CN109459565B - 人血清C4BP-a蛋白作为预测氯吡格雷抵抗的生物标志物及其临床应用 - Google Patents

人血清C4BP-a蛋白作为预测氯吡格雷抵抗的生物标志物及其临床应用 Download PDF

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CN109459565B
CN109459565B CN201811515436.9A CN201811515436A CN109459565B CN 109459565 B CN109459565 B CN 109459565B CN 201811515436 A CN201811515436 A CN 201811515436A CN 109459565 B CN109459565 B CN 109459565B
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c4bp
clopidogrel
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谢红光
吉金子
黄贝贝
邰婷
米琼宇
谢文剑
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Puling biological (Nanjing) Co.,Ltd.
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Abstract

本发明公开了预测氯吡格雷抵抗的生物标志物及其临床应用。人血清中C4BP‑a蛋白作为检测靶标或标准品在制备预测氯吡格雷抵抗的试剂盒中的应用。一种预测氯吡格雷抵抗的试剂盒，包含检测人血清C4BP‑a的抗体及其它相关试剂。在临床上，通过对人血清C4BP‑a的检测可预测氯吡格雷抵抗，为指导临床合理用药、帮助临床医生诊断或预测氯吡格雷的临床疗效和预后提供了全新的检测方法。

Description

人血清C4BP-a蛋白作为预测氯吡格雷抵抗的生物标志物及其 临床应用
技术领域
本发明属于生物检测领域，涉及人血清C4BP-a蛋白作为预测氯吡格雷抵抗的生物标志物及其临床应用。
背景技术
作为一种已经入选WHO基本药物目录的抗血小板药物，氯吡格雷是急性冠脉综合征(ACS)或经皮冠脉介入术(PCI)的冠心病病人在植入药物涂层支架后用来预防心肌缺血再次发生或支架内血栓形成的最常用药物，与阿司匹林合用所组成的“双抗血小板治疗”方案是ACS或PCI植入支架术后的“金标准”抗血小板治疗方案，为全世界所认可和采纳。
氯吡格雷抵抗(或耐受)是指病人在服用推荐(或标准)剂量的氯吡格雷后达不到预期的抗血小板作用，导致药物的临床疗效较差或无效的现象。据估计，约10％–45％的服药病人有氯吡格雷抵抗。
现有的判断氯吡格雷临床疗效的替代指标是用服药前后ADP诱导血小板聚集的抑制率大小来反映氯吡格雷抵抗的有无或程度。但是，血小板聚集率检测方法的重复性较差，难以精准指导临床合理使用氯吡格雷。
此外，目前临床采用的病人外周血白细胞DNA检测CYP2C19基因型可视为指导病人合理用药的依据之一。但是，CYP2C19基因型的个体间差异仅能反映病人在服用氯吡格雷后临床疗效个体差异的12％。我们最近的临床研究发现，用CYP2C19基因型区分氯吡格雷抵抗与否的诊断敏感性仅为30.4％，ROC曲线下面积是0.617，显示其临床诊断价值或预测意义十分有限。病人产生氯吡格雷抵抗的原因较多，但产生的机制并不十分清楚，临床上也缺乏有效的预测氯吡格雷抵抗的生物标志物。
发明内容
本发明的目的是针对现有相关技术的上述不足，提供一种全新的预测氯吡格雷抵抗的生物标志物及其临床应用。
本发明的目的可通过以下技术方案来实现：
人血清补体抑制剂C4结合蛋白alpha(C4b-binding protein alpha,C4BP-α或C4BP-a或C4BPa)作为检测靶标或标准品在制备预测氯吡格雷抵抗的试剂盒中的应用。
检测人血清中C4BP-a蛋白的试剂在制备预测氯吡格雷抵抗的试剂盒中的应用。
一种预测氯吡格雷抵抗的试剂盒，包含检测人血清中C4BP-a蛋白的抗体。
所述的检测人血清C4BP-a的试剂，优选包括ELISA法检测人血清中C4BP-a蛋白的相关试剂。
有益效果
本发明发现了人血清中C4BP-a(蛋白)的水平高低能准确预测氯吡格雷抵抗(见图1)。基于上述发现，本发明提供了一种预测氯吡格雷抵抗的全新的标志物人血清C4BP-a蛋白，该标志物作为检测靶点可预测或辅助判断病人服药后的临床疗效或预后。临床研究结果显示：人血清C4BP-a(蛋白)检测对诊断氯吡格雷抵抗的ROC曲线下面积是0.852，敏感性是88.6％，特异性为75％(见图2)。
