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目的:构建人源性抗血小板膜糖蛋白Ⅱb/Ⅲa(GPI-Ib/IIIa)Fab噬菌体抗体库,筛选抗GPⅡb/Ⅲa特异性的噬菌体抗体,并对其功能进行研究。方法:取血小板膜糖蛋白抗体(抗GPIIb/IIIa)阳性的特发性血小板减少性紫癜(ITP)患者脾细胞,采用噬菌体抗体库技术构建人源性抗GPⅡb/Ⅲa Fab噬菌体抗体库,以表达GPⅡb/Ⅲa的CHO123细胞筛选抗体库,并以ELISA法检测噬菌体抗体;用Western blot对抗体进行鉴定,并测定其与血小板抗原的结合;观察筛选到Fab抗体对血小板聚集的影响。结果:筛选出2株能够与血小板膜GPⅡb/Ⅲa特异性结合的Fab抗体,其序列与人免疫球蛋白轻、重链可变区序列具有高度同源性,表达纯化的Fab抗体能抑制血小板聚集。结论:成功地从人源性抗血小板膜糖蛋白Ⅱb/Ⅲa Fab抗体库筛选出特异性识别GPⅡb/Ⅲa,具有抑制血小板聚集作用的人源性抗体。
OBJECTIVE: To construct a phage display library of human anti-platelet glycoprotein Ⅱb / Ⅲa (GPI-Ib / IIIa) phage and screen its anti-GPⅡb / Ⅲa specific phage antibody. Methods: Splenocytes from patients with idiopathic thrombocytopenic purpura (ITP) who were positive for platelet glycoprotein (anti GPIIb / IIIa) and phage antibody library were used to construct human anti-GPⅡb / Ⅲa Fab phage antibody library for expression Antibodies were screened from CHO123 cells of GPⅡb / Ⅲa and phage antibodies were detected by ELISA. Antibodies were identified by Western blot and assayed for their binding to platelet antigens. The effects of Fab antibodies on platelet aggregation were observed. RESULTS: Two Fab antibodies that could specifically bind to platelet membrane GPⅡb / Ⅲa were screened out. The sequences of the Fab antibodies were highly homologous to the sequences of human immunoglobulin light and heavy chain. The purified Fab antibody could inhibit platelet aggregation . Conclusion: The human anti-platelet glycoprotein Ⅱb / Ⅲa Fab antibody library was successfully screened for human antibodies that specifically recognize GP Ⅱ b / Ⅲ a and inhibit platelet aggregation.