小鼠抗人胰岛素受体单克隆抗体的制备、纯化及鉴定

 M11D杂交瘤细胞株是由人胎盘细胞膜纯化所得胰岛素受体免疫BALB/C小鼠后,取其脾细胞与同系小鼠骨髓瘤细胞株NS-1细胞融合所得。该杂交瘤细胞分泌的抗体经ELISA及放射免疫沉淀法证实为胰岛素受体特异的单克隆抗体。该抗体经Protein A-Sepharose亲和层析分离、纯化,SDS-聚丙烯酰胺梯度凝胶电泳鉴定得分子量分别为53000及23000的两条区带,免疫双扩证明为IgGl。该抗体特异地沉淀125Ⅰ-人胎盘细胞膜胰岛素受体,沉淀经SDS-聚丙烯酰胺凝胶电泳后放射自显影得分子量为135000的特异显影带,与胰岛素受体α亚基分子量相同,说明M11D为抗胰岛素受体α亚基的单克隆抗体。

Preparation, Purification and Characterization of Monoclonal Antibody to Human Insulin Receptor

Monoclonal antibodies against the insulin receptor were prepared in BALB/ C mice by immunization with purified insulin receptor from human placenta membrane. The spleen of the mouse was then removed, and its lymphocytes were fused with a mouse myeloma cell line, NS-1 cells. After screening over 40 resulting hybrids, one stable hybrid was obtained that produced IgG1 antibodies against the insulin receptor. These monoclonal antibodies were purified by protein A-sepharose chromatography and then electrophoresed on a SDS-polyacrylamide gel. Two purified bands were shown which correspond to the light and heavy chain of the immunoglo-bulin molecule respectively. This antibody immunoprecipitated 125I-placenta membrane, the immunoprecipitates were electrophoresed on a 7,5% SDS-polyacrylamide gel and then autoradiographed. One specific polypeptide band was shown with molecular weight of 135000 on the autoradiogram. The molecular weight of this band was identical to that of α-subunit of the insulin receptor. So the monoclonal antibody we obtained was specific to the α-subunit of the insulin receptor.