抗血小板膜糖蛋白Ib单克隆抗体的制备、鉴定及其功能的初步研究

Development,identification and function assay of monoclonal antibody against platelet membrane glycoprotein Ib

目的:制备抗人血小板膜糖蛋白Ib(GPIb)单克隆抗体(mAb),并进行生化鉴定与初步探讨其临床应用。方法:采用血小板免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞融合。经间接ELISA法筛选和克隆化培养,获得1株抗人GPIbmAb的杂交瘤细胞,纯化其腹水获得mAb;免疫双扩散鉴定其抗体亚类;流式细胞术和免疫放射法鉴定其识别的抗原;ELISA法检测该抗体对血浆血管性血友病因子(vWF)的瑞斯托霉素辅因子活性的影响。结果:成功制备了1株抗人GPIbmAb,命名为SZ-151。ELISA法测定其腹水效价为1∶20 000,免疫双扩散鉴定其为IgG1亚类。免疫放射法和流式细胞术检测结果证实SZ-151和mAb SZ-2识别同一抗原GPIb,且作用位点不同。ELISA结果显示,SZ-151对瑞斯托霉素辅因子活性无抑制作用。结论:获得1株新的抗血小板膜糖蛋白Ib的mAb,可用于血浆vWF的瑞斯托霉素辅因子活性检测,为血管性血友病的诊断和分型提供了有效的工具。

AIM： To prepare and identify a monoclonal antibody（mAb） against human platelet glycoprotein Ib and make its application.METHODS： BALB/c mice were immunized with human platelets washed,and the spleen cells of them were fused with myloma cells.A hybridoma cell was screened by indirect ELISA and cloned,and the mAb were purified from the ascites of mice.Ig subclass was analysed by double immunodiffusion.The antigen recognized by monoclonal antibody was identified by flow cytometry and radioimmunoassy,respectively.The inhibition of mAb on plasma von Willebrand factor ristocetin cofactor activity（vWF∶Rcof） was investigated by enzyme linked immunosorbent assay（ELISA）.RESULTS： A murine mAb against human platelet membrane glycoprotein（GP） Ib was developed and denominated as SZ-151.SZ-151 belonged to IgG1 subclass and its titer in ascites was 1∶20 000.Flow cytometry and radioimmunoassy showed that the antigen recognized by monoclonal antibody SZ-151 was platelet membrane GPIb.ELISA showed that SZ-151 did not inhibit plasma von Willebrand factor ristocetin cofactor activity.CONCLUSION： A mAb,SZ-151 against platelet glycoprotein Ib was developed,which could be useful in assays of plasma von Willebrand factor ristocetin cofactor activity（vWF∶Rcof） and can be used for diagnose patients with vWD.