单克隆抗体捕捉猪瘟病毒抗原ELISA方法的建立

畜牧兽医学报 ›› 2006, Vol. 37 ›› Issue (5): 474-479.

单克隆抗体捕捉猪瘟病毒抗原ELISA方法的建立

刘建文，余兴龙，张丽颖，涂长春*

军事医学科学院军事兽医研究所，长春 130062

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2006-05-25 发布日期:2006-05-25

Establishment of Antigen-captured ELISA Using Monoclonal Antibodies to Detect Classical Swine Fever Virus

LIU Jianwen， YU Xinglong， ZHANG Liying， TU Changchun*

The Institute of Veterinary Sciences, Academy of Military Medicine Sciences, Changchun 130062, China

Received:1900-01-01 Revised:1900-01-01 Online:2006-05-25 Published:2006-05-25

摘要/Abstract

摘要： 分别用原核表达的猪瘟病毒(CSFV)主要抗原 E2蛋白和猪瘟基因疫苗免疫BALB/c小鼠，通过细胞融合与克隆筛选出5株稳定分泌CSFV抗体的杂交瘤细胞株1E2、1G7、3A2、3B7和4B6。间接免疫荧光和Western-blotting试验结果表明，5株单抗均与CSFV E2蛋白和全病毒抗原反应。将筛选的CSFV特异性单抗1E2、3B7和4B6纯化后等量混合后包被酶标板（捕捉抗体），与兔抗CSFV IgG（检测抗体）联合应用，建立起CSFV抗原捕捉ELISA(AC-ELISA)方法。随后采用方阵滴定法确定了单抗与多抗的最适工作浓度及判定检测结果的OD450临界值。最后以建立的AC-ELISA检测CSFV细胞培养物、CSFV攻毒死亡猪的病料和临床猪瘟组织样品，结果表明，该方法敏感、特异、重复性好，与病毒分离和RT-PCR方法符合率分别为86.2%和90.3%。

关键词: 猪瘟病毒, 单克隆抗体, 抗原捕捉ELISA

Abstract: The five monoclonal antibodies (mAbs) against classical swine fever virus were generated by immunization of BALB/c mice respectively with CSFV DNA vaccine plasmid (expressing glycoprotein E2) and E.coli-expressed E2 protein. These mAbs could react with CSFV growing in PK-15 cells in indirect immunofluorescent assay (IFA), as well as with expressed E2 protein in western-blotting. Of them, the mAbs 1E2, 3B7 and 4B6 are CSFV specific antibody without cross reaction with E2 protein of BVDV, therefore have been used, along with rabbit anti-CSFV IgG, to develop the antigen-captured ELISA(AC-ELISA) for detecting CSFV antigen directly from clinical tissue samples. The cut-off value was determined by testing negative tissue samples of 24 healthy pigs. The method was testified by comparison with virus isolation and RT-PCR of 31 positive tissue samples from piglets experimentally infected with CSFV shimen strain,11 clinically CSF cases and 12 healthy samples. The results showed that the AC-ELISA is sensitive, specific and has high consistency with virus isolation and RT-PCR.

Key words: CSFV, mAbs, antigen captured ELISA

刘建文;余兴龙;张丽颖;涂长春. 单克隆抗体捕捉猪瘟病毒抗原ELISA方法的建立[J]. 畜牧兽医学报, 2006, 37(5): 474-479.

LIU Jianwen;YU Xinglong;ZHANG Liying;TU Changchun. Establishment of Antigen-captured ELISA Using Monoclonal Antibodies to Detect Classical Swine Fever Virus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2006, 37(5): 474-479.

