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目的制备抗delta-like-1的单克隆抗体并鉴定其特异性。方法用delta-like-1多肽-BSA蛋白复合物作为免疫原免疫Balb/c小鼠,取其脾细胞与小鼠骨髓瘤细胞(SP2/0)融合,经多次筛选及克隆化,建立可以稳定分泌抗delta-like-1单抗的杂交瘤细胞株。用ELISA、Western blot、细胞免疫组化对此抗体特性进行鉴定。结果筛选到两株能稳定分泌抗delta-like-1单抗的细胞株3H11C11A9E3和2D5D11F8F1,亚类鉴定两株均为IgG1,轻链为kappa链;ELISA法测定两株细胞腹水抗体效价分别为1:4.096×105和1:1.024×105,抗体亲和常数分别为3.98×107、3.28×107L.mol-1;Western blot显示此单抗能特异性识别神经胶质瘤细胞株U251中的天然delta-like-1蛋白;细胞免疫组化进一步显示delta-like-1分布于细胞核中。结论成功制备了抗delta-like-1单克隆抗体,可为研究delta-like-1与脑胶质瘤等神经系统疾病的关系奠定基础。
Objective To prepare monoclonal antibodies against delta-like-1 and identify its specificity. Methods Balb / c mice were immunized with delta-like-1 polypeptide-BSA protein complex and their spleen cells were fused with mouse myeloma cells (SP2 / 0). After multiple screening and cloning, Stable hybridoma cell line secreting anti-delta-like-1 monoclonal antibody. The characteristics of this antibody were identified by ELISA, Western blot and immunohistochemistry. Results The two cell lines, 3H11C11A9E3 and 2D5D11F8F1, which could secrete anti-delta-like-1 monoclonal antibody were screened, IgG1 subclass and kappa chain were light chain. The antibody titer of ascites was determined by ELISA 1: 4.096 × 105 and 1: 1.024 × 105, the antibody affinity constants were 3.98 × 107,3.28 × 107L.mol-1, respectively. Western blot showed that this monoclonal antibody could specifically recognize the natural delta-like-1 protein. Cellular immunohistochemistry further showed that delta-like-1 was distributed in the nucleus. Conclusion The anti-delta-like-1 monoclonal antibody was successfully prepared, which could provide a basis for the study of the relationship between delta-like-1 and other nervous system diseases such as gliomas.