CN112611877B - 一款利用angptl4预测急性呼吸窘迫综合征患病及预后的试剂盒 - Google Patents
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Abstract
本发明属于急性呼吸窘迫综合征检测领域。本发明提供了检测血清中ANGPTL4蛋白的试剂在制备预测急性呼吸窘迫综合症患病及预后的试剂盒中的用途。本发明还提供了对应的预测急性呼吸窘迫综合症患病及预后的试剂盒。本发明可借助检测ANGPTL4蛋白在血清中的含量,实现急性呼吸窘迫综合征诊断和预后的快速预测,应用前景良好。
Description
技术领域
本发明属于急性呼吸窘迫综合征诊断及预后评估领域。
背景技术
急性呼吸窘迫综合征(ARDS)是由肺内原因和/或肺外原因引起的,以顽固性低氧血症为显著特征的临床综合征,因高病死率而倍受关注。准确预测ARDS的发生和预后,对ARDS患者中的高危人群的识别和尽早治疗意义重大。
王冉等通过研究人口学资料、APACHE II评分、氧合指数、血常规、凝血六项、炎症标志物等数据,发现可能导致ARDS死亡的独立危险因素为:低蛋白血症、血小板计数、氧合指数;以低蛋白血症、氧合指数≤150mmHg、血小板≤100×109个/L等三个指标的至少一个对ARDS患者死亡预测效果的约登指数(特异度+敏感度-1)范围在0.2619~0.3849(急性呼吸窘迫综合症进展及预后的危险因素研究,解放军医学杂志,2017年,第42卷第5期)。然而,目前常用的评分表(如:SOFA评分,APACHE II评分等)主要针对多器官衰竭和整体危重度打分,较依赖主观判断,有耗时较长,灵敏度和特异性欠佳等缺点,探索新的指标或标志物将有助于快速准确地判断ARDS严重程度及评估预后和死亡率,进而对ARDS患者进行分级管理、智能管理,从而优化紧张的医疗资源分配。在ARDS病情进展早期阶段就能介入干预,改善预后降低死亡率。最近,曾宪飞等对血管生成素2(Ang-2)、II型肺泡细胞表面抗原6(KL-6)、肺表面活性蛋白D(SP-D)、血管性血友病因子(vWF)、白介素8(IL-8)等5种血清学标志物在ARDS预后预测中的价值进行了研究,发现单一标志物诊断ARDS效果最好的是KL-6,其ROC曲线的AUC为0.816;单一标志物预测死亡效果最好的是IL-8,ROC曲线的AUC为0.764(5种血清学标志物对急性呼吸窘迫综合征的诊断和预后预测价值,西安交通大学学报(医学版),2019年,第40卷第4期)。
血管生成素样蛋白4(Angiopoietin-related protein 4,ANGPTL4),在UniProtKB数据库编号为P51884,是一种分泌型糖蛋白,可介导脂蛋白脂酶LPL的失活,从而在调节血清中甘油三酯清除和脂质代谢中起作用。研究表明非小细胞肺癌组织中ANGPTL4表达量与肺腺癌预后关系紧密(ANGPTL4Correlates with NSCLC Progression and RegulatesEpithelial-Mesenchymal Transition via ERK Pathway.Lung.2016Aug;194(4):637-46.)。而ANGPTL4与ARDS诊断及预后的关系,目前还未见报道。
发明内容
本发明的目的是提供一种预测ARDS患病及评估其预后的试剂盒。
本发明的技术方案包括:
检测血清中ANGPTL4蛋白含量的试剂在制备预测急性呼吸窘迫综合症患病及预后的试剂盒中的用途。
该试剂盒可同时或分别用于预测急性呼吸窘迫综合症的患病,以及预测急性呼吸窘迫综合症的预后。
如前述的用途,所述检测血清中ANGPTL4蛋白含量的试剂为酶联免疫吸附检测试剂。
如前述的用途,所述检测血清中ANGPTL4蛋白含量的试剂为液相色谱方法用试剂或胶体金检测试剂。
如前述的用途,所述检测血清中ANGPTL4蛋白含量的试剂为R&D Systems Luminex液相芯片检测试剂。
一种预测急性呼吸窘迫综合症患病及预后的试剂盒,它包括检测血清中ANGPTL4蛋白含量的试剂。
该试剂盒可同时或分别用于预测急性呼吸窘迫综合症的患病,以及预测急性呼吸窘迫综合症的预后。
如前述的试剂盒,所述检测血清中ANGPTL4蛋白含量的试剂为酶联免疫吸附检测试剂。
如前述的试剂盒,所述检测血清中ANGPTL4蛋白含量的试剂为液相色谱方法用试剂或胶体金检测试剂。
如前述的试剂盒,所述检测血清中ANGPTL4蛋白含量的试剂为R&D SystemsLuminex液相芯片检测试剂。
本发明的核心思想是:相较于正常对照,ARDS患者血清中的ANGPTL4蛋白的含量显著升高,且ARDS的预后与血清中ANGPTL4蛋白含量的高低也存在显著相关性,即ANGPTL4蛋白含量越高,则ARDS患者的预后越差。因此,常规蛋白检测方法例如液相色谱(比如高效液相色谱、超高效液相色谱)、酶联免疫吸附检测、胶体金均可对ANGPTL4蛋白进行检测,即可进行ARDS诊断及预后的预测。依据本发明的核心思想进行的,通过检测人血清中ANGPTL4含量而对ARDS诊断及预后进行预测的技术均在本发明的范围内。
进一步地,本发明实施例2中使用的R&D Systems Luminex液相芯片,检测了正常对照及ARDS患者血清中的ANGPTL4蛋白的含量,并且分析了诊断及预后与ANGPTL4蛋白水平的关系,并做了ROC分析。得到的诊断ARDS的曲线下面积(AUC)为0.950,当检测临界值(cut-off值)为159.47ng/ml时,预测ARDS发生的敏感度为87.5%,特异度为100%,准确性很高。分析得到的评估ARDS预后的AUC为0.746,当cut-off值为1009.89ng/mL时,其对患者28天内死亡预测的特异性为100%,灵敏度为59.39%,也有较高的准确度。
