BA-ELISA定量检测人血小板膜糖蛋白Ⅱb／Ⅲa、CD62p的表达及其临床应用被引量：1

Detection of platelet membrane glycoprotein Ⅱb/Ⅲaand CD62p by biotin-avidin enzyme-linked immunosorbent assay and its clinical application

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摘要目的建立测定血小板膜糖蛋白功能的生物素一亲和素-酶联免疫吸附（BA-ELISA）方法，并探讨其临床应用价值。方法采用抗血小板膜糖蛋白Ⅱb／Ⅲa（membraneglycoproteinⅡb／Ⅲa，GPⅡb／Ⅲa）单抗7E3和抗血小板CD62p单抗SZ51及生物素一亲和素系统建立BA-ELISA方法，并应用该方法检测50名健康志愿者、30名急性心肌梗死（AMI）、30名急性脑梗死（ACI）和30名糖尿病（DM）患者的血小板膜糖蛋白功能，并与流式细胞术（FCM）测定结果进行比较。同时，应用该方法对抑制性单抗SZ21和阿司匹林抑制血小板活化的功能效果进行测定。结果该方法检测血小板计数敏感度低达3．13×10^9／L（7E3包被板）和6．25×10^9／L（SZ51包被板），批内和批间变异系数分别为6．23％～8．35％和4．38％-5．78％。测得AMI、ACI和DM患者ADP诱导的（未诱导的）血小板的GPⅡb／ⅢaCD62p表达量均显著高于健康志愿者（P〈0．01）。BA-ELISA测定表明，SZ21和阿司匹林可明显抑制ADP诱导的血小板功能。该方法和FCM同时测定健康志愿者、AMI、ACI和DM患者的GPⅡb／Ⅲa和CD62p表达水平。测定数据经直线回归分析得r=0．86。结论BA-ELISA检测血小板的膜糖蛋白，可精确判断血小板的功能状态，为指导临床预防、诊治、评估疾病提供简便、可行的方法。活化的血小板膜糖蛋白测定可从凝血的角度对上述疾病的早期诊断、病情进展的判断、抗血栓药物的评估以及判断疾病的预后起到辅助作用。 Objective To develop a biotin-avidin enzyme-linked immunosorbent assay （BA-ELISA） method for detecting platelet membrane glycoprotein function, and evaluate its clinical application. Methods With the monoclonal antibodies （mAb） recognizing GPⅡb/Ⅲa （7E3）, CD62p （SZ51） and biotin-avidin system, to developed a biotin-avidin-ELISA （BA-ELISA）. The levels of GPⅡb/Ⅲa and CD62p were measured in patients with acute myocardial infarction （AMI）, acute cerebral infarction （ACI）, diabetes mellitus （DM） or healthy people. This BA-ELISA was compared with flow cytometry （FCM）. Inhibition of membrane glycoprotein expression was evaluated with inhibitory mAb, SZ21 and aspirin （acetylsalicylic acid, ASA） respectively. Results The BA-ELISA detected platelet count as low as 3.13 ×10^9/L or 6.25 × 10^9/L, respectively, in platelet-rich plasma （PRP） of the specimens. Both of the interassay and intraassay coefficient variation （CV） were less than 10%. Adenosine diphosphate （ADP）-induced, or non-ADP-induced GPⅡb/Ⅲa and CD62p expression in AMI, ACI, DM were significantly higher than those in controls （all P 〈 0.01）, respectively. Either SZ21 or aspirin inhibited the expression of GPⅡb/Ⅲa and CD62p induced by ADP. A high correlation was showed between BA-ELISA and FCM methods. Conclusion These observations indicate that BA-ELISA is a sensitive and high-throughput assay for evaluating platelet membrane glyeoprotein expression and activation extent. This method is suitable to screen inhibitors/activators ofplatelet activation and has a potential in use for diagnostic purooses.