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目的制备特发性血小板减少性紫癜(ITP)患者血浆 IgG 及其酶切片段,探讨其与血小板 GPⅡb/Ⅲa 和(或)GP Ⅰb/Ⅸ结合的免疫活性及其对正常人血板小板聚集功能的影响。方法用改良 MAIPA 法和比浊法血小板聚集试验筛选出自身抗体阳性并且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,改良单克隆抗体俘获血小板抗原技术(MAIPA)检测完整抗体及其酶切片段与血小板膜糖蛋白的结合活性;比浊法血小板聚集试验对比观察 ITP 患者血浆、纯化的 IgG 抗体及其酶切片段对正常人血小板聚集功能的影响。结果①68例慢性ITP 患者中,34例(53.6%)血浆中抗 CPⅡb/Ⅲa 和(或)CPⅠb/Ⅸ自身抗体阳性,其中5例(14.7%)明显抑制了二磷酸腺甘(ADP)或瑞斯托霉素对血小板聚集的诱导作用;②用蛋白 A 柱结合蛋白酶酶切成功获得了纯化的 IgG 及 F(ab′)_2片段;③患者纯化的 IgG 及 F(ab′)_2片段均具有抗 GPⅡb/Ⅲa 或GP Ⅰb/Ⅸ活性,但去除 IgG 的血浆丧失了与 GPⅡb/Ⅲa 或 CP Ⅰb/Ⅸ的结合活性;④2例患者纯化的IgG 及其 F(ab′)_2片段抑制 ADP 诱导的血小板聚集。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并可抑制血小板聚集功能,其抑制聚集作用呈剂量依赖性。
Objective To prepare plasma IgG and its digested fragments in patients with idiopathic thrombocytopenic purpura (ITP) and investigate its immunological activity in combination with platelet GPⅡb / Ⅲa and / or GP Ⅰb / Ⅸ and its effect on normal human platelet aggregation Effect of function. Methods Patients with positive autoantibody and platelet aggregation were screened by improved MAIPA assay and turbidimetric platelet aggregation test. The plasma IgG antibody was purified by protein A column and F (ab ’) _ 2 fragment was prepared by pepsin. The modified monoclonal antibody Capture platelet antigen technology (MAIPA) detection of intact antibody and its digested fragments and platelet membrane glycoprotein binding activity; turbidimetric platelet aggregation test compared ITP patients with plasma, purified IgG antibody and its digested fragments of normal human platelet aggregation Effect of function. RESULTS: Of the 68 patients with chronic ITP, 34 (53.6%) had positive anti-CPⅡb / Ⅲa and / or CPⅠb / Ⅸ autoantibodies in plasma, of which 5 (14.7%) significantly inhibited adenosine diphosphate (ADP) Staphylococcus aureus platelet aggregation induced; ② protein A column with protease digestion and purification of IgG and F (ab ’) _ 2 fragments obtained; ③ patients with purified IgG and F (ab’) _ 2 fragments have The activity of GPⅡb / Ⅲa or GP Ⅰ b / Ⅸ was inhibited, but the activity of GP Ⅱb / Ⅲa or CP Ⅰb / Ⅸ was lost in the plasma of IgG; ④ The purified IgG and F (ab ’) _ 2 fragments of 2 patients inhibited ADP-induced Platelet aggregation. Conclusion F (ab ’) _2 fragment is a functional fragment of IgG autoantibody. It not only retains good antigen binding activity but also inhibits platelet aggregation, which inhibits the aggregation in a dose - dependent manner.