CN111443208B - 鉴别活动性结核病和潜伏性结核病的组合物 - Google Patents
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Abstract
本发明涉及分子生物学和免疫学领域,具体涉及Rv1733c用于制备鉴别活动性结核病和潜伏性结核感染的试剂盒的应用。本发明应用荧光免疫斑点法,检测经结核分枝杆菌潜伏相关抗原Rv1733c刺激后引起的特异性Th1细胞免疫反应,鉴别活动性结核病和潜伏性结核感染。通过实验发现,经新型潜伏感染相关抗原Rv1733c刺激后产生IL‑2的T细胞斑点数对鉴别活动性结核病和潜伏性结核感染有一定的价值,能够提高鉴别的准确性。
Description
技术领域
本发明涉及分子生物学和免疫学领域,具体涉及Rv1733c用于制备鉴别活动性结核病和潜伏性结核感染的试剂盒的应用。
背景技术
结核病(tuberculosis,TB)严重威胁着人类健康。目前全球约1/3人口潜伏感染结核分枝杆菌(Mycobacterium tuberculosis,Mtb),其中5%-10%有可能发展为结核病。当潜伏感染者免疫力下降时,体内的MTB激活、增殖、播散,最终发展为活动性结核病。
结核分枝杆菌在潜伏感染期主要以休眠菌状态存在。结核分枝杆菌休眠菌为了适应宿主环境和逃避机体免疫作用而特异性转录表达一组由休眠调节子调控的休眠相关抗原。休眠相关抗原的表达对结核分枝杆菌在体内的潜伏感染和持续存活具有重要意义。其中休眠相关抗原Rv1733c是Mtb潜伏感染人群中外周血T淋巴细胞识别率较高的一个蛋白,该蛋白能够有效刺激T淋巴细胞分泌IFN-γ。目前Rv1733c被更多用于制备结核病相关疫苗,如专利申请CN108239660A;WO2012/210018A1;WO2006/104389A1。
目前临床诊断和检测中,很难快速准确的将结核病(active Tuberculosis,ATB)和潜伏性结核感染(Latent TB infection,LTBI)区分开来。如ELISPOT技术的T-SPOT.TB检测已被临床广泛用于诊断结核感染,但其无法鉴别诊断活动性结核病和潜伏结核感染。主要原因包括:1)T-SPOT.TB检测所应用的抗原仅与结核菌毒力相关;2)仅检测单一效应细胞因子IFN-γ的分泌。因其应用的ELISPOT技术,采用化学显色法,在检测多因子时,会由于斑点混色造成无法识别,影响其临床应用。
荧光免疫斑点法(FluoroSpot)目前被用于鉴别活动性结核病和非活动性结核病(Lifan Zhang;Application of IFN-γ/IL-2FluoroSpot assay for distinguishingactive tuberculosis from non-active tuberculosis:A cohort study;ClinicaChimica Acta 499(2019)64–69),但其使用的刺激源为ESAT-6抗原和CFP-10抗原,灵敏度和特异性并不理想。因此,需要一种能够准确、灵敏的鉴别活动性结核病和潜伏性结核感染的试剂盒及方法。
发明内容
为了提高活动性结核病和潜伏性结核感染鉴别的灵敏度和特异性,本发明旨在应用荧光免疫斑点法(FluoroSpot),检测经结核分枝杆菌潜伏相关抗原Rv1733c刺激后引起的特异性Th1细胞免疫反应,鉴别ATB和LTBI。通过实验发现,经新型潜伏感染相关抗原Rv1733c刺激后产生IL-2的T细胞斑点频数对鉴别LTBI和ATB有一定的价值,可辅助进行鉴别,提高鉴别的准确性。
具体而言,本发明涉及一种组合物,其含有Rv1733c抗原、ESAT-6抗原和CFP-10抗原;所述组合物用于诊断结核病;进一步的,用于鉴别活动性结核病和潜伏性结核感染。
进一步的,所述组合物中还可以含有IFN-γ和IL-2单抗;也可以包含荧光免疫斑点法所使用到的主要试剂;所述试剂可以是抗IFN-γ和抗IL-2的单抗,和/或荧光素标记的单抗IFN-γ-FITC和IL-2-biotin,和/或二抗anti-FITC-490和SA-550等。
