首页|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

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

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  • 维普
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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 GP Ⅱ b/ Ⅲ a 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 GP Ⅱ b/ Ⅲ a 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 GP H b/ M a 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.

plateletmembrane glycoproteinbiotin-avidin systemenzyme-linked immunosorbent assayacute myocardial infarctionacute cerebral infarctiondiabtes mellitus

张有涛、赵益明、朱明清、蒋敏、程寅峰、史进方、顾国浩、阮长耿

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苏州大学附属第一医院检验科,江苏苏州215006

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

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

江苏省高校自然科学基金资助项目江苏省卫生厅“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

08KJD310008WKF07009

2012

苏州大学学报(医学版)
苏州大学

苏州大学学报(医学版)

CSTPCD
影响因子：0.499
ISSN：1673-0399
年,卷(期)：2012.32(4)
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