附图说明
图1.人血清C4BP-a的蛋白定量及其在氯吡格雷敏感者(S)和抵抗者(R)两组间的差异(mean±SEM；19.10±1.30vs 11.03±0.51；S vs R；p<0.001)。
图2.人血清C4BP-a(蛋白)诊断氯吡格雷抵抗的ROC曲线。
具体实施方式
人血清C4BP-a蛋白检测在诊断氯吡格雷抵抗中的应用
一、检测人血清中C4BP-a(蛋白)水平的试剂盒，包括如下成分：
去离子水
PBS(1×)
96孔板(预包被)
孔板覆膜
标准品
C4BP-a标准品稀释液
检测溶液A
检测稀释液A
检测溶液B
检测稀释液B
TMB底物溶液
终止液
浓洗涤液(30×)
二、试剂盒使用方法
用市售ELISA试剂盒(武汉云克隆科技股份有限公司产品)检测病人血清中C4BP-a含量。具体操作如下。
1、C4BP-a标准曲线的制备(临用前15min内制备)：
1)每瓶标准品加入C4BP-a标准品稀释液1mL，室温静置10min，同时反复颠倒/搓动以助溶解，涡旋混匀。
2)依次梯度稀释各为10、5、2.5、1.25、0.625、0.312、0.156、0ng/mL共8个标准品稀释液(见下)用于建立标准定量曲线。
2、样品处理
1)血清至冰上融化，轻摇混匀；
2)取10μL血清加入90μL PBS混匀；
3)取10μL(2)液，加入990μL PBS混匀；
4)取10μL(3)液，加入990μL PBS混匀，形成样品稀释液。
3、人血清C4BP-a定量
1)加样(标准品或样本)：分别设置标准样品孔、待测样品孔。设标准样品孔8孔，依次加入100μL不同浓度的标准品样品(见上)，其余各孔各加待测样品100μL，酶标板覆膜，37℃温水孵育(下同)，1h。
2)弃去液体，甩干。
3)每孔加检测溶液A工作液100μL(现配)，覆膜，37℃孵育1h。
4)弃去孔内液体，每孔用350μL的洗涤液(工作液)洗涤，浸泡2min，甩干酶标板内的液体。
5)在实验台上铺垫吸水纸，酶标板朝下用力拍打，重复洗板3次。在最后一次洗涤后，要把孔内的洗涤液完全甩干。
6)加检测溶液B工作液(现配)100μL，加上覆膜，37℃，30min。
7)弃去液体，甩干，洗板5次，方法同步骤5。
8)加TMB底物溶液90μL，覆膜，37℃避光显色20min(标准品变蓝)。
9)每孔加终止溶液50μL，终止反应，此时溶液由蓝色立即变为黄色。
10)使用TECAN酶标仪在450nm波长下测定各孔的吸光度。
11)根据标准曲线和各待测样品孔的吸光度计算各样品的C4BP-a浓度。
三、有效性
选取88名经皮冠脉介入术(PCI)植入药物涂层支架后进行冠状动脉造影复查的病人，口服氯吡格雷75mg/天长达至少6个月。造影前抽取病人早晨空腹静脉血1管(约1mL)，枸橼酸抗凝(蓝盖管)用于ADP诱导的全血血小板聚集率测定，剩余全血离心取上清，冻存于–80℃冰箱，备用。
根据测得的全血血小板聚集率将病人分为两组，其中：
阴性组：对氯吡格雷反应敏感者(S)，血小板聚集率为0–1欧姆，共44名；
阳性组：对氯吡格雷反应不敏感者(R)，血小板聚集率为≥10欧姆，共44名。
根据上述实验方法提取病人血清，用市售ELISA试剂盒(见前)检测其中C4BP-a的蛋白含量。本发明以44名氯吡格雷反应敏感者血清中C4BP-a蛋白含量作为(氯吡格雷抵抗)阴性组，以44名氯吡格雷反应不敏感者血清中C4BP-a蛋白含量作为(氯吡格雷抵抗)阳性组，分别检测并比较两组之间血清C4BP-a蛋白水平的差异(见图1)；建立血清C4BP-a蛋白含量诊断氯吡格雷抵抗的ROC曲线(见图2)。结果显示，ROC曲线下面积(AUC)为0.852(95％CI:0.771–0.933；p<0.001)。对氯吡格雷抵抗诊断的敏感性(真阳性率)为88.6％，特异性(真阴性率)为75％，约登(Youden)指数J为0.636，诊断氯吡格雷抵抗的最佳临界(cutoff)值为11.733。

Claims (2)

1.人血清C4BP-a蛋白作为检测靶标或标准品在制备预测氯吡格雷抵抗的试剂盒中的应用。
2.检测人血清中C4BP-a蛋白的相关试剂在制备预测氯吡格雷抵抗的试剂盒中的应用。
CN201811515436.9A 2018-12-12 2018-12-12 人血清C4BP-a蛋白作为预测氯吡格雷抵抗的生物标志物及其临床应用 Active CN109459565B (zh)

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