CN113533748B - 一种预测儿童哮喘发作的组合试剂盒及其应用 - Google Patents
一种预测儿童哮喘发作的组合试剂盒及其应用 Download PDFInfo
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Abstract
本发明公开了一种预测儿童哮喘发作的组合试剂盒及其应用,本发明通过采集哮喘患儿急性期、缓解期外周血,经TMT TM标记的定量蛋白质组学技术检测,生物信息学分析差异表达的蛋白质,获得目标蛋白、建库,然后再经PRM靶向蛋白质组学技术对靶标蛋白进行定量分析,获得一个血浆蛋白标志物组合,通过用ELISA方法检测哮喘患儿外周血清确定各标志物及其组合在临床上的应用价值,在此基础上,本发明通过了一种组合试剂盒用于检测血浆标志物组合,通过该检测结果来预测哮喘发作,该预测结果具有较高的诊断灵敏度和特异度,有望成为哮喘发作的实验室诊断指标。
Description
技术领域
本发明属于生物医药开发技术领域,尤其涉及一种预测儿童哮喘发作的组合试剂盒及其应用。
背景技术
哮喘是一种慢性气道疾病,表现为可逆性气流阻塞、呼吸困难、喘息、咳嗽、不同程度的气道炎症和支气管高反应性等。尽管人们已经在数百年前就知道哮喘病,但是仍然没有金标准用于哮喘病诊断。
气道高反应性(airway hyperresponsiveness,AHR)检测有助于有症状患者的诊断,但是AHR的作用是有限的:尽管AHR检测可用于排除哮喘,但是对于非哮喘患者,对于无症状但有变应性炎症患者,其气道也呈高反应性。有报道20%~30%成人哮喘患者为过度诊断。
纤维支气管镜曾经被认为是诊断哮喘病最重要的手段,尽管其能够精确地侵入气道进行检测,但是价格昂贵、操作繁琐,稍微操作不当就会造成组织穿孔和出血,不能实施批量检测,也不能在初级医疗机构常规开展。
长期以来,寻找血清学指标成为诊断哮喘病的重要研究方向之一,因为外周血样本容易获得,容易在临床推广应用。迄今为止,任何单个血清标志物均不能用于准确判断患者病情程度。尽管文献报道了许多血清标志物用于哮喘病诊断,但是其临床转化应用鲜见报道。数个队列研究使用数据驱动的方法(data-driven approaches)鉴定出不同临床表型的哮喘患者,但是不同研究获得的危险因素不一致,即设定的同一表型仍然具有异质性;数个文献使用数据驱动技术辨别不同表型哮喘病的治疗效果,结果表明它们的治疗效果相似。敏感度好、特异度好的血清标志物不仅可以鉴别不同表型的儿童哮喘,还有助于个性化用药的选择,以及预测治疗效果。因此,全面系统阐述哮喘发生发展,包括临床症状、与遗传相关的血清标志物、与肺功能相关的血清标志物,对有助于开发更加有效的治疗策略。
蛋白质组学(Proteome)概念诞生于20世纪90年代,是指利用高分辨的蛋白质分离技术和高效的蛋白质鉴定技术在蛋白质水平全面、动态和定量地观察生命现象及规律,包括蛋白质的表达水平、翻译后修饰、蛋白质与蛋白质的相互作用,对映于基因组表达的所有蛋白质的集合,即细胞、组织或机体全部蛋白质的存在及其活动方式。TMT技术是常用的差异蛋白质组学技术,比较不同样品中差异表达的蛋白质,在疾病标记物筛选、药物作用靶点等领域都有广泛应用。PRM是一种基于高分辨、高精度质谱的离子监视技术,能够对目标蛋白质、目标肽段进行选择性检测,从而实现对目标蛋白质/肽段进行绝对定量。
发明内容
本发明的首要目的在于提供一种预测儿童哮喘发作的组合试剂盒。
本发明的再一目的在于提供上述组合试剂盒在作为预测儿童哮喘发作的试剂中的应用。
本发明是这样实现的,一种预测儿童哮喘发作的组合试剂盒,该组合试剂盒由若干分别用于检测抗人α-1抗胰糜蛋白酶(AACT)、血清免疫球蛋白A(IgA)、血清淀粉样蛋白A(SAA)、血红蛋白(HBB)的子试剂盒构成。
优选地,所述组合试剂盒包括:用于检测蛋白AACT的alpha 1antichymotrypsinhuman ELISA kit(ab157706)子试剂盒,用于检测蛋白IgA的an IgA human SimpleStepELISA kit(ab196263)子试剂盒,用于检测蛋白SAA的a human SAA ELISAkit(ab100635)子试剂盒,以及用于检测蛋白HBB的an HBB human ELISAkit(ab157707)子试剂盒。
本发明进一步公开了上述组合试剂盒在作为预测儿童哮喘发作的试剂中的应用。
