草鱼呼肠孤病毒抗原捕获ELISA检测方法的建立  

Establishment of Antigen-Capture ELISA for Detection of Grass Carp Reovirus

作　　者：景宏丽[1] 曹欢 张旻[1] 王娜[1] 林祥梅[1] 张利峰[3] 吴绍强[1] 

机构地区：[1]中国检验检疫科学研究院动物检疫研究所,北京100029 [2]北京市水产技术推广站,北京100021 [3]北京出入境检验检疫局检验检疫技术中心,北京101113

出　　处：《中国动物检疫》2014年第12期78-81,83,共5页China Animal Health Inspection

基　　金："十二五"国家科技支撑计划项目<重大外来与新发水生动物疫病识别与监测技术研究及示范>(2013BAD12B02)

摘　　要：研究以纯化的羊抗草鱼呼肠孤病毒多克隆抗体作为捕获抗体,抗草鱼呼肠孤病毒的单克隆抗体为检测抗体,建立草鱼呼肠孤病毒抗原捕获ELISA检测方法,其最佳反应条件是:羊多克隆抗体包埋浓度为2.5μg/m L,抗草鱼呼肠孤病毒单克隆抗体工作浓度为1:400稀释,以3%牛血清白蛋白作为封闭液。该方法的检测限为5×105 pfu/m L,且能够特异性检出发病草鱼内脏组织中的草鱼呼肠孤病毒。特异性试验结果表明该方法具有较高的特异性,与病毒性出血性败血症病毒、传染性造血器官坏死病毒和鲤春血症病毒等水产动物病毒株均不反应。该方法还具有较好的稳定性。An antigen-capture enzyme-linked immunoassay（ELISA）was developed for the detection of grass carp reovirus（GCRV）with puriifed polyclonal antiserum against GCRV as capture antibody and monoclonal antibodies against GCRV as detection antibody. The 96-well enzyme immunoassay（EIA）plates were coated with 100μL puriifed polyclonal antiserum at concentration of 2.5μg/mL and blocked with 3%？bovine serum albumin（BSA）. The working concentration of monoclonal antibody was diluted to 1：400 with distilled water. GCRV was detected in culture supernatants（5×10^5 pfu/mL）and in the extracts of kidney and spleen of infected grass carp. The assay was highly speciifc：viral hemorrhagic septicemia virus,infectious haematopoietic necrosis virus,spring viraemia of carp virus, epizootic haematopoietic necrosis virus,chinook salmon reovirus could not be detected by the developed ELISA and the method was also of good stability.

关 键 词：草鱼呼肠孤病毒 抗原捕获ELISA 单克隆抗体 

分 类 号：S941.41[农业科学—水产养殖]