AsiaI口蹄疫vp2蛋白单克隆抗体的制备和单抗竞争.PDFVIP

生 物 工 程 学 报 Chin J Biotech 2008, September 25; 24(9): 1664-1669 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 技术与方法 Asia I 口蹄疫vp2 蛋白单克隆抗体的制备及单抗竞争 ELISA 方法的建立 向敏, 张克山, 卢顺，蔡利军, 罗勇, 张建民, 何华, 王勤刚, 吴斌 华中农业大学动物医学院 农业微生物学国家重点实验室, 武汉 430070 摘 要: 制备Asia I 口蹄疫病毒vp2 单克隆抗体(mAb)并建立了单抗竞争ELISA 方法。用纯化的Asia I 型口蹄疫病毒 vp2 重组蛋白免疫BALB/c 小鼠, 将免疫小鼠的脾细胞与骨髓瘤SP2/0 细胞融合, 采用间接ELISA 和有限稀释法筛选杂 交瘤细胞。分别用ELISA 、Western blotting 检测mAb 腹水的效价及其特异性。筛选到杂交瘤细胞2 株, 腹水效价均在 100×29 以上; 以纯化后的Asia I 型口蹄疫病毒vp2 重组蛋白作为抗原, 利用Asia I 型口蹄疫病毒vp2 单抗酶标物建立了 竞争ELISA 方法用来检测Asia I 型口蹄疫抗体。临床应用表明, 该方法与UBI 公司的口蹄疫全病毒抗体检测试剂盒总 符合率达 89.0%, 和荷兰赛迪公司的口蹄疫病毒LPB-ELISA 抗体检测试剂盒总符合率达86.5%。 关键词: 口蹄疫, 单抗竞争 ELISA, 单克隆抗体, vp2 蛋白 Preparation of Monoclonal Antibodies Against VP2 Protein of Asia I Type Foot-and-mouth Disease Virus and Establishment of a Competitive ELISA for the Detection of Antibodies Min Xiang, Keshan Zhang, Shun Lu ，Lijun Cai , Yong Luo, Jianmin Zhang, Hua He, Qingang Wang, and Bin Wu State Key Laboratory of Agriculture Microbiology, College of Veterinary Medicine, Huazhong Agriculture University, Wuhan 430070, China Abstract: Monoclonal antibodies against FMDV vp2 protein were prepared and a competitive ELISA based on the monoclonal antibodies and vp2 protein was established. Balb/c mice were immunized with Escherichia coli expressed fusion protein. The splenocytes from immunized mice were fused with myeloma cells SP2/0. The hybridism cells were screened by indi