人血小板功能抗体及其片段的识别

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.

Identification of human platelet specific functional antibody and its fragments

OBJECTIVE: To study the immunoreactivity of the specific anti-platelet glycoprotein (GP) IgG antibody and its F(ab')2 fragments from patients with chronic idiopathic thrombocytopenic purpura (ITP) and to investigate their effects on platelet aggregation function. METHODS: Peripheral blood samples were collected from 84 patients with ITP. Modified monoclonal antibody immobilization of platelet antigen assays was used to detect the IgG antibodies specific for GP I b/II a, GP I b/IX and GP VI. The IgG antibody and its F(ab')2 fragments in the positive plasma inhibiting platelet aggregation function were prepared and purified. Plate-rich Peripheral blood sample was collected from a normal person with O type blood and platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared. Plasma pf ITP patient or purified IgG or F(ab')2 fragments of different concentrations were added into PRP, and then inducers of platelet aggregation ADP, ristocetin, or collagen were added. The platelet aggregation was measured. Platelet GP II b/III a specific human-rat chimeric antibody 7E3 and GP I b specific antibody SZ2 were used as positive controls and PBS was used as negative control. RESULTS: GP II b/III a and/or GP I b/IX and/or GP VI specific antibodies were found in 48 (57.1%) patients. The plasma, purified IgG and F(ab')2 fragments of 7 of these 48 patients (14.6%) with positive autoantibody showed significant activity against GP II b/III a (4 patients), GP I b/IX (2 patients), or GP VI (one patient). The purified IgG and F(ab')2 fragments of 2 patients positive in GP II b/ III a autoantibody out of the 7 patients significantly inhibited the platelet aggregation induced by ADP, the purified IgG and F(ab')2 fragments of 1 patients positive in GP I b/IX out of the 7 patients significantly inhibited the platelet aggregation induced by ristocetin, and the purified IgG and F(ab')2 fragments of 1 patients positive in GP VI out of the 7 patients significantly inhibited the platelet aggregation induced by collagen. CONCLUSION: A functional fragment, F(ab')2 portion of IgG is responsible for the autoantibody interaction with platelet GPs in ITP, and some of them also affect the platelet function. It can be used to develop completely humanized anti-GP small molecular phage antibody.