本发明涉及一种诊断结核病的试剂盒,试剂盒含有Rv1733c抗原;进一步,试剂盒中还可以含有ESAT-6抗原和CFP-10抗原;所述抗原为肽段抗原;进一步的,试剂盒中还含有IFN-γ和IL-2单抗。
本发明还涉及鉴别活动性结核病和潜伏性结核感染的试剂盒,试剂盒含有Rv1733c抗原;进一步,试剂盒中还可以含有ESAT-6抗原和CFP-10抗原;所述抗原为肽段抗原;进一步的,试剂盒中还可以含有IFN-γ和IL-2单抗等。
本发明涉及一种诊断结核病的试剂盒,试剂盒含有Rv1733c抗原,试剂盒中还包含荧光免疫斑点法所使用到的主要试剂;所述试剂包括抗IFN-γ和抗IL-2的单抗,和/或荧光素标记的单抗IFN-γ-FITC和IL-2-biotin,和/或二抗anti-FITC-490和SA-550。
本发明涉及一种鉴别活动性结核病和潜伏性结核病的试剂盒,试剂盒含有Rv1733c抗原,试剂盒中还包含荧光免疫斑点法所使用到的主要试剂;所述试剂包括抗IFN-γ和抗IL-2的单抗,和/或荧光素标记的单抗IFN-γ-FITC和IL-2-biotin,和/或二抗anti-FITC-490和SA-550。
本发明还涉及Rv1733c在制备诊断结核病的试剂盒中的用途,进一步,试剂盒中还含有ESAT-6抗原和CFP-10抗原。
本发明还涉及Rv1733c用于制备鉴别活动性结核病和潜伏性结核感染的试剂盒的用途,进一步,试剂盒中还含有ESAT-6抗原和CFP-10抗原。
本发明还涉及Rv1733c抗原、ESAT-6抗原和CFP-10抗原联用在制备鉴别活动性结核病和潜伏性结核感染的试剂盒的用途。
进一步的,前述Rv1733c抗原为Rv1733c SLP肽段库。SLP为合成长肽(Syntheticlong peptides)。
进一步的,所述Rv1733c SLP肽段库每条肽段长28个氨基酸,两端重叠14个氨基酸。
进一步的,所述Rv1733c SLP肽段库是如下肽段或其组合:
Rv1733c p1-28 MIATTRDREGATMITFRLRLPCRTILRV;
Rv1733c p16-43 FRLRLPCRTILRVFSRNPLVRGTDRLEA;
Rv1733c p29-56 FSRNPLVRGTDRLEAVVMLLAVTVSLLT;
Rv1733c p43-70 AVVMLLAVTVSLLTIPFAAAAGTAVQDS;
Rv1733c p57-84 IPFAAAAGTAVQDSRSHVYAHQAQTRHP;
Rv1733c p71-98 RSHVYAHQAQTRHPATATVIDHEGVIDS;
Rv1733c p85-112 ATATVIDHEGVIDSNTTATSAPPRTK IT;
Rv1733c p99-126 NTTATSAPPRTKITVPARWVVNGIERSG;
Rv1733c p113-140 VPARWVVNGIERSGEVNAKPGTKSGDRV;
Rv1733c p125-152 SGEVNAKPGTKSGDRVGIWVDSAGQLVD;
Rv1733c p141-168 GIWVDSAGQLVDEPAPPARAIADAALAA;
Rv1733c p169-196 LGLWLSVAAVAGALLALTRAILIRVRNA。
进一步的,所述Rv1733c SLP肽段库也可以是现有技术中Rv1733c SLP肽段库中任意肽段的组合或其变体。
进一步的,所述ESAT-6抗原为ESAT-6肽段库。
进一步的,所述CFP-10抗原为CFP-10肽段库。
本发明还涉及鉴别活动性结核病和潜伏性结核感染的方法,具体包括样本采集、预包被、抗原刺激和孵育检测。
所述样本采集步骤包括:采集静脉血,分离获得外周血单个核细胞,用AIM-V培养基配置细胞悬液。
所述预包被步骤包括:反应板底预包被抗IFN-γ和抗IL-2的单抗;AIM-V细胞培养液作为空白对照;植物血凝素为阳性对照。
所述抗原刺激步骤包括:加入ESAT-6肽段库、CFP-10肽段库、Rv1733c肽段库;加入外周血单个核细胞和anti-CD28。
