白斑综合征病毒单克隆抗体的制备及BA-ELISA方法的建立  

Preparation and establishment of BA-ELISA of monoclonal antibodies against white spot syndrome virus

作　　者：刘问[1,2] 钱冬[2] 严小军[1] 

机构地区：[1]宁波大学生命科学与生物工程学院,浙江宁波315211 [2]浙江省淡水水产研究所鱼病研究室,浙江湖州313001

出　　处：《大连海洋大学学报》2009年第S1期49-52,共4页Journal of Dalian Ocean University

基　　金：浙江省重大科技攻关项目(2005C12038);浙江省科技厅院所重点资助项目(2006F12005)

摘　　要：白斑综合征病毒(white spot syndrom virus,WSSV)是甲壳类动物的重要病原,作者采用提纯的WSSV免疫BALB/c小鼠,取小鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,用间接ELISA法筛选阳性孔,经有限稀释法克隆,共得到3株能特异分泌抗WSSV的单克隆抗体(Mab)的杂交瘤细胞(1E9、1E10、4F5)。将3株杂交瘤细胞注射小鼠,制备腹水单克隆抗体,ELISA效价为1∶104~105。1E9、4F5的抗体亚型均为IgG1,1E10的抗体亚型为IgG2b。特异性实验表明:3株单抗与虾类病原桃拉综合征病毒(TSV)、罗氏沼虾诺达病毒(MrNV)、哈维氏弧菌(V.harveyi)均无交叉反应。Western bolt分析表明:单抗1E9与病毒28kD外膜蛋白特异性结合。采用生物素标记纯化后的单抗1E9,并建立了生物素-亲和素酶联免疫吸附法(Biotin-Adivin ELISA,BA-ELISA)用于WSSV的检测,该方法对提纯病毒的检测灵敏度约为4 ng,对病虾血淋巴的检测灵敏度达1∶25000。White spot syndrome virus(WSSV) is currently the most serious viral pathogen in farmed shrimp worldwide.Mouse myeloma cells(SP2/0) were fused with spleen cells from BALB/c immunized with purified WSSV.Positive colonies were selected by indirect ELISA,and after 2-3 times limited dilution cloning,three hybridoma cell lines secreting monocolonal antibodies(Mabs) against WSSV were produced.The titres of ascitic fluids of three Mabs was 1∶104-105.Isotypes of Mabs were tested by SBA clonotyping system,the result showed that 1E9 and 4F5 were characterized as IgG1,and 1E10 IgG2b.The three Mabs did not cross react with pathogens of shrimp TSV,MrNV and V.harveyi.The method of BA-ELISA was established using the hybridoma cell 1E9,which can react with 28 kD coat protein of WSSV by Western blot.The sensitivity for detection WSSV by BA-ELISA from the purified virus reached over 4 ng and from the diseased shrimp reached over 1∶25000.

关 键 词：白斑综合征病毒 单克隆抗体 BA-ELISA 

分 类 号：S945.4[农业科学—水产养殖]