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目的制备抗O1群小川型霍乱弧菌(VibriocholeraeO1serotypeOgawa)特异性单克隆抗体(McAb),为霍乱的早期快速诊断提供有力的抗体工具。方法以灭活的O1群小川型霍乱弧菌免疫Balbc小鼠,通过杂交瘤技术制备针对O1群小川型霍乱弧菌的McAb,以间接ELISA法对所需的杂交瘤细胞株进行筛选,分析其亚类,检测其效价及相对亲和力,以间接ELISA和Westernblot鉴定McAb特异性,并进行McAb结合表位分析。结果融合了602株能分泌抗O1群小川型霍乱弧菌McAb的杂交瘤细胞株,最后得到5株能稳定分泌特异性的针对该McAb的细胞株,其抗体亚类分别为3株IgG1,1株IgG2b,1株IgG3;腹水效价均达1×10-6;亲和常数在1×108～1×109之间。间接ELISA法及Westernblot证实所获的McAb可与O1群小川型霍乱弧菌发生特异性反应。ELISA相加实验结果显示除有2株McAb识别相同的抗原表位外,其余均识别不同的抗原表位。结论获得霍乱弧菌O1群小川型特异性McAb,为O1群小川型霍乱早期快速诊断和发病机理的研究提供基础。
Objective To prepare monoclonal antibodies against Vibrio cholerae O1 serotype Ogawa (O1), and to provide a powerful antibody tool for rapid early diagnosis of cholera. Methods Balbc mice were immunized with inactivated O1 group Ogawa-type Vibrio cholerae and McAbs against O1 group Ogawa-type Vibrio cholerae were prepared by hybridoma technique. The desired hybridoma cell strains were screened by indirect ELISA and analyzed Subclass to detect its potency and relative affinity. McAb specificity was identified by indirect ELISA and Western blot, and McAb binding epitope analysis was performed. Results A total of 602 hybridoma cell lines secreting McAbs against O1 group Ogawa-type Vibrio cholerae were obtained. Finally, five cell lines that could stably secrete specific McAbs were obtained. The antibody subtypes were 3 IgG1,1 Strains of IgG2b, 1 strain of IgG3; ascites titer reached 1 × 10-6; affinity constant in 1 × 108 ~ 1 × 109 between. Indirect ELISA and Westernblot confirmed that the McAbs obtained could react specifically with O1 group Ogawa-type Vibrio cholerae. ELISA addition experiments showed that in addition to two McAb recognition of the same epitope, the rest are recognized by different epitopes. Conclusion Obtaining Ogawa type specific McAb of Vibrio cholerae O1 group provides the basis for rapid diagnosis and pathogenesis of Ogawa Ogawa cholera in the early stage.