所述孵育检测步骤包括:孵育;加入荧光素标记的单抗IFN-γ-FITC和IL-2-biotin;避光干燥室温下孵育2h;加入二抗anti-FITC-490和SA-550;避光干燥室温下孵育1h;对分泌IL-2的特异性T细胞进行计数。
本发明具有以下优点:
首先,本发明采用了荧光免疫斑点法:应用IFN-γ/IL-2免疫荧光斑点法,在单细胞水平同时检测IFN-γ和IL-2两种细胞因子的分泌,进行联合诊断,最大程度的节约了人力和血样。
其次,本发明采用了新型潜伏感染相关抗原,选择具代表性的结核分枝杆菌潜伏感染抗原Rv1733c,通过克隆表达及纯化完成抗原肽段库的合成,并作为刺激原致敏T淋巴细胞产生细胞因子,最后通过检测分泌胞内因子IFN-γ和IL-2的结核分枝杆菌特异性T细胞数的差异,证明在ESAT-6&CFP-10FluoroSpot的基础上,联合应用潜伏感染相关抗原Rv1733c刺激后单分泌IL-2的T细胞斑点频数可提高鉴别诊断ATB和LTBI的灵敏度和特异性。
附图说明
图1RV1733SLP抗原刺激后单分泌IL-2的T细胞斑点频数;
图2ESAT-6&CFP-10抗原刺激后单分泌IFN-γ的T细胞斑点频数;
图3ESAT-6&CFP-10抗原刺激后单分泌IFN-γ的T细胞斑点比例;
图4ESAT-6&CFP-10联合应用Rv1733c SLP抗原刺激后单分泌IL-2的T细胞斑点频数
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用的试剂可以商业购买得到。
实施例一:样本采集
纳入病原确诊的活动性结核病患者和无症状的潜伏结核感染者,以下为病原样本的筛选标准。
1.活动性结核病组纳入标准为:
1)年龄18-75岁;
2)有发热、咳嗽等活动性结核表现;
3)MTB涂片抗酸染色或培养阳性、或MTB核酸、或Xpert MTB/RIF检测阳性;
4)未经抗结核治疗。
2.潜伏性结核感染组纳入标准为:
1)年龄18-75岁;
2)无发热、咳嗽等活动性结核表现;
3)既往无结核病史,胸片无陈旧结核改变;
4)T-SPOT.TB阳性。
排除标准:1)妊娠或哺乳期;2)HIV抗体阳性。
实施例二:潜伏期抗原肽段库合成
参考现有技术中Rv1733c SLP(Synthetic Long Peptide)肽段库的序列(Coppolaet al;Synthetic Long Peptide Derived from Mycobacterium tuberculosis LatencyAntigen Rv1733c Protects against Tuberculosis;Clin Vaccine Immunol.2015Sep;22(9):1060-9),克隆表达及纯化完成结核分枝杆菌缺氧相关潜伏期抗原Rv1733c SLP肽段库,每条肽段长28个氨基酸,两端重叠14个氨基酸。合成如下Rv1733c SLP肽段库:
Rv1733c p1-28 MIATTRDREGATMITFRLRLPCRTILRV;
Rv1733c p16-43 FRLRLPCRTILRVFSRNPLVRGTDRLEA;
Rv1733c p29-56 FSRNPLVRGTDRLEAVVMLLAVTVSLLT;
Rv1733c p43-70 AVVMLLAVTVSLLTIPFAAAAGTAVQDS;
Rv1733c p57-84 IPFAAAAGTAVQDSRSHVYAHQAQTRHP;
Rv1733c p71-98 RSHVYAHQAQTRHPATATVIDHEGVIDS;
Rv1733c p85-112 ATATVIDHEGVIDSNTTATSAPPRTK IT;
Rv1733c p99-126 NTTATSAPPRTKITVPARWVVNGIERSG;
Rv1733c p113-140 VPARWVVNGIERSGEVNAKPGTKSGDRV;
Rv1733c p125-152 SGEVNAKPGTKSGDRVGIWVDSAGQLVD;
Rv1733c p141-168 GIWVDSAGQLVDEPAPPARAIADAALAA;
Rv1733c p169-196 LGLWLSVAAVAGALLALTRAILIRVRNA。