本发明克服现有技术的不足,提供一种预测儿童哮喘发作的组合试剂盒及其应用,本发明通过采集哮喘患儿急性期、缓解期外周血,经TMT TM标记的定量蛋白质组学技术检测,生物信息学分析差异表达的蛋白质,获得目标蛋白、建库,然后再经PRM靶向蛋白质组学技术对靶标蛋白进行定量分析,获得血清标志物一个血浆蛋白标志物组合,通过用ELISA方法检测哮喘患儿外周血清确定各标志物及其组合在临床上的应用价值,在此基础上,本发明通过了一种组合试剂盒用于检测血浆标志物组合,通过该检测结果来预测哮喘发作。
相比于现有技术的缺点和不足,本发明具有以下有益效果:本发明组合试剂盒用于检测血浆标志物组合,通过该检测结果来预测哮喘发作,该预测结果具有较高的诊断灵敏度和特异度,有望成为哮喘的实验室诊断指标。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
一、材料和方法
1、血浆样品
本发明的研究过程获得了无锡市儿童医院伦理委员会的批准。训练集包括:临床缓解期哮喘儿童4例、急性发作期哮喘儿童4例、和健康对照组儿童4名(表1)。验证实验包括:临床缓解期哮喘儿童16例、急性发作期哮喘儿童16例、和健康对照组儿童16名(表1)
表1.Participant demographic and clinical informationa
aPlasma was used forTMT and PRM proteomic analyses.
bAbbreviations:M,male;F,female;DF-sIgE,D.farinae-specific IgE;DP-sIgE,D.pteronyssinus-specific IgE.
哮喘的诊断依据患者病史、体检检查和肺功能测试,具体见指南(中华医学会儿科学分会呼吸学组.儿童哮喘诊断与防治指南.中华儿科杂志,2016;54(3):167-181.)。
排除标准:具有其他肺部疾病或系统性疾病症状或体征、接受口服激素治疗的患者。三组入选对象在体重、身高和体质量指数(body mass index,BMI)。健康对照组儿童无呼吸疾病史、无过敏史。所有实验均经无锡市儿童医院伦理委员审批。所有受试者均于清晨空腹静脉血3mL。
2、蛋白提取
(1)每个样品取40μL血液,以结合缓冲液(试剂盒:Binding Buffer)10倍稀释;
(2)除去柱子上的盖子,以纸吸去贮存缓冲液;
(3)除去柱底部的尖咀,放入大小适合的收集管;
(4)加入结合缓冲液,让其靠重力流过柱体;
(5)将柱子放入一个新的大小适合的收集管中;
(6)加入稀释后的样本,让其靠重力流过柱体;
(7)以600μL结合缓冲液清洗柱体;
(8)再次以600μL结合缓冲液清洗柱体收集上三步的洗脱组份,即为去除白蛋白/IgG后的样本真空冷冻干燥后备用;
(9)将冻干的样品加入300μL SDS裂解进行复溶;
(10)将溶液在室温下12000×g离心10min,上清,并再次离心取上清。
上清即为样品的总蛋白溶液,进行蛋白浓度测定并分装后储存于-80℃备用。
3、样品浓度测定
采用BCA蛋白浓度测定方法(Smith PK,Krohn RI,Hermanson GT,etal.Measurement of protein using bicinchoninic acid.Analytical Biochemistry,1985,150(1):76-85.)。
4、SDS-聚丙烯酰胺凝胶电泳
(1)每个样品取10μg蛋白,采用12%SDS-PAGE进行分离;
(2)分离后的凝胶采用考马斯亮兰染色法进行染色,参照Candiano等(CandianoG,Bruschi M,Musante L,et al.Blue silver:A very sensitive colloidal CoomassieG-250staining for proteome analysis.Electrophoresis,2004,25(9):1327-1333)的实验方法。具体操作如下:A.固定2h;B.染色12h;C.水洗至背景清晰;
(3)染色后的凝胶应用ImageScanner扫描仪进行扫描,扫描模式为全彩模式,光密度值为300dpi。
5、胰蛋白酶酶解及标记
参考FASP方法(Wisniewski JR,Zougman A,Nagaraj N,et al.Universal samplepreparation method for proteome analysis.Nature Methods,2009,6(5):359-362.)酶解蛋白:
(1)蛋白定量后每个样品各取100μg于10K的超滤管中,取120μL还原剂缓冲液(10mM DTT,8M尿素,100mM TEAB,pH 8.0),60℃反应1h;
(2)加入IAA至终浓度为50mM,室温避光反应40min;
(3)4℃,12000rpm离心20min,弃掉收集管底部溶液;