[1]	王盼举, 樊新丽, 张梦圆, 宋晶晶, 李敏, 吾力江, 巴音查汗. 地方株马驽巴贝斯虫Bc48截短体单克隆抗体的制备及应用[J]. 畜牧兽医学报, 2019, 50(9): 1882-1887.
[2]	胡永新, 赵永刚, 张永强, 刘拂晓, 樊晓旭, 吴晓东, 王志亮. 表达非洲猪瘟病毒P72蛋白复制缺陷型重组腺病毒的构建及鉴定[J]. 畜牧兽医学报, 2019, 50(8): 1635-1641.
[3]	胡永新, 赵永刚, 张永强, 刘拂晓, 樊晓旭, 吴晓东, 王志亮. 表达非洲猪瘟病毒P72蛋白复制缺陷型重组腺病毒的构建及鉴定[J]. 畜牧兽医学报, 2019, 50(8): 1635-1641.
[4]	唐泽群, 罗海燕, 葛铭, 郭荣, 许丹蕾, 张瑞莉. 抗chTLR3单克隆抗体制备及其初步应用[J]. 畜牧兽医学报, 2019, 50(6): 1292-1300.
[5]	唐泽群, 罗海燕, 葛铭, 郭荣, 许丹蕾, 张瑞莉. 抗chTLR3单克隆抗体制备及其初步应用[J]. 畜牧兽医学报, 2019, 50(6): 1292-1300.
[6]	梁文花, 贾云慧, 许程志, 吴运谱, 陈艳, 杨焕良, 乔传玲, 陈化兰. 两株H1N1亚型猪流感病毒HA蛋白中和性单克隆抗体的制备及抗病毒活性研究[J]. 畜牧兽医学报, 2018, 49(7): 1460-1466.
[7]	梁文花, 贾云慧, 许程志, 吴运谱, 陈艳, 杨焕良, 乔传玲, 陈化兰. 两株H1N1亚型猪流感病毒HA蛋白中和性单克隆抗体的制备及抗病毒活性研究[J]. 畜牧兽医学报, 2018, 49(7): 1460-1466.
[8]	李文良, 毛立, 邓加武, 郝飞, 李基棕, 杨蕾蕾, 张纹纹, 江杰元. 抗病毒蛋白Viperin抑制猪瘟病毒在PK-15细胞上的复制[J]. 畜牧兽医学报, 2018, 49(1): 139-146.
[9]	李文良, 毛立, 邓加武, 郝飞, 李基棕, 杨蕾蕾, 张纹纹, 江杰元. 抗病毒蛋白Viperin抑制猪瘟病毒在PK-15细胞上的复制[J]. 畜牧兽医学报, 2018, 49(1): 139-146.
[10]	梁武龙，方佳，林鸷，郑敏萍，鲍长磊，王涛，张彦明. 猪瘟病毒通过网格蛋白介导的内吞途径入侵ST细胞[J]. 畜牧兽医学报, 2017, 48(1): 140-149.
[11]	梁武龙，方佳，林鸷，郑敏萍，鲍长磊，王涛，张彦明. 猪瘟病毒通过网格蛋白介导的内吞途径入侵ST细胞[J]. 畜牧兽医学报, 2017, 48(1): 140-149.
[12]	张永宁，宋姗姗，吴绍强，林祥梅. 施马伦贝格病毒核衣壳蛋白的表达及单克隆抗体制备[J]. 畜牧兽医学报, 2016, 47(6): 1280-1286.
[13]	张永宁，宋姗姗，吴绍强，林祥梅. 施马伦贝格病毒核衣壳蛋白的表达及单克隆抗体制备[J]. 畜牧兽医学报, 2016, 47(6): 1280-1286.
[14]	姚静静，胡骁飞，韩俊岭，李青梅，王方雨，邓瑞广，张改平. 伏马菌素B₁单克隆抗体的制备及免疫学检测方法初步应用[J]. 畜牧兽医学报, 2016, 47(5): 1009-1017.
[15]	姚静静，胡骁飞，韩俊岭，李青梅，王方雨，邓瑞广，张改平. 伏马菌素B₁单克隆抗体的制备及免疫学检测方法初步应用[J]. 畜牧兽医学报, 2016, 47(5): 1009-1017.