WO2018076404A1 - 一种体外检测活动性结核的方法 - Google Patents

一种体外检测活动性结核的方法 Download PDF

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WO2018076404A1
WO2018076404A1 PCT/CN2016/105469 CN2016105469W WO2018076404A1 WO 2018076404 A1 WO2018076404 A1 WO 2018076404A1 CN 2016105469 W CN2016105469 W CN 2016105469W WO 2018076404 A1 WO2018076404 A1 WO 2018076404A1
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tuberculosis
detection
active
ifn
active tuberculosis
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黄曦
胡鹏男
李妍
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广州迪澳医疗科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases

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  • the invention belongs to the field of detection, and particularly relates to a method for detecting active tuberculosis in vitro.
  • Tuberculosis is a common chronic infectious disease caused by Mycobacterium tuberculosis, which can invade many organs and is most common in tuberculosis of the lungs. Nearly one-third of the world's people have been infected with tuberculosis, and statistics show that 1.5 million people died of tuberculosis and 9 million new cases in 2013. Tuberculosis has become one of the major diseases in adults worldwide due to infectious diseases. . China is one of the 22 countries with high burden of tuberculosis in the world, and the number of active tuberculosis patients ranks second in the world.
  • tuberculosis The main cause of tuberculosis is that a tuberculosis patient can infect up to 10-15 people through close contact during the year before the patient is undetected and treated. Tuberculosis is transmitted through the respiratory tract. There are a large number of tubercle bacilli in the lungs of tuberculosis patients. If he coughs or sneezes on healthy people, it is very likely to spread the bacteria to healthy people. Before the patient was found, the patient did not take any preventive measures. In close contact with family members, colleagues, classmates, etc., the contacts were easily infected by tuberculosis.
  • BCG vaccination is currently the most internationally recognized immunization method for the prevention of tuberculosis. China's planned immunization program requires newborns to be vaccinated with BCG, but its protection efficiency has not reached 100%. Therefore, the primary measures to prevent tuberculosis It is early detection of new people who are hidden in the crowd.
  • the imaging method is poorly specific, the diagnosis process is closely related to the doctor's experience, and the diagnosis results are strongly subjective; the sputum culture method is the gold standard for tuberculosis diagnosis, but the sensitivity is low. It takes a long time (2-8 weeks), which is not conducive to the early diagnosis of tuberculosis; the sensitivity of acid-fast staining is only 20-30%; the molecular diagnostic method (such as PCR) has poor repeatability and is cumbersome to operate. Certain requirements; the tuberculin test (TST) is susceptible to BCG vaccination, resulting in false positives in the test results.
  • Mycobacterium tuberculosis is a typical intracellular infection, and the body's immune mechanism is mainly immune to T cells.
  • M. tuberculosis invades the human body, when the body's immunity is low, macrophages cannot prevent the growth of the phagocytosis of M. tuberculosis, but can bring the phagocytic M. tuberculosis to other places and present the antigen.
  • Mycobacterium tuberculosis multiplies and sensitizes surrounding T lymphocytes, and sensitized T lymphocytes produce a variety of cytokines (such as INF- ⁇ , IL-2 and TNF- ⁇ , etc.), these cytokines work together to kill Mycobacterium tuberculosis in the lesion.
  • cytokines such as INF- ⁇ , IL-2 and TNF- ⁇ , etc.
  • the present invention has found that IFN- ⁇ and IL-2 secreted by peripheral blood mononuclear cells induced by specific stimulation of antigens are closely related to the disease state of patients through a large number of screening experiments. By detecting the secretion of these two factors, it is possible to accurately judge whether the patient is active tuberculosis, and it is more conducive to the clinician to make a timely diagnosis of the patient's disease state.
  • the quantitative detection reagent comprises an enzyme used in an enzyme-linked immunosorbent assay, an enzyme-linked immunospot assay, a chemiluminescence method, or a liquid chip method.
  • the tuberculosis is active tuberculosis.
  • active tuberculosis includes tuberculosis, active lymphatic tuberculosis, active intestinal tuberculosis, active bone tuberculosis, active tuberculosis, and the like.
  • the specific antigen comprises at least one of three proteins, ESAT-6, CFP-10 and Rv1985c, in a form selected from the group consisting of fusion proteins, protein mixtures or peptides of the three proteins.
  • the method used for the detection includes an enzyme-linked immunosorbent assay, an enzyme-linked immunospot assay, a chemiluminescence method, and a liquid chip method.
  • the present invention provides a method for effectively determining active tuberculosis by stimulating peripheral blood mononuclear cells to produce tuberculosis-related cytokines, detecting secretion amount of IFN- ⁇ and IL-2, and analyzing the results, and drawing conclusions .
  • this method is more accurate and specific in the judgment results of active tuberculosis.
  • the active tuberculosis detection method provided by the invention has greatly shortened the detection time, and provides a basis for the clinician to timely diagnose the patient and guide the medication, and has a very positive significance for the control of tuberculosis.
