禽呼肠孤病毒特异性单克隆抗体的制备及阻断ELISA检测方法的建立  

Preparation of Avian Reovirus Specific Monoclonal Antibody and Establishment of Blocking ELISA Method

作　　者：赵璐 黄聪 黄远玲 韩靖宜 李鸿昌 陈鸿军[1] 陈宗艳[1] ZHAO Lu;HUANG Cong;HUANG Yuanling;HAN Jingyi;LI Hongchang;CHEN Hongjun;CHEN Zongyan(Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Science,Shanghai 200241)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国家禽》2024年第12期60-68,共9页China Poultry

基　　金：“十四五”国家重点研发计划项目(2022YFD1801000)。

摘　　要：为建立检测禽呼肠孤病毒(ARV)血清抗体阻断ELISA方法,试验采用浓缩的ARV-AH20株病毒粒子免疫5周龄BALB/c小鼠,利用细胞融合技术制备杂交瘤细胞,并通过间接ELISA和免疫荧光试验(IFA)进行阳性杂交瘤细胞亚克隆筛选,获得两株稳定分泌针对σC蛋白的单克隆抗体杂交瘤细胞株H9B2D77、B4E4B10。选择H9B2D77株作为检测抗体、重组σC蛋白作为包被抗原,经条件优化,建立了一种检测ARV抗体的阻断ELISA方法。结果显示:该方法与常见鸡源病原的抗体阳性血清均不存在交叉反应,批间和批内变异系数均小于6%;对48份鸡血清样品的检测,该方法与IFA具有100%的总体符合率。综上所述,基于禽呼肠孤病毒特异性单克隆抗体建立的阻断ELISA方法特异性高,重复性好,可用于ARV抗体检测。In order to establish a blocking ELISA method for the detection of avian reovirus(ARV)antibodies,five-week-age BALB/c mice were immunized with concentrated ARV strain AH20 in this study.Hybridoma cells were produced by cell fusion and screened by indirect ELISA and immunofluorescence assay(IFA)during subcloning,then the hybridoma cell lines H9B2D77 and B4E4B10 with stable secretion of monoclonal antibody were screened finally.The H9B2D77 monoclonal antibody was used as the detection antibody,the recombinantσC protein was used as the coating antigen and the conditions were continuously optimized to establish a blocking ELISA method for detecting ARV antibody.The reults showed that the established method did not cross-react with antibody-positive sera of common chicken-origin pathogens,which had good repeatability,and the intra-and inter-batch coefficients of variation were less than 6%,and the overall coincidence rate of this method with IFA was 100%in the detection of 48 serum samples.In summary,the blocking ELISA method based on specific monoclonal antibody of ARV provided high specificity and good repeatability,which could be used for the detection of ARV antibody.

关 键 词：禽呼肠孤病毒 σC蛋白 单克隆抗体 阻断ELISA 

分 类 号：S855.3[农业科学—临床兽医学]