实施例三:荧光免疫斑点法(FluoroSpot)
1.样本采集
受试者采集4ml静脉血,肝素抗凝,室温下4小时内经密度梯度离心分离获得外周血单个核细胞(PBMC),用AIM-V培养基(GibcoTM AIM V Medium liquid,Invitrogen,USA)配置浓度为2.5×106PBMCs/ml的细胞悬液。
2.预包被
96孔Human IFN-γ/IL-2FluoroSpot反应板,板底预包被抗IFN-γ和抗IL-2的单抗。单孔加入50ul AIM-V细胞培养液作为空白对照;复孔加入浓度5μg/ml植物血凝素(PHA)为阳性对照。
3.抗原刺激
复孔分别加入ESAT-6肽段库、CFP-10肽段库、Rv1733c SLP肽段库作为刺激抗原。每孔加入2.5×105个外周血单个核细胞(PBMC)和anti-CD28(0.5μg/mL,AID,Straβberg,Germany)。
4.孵育检测
将反应板放置5%CO2细胞培养箱、37℃孵育16-20h。加入荧光素标记的单抗IFN-γ-FITC和IL-2-biotin(终浓度0.5mg/ml)。避光干燥室温下孵育2h。加入二抗anti-FITC-490和SA-550。避光干燥室温下孵育1h。加入荧光增强剂。应用荧光iSPOT分析仪对分泌IFN-γ、IL-2、IFN-γ&IL-2的特异性T细胞分别进行计数。
实施例四:统计学分析
应用SPSS24.0进行统计分析,使用Kolmogorov-Smirnov检验变量是否服从正态分布。正态分布的计量资料用均数±标准差(Mean±SD)表示,非正态分布的计量资料用中位数,四分位间距(Median,IQR)表示。计数资料用百分比,95%可信区间(%,95%CI)表示。两组间斑点形成细胞频数比较采用两个独立样本秩和检验。
Rv1733c SLP肽段库刺激后的分泌不同细胞因子的斑点形成细胞频数绘制受试者工作特性曲线(Receiver operating characteristic,ROC),比较ROC曲线下面积(areaunder the ROC curve,AUROC)。界定Rv1733c SLP特异性的FluoroSpot鉴别诊断ATB和LTBI的最佳cutoff值。计算敏感性,特异性,阳性预测值(PPV),阴性预测值(NPV),阳性似然比(PLR)和阴性似然比(NLR)。p值<0.05被认为是统计学上显著的。
实施例五:实验结果
采用病例对照研究设计,纳入2017年1至12月北京协和医院和北京胸科医院住院的病原确诊的活动性结核病患者作为病例组,同期潜伏性结核感染者作为对照组。应用荧光免疫斑点法检测经结核分枝杆菌潜伏期抗原Rv1733c SLP刺激后特异性T细胞IFN-γ和IL-2的分泌。联合ESAT-6&CFP-10FluoroSpot检测,评价鉴别诊断活动性结核病与潜伏结核感染的敏感性、特异性、预测值和似然比。
研究纳入病原确诊的活动性结核病20例、潜伏性结核感染28例。以结核分枝杆菌潜伏相关抗原Rv1733c SLP抗原刺激后单分泌IL-2的T细胞斑点频数绘制ROC曲线,AUROC最大为0.711(95CI 0.566-0.856),诊断界值为0(SFCs/250,000PBMCs)时,鉴别诊断ATB和LTBI的灵敏度和特异度分别为75%(95CI 50.90%to 91.34%)和60.71%(95CI 40.58%to 78.50%),如图1。
利用ESAT-6&CFP-10抗原刺激后单分泌IFN-γ的T细胞斑点频数鉴别诊断ATB和LTBI,灵敏度和特异度为70%(95%CI 45.72%to 88.11%)和64.29%(95%CI 44.07%to81.36%),如图2。
利用ESAT-6&CFP-10抗原刺激后单分泌IFN-γ的T细胞斑点比例数鉴别诊断ATB和LTBI,灵敏度和特异度为85%(95%CI 62.11%to 96.79%)和71.43%(95%CI 51.33%to86.78%),如图3。