(4)加入100μL 300mM TEAB缓冲液,12000rpm离心20min,2次;
(5)更换新收集管,在超滤管中加入100μL 300mM TEAB缓冲液,并加入2μL 1μg/μL的测序级胰蛋白酶溶液,37℃反应12h;
(6)12000rpm离心20min,收集酶解后肽段,在超滤管中再加入50μL200mM TEAB缓冲液,12000rpm离心20min,收集管底溶液并冻干。
(7)向冻干样品中加入100μL 200mM TEAB缓冲液,涡流混匀,取出40μL样品于1.5mL Ep管中进行标记反应;
(8)从冰箱中取出TMT试剂,平衡到室温,加入41μL无水乙腈,涡流5min,离心;
(9)取41μL TMT试剂加到样品中,涡流混匀,室温放置1h;
加入8μL 5%羟胺终止反应15min,冻干后于-80℃保存。
6、反相色谱分离
液相色谱:Agilent 1100HPLC;
色谱柱:Agilent Zorbax Extend–C18窄径柱,2.1×150mm,5μm;
检测波长:紫外210nm和280nm;
流动相A相:ACN-H2O(2:98,v/v);
流动相B相:ACN-H2O(90:10,v/v);
流速:300μL/min;
梯度洗脱条件:0~8min,98%A;8~8.01min,98%~95%A;8.01~48min,95%~75%A;48~60min,75~60%A;60~60.01min,60~10%A;60.01~70min,10%A;70~70.01min,10~98%A;70.01~75min,98%A。
收集8~60分钟的样品,依次每隔一分钟收集洗脱液到1~15号离心管中,按此顺序循环收样至梯度结束,收集好后真空冷冻干燥抽干,样品冷冻保存待上质谱。
7、色谱条件与质谱条件
色谱条件:
样品以300nL/min的流速上样到预柱Acclaim PepMap100100μm×2cm(RP-C18,Thermo Fisher),再经分析柱Acclaim PepMap RSLC,75um×15cm(RP-C18,Thermo Fisher)分离。
流动相A相:H2O-FA(99.9:0.1,v/v);
流动相B相:ACN-H2O-FA(80:19.9:0.1,v/v/v);
梯度洗脱条件:梯度洗脱条件:0~40min,5~30%B;40~54min,30~50%B;54~55min,50~100%B;55~60min,100%B。
质谱条件:
一级MS质量分辨率设为70000,自动增益控制值设为1e6;质谱扫描设定为全扫描荷质比m/z范围300~1600,并对其中10个最高峰进行MS/MS扫描;所有MS/MS图谱采集使用数据依赖型的正离子模式下的高能碰撞裂解完成,碰撞能量设为32;MS/MS的分辨率设为17500,自动增益控制设为2e5,离子最大累积时间为80ms;动态排除时间设为30s。
二、数据处理
实验数据采用Proteome DiscovererTM 2.2(美国Thermo公司)软件分析,所用数据库为来自于UniProt的人数据库。肽段鉴定的假阳性率控制在1%以下,具体搜库参数设置如表2所示。
表2质谱检索参数
样本类型 | TMT6plex(PeptideLabeled) |
6plex(PeptideLabeled) | Iodoacetamide |
Digestion: | Trypsin |
Instrument: | QExactive |
Database: | Homosapiens.fasta |
PRM(平行反应监视,parallel reaction monitoring)
PRM是一种基于高分辨、高精度质谱的离子监视技术,该技术结合了四级杆的高选择性以及Orbitrap/或ToF质谱分析器的高分辨、高精度特异性,能够对目标蛋白、目标肽段进行选择性定量检测。PRM靶向定量蛋白质组学技术已经成为一种非常重要的质谱定量方法,主要步骤包括:蛋白质提取、蛋白质定量、蛋白质酶解、LC-PRM/MS分析、PRM数据Skyline分析。
1、仪器和试剂
1)UA buffer(8M Urea,150mM Tris-HCl,pH8.0)
2)NH4HCO3(Sigama,A6141)
3)Acetonitrile(Merck,1499230-935)
4)Q-exactive Plus(Thermo Scientific)
5)Easy-nLC1200(Thermo Scientific)
6)Trap column(Reverse-Phase),100um×20mm(5um,C18)
7)Thermo Scientific EASY column(Reverse-Phase),75um×120mm(3um,C18).
