传染性胰坏死病毒双抗体夹心ELISA检测方法的建立  

Development of a double antibody sandwich ELISA for detection of infectious pancreatic necrosis virus

作　　者：张琳琳[1] 连科迅[2] 张英[1] 蒋烨[1] 姜艳萍[2] 崔文[2] 乔薪瑗[2] 唐丽杰[2] 李一经[2] 刘敏[1] 

机构地区：[1]东北农业大学动物科学与技术学院,哈尔滨150030 [2]东北农业大学动物医学学院,哈尔滨150030

出　　处：《淡水渔业》2014年第4期57-62,72,共7页Freshwater Fisheries

基　　金：国家支撑计划(2013BAD12B02)

摘　　要：以纯化的兔抗传染性胰坏死病病毒(IPNV)VP2重组蛋白多克隆抗体为包被抗体,抗IPNV VP2单克隆抗体为检测抗体建立了IPNV双抗体夹心ELISA方法。优化反应条件为:兔抗IPNV VP2重组蛋白多克隆抗体包被浓度为1.28μg/mL,抗IPNV VP2单克隆抗体的工作浓度为1.34μg/mL,酶标二抗稀释比例为1∶2 000,以P/N>2,且OD490 nm>0.101 494作为阳性判定标准。该方法的重复性变异系数均小于10%,与传染性造血器官坏死病毒(IHNV)、病毒性出血败血症病毒(VHSV)、鲤春病毒血症病毒(SVCV)、轮状病毒(HRV)无交叉反应。对41份虹鳟肝脏样品分别进行双抗体夹心ELISA和RT-PCR检测,结果双抗体夹心法与RT-PCR法检测符合率为97%,表明本实验建立的双抗体夹心ELISA检测方法检测IPNV具有较高的敏感性和特异性,可用于IPNV的病原学检测。A double-antibody sandwich ELISA （ DAS-ELISA） was developed using purified polyclonal antibody and mono-clonal antibody （ MAb） against viral infectious pancreatic necrosis virus （ IPNV） VP2 protein as capture and detector anti-body, respectively.The optimized reaction conditions included coating with 100μL/well of purified polyclonal antibody at concentration of 1.28 μg/mL, probing with the working concentration of 1.34 μg/mL IgG from the MAb diluting HRP-conjugated antibody with the proportion of 1∶2 000 and judging with P/N〉2 and OD490 nm =0.101 494 as positive criteria. The DAS-ELISA was specific detection of IPNV , but no cross-reaction with IHNV, VHSV, SVCV, HRV and inter-assay coefficient of variability were within 10%.In addition, a total of 41 livers of rainbow trout samples were tested by the DAS-ELISA and PT-PCR, and the coincidence was 97%between DAS-ELISA and PT-PCR.These data demonstrated that the DAS-ELISA was specific and sensitive , and provided a useful tool for diagnosis of IPNV infection .

关 键 词：传染性胰坏死病毒 双抗体夹心ELISA检测方法 单克隆抗体 兔抗血清 

分 类 号：S941.41[农业科学—水产养殖]