本发明的试剂盒是根据前述发现发明的试剂盒,该试剂盒可以通过检测血清中ANGPTL4蛋白,实现ARDS的诊断和预后的客观、准确、快速的预测。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1是正常对照和ARDS患者中ANGPTL4蛋白的水平比较的散点图。
图2是ANGPTL4蛋白浓度与ARDS诊断的ROC曲线图
图3是两组患者血清中ANGPTL4蛋白的水平比较的散点图。
图4是ANGPTL4蛋白浓度与ARDS预后的ROC曲线图。
具体实施方式
实施例1本发明的试剂盒
1.本发明试剂盒的组成
ANGPTL4特异性抗体为R&D Systems(厂家)生产的山羊抗人ANGPTL4多克隆抗体(货号AF3485)。
2.试剂盒使用方法
(1)向96孔板中每孔内加入50μL标准品或血清样本,封膜后,室温摇晃孵育2小时;
(2)100μL洗板液洗板3次;
(3)每孔内加入50μL生物素标记的二抗,封膜后室温摇晃孵育30min;
(4)100μL洗板液洗板3次;
(5)向每孔中加入50μL显色底物,室温摇晃混匀2min;
(6)酶标仪读数。
当患者血清中ANGPTL4蛋白的检测值为159.47ng/ml(即cut-off值)时,预测发展成为ARDS的可能性较大,当其含量在1009.89ng/mL以上,可判定患者28天内预后不佳(死亡风险高)。
实施例2 ARDS患者血清中ANGPTL4蛋白检测
1.被检对象
正常受试者32人,ARDS患者56人,并随访28天后死亡的ARDS患者为22人,28天后存活的患者为34人。被检对象人群在实验前均已经知情并同意。
2.方法
留取被检对象的血液,收集血清,针对ANGPTL4蛋白进行R&D Systems Luminex液相芯片检测,具体地,包括如下步骤:
(1)重悬混匀稀释的检测混合液(含标记ANGPTL4抗体的微球),向每个酶标板孔内加入50μL混合液;
(2)加入50μL标准品或样本,封膜后,室温摇晃孵育2小时;
(3)100μL洗板液洗板3次;
(4)每孔内加入50μL生物素标记的抗体混合物,封膜后室温摇晃孵育1小时;
(5)100μL洗板液洗板3次;
(6)每孔内加入50μL含链霉亲和素的混合物,封膜后室温摇晃孵育30min;
(7)100μL洗板液洗板3次;
(8)向每孔中加入100μL缓冲液,室温摇晃混匀2min;
(9)90min内读板(Bio-Plex 200,Bio-Rad)。
3.结果
(1)ARDS患者血清中的ANGPTL4的水平显著高于正常受试者。
如图1所示,相对于正常对照,ARDS患者血清中ANGPTL4的水平显著升高(471.64[200.17–939.32]ng/mL vs.81.78[58.41–103.80]ng/mL),统计P值<0.0001。
(2)血清中ANGPTL4蛋白含量在ARDS诊断中有较大应用价值。
如图2所示,诊断ARDS的ROC分析结果显示,ROC的曲线下面积(AUC)高达0.950;当检测临界值(cut-off值)为159.47ng/ml时,预测ARDS发生的敏感度为87.5%,特异度为100%,展示出较高的临床诊断价值。
(3)28内死亡的ARDS患者血清中ANGPTL4水平显著高于28内存活的患者
对存活和死亡患者两组血清中ANGPTL4蛋白的检测结果如图3所示,28内死亡的ARDS患者血清中ANGPTL4水平显著高于28内存活的患者(1055.39±731.79ng/mLvs.412.13±273.76ng/mL),统计P值<0.0001;
(4)血清ANGPTL4蛋白在ARDS预后评估中具有较大价值
对存活和死亡患者血清中ANGPTL4蛋白含量的ROC分析结果如图4所示,ROC的AUC高达0.746;当cut-off值为1009.89ng/mL时,其特异性为100%,灵敏度为59.39%。
实验结果说明,通过检测血清中ANGPTL4蛋白的水平,理论上当其高于1009.89ng/mL时,其预后差(死亡)的可能性为100%,临床意义较大;本发明的试剂盒性能十分优异。
综上,本发明的试剂盒通过对ANGPTL4蛋白的定量检测,可实现对ARDS早期诊断预警及预后的快速、准确的预测,应用前景良好。
Claims (5)
1.检测血清中ANGPTL4蛋白含量的试剂在制备预测急性呼吸窘迫综合症预后的试剂盒中的用途,其特征在于,当血清中ANGPTL4蛋白含量在1009.89ng/mL以上,可判定患者28天内预后不佳,死亡风险高。
2.如权利要求1所述的用途,其特征在于,所述检测血清中ANGPTL4蛋白含量的试剂为酶联免疫吸附检测试剂。
3.如权利要求1所述的用途,其特征在于,所述检测血清中ANGPTL4蛋白含量的试剂为液相色谱方法用试剂。
4.如权利要求1所述的用途,其特征在于,所述检测血清中ANGPTL4蛋白含量的试剂为胶体金检测试剂。
5.如权利要求1所述的用途,其特征在于,所述检测血清中ANGPTL4蛋白含量的试剂为R&D Systems Luminex液相芯片检测用试剂。
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