鱼肠道弧菌单克隆抗体的制备及应用

摘要

Five monoclonal antibodies (MAbs) (1 C6,1D8,2D7,2D10,2E3) against Vibrio ichthyoenteri as a pathogen in marine animals were produced from hybridoma cells through Enzyme Linked Immunosorbent Assay (ELISA) and Indirect Immunofluorescence Assay Test (IFAT) by immunised Balb/C mice.The western blotting revealed that the protein of V.ichthyoenteri was shown to have molecular weight of 44 200 and 36 700,and the flowing cytometry (FCM) analysis showed that the MAb 2D10 reacted with positive cells had positive percent of 13.63 ％,and negative percent of less than 1％.Artificial infection experiment proved that the application of monoclonal antibody provided a method for the early diagnosis of V.ichthyoenteri.%以鱼肠道弧菌Vibrio ichthyoenteri为抗原免疫小鼠,用PEG法将小鼠脾细胞与SP2/0骨髓瘤细胞融合,分别采用酶联免疫吸附(ELISA)技术和间接免疫荧光抗体(EFAT)技术筛选强阳性杂交瘤细胞株,亚克隆阳性细胞株,获得5株抗鱼肠道弧菌单克隆抗体(1C6、1D8、2D7、2D10、2E3).Western blotting 分析结果显示,单抗2D10与相对分子质量为44 200、36 700的鱼肠道弧菌蛋白发生特异性结合；用流式细胞术(FCM)检测单抗强度,结果显示阳性荧光比例为13.63％,阴性对照荧光比例不足1％；人工感染牙鲆试验结果表明,运用该单抗能够建立鱼肠道弧菌的快速诊断方法.