在前期ESAT-6&CFP-10Fluorospot的基础上,联合应用Rv1733c SLP抗原刺激后单分泌IL-2的T细胞斑点频数鉴别诊断ATB和LTBI,平行实验可使敏感性提高至100%(95CI83.16%to 100.00%),序列试验可使特异性提高至92.86%(95CI 71.77%to 97.73%),如图4。
表1
结论:Rv1733c SLP可作为以T细胞为基础的结核诊断试验的备选抗原,与ESAT-6、CFP-10抗原联合,有助于辅助鉴别诊断活动性结核病和潜伏性结核感染。在ESAT-6&CFP-10FluoroSpot的基础上,联合应用潜伏感染相关抗原Rv1733c SLP刺激后单分泌IL-2的T细胞斑点频数可提高鉴别诊断ATB和LTBI的灵敏度和特异性。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (2)
1.一种组合物在制备鉴别潜伏性结核感染和活动性结核病试剂中的应用,其特征在于,所述组合物为Rv1733c SLP抗原、ESAT-6和CFP-10抗原,所述组合物通过刺激后单分泌IL-2的T细胞斑点频数的荧光免疫斑点法实现所述应用,在所述ESAT-6和CFP-10抗原刺激后单分泌IFN-γ斑点的频数和比例的基础上,联合应用所述Rv1733c SLP刺激后单分泌IL-2的T细胞斑点频数可提高鉴别诊断活动性结核病和潜伏性结核感染的灵敏度和特异性,所述Rv1733c SLP为潜伏性结核感染相关抗原,所述的Rv1733c SLP肽段库为:
Rv1733c p1-28 MIATTRDREGATMITFRLRLPCRTILRV;
Rv1733c p16-43 FRLRLPCRTILRVFSRNPLVRGTDRLEA;
Rv1733c p29-56 FSRNPLVRGTDRLEAVVMLLAVTVSLLT;
Rv1733c p43-70 AVVMLLAVTVSLLTIPFAAAAGTAVQDS;
Rv1733c p57-84 IPFAAAAGTAVQDSRSHVYAHQAQTRHP;
Rv1733c p71-98 RSHVYAHQAQTRHPATATVIDHEGVIDS;
Rv1733c p85-112 ATATVIDHEGVIDSNTTATSAPPRTKIT;
Rv1733c p99-126 NTTATSAPPRTKITVPARWVVNGIERSG;
Rv1733c p113-140 VPARWVVNGIERSGEVNAKPGTKSGDRV;
Rv1733c p125-152 SGEVNAKPGTKSGDRVGIWVDSAGQLVD;
Rv1733c p141-168 GIWVDSAGQLVDEPAPPARAIADAALAA;
Rv1733c p169-196 LGLWLSVAAVAGALLALTRAILIRVRNA。
2.根据权利要求1所述的应用,其特征在于,所述的荧光免疫斑点法包括如下步骤:
(1)样本采集
受试者采集4ml静脉血,肝素抗凝,室温下4小时内经密度梯度离心分离获得外周血单个核细胞PBMC,用AIM-V培养基配置浓度为2.5×106PBMCs/ml的细胞悬液;
(2)预包被
96孔Human IFN-γ/IL-2FluoroSpot反应板,板底预包被抗IFN-γ和抗IL-2的单抗,单孔加入50ul AIM-V细胞培养液作为空白对照;复孔加入浓度5μg/ml植物血凝素PHA为阳性对照;
(3)抗原刺激
复孔分别加入ESAT-6肽段库、CFP-10肽段库、Rv1733c SLP肽段库作为刺激抗原,每孔加入2.5×105个外周血单个核细胞PBMC和0.5μg/mL的anti-CD28;
(4)孵育检测
将反应板放置5%CO2细胞培养箱、37℃孵育16-20h,加入荧光素标记的单抗IFN-γ-FITC和IL-2-biotin,避光干燥室温下孵育2h,加入二抗anti-FITC-490和SA-550,避光干燥室温下孵育1h,加入荧光增强剂,应用荧光iSPOT分析仪对分泌IFN-γ、IL-2、IFN-γ和IL-2的特异性T细胞分别进行计数。
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