CN110687231A - 慢性阻塞性肺疾病分型检测试剂盒 - Google Patents
慢性阻塞性肺疾病分型检测试剂盒 Download PDFInfo
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Abstract
本发明属于慢性阻塞性肺疾病代谢组领域。本发明提供了检测血清中的Kazinol H的试剂在制备慢性阻塞性肺疾病分型试剂盒中的用途;所述分型指区分慢性支气管炎型或肺气肿型慢性阻塞性肺疾病。本发明还提供了对应的慢性阻塞性肺疾病分型检测试剂盒。本发明可借助检测Kazinol H在血清中的含量,实现慢性阻塞性肺疾病的有效分型。
Description
技术领域
本发明属于慢性阻塞性肺疾病代谢组领域,尤其涉及一种慢性阻塞性肺疾病分型检测试剂盒。
背景技术
慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)是一种具有气流阻塞特征的慢性支气管炎和(或)肺气肿,可进一步发展为肺心病和呼吸衰竭的常见慢性疾病。
慢性阻塞性肺病的确切病因不清楚,一般认为与慢支和阻塞性肺气肿发生有关的因素都可能参与慢性阻塞性肺病的发病。已经发现的危险因素大致可以分为外因(即环境因素)与内因(即个体易患因素)两类。外因包括吸烟、粉尘和化学物质的吸入、空气污染、呼吸道感染及社会经济地位较低的人群(可能与室内和室外空气污染、居室拥挤、营养较差及其他与社会经济地位较低相关联的因素有关)。内因包括遗传因素、气道反应性增高、在怀孕期、新生儿期、婴儿期或儿童期由各种原因导致肺发育或生长不良的个体。
COPD的致残率和病死率很高,全球40岁以上发病率已高达9%~10%。
目前COPD的诊断主要依靠肺功能检查、胸部X线检查、胸部CT检查等,但是肺功能检查(肺活量测定法),是一种费时且昂贵的规程,只能由专科肺内科医师实施。而且,COPD作为一种兼具复杂性和异质性的疾病,单纯依靠肺功能并不能客观反映出COPD的特性。因此,COPD进一步被分为慢性支气管炎型(慢支炎型)和肺气肿型2种表型,前者表现为长期产痰性咳嗽、气道阻塞严重、气管壁增厚;后者主要表现为肺过度充气。缺乏经验的临床医生在对患者COPD分型时,较为主观,对症状不典型的COPD难以准确分型,导致用药不准确。
因此,急需一种能够对COPD准确分型的工具。
发明内容
本发明的目的在于提供一种COPD分型标志物和试剂盒,本发明所述分型均指区分慢性支气管炎型或肺气肿型慢性阻塞性肺疾病。
本发明的技术方案如下:
本发明提供了检测血清中Kazinol H的试剂在制备慢性阻塞性肺疾病分型试剂盒中的用途。
如前述的用途,所述检测血清中Kazinol H的试剂为液相色谱-质谱联用(LC-MS)方法用试剂。
如前述的用途,所述检测血清中Kazinol H含量的试剂为液相色谱-质谱联用代谢组学方法用试剂。
进一步地,所述检测血清中Kazinol H含量的试剂为液相色谱-质谱联用非靶向代谢组学方法用试剂。
如前述的用途,所述检测血清中Kazinol H含量的试剂为液相色谱方法用试剂。
本发明还提供了一种慢性阻塞性肺疾病分型试剂盒,它包括检测血清中KazinolH含量的试剂。
如前述的分型试剂盒,所述检测血清中Kazinol H含量的试剂为液相色谱-质谱联用方法用试剂。
如前述的分型试剂盒,所述检测血清中Kazinol H含量的试剂为液相色谱-质谱联用代谢组学方法用试剂。
进一步地,所述检测血清中Kazinol H含量的试剂为液相色谱-质谱联用非靶向代谢组学方法用试剂。
如前述的分型试剂盒,所述检测血清中Kazinol H含量的试剂为液相色谱方法用试剂;使用时,需要以全反式七苯基二磷酸酯标准品作为参照。
发明人发现,不同表型COPD(慢支炎型/肺气肿型)患者血清中KazinolH的含量存在显著性区别,慢支炎型COPD患者血清中Kazinol H含量远高于肺气肿型COPD患者。常规化合物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)、液相色谱-质谱联用以及液相色谱-质谱联用代谢组学方法均可对Kazinol H进行检测,用已有的液相色谱、液相色谱-质谱联用或液相色谱-质谱联用代谢组学(LC-MS代谢组学,包括靶向LC-MS代谢组学和LC-MS非靶向代谢组学)方法检测数据作为参照,即可进行COPD分型判断。
进一步地,本发明实施例1中使用的LC-MS非靶向代谢组学方法,检测了不同表型COPD(慢支炎型/肺气肿型)患者血清中Kazinol H的具体量化的数值,并作出了ROC分析,得到的曲线下面积(AUC)为0.697,当检测临界值(cut-off值)为18.154时,其特异性为0.867,灵敏度为0.5。因此,如果以LC-MS非靶向代谢组学方法用试剂盒对COPD进行分型检测,即可以Kazinol H检测值与18.154的大小关系为分型判定依据,当检测值大于18.154时,判定为慢支炎型COPD,当检测值小于18.154时,判定为肺气肿型COPD。
本发明的COPD分型检测试剂盒,可以通过检测血清中Kazinol H,实现COPD客观、准确、快速的分型。
