摘要
通过建立一种检测水貂细小病毒(mink enteritis virus,MEV)IgG抗体的间接ELISA方法,旨在为MEV感染检测与免疫效果评价提供技术手段。以纯化的重组MEV VP2蛋白作为包被抗原,以HRP-羊抗雪貂IgG为二抗,通过优化反应条件,建立了检测MEV特异性IgG抗体的间接ELISA方法,并对该方法进行了特异性、敏感性及重复性试验;通过临床样品检测评价ELISA方法与HI试验的符合率,制备阳性参考品并设定酶联免疫单位(enzyme immune units,EIU)的计算方法,以测定样品中抗MEV IgG抗体的相对含量,分析ELISA方法检测血清特异性IgG抗体水平与中和抗体的相关性。结果显示,ELISA方法的最佳抗原包被质量浓度为1.425mg/L,待检血清和酶标抗体的工作浓度分别为1∶80与1∶2000倍稀释。结果表明,该ELISA方法能够特异地检测MEV抗体,与水貂犬瘟热病毒(canine distemper virus,CDV)、水貂阿留申病毒(Aleutian disease virus,ADV)的抗血清无交叉反应,批内和批间重复性试验的变异系数低于5%,血清特异性IgG抗体水平与中和抗体效价之间存在线性关系,呈显著的正相关(r=0.9461,P<0.001)。
The object of this study is to establish an indirect ELISA method for detecting IgG antibody against mink enteritis virus(MEV)and to provide a technique for MEV detection and immune effect evaluation.The VP2protein was purified and used as the coating antigen,the HRPconjugated goat anti-ferret IgG was used as the secondary antibody.By optimizing the reaction conditions,an indirect ELISA method for detection of anti-MEV IgG antibody was established,the specificity,sensitivity and repeatability of the method were tested,the coincidence rate between the ELISA and the HI test was evaluated by clinical sample detection.The IgG antibody positive reference material was prepared,and the relative content of IgG in sample was determined according to the calculation method of enzyme immune units.The correlation between the specific IgG and the neutralizing antibodies in serum was analyzed.The results showed that the optimal antigen coating concentration of the ELISA was 1.425mg/L,and the working concentrations of tested serum and enzyme-labeled antibody were 1∶80and 1∶2000,respectively.The method could be used to detect the MEV specific antibodies without cross reactions with those antibodies against canine distemper virus(CDV)and Aleutian disease virus(ADV).The coefficients of variation(CV)of intra-and inter-batch repetitive tests of the ELISA were less than 5%.The coincidence rate was 96.5%when the same serum samples were detected using the ELISA and HI test for detecting MEV antibody.The level of specific IgA antibody was positively correlated with that of the neutralizing antibody in serum(r=0.9227,P<0.01)
作者
朱翔宇
蔡熙姮
鲁荣光
卜研
柏玲
朱言柱
廉士珍
白雪
闫喜军
胡博
ZHU Xiangyu;CAI Xiheng;LU Rongguang;BU Yan;BAI Ling;ZHU Yanzhu;LIAN Shizhen;BAI Xue;YAN Xijun;HU Bo(Institue of Special Wild Economic Animal and Plant Science,Chinese Academy of Agricultural Science,Changchun 130112,China;Key Laboratory of Economic Animal Epidemic Disease,Ministry of Agriculture and Rural Affairs,Changchun 130112,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2021年第4期661-669,共9页
Chinese Journal of Veterinary Science
基金
国家重点研发计划资助项目(2017YFD0501600)。
