人血小板膜糖蛋白功能测定的酶联免疫分析方法的建立  

Establishment of Enzyme-Linked Immunosorbent Assay to Study Platelet Membrane Glycoprotein Function and Its Significance

作　　者：张有涛[1] 赵益明[1] 何杨[1] 朱明清[1] 阮长耿[1] 

机构地区：[1]苏州大学附属第一医院江苏省血液研究所,卫生部血栓与止血重点实验室,江苏苏州215006

出　　处：《苏州大学学报（医学版）》2010年第2期291-295,共5页Suzhou University Journal of Medical Science

基　　金：江苏省高校自然科学基金(08KJD310008);江苏省卫生厅"科教兴卫工程"临床医学中心血液病学开放课题基金(WKF07009)资助项目

摘　　要：目的建立测定血小板膜糖蛋白(GP)功能的酶联免疫分析(ELISA)方法 ,并对其灵敏度、批内和批间差异进行方法学考核。方法以抗血小板CD62P单抗SZ51和抗血小板膜糖蛋白Ⅱb/Ⅲa(GPⅡb/Ⅲa)单抗7E3分别包板,加入经ADP诱导的或未诱导的正常人富含血小板血浆(PRP),以生物素标记的抗GPIb单抗SZ2(Biotin-SZ2)反应后,加入亲和素标记的辣根过氧化物酶(Avdin-HRP)检测,邻苯二胺(OPD)显色后,酶标仪读取吸光度(A)值。对其灵敏度及批内、批间差异作方法学评估,应用该方法对抑制性单抗SZ-21和阿司匹林抑制血小板活化的功能效果进行测定,并与流式细胞术(FCM)测定结果进行比较。结果该方法可检测血小板计数低达6.25×109/L(SZ51包被板)和3.13×109/L(7E3包被板)的标本,批内和批间变异系数均小于10%。测得50名健康正常人ADP诱导的(未诱导的)血小板的CD62P和GPⅡb/Ⅲa的A值分别为0.68±0.21(0.18±0.09)和0.87±0.29(0.44±0.14)。ELISA测定结果表明,SZ21和阿司匹林可明显抑制ADP诱导的血小板功能。流式细胞仪测定ADP诱导的(未诱导的)人血小板表面CD62P和GPⅡb/Ⅲa的阳性细胞百分率分别为(60.1±7.12)%[(2.56±0.61)%]和(86.32±17.82)%[(20.25±14.56)%],其批内和批间变异系数均<10%。结论该方法可以测定血小板功能活化剂或抑制剂对血小板功能的影响,可以测定血栓性疾病或血栓形成相关性疾病引起的血小板活化程度。Objective To develop an enzyme-linked immunosorbent assay（ ELISA） method for detecting platelet membrane glycoprotein（GP） function,and evaluate its sensitivity,inter-assay and intra-assay coefficient of variation （CV）.Methods A monoclonal antibody （mAb） to GPⅡb/Ⅲa,7E3 and a mAb to CD62P,SZ51 were immobilized onto microtiter plate wells,respectively,and then platelet-rich plasma （PRP） with or without ADP was added to the wells,respectively.After incubated with biotin-labeled anti-GPIb mAb,SZ2 （biotin-SZ2）,the conjugated biotin-SZ2 was checked by avdin-labeled horseradish peroxidase （Avdin-HRP）.The sensitivity and repeatability of ELISA for platelet function were evaluated with flow cytometry （FCM）.Inhibition of platelet membrane glycoprotein function was evaluated with inhibitory mAb,SZ21 and aspirin.Results The ELISA can detect platelet count as low as 6.25 × 109 /L （SZ 51 coated plates） and 3.13 × 109 /L （7E3 coated plates） in PRP of the specimens.Both of the inter-assay and intra-assay coefficient variation（C V） were less than 10%.CD62P and GPⅡb/Ⅲa of the 50 ADP-induced （non-induced） healthy platelets in PRP were detected by ELISA.The absorbance （A492/620nm） value were 0.68±0.21（0.18±0.09）and 0.87±0.29（0.44±0.14）.The range of the ADP-induced （non-induced） CD62P and GPⅡb/Ⅲa of platelet membrane detected by FCM is （60.1±7.12）%[（2.56±0.61）%] and （86.32 ±17.82）%[（20.25±14.56）%],and both of the inter-assay and intra-assay CV were less than 10%.Conclusion This method can be used to evaluate the influence of platelet activators/inhibitors on platelet function and detect platelet activation level caused by thrombus or its associated diease.

关 键 词：血小板 膜糖蛋白 酶联免疫吸附试验 

分 类 号：R331.143[医药卫生—人体生理学]