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Kazinol H浓度与COPD分型的ROC曲线图。
具体实施方式
实施例1不同表型COPD(慢支炎型/肺气肿型)患者血清中Kazinol H检测
1.被检对象
慢支炎型COPD患者(A组)30人,肺气肿型COPD患者(B组)30人。被检对象人群在实验前均已经知情并同意。
2.方法
将慢支炎表型与肺气肿表型COPD患者血清各30例分别进行样品预处理、代谢物提取、LC-MS全扫描检测、数据预处理及统计分析。基于UPLC-VION IMS Q-Tof高分辨质谱仪的非靶向代谢组学功能,结合代谢组学数据处理软件Progenesis QI v2.3,对原始数据进行定性及相对定量分析,并对原始数据进行标准化预处理。
具体实验步骤:
(1)样本前处理
1)将-80℃下保存的样本取出,在室温下解冻,移取100μL血清,加入内标(L-2-氯苯丙氨酸,0.3mg/mL;C-17,0.01mg/mL,均为甲醇配置)各10μL,涡旋震荡10s;
2)加入300μL的蛋白沉淀剂甲醇-乙腈(V∶V=2∶1),涡旋震荡1min;
3)冰水浴中超声提取10min;
4)-20℃下静置30min;
5)离心10min(13000rpm,4℃),用注射器吸取200μL的上清液,使用0.22μm的有机相针孔过滤器过滤后,转移到LC进样小瓶,-80℃下保存,直到进行LC-MS分析。
6)质控样本(QC)由所有样本的提取液等体积混合制备而成,每个QC的
体积与样本相同。
备注:所有提取试剂使用前均在-20℃进行预冷。质控(QC)样本:等量取各组样本混合为QC。每10个样本中插入一个QC,QC样本用于评价整个实验过程中系统稳定性。
(2)液相色谱-质谱分析条件
本次实验的分析仪器为沃特世的I-Class超高效液相串联VION IMSQ-Tof高分辨质谱仪组成的液质联用系统。
色谱条件:
色谱柱:ACQUITY UPLC BEH C18(100mm×2.1mm,1.7um);柱温:45℃;流动相:A-水(含0.1%甲酸),B-乙腈/甲醇(2/3)(v/v)(含0.1%甲酸);流速:0.4mL/min;
进样体积:1μL。
质谱条件:离子源:ESI;样品质谱信号采集分别采用正负离子扫描模式。
3.结果
A、B组血清中Kazinol H的检测结果如下表所示:
备注:FC(fold change)是指的该蛋白在慢支炎表型的血清中的含量/肺气肿表型的血清中的含量。
可见,慢支炎型COPD患者血清中的Kazinol H的含量显著高于肺气肿型COPD患者。
对A、B组血清中Kazinol H含量的ROC分析结果如图1所示,ROC的曲线下面积(AUC)为0.697;当检测临界值(cut-off值)为18.154时,其特异性为0.867,灵敏度为0.5。
基于Kazinol H在COPD患者血清中的含量的检测,可开发出COPD分型试剂盒。如果以LC-MS非靶向代谢组学方法(如本实施例)用试剂盒对COPD进行分型检测,即可以KazinolH检测值与18.154的大小关系为分型判定依据,当检测值大于18.154时,判定为慢支炎型COPD,当检测值小于18.154时,判定为肺气肿型COPD。
当然,LC-MS非靶向代谢组学方法仅仅是检测化合物的常规手段,理论上,能够检测Kazinol H在COPD患者血清中的含量的各种试剂盒(例如液相色谱方法试剂盒,液相色谱-质谱联用方法试剂盒)都能实现COPD分型。以液相色谱方法试剂盒为例,只需事先检测已知的慢支炎型和肺气肿型COPD患者各自的血清内Kazinol H含量,作为参照标准;用同样的试剂盒检测未知分型的COPD患者血清得到Kazinol H含量数值,与参考标准相比较,即可进行COPD分型判断。
实施例2本发明的试剂盒
1.本发明试剂盒的组成
甲醇、甲酸、乙腈和L-2-氯苯丙氨酸。
2.试剂盒使用方法
实验操作同实施例1的第2节。
当血清中Kazinol H的检测值为18.154以上,可判别为慢支炎型COPD;否则,可判别为肺气肿型COPD。
综上,本发明的试剂盒通过对Kazinol H的定量检测,可实现对慢支炎型或肺气肿型COPD的有效分型,可简单快捷地为临床医生提供有价值的参考信息,便于对症下药,应用前景良好。
Claims (6)
1.检测血清中Kazinol H含量的试剂在制备慢性阻塞性肺疾病分型试剂盒中的用途;所述分型指区分慢性支气管炎型或肺气肿型慢性阻塞性肺疾病。
2.如权利要求1所述的用途,其特征在于,所述检测血清中Kazinol H含量的试剂为液相色谱-质谱联用方法用试剂。
3.如权利要求1所述的用途,其特征在于,所述检测血清中Kazinol H含量的试剂为液相色谱方法用试剂。
4.一种慢性阻塞性肺疾病分型试剂盒,其特征在于,它包括检测血清中Kazinol H含量的试剂;
所述分型指区分慢性支气管炎型或肺气肿型慢性阻塞性肺疾病。
5.如权利要求4所述的分型试剂盒,其特征在于,所述检测血清中Kazinol H含量的试剂为液相色谱-质谱联用方法用试剂。
6.如权利要求4所述的分型试剂盒,其特征在于,所述检测血清中Kazinol H含量的试剂为液相色谱方法用试剂。
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