  • the tuberculosis used in the detection of the present invention is for all tuberculosis, including tuberculosis and extrapulmonary tuberculosis (such as lymphatic tuberculosis, intestinal tuberculosis, bone tuberculosis, kidney tuberculosis, etc.), and has wider coverage and utility.
  • tuberculosis including tuberculosis and extrapulmonary tuberculosis (such as lymphatic tuberculosis, intestinal tuberculosis, bone tuberculosis, kidney tuberculosis, etc.), and has wider coverage and utility.
  • Figure 1 shows the level of secretion of IFN- ⁇ and IL-2 by ELISA
  • Figure 2 shows ELISPOT detection of IFN- ⁇ and IL-2 secretion levels
  • Figure 3 shows the level of secretion of IFN- ⁇ , IL-2 and IL-10 by ELISA.
  • Active tuberculosis refers to tuberculosis, which proves that M. tuberculosis is discharged. The lesion is active. There are often patchy shadows or tuberculous cavities on the chest radiograph, or disseminated lesions, indicating that M. tuberculosis is active and poisonous. Strong. Active tuberculosis patients are the main source of tuberculosis. If they cannot be diagnosed and treated in time, it will cause widespread spread of tuberculosis. Rapid and accurate detection of active tuberculosis patients is the key to reducing the incidence of tuberculosis and mortality.
  • Active tuberculosis includes, but is not limited to, active tuberculosis, active lymphatic tuberculosis, active intestinal tuberculosis, active bone tuberculosis, and active tuberculosis.
  • Example 1 Separation of human peripheral blood mononuclear cells (PBMC) by density gradient centrifugation
  • the blood vessels of fresh heparin lithium or heparin sodium are used to aseptically take 4-5 ml of peripheral blood of the person to be examined, and the anticoagulant and blood are mixed upside down for use.
  • Two 15 ml centrifuge tubes were prepared, and an equal volume of saline and lymphocyte separation solution for injection was added. After mixing fresh heparin anticoagulation with physiological saline, it is slowly added to the separation solution at a constant rate. The separation solution and the blood should be separated when it is added, and then centrifuged at 1800 g for 20 min at 22 ° C. After centrifugation, PBMCs cells can be seen in the cloud. In the layer.
  • the PBMCs cell layer was pipetted into a new 15 ml centrifuge tube, supplemented to 12 ml with RPMI-1640 medium, and centrifuged at 600 g for 10 min at 22 °C.
  • the supernatant was decanted, supplemented with 5 ml of RPMI-1640 medium, gently resuspended by pipetting, and centrifuged at 350 g for 10 min at 22 °C.
  • the supernatant was discarded and the cells were resuspended by adding 0.5 ml of serum-free medium. Manual counting by trypan blue staining or automatic counting using suitable equipment. Based on the count results, it was diluted with serum-free medium to a desired concentration of PBMC cell suspension.
  • Example 2 Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of IFN- ⁇ and IL-2 secreted by PBMC after induction of clinically diagnosed active tuberculosis patients and healthy persons.
  • ELISA Enzyme-linked immunosorbent assay
  • test results are shown in Figure 1. It can be seen that compared with healthy people, PBMC of active tuberculosis patients have IFN- ⁇ and IL-2 cytokines secreted after induction by specific stimulating antigen. Significantly improved. With IFN- ⁇ secretion >20 and IL-2 secretion >15 as the standard, the samples with positive results of both cytokines can be judged as active tuberculosis. According to this determination scheme, 10 samples of active tuberculosis patients detected by this method were all determined to be active tuberculosis, and 10 healthy human samples were judged as inactive tuberculosis.
  • Example 3 Enzyme-linked immunospot assay (ELISPOT) was used to detect the levels of IFN- ⁇ and IL-2 secreted by PBMC after induction of clinically diagnosed active tuberculosis patients and healthy persons.
  • PBMC peripheral venous blood samples
  • test plate Take out the test plate, discard the culture supernatant, rinse the test plate with PBS for 3-4 times, then wash the plate 1-2 times with PBS, gently shake it 5 times each time, and pat dry after the last wash.
  • a labeled antibody working solution of the corresponding cytokine was added to each well and incubated at 37 ° C for 1 hour. Discard the liquid, wash the plate with PBS for 3-5 times, gently shake it 5 times each time. After the last wash, pat dry, add the enzyme conjugate working solution to each well, and incubate at 37 ° C for 1 hour.
  • Example 4 induced stimulation of PBMC in active tuberculosis patients, latent infections and healthy people, and compared the secretion of IFN- ⁇ , IL-2 and IL-10.
  • PBMC peripheral venous blood samples
  • serum-free culture was performed after cell counting.
  • Base the PBMC to a concentration of 2.5 ⁇ 10 6 /ml inoculate the cells into a 96-well plate, add 2.5 ⁇ 10 5 cells per well, and add a specific stimulating antigen diluted in serum-free medium (concentration 2 ⁇ g). /ml), incubate for 18 hours at 37 ° C, 5% CO 2 , and take cell culture medium for detection of IFN- ⁇ , IL-2 and TNF- ⁇ cytokines.
  • the detection method in the present invention has high accuracy and can help clinicians to quickly determine active tuberculosis ( It can also be combined with other small indicators to make a comprehensive judgment.
  • the samples determined to be negative in the method of the present invention can be further tested by other methods to finally determine the result.

