骨形态发生蛋白-2单克隆抗体的制备及评价

目的：制备骨形态发生蛋白-2（BMP-2）单克隆抗体，并对其进行评价。方法：用重组人源BMP-2蛋白（rhBMP-2）对小鼠进行免疫；确认小鼠血清BMP-2抗体检测阳性后，取其脾细胞与小鼠骨髓瘤细胞按3∶1的比例融合得到杂交瘤细胞株；用间接竞争ELISA法筛选BMP-2阳性单克隆杂交瘤细胞；用常规Protein G进行纯化得到BMP-2单克隆抗体，并对其纯度、表位构相，特异性进行评价。结果：成功制备BMP-2单克隆抗体，其亚型为IgG2a，κ，纯化后的SDS-PAGE纯度大于90%。评价结果表明，本课题组研制的BMP-2抗体与rhBMP-2的结合位点为非线性表位，且特异性优于市售抗体，与BMP家族的BMP-4、BMP-5、BMP-7无交叉反应。结论：本课题组研制的单克隆抗体特异性好，效价高，可用于建立酶联免疫方法，检测骨修复材料中BMP-2含量。

Objective:To develop and evaluate a specific monoclonal antibody against bone morphogenetic protein-2 (BMP-2). Methods:Mice were immunized with the previously developed recombined human BMP-2 (rhBMP-2) protein. When the serum of the mice was tested positive with the BMP-2 antibody,the spleen cells from the mice were isolated and fused with myeloma cells at a ratio of 3:1. Monoclonal hybridoma cells were selected by indirect ELISA using rhBMP-2 as antigen. The BMP-2 antibodies were purified with routine protein G. The purity,epitope conformation and specificity were evaluated. Results:The isotype IgG2a,κ monoclonal antibody against BMP-2 was successfully obtained. The purity of the post-purified product was up to 90% detected by SDS-PAGE. The binding sites of our BMP-2 antibody to rhBMP-2 are linear epitopes,and the specificity of the monoclonal antibody is better than that of the monoclonal antibody available on the market. There were no cross-reactions detected with BMP-4,BMP-5,BMP-7 of the BMP family. Conclusion:The BMP-2 monoclonal antibody developed in this study has high specificity and potency. It can be used for establishing enzyme-linked immunoassay for detecting the content of BMP-2 in bone graft materials.