草鱼呼肠孤病毒HZ08株抗体IPMA检测方法的建立及应用  

Establishment and application of immunoperoxidase monolayer assay for detection of the antibody against grass carp reovirus HZ08

作　　者：曾伟伟[1] 王庆[1] 王英英[1] 李莹莹[1] 郝贵杰[2] 石存斌[1] 沈锦玉[2] 

机构地区：[1]中国水产科学研究院珠江水产研究所,农业部渔药创制重点实验室,广东广州510380 [2]浙江省淡水水产研究所农业部淡水渔业健康养殖重点实验室,浙江湖州313001

出　　处：《水产学报》2017年第1期142-149,共8页Journal of Fisheries of China

基　　金：农业部淡水渔业健康养殖重点实验室开放课题(ZJK201301);江西省科技计划项目(20152ACF60021);“十二五”国家科技支撑计划(2012BAD12B02)~~

摘　　要：为建立一种检测草鱼呼肠孤病毒HZ08株(HZ08)抗体的免疫学方法,本实验以接种HZ08病毒的细胞培养板为抗原板,以免疫弱毒疫苗的草鱼血清为抗体,通过反应条件优化,建立了检测GCRV HZ08血清抗体的免疫过氧化物酶单层细胞实验(IPMA)方法,并对该方法特异性、敏感性、重复性、与ELISA检测结果的符合率及对临床血清样品的检测效果等进行了验证。结果显示,HZ08株种毒按照1×103拷贝/μL浓度接种后72h固定,HRP-Ig G二抗1∶1000稀释,待检血清1∶100稀释时效果最佳;该IPMA方法与草鱼阴性血清、免疫细菌三联疫苗的草鱼血清、感染GCRV JX0901(GCRVⅠ型)的草鱼血清无交叉反应;GCRV HZ08株阳性血清稀释至1∶800时仍能检出;批内和批间重复性实验显示,该方法具有良好的重复性;符合性实验结果显示,该IPMA方法与ELSIA方法的阳性和阴性符合率分别为95.7%和87.5%;用建立的IPMA方法检测草鱼出血病弱毒疫苗免疫草鱼,免疫2周后抗体水平达到峰值;对现地送检的126份草鱼血清样本进行检测,平均检出阳性率为72.2%。研究表明,建立的HZ08株IPMA方法特异性强、重复性好、敏感性高,为GCRVⅡ型的流行病学调查、疫苗免疫效果评价及抗原抗体检测提供了一种有效的检测手段。In order to develop an immunological method for detection of the antibody against grass carp reovirus HZ08,using the cell culture plate which infected with the the GCRV HZ08 as the antigen,and serum collected from the vaccine immune grass carp used as the antibody,an immunoperoxidase monolayer assay（IPMA） was developed to detect antibody against grass carp reovirus HZ08 based on the optimized reaction condition.The specificity,sensitivity,reproducibility and using for clinical application of IPMA were evaluated,and compared with that of ELISA.The results showed that in the optimized IPMA,the virus was diluted at 1×103 copies/μL after72 hours,HRP-Ig G was diluted at 1：1000,and grass carp serum samples were diluted at 1：100,there was no crossreaction with serum from grass carp against other pathogeny. The sensitivity test showed that the positive serum could be detected at 1：800. Reproducibility tests proved that the established IPMA had good reproducibility. The IPMA method had 95.7% positive coincident rate and 87.5% negative coincident rate with ELISA method. The results of the IPMA for detection of the immunized grass carp serum showed that the antibody titer reached peak value in the 2nd week post-inoculation.A total of 126 field serum samples were examined using this method,the average positive rate was 72.2 %. The IPMA is a highly specific,sensitive,and stable method with high repeatability,and it offered a convenient tool for the epidemiological investigation,vaccine immunization effect assessment,and antigen and antibody detection.

关 键 词：草鱼 呼肠孤病毒 免疫过氧化物酶单层细胞实验 抗体检测 

分 类 号：S941.41[农业科学—水产养殖]