis Virus

作　　者：张小荣[1,2] 张林[1,2] 刘怡[1,2] 黄海琼[1,2] 吴艳涛[1,2] 

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏省重要动物疫病与人兽共患病协同创新中心,江苏扬州225009

出　　处：《中国家禽》2017年第9期16-19,共4页China Poultry

基　　金：现代农业产业技术体系建设专项资金(CARS-41-k08);江苏省科技支撑计划(BE2014354/01);江苏省高校优势学科项目;扬州大学高端人才支撑计划

摘　　要：通过将传染性喉气管炎病毒(ILTV)糖蛋白g D基因78~600 nt抗原编码区在大肠杆菌中进行高效可溶性表达,以重组蛋白免疫BALB/c小鼠,通过淋巴细胞杂交瘤技术筛选获得2株特异性单克隆抗体4D6和7D9,经亚类鉴定均为IgG1,轻链为κ链。经交叉配对试验,建立了以7D9为捕获抗体,以HRP偶联7D9(HRP-7D9)作为检测抗体的ILTV抗原检测夹心ELISA方法。经测定,该方法具有良好的特异性和敏感性,对ILTV抗原的最低检测限为10~2TCID_(50)/m L。应用该方法对40份临床样品进行双盲测试,以PCR方法同步检测进行对照,结果显示两种方法之间的符合率为87.5%。In this study,a soluble recombinant protein coding by infectious laryngotracheitis virus （ILTV）gD gene （78~600 nt region）was expressed in Escherichia coll. BALB/c mice were immunized with recombinant protein,and two gD-specific monoclonal antibodies （MAb）, 4D6 and 7D9 were screened by hybridoma technique. Both MAbs were belonging to IgG1 subclass with Kappa light chain. A sandwich ELISA method for ILTV viral antigen detection was developed by cross matching test based on MAbs. MAb 7D9 was used as capture antibody to coat ELISA plate, and the HRP conjugated MAb 7D9 （HRP-7D9）was as detecting antibody. The developed sandwich ELISA assay showed good sensitivity and specificity,and the detection limit was l0s TCID550/mL for ILTV viral antigen. Forty clinical samples were used to evaluate the performance of the sandwich ELISA in a double-blind test, and the PCR method was set as control. The results demonstrated that the coincidence rate was 87.5% between these two methods.

关 键 词：传染性喉气管炎病毒 单克隆抗体 夹心ELISA 

分 类 号：S855.3[农业科学—临床兽医学]