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  

A biotin-avidin enzyme-linked immunosorbent assay for studying platelet membrane glycoprotein Ⅱb/Ⅲa,CD62p expression and its clinical application

作　　者：张有涛[1] 赵益明[2] 朱明清[2] 蒋敏[1] 程寅峰[1] 史进方[1] 顾国浩[1] 阮长耿[2] 

机构地区：[1]苏州大学附属第一医院检验科,江苏苏州215006 [2]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）》2012年第4期524-527,共4页Suzhou University Journal of Medical Science

基　　金：江苏省高校自然科学基金资助项目(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资助项目(WKF07009)

摘　　要：目的建立测定血小板膜糖蛋白功能的生物素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(GPⅡb/Ⅲa)单抗7E3和抗血小板CD62p单抗SZ51及生物素亲和素系统建立BA-ELISA方法,并应用该方法检测50名健康志愿者、30例急性心肌梗死(AMI)、30例急性脑梗死(ACI)和30例糖尿病(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3.13×109/L(7E3包被板)和6.25×109/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/Ⅲa和CD62p表达量均显著高于健康志愿者(均P<0.01)。BA-ELISA测定表明,SZ21和阿司匹林可抑制ADP诱导的血小板功能(但P>0.05)。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、抗血栓药物的评估及疾病预后的判断起到辅助作用。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 develop 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 in the 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 were as low as 3.13 × 109 /L or 6.25 × 109 /L in the 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 the controls（all P 〈 0.01）.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 glycoprotein expression and activation extent.This method is suitable to screen inhibitors/activators of platelet activation and has a potential in use for diagnostic purposes.

关 键 词：血小板 膜糖蛋白 生物素亲和素系统 酶联免疫吸附试验 急性心肌梗死 急性脑梗死 糖尿病 

分 类 号：R446.113[医药卫生—诊断学]