抗早孕因子单克隆抗体的制备与鉴定

目的建立分泌抗EPF(Early pregnancy factor,早孕因子)单克隆抗体的杂交瘤细胞株,纯化单抗并鉴定.方法用本实验室已纯化的早孕和肿瘤源性EPF作为抗原刺激Balb/c小鼠,用免疫后的小鼠脾细胞与同系小鼠骨髓瘤细胞(NS-1)融合,经4次克隆化,获得可稳定分泌抗EPF单克隆抗体的细胞株,注入Balb/c小鼠腹腔制备腹水型单抗,Protein-A亲和层析纯化,SDS电泳和Western-blot等方法分析纯化结果.结果融合后获得一株稳定分泌抗EPF抗体的细胞株(C3D11),克隆化后,获得稳定分泌抗EPF单克隆抗体的细胞株,将增殖后的细胞注射Balb/c小鼠腹腔获得腹水型单抗,以亲和层析法纯化,SDS-PAGE分析显示纯化后去掉了大部分杂蛋白,免疫印迹分析抗体纯度较高,与抗原匹配性良好.结论本研究制备的EPF单克隆抗体为特异性抗EPF抗体.

Preparation and identification of anti-EPF monoclonal antibodies

Objective To establish hybridoma cell lines secreting anti-early pregnancy factor (EPF) antibodies, and to select monoclonal antibodies of high affinity and specificity from the cell lines. Methods Spleen cells from BALB/C mice immunized with purified EPF were fused with murine NS-1 myeloma cells. Ascite fluid containing the monoclonal antibodies against EPF was obtained by ventral injection. Monoclonal antibodies were purified by protein-A affinity chromatography. Results One hybridoma cell line (C3D11) of anti-EPF was obtained, which could stably secrete the monoclonal antibodies. Ascites containing the antibodies was obtained through subcloning the obtained cell lines for4 cycles. With a high purity, the obtained antibodies had and could match the specific antigens perfectly. Conclusion The monoclonal antibodies are aimed at EPF.