2、样品制备
取每份样品约200ug蛋白进行溶液内酶解,酶解步骤如下:加入适量50mM NH4HCO3buffer稀释10倍。加二硫苏糖醇(Dithiothreitol,简称为DTT)至终浓度为10mM,37℃保温1hr,冷却至室温;加入适量1M(吲哚-3-乙酸indole-3-acetic acid,IAA)使其终浓度为50mM,避光室温30min。然后往样中加入2ug Trypsin,37℃16h。酶解后的肽段脱盐冻干,然后用0.1%FA复溶,OD280测定肽段浓度。
3、LC-PRM/MS分析
根据TMT结果,选择3组间差异表达显著的,且单个蛋白表达丰度高的,最终选择每个目标蛋白中的各1~3条肽段具有可靠的鉴定信息及良好的色谱分离行为(色谱洗脱峰尖锐对称),可以用于PRM定量分析。将适合PRM分析的肽段信息导入软件Xcalibur中进行PRM方法设置。
取每例样品的肽段2g进行LC-PRM/MS分析:上样后使用纳升流速Eaxy nLC 1200色谱系统(Thermo Scientific)进行色谱分离。缓冲液:A液为0.1%甲酸水溶液,B液为0.1%甲酸、乙腈和水混合溶液(其中乙腈为95%)。色谱柱以95%的A液平衡。样品进样到TrapColumn(100m20 mm,5m,C18,Dr.Maisch GmbH)后经过色谱分析柱(75m 150mm,3m,C18,Dr.Maisch GmbH)进行梯度分离,流速为300nl/min。液相分离梯度如下:0~5分钟,B液线性梯度从2%到5%;5~45分钟,B液线性梯度从5%到23%;45分钟到50分钟,B液线性梯度从23%至40%;50分钟~52分钟,B液线性梯度从40%到100%;52分钟~60分钟,B液维持在100%。肽段分离后用Q-Exactive Plus质谱仪(Thermo Scientific)进行靶向PRM质谱分析。分析时长为60min,检测模式:正离子,母离子扫描范围:350~1500m/z,一级质谱分辨率为70,000@m/z200,AGC target:3e6,一级质谱Maximun IT:200ms。肽段二级质谱分析按照下列方法采集:每次全扫描(Full MS scan)后根据Inclusion列表依次选择目标肽段的Precursor m/z进行二级质谱扫描分析(MS2),其中MS2的分辨率为17,500@m/z 200,AGCtarget:3e6,二级质谱Maximun IT:100ms,MS2ActivationType:HCD,Isolationwindow:2.0Th,Normalized collision energy:27。得到的质谱原始RAW文件使用软件Skyline 4.1进行PRM数据分析。
4、ELISA验证
根据TMT和PRM检测结果,对二者检测结果一致的蛋白,用ELISA试剂盒进行联合检测,明确诊断性能。TMT和PRM检测结果一致的有7个蛋白,但是只有AACT、IgA、SAA和HBB有市售试剂盒。
根据TMT和PRM检测结果,对二者检测结果一致的蛋白,用ELISA试剂盒进行联合检测,明确诊断性能。TMT和PRM检测结果一致的有7个蛋白,但是只有AACT,IgA,SAA以及HBB有市售试剂盒,所以购买a commercial alpha 1antichymotrypsinhuman ELISAkit(ab157706),an IgAhuman SimpleStep ELISAkit(ab196263),ahuman SAAELISAkit(ab100635),and an HBB human ELISA kit(ab157707),根据试剂盒说明书进行操作。使用酶标仪为iMark Microplate Reader S/N 10288,波长为450nm,测定OD(Optical density)值。
5、统计学分析
检测结果以(SD)表示,用SPSS 19.0(SPSS,Chicago,IL,USA)进行数据统计和分析,线性回归分析、Pearson相关性分析等。两组均数间用Mann-Whitney U-test,P<0.05为差异具有统计学意义。计算受试者工作曲线(Receiveroperator characteristic,ROC),评价候选蛋白的诊断性能。
三、结果
1、TMT鉴定哮喘患儿外周血差异表达的蛋白质
用TMT检测4例急性哮喘患儿、4例缓解期哮喘患儿、4名正常儿童血浆,检测出347个差异表达的蛋白。急性期和对照组,有125个差异表达的蛋白(FC>1.2,P<0.05),包括50个上调、75个下调蛋白。缓解期和对照组,有142个差异表达的蛋白,包括72个上调、70个下调。急性期和缓解析,有55个差异表达的蛋白质,包括22个上调和33个下调。
2、PRM检测哮喘患儿外周血差异表达的蛋白质
根据TMT检测结果,依据差异表达的倍数、P值以及单个蛋白在血浆中表达的丰度,挑选出11个蛋白质,依次为Pigment epithelium-derived factor(PEDF)、Immunoglobulinheavy constant delta、Immunoglobulin heavy constant gamma 4、Cofilin-1、Alpha-1-antichymotrypsin、Immunoglobulin heavy constant alpha 1、Serum amyloid A-1protein、Ubiquitin-40S ribosomal protein S27a、Hemoglobin subunitbeta、Hemoglobin subunit alpha(Hemoglobin alpha chain),采用LC-PRM/MS进行定量分析。PRM检测结果显示有7个蛋白表达水平与TMT检测结果一致,具体见表3。
