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为了研制骨桥蛋白(OPN)特异性单克隆抗体(mAb),并鉴定其特异性。本研究在毕赤酵母中表达具有良好免疫原性的重组人OPN蛋白的基础上,用重组人骨桥蛋白(rhOPN)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,间接ELISA筛选杂交瘤细胞,并结合免疫印迹对抗体的特异性进行鉴定,通过竞争抑制试验对单克隆抗体识别的抗原位点进行分析。结果共获得4株能够识别OPN不同抗原位点的mAb,亚类测定显示,3株为IgG1,1株为IgG2a。这些mAb能与重组人OPN特异性结合。本研究的四株抗体中,只有4G2B5能够检出与肿瘤转移密切相关的OPN-c的条带,预示该抗体可用于判断肿瘤预后和高转移肿瘤的临床检测。本研究成功获得了针对骨桥蛋白的特异性mAb,同时为进一步研究OPN蛋白的结构和功能提供了重要工具。
In order to develop osteopontin (OPN) -specific monoclonal antibodies (mAbs), and to identify their specificity. In this study, recombinant human OPN protein with good immunogenicity was expressed in Pichia pastoris. BALB / c mice were immunized with rhOPN and the spleen cells were fused with mouse myeloma cells. The hybridoma cells were screened by ELISA, and the specificity of the antibodies was identified by Western blotting. Antigen sites recognized by the monoclonal antibodies were analyzed by competitive inhibition assay. Results A total of four mAbs that recognized different OPN epitopes were obtained. Subgroups of the mAbs were identified as IgG1 and 3 strains as IgG2a. These mAbs specifically bind to recombinant human OPN. Of the four antibodies in this study, only 4G2B5 was able to detect a band of OPN-c closely related to tumor metastasis, indicating that the antibody can be used to determine the prognosis of tumors and the clinical detection of highly metastatic tumors. In this study, we obtained the specific mAb targeting osteopontin and provided an important tool for further study on the structure and function of OPN protein.