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目的:制备抗人磷脂酰蛋白聚糖3(Glypican3,GPC3)N端蛋白的单克隆抗体(mAb),并进行特性鉴定。方法:以纯化的GPC3 N端融合蛋白免疫BALB/c小鼠,应用淋巴细胞杂交瘤技术制备抗人GPC3N端mAb并进行纯化。经Western blot和ELISA方法鉴定其特异性、Ig亚类和效价。结果:获得2株可稳定分泌抗人GPC3N端mAb的杂交瘤细胞株(Ⅲ4B,Ⅻ6C)。两株mAb均为Ig1亚类,其腹水mAb的效价分别为1∶9 600及1∶7 200,均可特异性地识别GPC3蛋白。结论:成功地建立可稳定分泌抗人GPC3N端mAb的杂交瘤细胞株,其分泌的mAb特异性强、效价高。
OBJECTIVE: To prepare a monoclonal antibody (mAb) against N-terminal protein of Glypican3 (GPC3) and to characterize it. Methods: BALB / c mice were immunized with purified GPC3 N-terminal fusion protein. Anti-human GPC3N mAb was prepared by using lymphocyte hybridoma technique and purified. Specificity, Ig subclass and titer were identified by Western blot and ELISA. Results: Two hybridoma cell lines stably secreting anti-human GPC3N mAb (Ⅲ4B, Ⅻ6C) were obtained. The two mAbs were all Ig1 subclasses. The titer of ascites mAb was 1: 900 and 1: 200, respectively, which could specifically recognize GPC3 protein. Conclusion: The hybridoma cell line that can stably secrete anti-human GPC3 N-terminal mAb was successfully established. The mAb secreted by the mAb was highly specific and high titer.