变异型禽呼肠孤病毒抗体间接ELISA检测方法的建立  

Development of an Indirect ELISA Assay for Antibody Detection of Avian Reovirus Variant

作　　者：牛世奇 武子华 马天馨 李雪松[1] 滕巧泱[1] 苑纯秀[1] 李泽君[1] 刘芹防[1] NIU Shiqi;WU Zihua;MA Tianxin;LI Xuesong;TENG Qiaoyang;YUAN Chunxiu;LI Zejun;LIU Qinfang(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2024年第6期50-56,共7页Chinese Journal of Animal Infectious Diseases

基　　金：自然科学基金面上项目(32172835)。

摘　　要：随着变异型禽呼肠孤病毒(ARV)不断分离出来,其严重威胁着我国养鸡产业发展,目前尚没有针对变异型ARV的血清学检测方法,且商品化试剂盒(INDEXX)检测不出变异株感染鸡后的阳性血清,因此为了建立能快速检测ARV血清抗体的ELISA检测方法。本实验利用分离到的ARV变异株在LMH细胞上增殖,对病毒进行灭活浓缩后作为包被抗原,对各种条件进行了优化,建立了检测ARV血清抗体的间接ELISA方法。结果显示:ARV的最佳包被浓度为1.25μg/mL,血清最佳稀释倍数为1∶800,最佳包被条件为37℃、2 h后4℃过夜进行孵育,封闭液用1%BSA,血清最佳孵育条件为37℃、1.5 h,HRP标记羊抗鸡二抗的最佳稀释倍数为1∶8000,最佳孵育条件为37℃、1.5 h,并确定了临界值为0.442。该检测方法的特异性和敏感性较好,批内、批间重复率变异系数最大在5%左右,具有良好的重复性。变异型禽呼肠孤病毒抗体间接ELISA检测方法的建立为临床快速检测变异型ARV的抗体及疫苗免疫效价评估奠定基础。The increasing occurrence of avian reovirus(ARV)variants imposes a serious threat to chicken industry in China.Currently,there is no serological method for detection of ARV variants as the commercial kit(INDEXX)cannot detect the positive serum of chickens infected with the mutant strains.The objective of the presnt study was to develop an ELISA assay that can rapidly detect ARV serum antibodies so that infection with ARV variant strains could be detected as early as possible.In this study,the ARV variant was propagated on LMH cells followed by inactivation,and purification.The concentrated virus was used as the coating antigen for developing an indirect ELISA method for detection of ARV serum antibodies.The method was also optimized for its reaction conditions.The experimental data included the optimal coating concentration at 1.25μg/mL,serum dilution at 1:800,coating condition at 37℃for 2 h followed by overnight incubation at 4℃,blocking solution of 1%BSA,serum incubation at 37℃for 1.5 h,HRP labeled goat antichicken antibody dilution at 1:8000 and incubation at 37℃for 1.5 h,and cut value at 0.442.The method also had good specificity and sensitivity.The coefficients of variation between intra-assays and inter-assays was about 5%,indicating good repeatability.Taken together,the indirect ELISA assay developed in this study provided a rapid antibody detection method for ARV variants and might be used for clinic diagnosis and assessment of vaccine efficany.

关 键 词：禽呼肠孤病毒 间接ELISA 检测方法 

分 类 号：S852.65[农业科学—基础兽医学]