摘要
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 ITP plasma IgG and its F （ab＇）2 fragments and investigate their immunoreactivity to platelet GP Ⅱ b/Ⅲ a and/or GP Ⅰ b/Ⅸ and their effects on platelet aggregation function. Methods The ITP patients having inhibitory autoantibody to the platelet aggregation were selected by modified MAIPA and platelet aggregation test with turbidomitry. Plasma IgG and its F（ab＇） 2 fragments were prepared by streptococcal protein A affinity column and pepsin digestion. The immunoreactivity and the effects on platelet aggregation function of the whole antibody and its fragments were detected by modified MAIPA and platelet aggregation test, respectively. Results ①Anti-platelet GP Ⅱ b/Ⅲ a and/or GP Ⅰb/Ⅸ autoantibodies were detected in 34 of 68 （53.6%） ITP patients＇ plasmas and that from 5 patients significantly inhibited the platelet aggregation induced by ADP or ristocetin. ②By using protein A column combined with protease digestion, pure IgG and its F （ab＇）2 fragments were successfully obtained. ③The purified IgG and its F（ab＇） 2 fragments retained the ability to bind to their respective glycoproteins and inhibited the platelet aggregation function, whereas the IgG depleted plasma lost the ability of binding to the platelet GPs. Conclusions F（ab＇） 2 fragment of the IgG antibody is a functional fragment, which not only has the binding ability to the platelet GPs but also inhibits the platelet aggregation function in a dose-dependent manner.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2006年第3期158-161,共4页
Chinese Journal of Hematology
基金
国家自然科学基金(30470742
30300312)
山东省自然科学基金(Y2003C06)
