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目的研究特发性血小板减少性紫癜(ITP)患者血小板膜糖蛋白(GP)特异性IgG抗体及其片段的免疫活性及对血小板聚集功能的影响。方法用改良单克隆抗体特异性俘获血小板抗原技术(MAIPA)检测84例慢性ITP患者血浆中抗GPⅡb/Ⅲa、GPⅠb/Ⅸ及GPⅥ自身抗体,比浊法血小板聚集试验筛选出自身抗体阳性并能抑制血小板聚集的患者,用蛋白A柱纯化其血浆IgG抗体并用胃蛋白酶制备F(ab′)2片段。检测纯化的IgG抗体及其酶切片段与血小板GP的结合活性及对正常人血小板聚集功能的影响。结果(1)84例ITP患者血浆中,48例(57.1%)抗GPⅡb/Ⅲa和/或GPⅠb/Ⅸ和/或GPⅥ自身抗体阳性,其中7例(14.6%)明显抑制了二磷酸腺苷、瑞斯托霉素或胶原诱导的血小板聚集;(2)纯化的IgG及F(ab′)2片段具有与相应血小板GP的结合活性;(3)4例患者纯化IgG及F(ab′)2片段具有抑制血小板聚集的功能。结论F(ab′)2片段是IgG自身抗体的功能片段,它不但保留了良好的抗原结合活性且可部分抑制血小板聚集功能,为人源化血小板糖蛋白特异性抗体的制备奠定了基础。
Objective To investigate the immune activity of platelet glycoprotein (GP) specific IgG antibodies and their fragments in patients with idiopathic thrombocytopenic purpura (ITP) and their effects on platelet aggregation. Methods The anti-GPⅡb / Ⅲa, GPⅠb / Ⅸ and GPVI autoantibodies in plasma of 84 patients with chronic ITP were detected by modified monoclonal antibody specific capture platelet antigen technique (MAIPA). The turbidimetric platelet aggregation test screened for autoantibodies and inhibited In patients with platelet aggregation, their plasma IgG antibodies were purified with protein A columns and F (ab ’) 2 fragments were prepared with pepsin. To detect the binding activity of purified IgG antibody and its digested fragments with platelet GP and its effect on normal platelet aggregation. Results (1) Of the 84 patients with ITP, 48 (57.1%) were positive for GPⅡb / Ⅲa and / or GPⅠb / Ⅸ and / or GPVI autoantibodies, of which 7 (14.6%) significantly inhibited adenosine diphosphate, Ristocetin or collagen induced platelet aggregation; (2) purified IgG and F (ab ’) 2 fragments have binding activity to the corresponding platelet GP; (3) purified IgG and F (ab’) 2 Fragment has the function of inhibiting platelet aggregation. Conclusion F (ab ’) 2 fragment is a functional fragment of IgG autoantibodies. It not only retains good antigen binding activity but also partially inhibits platelet aggregation, which lays a foundation for the preparation of humanized platelet glycoprotein specific antibodies.