人胎盘层粘连蛋白单克隆抗体的制备与鉴定

Preparation and Identification of Monoclonal Antibody Against Human Laminin

目的获得能特异性识别人层粘连蛋白(hLN)的单克隆抗体(McAb)。方法用纯化的人层粘连蛋白(hLN)作抗原免疫Balb/C小鼠,以细胞融合、酶联免疫吸附实验(ELISA)筛选和克隆化技术获得抗hLN的杂交瘤细胞株;用生物学方法鉴定杂交瘤细胞,用免疫学方法鉴定McAb的特异性。结果获得4株稳定分泌抗人LN的杂交瘤细胞株(2A1、3B5、2C4、4D1),培养上清的ELISA效价分别为:1∶512、1∶1024、1∶512、1∶256;腹水效价分别为:1×106、1×107、1×106、1×105;采用ELISA相加法表明2A1、4D1与3B5、2C4识别的hLN上的抗原决定簇和识别的不同,4株单抗均属实IgG。结论成功建立了4株稳定分泌抗人LNMcAb的杂交瘤细胞株,它们分别识别hLN上2个不同的抗原位点,有望作为hLN定量检测的特异性抗体。

Objective To prepare a monoclonal antibody （McAb） against human laminin （hLN）. Methods Spleen cells from Balb/c mice immunized with human laminin （hLN） were fused with Sp2/0 myeloma ceils. The specific McAb were screened by ELISA and identified by western blot. Results Four hybridoma cell lines （2A1, 3B5, 2C4 and 4D1 ） secreting specific McAb agaionst human laminin （hLN） were selected and determined to recognize different epitops on human laminin. The titers of the culture supematant determined by ELISA of the four clones were 1：512, 1 ： 1 024, 1 ： 512,and 1 ： 256;of the ascites were 1×10^6、1×10^7、1×10^6、1×10^5. McAbs of 2A1 and 4D1 recognize one epitope on human laminin,whereas 3B5 and 2C4 recognize another epitope. 2A1, 4D1, 3B5 and 2C4 are IgG McAbs. Conclusion Four hybridoma cell lines producing high titer and specific McAbs against the human laminin were established.