猪血凝性脑脊髓炎病毒抗原捕获ELISA诊断方法的建立  

Development of antigen capture ELISA for detecting hemagglutinating encephalomyelitis virus of pigs

作　　者：赵传博[1] 王丽[1] 董波[1] 陆慧君[2] 赵魁[1] 贺文琦[1] 高丰[1,2] 

机构地区：[1]吉林大学动物医学学院,吉林长春130062 [2]吉林大学人兽共患病研究所/人兽共患病研究教育部重点实验室,吉林长春130062

出　　处：《中国兽医学报》2013年第4期526-531,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31072134;31172291;31272530)

摘　　要：采用实验室保存的杂交瘤细胞,通过扩大培养、接种小鼠、收集腹水、腹水纯化等程序得到了猪血凝性脑脊髓炎病毒(HEV)单克隆抗体,并对其效价进行了测定。用HEV单克隆抗体作为检测抗体,猪多抗作为捕获抗体,建立了检测HEV的抗原捕获ELISA方法,通过对各步反应条件的优化,最终获得最佳工作条件为:猪多抗1∶2 000稀释,单抗1∶4 000稀释,酶标抗体为1∶4 000稀释。特异性和敏感性试验结果表明:该方法对TGEV、HCV、PEDV和PRV等病毒无特异性交叉反应,其最低检测下线为3.75mg/L。与RT-PCR方法的对比试验证明,抗原捕获ELISA阳性检出结果与PCR方法相一致。上述结果说明,已成功建立特异、敏感的用于HEV检测的抗原捕获ELISA方法,为临床快速诊断HEV试剂盒的研制奠定了基础。A double-antibodise sandwich ELISA was development using mouse monoclonal antibody against hemagglutinating encephalomyelitis virus （HEV） as detecting antibody and polyclonal antisera （pig serum against HEV） as capture antibody for detecting HEV. The optimum conditions were achieved ： capture antisera was diluted for 1 ： 2 000,the mouse monoclonal antibody was diluted for 1 ： 4 000 and the enzyme-lable antibody was diluted for 1 ： 4 000. TGEV,HCV,PEDV and PRV were detected by the Ag-capture ELISA and the result showed that there was no crossing-reaction with HEV. The method has excellent specificity and could detected a minimum concentration of 3.75 mg/L of HEV. The results of positive detection by antigen capture ELISA were consistent with RT-PCR . The result showed that the Ag-capture ELISA was highly specific and sensitive. Development of A-g capture ELISA for detecting HEV was the basic for making HEV detected kit.

关 键 词：猪血凝性脑脊髓炎病毒 单克隆抗体 抗原捕获ELISA 

分 类 号：S852.651[农业科学—基础兽医学]