抗hCD177单克隆抗体的制备及生物活性研究

Preparation and biological activity study of monoclonal antibodies against human CD177

[目的]制备鼠抗人CD177(hCD177)单克隆抗体,对抗体的生物学活性进行初步研究。[方法]以过表达hCD177的HeLa细胞为免疫原,对Balb/c小鼠进行免疫;使用PEG作为诱导剂使小鼠骨髓瘤细胞SP2/0与脾细胞融合,采用细胞ELISA法和流式细胞术筛选阳性杂交瘤。通过腹水生产法,rProtein A柱亲和纯化hCD177单克隆抗体,细胞ELISA法对抗体的效价进行测定,流式细胞术和细胞免疫荧光定位对抗体生物学活性进行鉴定。[结果]获得1株产生抗hCD177单克隆抗体杂交瘤细胞,抗体亚型为IgG,细胞ELISA表明抗体的效价达到2×10^(6);流式细胞术表明抗体与过表达hCD177的HeLa细胞、L929细胞有较高的结合率;细胞免疫荧光定位试验表明抗体能特异性识别细胞表面的CD177蛋白;在对临床样品检测中,抗体能有效识别中性粒细胞,检测灵敏度高于商业化抗体。[结论]成功制备了hCD177单克隆抗体,该抗体能特异性识别细胞表面的CD177蛋白,可应用于细胞检测。

[Objective]To prepare mouse anti-human CD177(hCD177)monoclonal antibody and preliminary study of its biological activity.[Method]The hCD177-overexpressed HeLa cells were used as the immunogen to immunize Balb/c mice,and PEG induced fusion of mouse spleen cells with myeloma cells SP2/0.Positive hybridomas were screened by cell ELISA and flow cytometry.Anti-human CD177 monoclonal antibody was obtained by ascites production method and affinity purification by rProtein A column.The titer of the antibodies was determined using cell ELISA,and the antibodies were identified by flow cytometry and cellular immunofluorescence.[Result]One line of hybridoma cell secreting anti-human CD177 subtyped as IgG was successfully obtained with a high titer in ascites above 2×10^(6).Results of flow cytometry showed that the antibody had a high binding rate with the hCD177-overexpressed HeLa and L929 cells.Cellular immunofluorescence localization showed that the antibody can specifically recognize CD177 protein on the cell surface.In the detection of clinical samples,the antibody can effectively recognize neutrophils and the sensitivity was higher than a commercial antibody.[Conclusion]The anti-human CD177 monoclonal antibody was successfully prepared,can specifically recognize the CD177 protein on the cell surface and can beapplied for cell detection.