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为制备并鉴定番茄溃疡病菌(Clavibacter michiganensis subsp.michiganensis,Cmm)的单克隆抗体(McAbs),用全菌皮下免疫BALB/C小鼠,采用B细胞杂交瘤技术,经免疫、融合、间接ELISA筛选和克隆等,获得稳定分泌抗体的阳性杂交瘤细胞株,得到了抗番茄溃疡病菌的单克隆抗体。经免疫后获得3株单抗分别为1A4、1C3和1B7,经亚类鉴定分别是IgM、IgG1、IgG1;纯化腹水间接ELISA效价分别为1:3.2×106、1:8.1×105、1:3.2×106;与其他同属不同亚种无交叉反应。结果表明:3株单克隆抗体均具有较高特异性和敏感性,可作为番茄溃疡病菌的检测抗体,其中,1A4的效果最好。番茄溃疡病菌单克隆抗体的获得为进一步研发番茄溃疡病检测试剂盒奠定了基础。
To prepare and identify monoclonal antibodies (McAbs) against Clavibacter michiganensis subsp. Michiganensis (Cmm), BALB / C mice were immunized subcutaneously with whole bacteria and immunoblotted with B cell hybridoma technique by immunization, fusion and indirect ELISA And cloning, to obtain a positive hybridoma cell line stably secreting antibodies. A monoclonal antibody against tomato canker bacterium was obtained. The McAbs obtained after immunization were 1A4, 1C3 and 1B7, respectively, and were identified as IgM, IgG1 and IgG1 by subclasses respectively. Indirect ELISA titers of purified ascites were 1: 3.2 × 106, 1: 8.1 × 105, 3.2 × 106; no cross-reaction with other same sub-species. The results showed that all three monoclonal antibodies were highly specific and sensitive and could be used as detection antibodies against tomato canker. Among them, 1A4 was the best. Obtaining of monoclonal antibodies against tomato canker laid the foundation for the further development of a test kit for tomato canker.