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目的制备新型锌指蛋白ZBTB45的单克隆抗体,建立该分子的体内外免疫学检测方法,为研究其生物学功能提供技术手段。方法制备小鼠ZBTB45的多表位抗原融合蛋白,免疫小鼠,取其脾细胞与骨髓瘤细胞融合,经ELISA检测、多次有限稀释亚克隆建立分泌ZBTB45单克隆抗体的杂交瘤细胞株,从荷瘤小鼠的腹水中通过蛋白A柱纯化单克隆抗体,鉴定分析所制备抗体在免疫印迹、免疫沉淀和免疫组织化学中的应用效果。结果成功制备了6株抗ZBTB45的特异性单克隆抗体,其中3E5、2G4、4D2和1D4可成功用于免疫印迹和免疫沉淀分析,3E5、6D8和8E3可用于免疫组织化学检测内源性ZBTB45,具有较好的敏感性、特异性。结论首次成功制备了抗ZBTB45单克隆抗体,可应用于体外和体内的免疫学检测,为深入研究ZBTB45的生物学功能提供了技术支持。
OBJECTIVE To prepare a monoclonal antibody against a novel zinc finger protein ZBTB45 and to establish an immunological assay for the molecule in vitro and in vivo to provide technical means for studying its biological function. Methods The multi-epitope antigen fusion protein of mouse ZBTB45 was prepared and the spleen cells were fused with myeloma cells. The hybridoma cell lines secreting ZBTB45 monoclonal antibody were established by multiple dilution-limited subclones by ELISA. Monoclonal antibodies were purified by protein A column in ascites of tumor-bearing mice, and the effect of the prepared antibodies was analyzed by Western blotting, immunoprecipitation and immunohistochemistry. Results Six monoclonal antibodies against ZBTB45 were successfully prepared. Among them, 3E5, 2G4, 4D2 and 1D4 were successfully used in western blot and immunoprecipitation analysis. 3E5, 6D8 and 8E3 could be used for immunohistochemistry to detect endogenous ZBTB45, Has a good sensitivity and specificity. Conclusion The monoclonal antibody against ZBTB45 was successfully prepared for the first time and could be used for immunological detection in vitro and in vivo. It provided technical support for further study on the biological function of ZBTB45.