骨桥蛋白单克隆抗体的制备及其特异性分析

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。

Preparation and characterization of monoclonal antibodies against osteopontin

BALB/c mice were immunized with recombinant human osteopontin(rhOPN) and splenocytes of the immunized mice were fused with myeloma NS1 cells to produce hybridoma cell lines.ELISA assay was used to identify the monoclonal antibody against rhOPN protein and Western blotting was applied to identify the specificity of this antibody.It was demonstrated that 4 monoclonal antibodies(mAbs) recognizing different epitopes on OPN were obtained.These antibodies showed specific binding activity to recombinant OPN protein.The subtype of 3 antibodies was proved to be IgG1,and the other one was IgG2a.Only 4G2B5antibody among these 4 monoclonal antibodies could specifically recognise the OPN-c band protein which was closely related with the metastasis of tumors.This study suggests that 4G2B5 mAb could be applied for the high metastasis tumor prognosis monitor and the clinical diagnosis.