表3PRM和TMT方法检测11个候选蛋白的结果比较
3、ELISA检测应用
对TMT和PRM检测结果一致的7个蛋白,寻找市售试剂盒,结果购得alpha1antichymotrypsin human ELISA kit(ab157706),an IgA human SimpleStepELISAkit(ab196263),a human SAA ELISAkit(ab100635),and an HBB human ELISAkit(ab157707),分别用于检测AACT、IgA、SAA、HBB。共检测16例急性哮喘、16例缓解期哮喘、16名正常儿童血浆,具体结果如下表4~7所示。
表4三组受检对象血清AACT检测结果
注:三组间差异有统计学意义(F=4.231,P=0.021),其中:对照组和缓解期差异无统计学意义;急性期升高显著,与对照组和缓解期差异均有统计学意义。
表5三组受检对象血清SAA检测结果
SAA | CaseNumbers | 平均值 | 标准差 | 最高值 | 最低值 |
急性期 | 16 | 6.238 | 1.737 | 3.961 | 8.822 |
缓解期 | 16 | 5.315 | .689 | 4.150 | 6.749 |
对照组 | 16 | 4.657 | .509 | 3.976 | 5.688 |
注:三组间差异具有显著性(F=8.069,P=0.001),其中:对照组和缓解期差异无统计学意义;急性期升高显著,与对照组和缓解期差异均有统计学意义。
表6三组受检对象血清HBB检测结果
注:三组间差异有统计学意义(F=7.757,P=0.001),急性期和缓解期差异无统计学意义;均较对照组显著降低。
表7三组受检对象血清IgA检测结果
IgA | CaseNumbers | 平均值 | 标准差 | 最高值 | 最低值 |
急性期 | 16 | 27.090 | 8.836 | 12.458 | 41.074 |
缓解期 | 16 | 33.756 | 8.350 | 18.734 | 47.533 |
对照组 | 16 | 39.635 | 7.482 | 25.501 | 52.966 |
注:三组间差异均有统计学意义(F=9.282,P<0.001)。
与正常对照组比较,哮喘患儿(包括急性期和缓解期)血清AACT和SAA表达水平升高(P<0.05),HBB和IgA表达水平下降(P<0.01)。AACT、IgA、SAA和HBB的AUCs分别为0.688[95%confidence interval(CI),0.481–0.894]、0.707(95%CI,0.522–0.892)、0.623(95%CI,0.409–0.837)和0.525(95%CI,0.342–0.704),联合诊断曲线下面积(ROC curvearea),0.801for AACT+IgA+SAA,0.789for IgA+SAA,and 0.840forAACT+IgA+SAA+HBB。四个蛋白(AACT+IgA+SAA+HBB)联合诊断的曲线下面积最大。但是IgA、HBB具有最好的诊断灵敏度(87.50%),IgA+SAA、AACT+IgA+SAA具有最高的诊断特异度,为93.75%,具体见表8。
表8AACT、IgA、SAA和HBB检测水平的诊断性能
aAbbreviations:AUC,areaunderthecurve;SE,StandardError;95%CI,95%confidenceinterval.
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (1)
1.标志物组合在制备诊断儿童哮喘的试剂盒中的应用,所述标志物组合由人α-1抗胰糜蛋白酶、血清免疫球蛋白A、血清淀粉样蛋白A、血红蛋白HBB组成。
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Ricard F.Lockey 等.《Asthma:Comorbidities,Coexisting Conditions,and Differential Diagnosis》.2014,第115-138页. * |
Subtle Immunodefi ciency in Severe Asthma: IgA and IgG 2 Correlate with Lung Function and Symptoms;Silvana Balzar 等;《 Int Arch Allergy Immunol 》;20060323;第96-102页 * |
α_1-抗糜蛋白酶基因Bochum-1、Bonn-1突变型与儿童哮喘相关性研究;黄锐;曹虹;李海粟;罗放灵;;中国优生与遗传杂志;20100425(04);第21-23页 * |
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