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Abstract

一种体外检测活动性结核的方法，该方法是基于定量检测结核相关因子IFN-γ和IL-2的试剂在体外检测结核中的应用。与现有的活动性结核检测方法相比，这一方法在活动性结核的判断结果上更准确、特异性更高。与现有的金标准痰培养相比，检测时间大大缩短，为临床医生及时诊断病人、指导用药提供了依据，对结核病的控制具有非常积极的意义。用于检测的结核病是针对所有结核病，包括肺结核、淋巴结核、肠结核、骨结核、肾结核等，覆盖面和实用性更广。

Description

一种体外检测活动性结核的方法 技术领域
本发明属于检测领域，具体涉及一种体外检测活动性结核的方法。
背景技术
结核病是一种常见的由结核分枝杆菌引起的慢性传染病，可侵及许多脏器，以肺部结核感染最为常见。目前全球有近1/3的人已感染结核杆菌，统计数据表明在2013年有150万人死于结核病，900万新发病例，结核病已成为全世界成人因传染病而死亡的主要疾病之一。中国是全球22个结核病高负担国家之一，活动性肺结核病人数居世界第二位。结核病的传染主要是发生在病人未被发现并进行治疗之前，1名结核病患者一年中通过密切接触可传染多达10-15人。结核病的传播方式为经呼吸道传播，在肺结核病人的肺中有大量的结核杆菌，如果他对着健康人群咳嗽或打喷嚏的话就很有可能将病菌传播给健康人。而在未发现病患前，病人未采取任何预防手段，在与家庭成员、同事、同学等密切接触的过程中，接触者就容易被结核菌感染。现阶段结核病的主要治疗方法主为药物治疗，但是这样的治疗不能够完全阻止结核病的传播。接种卡介苗(BCG)是目前国际公认的最有效预防结核病的免疫方法，中国的计划免疫接种程序中规定新生儿须接种卡介苗，但是其保护效率也并未达到100％，因此，预防结核病的首要措施是尽早发现隐藏在人群中的新发病人。
目前临床常用于活动性结核诊断的方法中，影像学方法特异性差，诊断过程与医生的经验紧密相关，诊断结果带有较强的主观性；痰培养方法是结核诊断金标准，但敏感性低，且耗时长(2-8周)，不利于结核的早期诊断；抗酸染色法敏感性仅有20-30％；分子诊断方法(如PCR)重复性差，且操作繁琐，对实验室环境有一定的要求；结核菌素试验(TST)易受卡介苗(BCG)接种的影响，造成检测结果的假阳性。近年来出现了以T细胞为基础的γ-干扰素释放试验，但是该方法仅能判断患者是否感染了(过)结核分支杆菌，无法判断是否为活动性结核。以上检测方法中，痰培养、抗酸染色法和分子诊断方法虽然特异性好，但仅针对结核病患者中的菌阳结核(约占30％)而不能诊断菌阴结核，目前临床上仍然缺乏一种直接的方法对活动性结核进行检测。
结核分枝杆菌是一种典型的胞内感染菌，人体对其免疫机制主要是以T细胞为主的细胞免疫。结核分枝杆菌侵入人体后，在机体免疫力低下的时候，巨噬细胞不能防止所吞噬的结核分枝杆菌生长，但可将吞噬的结核分枝杆菌带到他处，提呈抗原，此时结核分枝杆菌大量繁殖，使周围T淋巴细胞致敏，致敏的T淋巴细胞可产生多种细胞因子(如INF-γ、IL-2与 TNF-α等)，这些细胞因子共同作用，杀死病灶中的结核分枝杆菌。
发明内容
为了解决上述存在的问题，本发明通过大量的筛选实验，发现经过特异性刺激抗原诱导的外周血单个核细胞所分泌的细胞因子中IFN-γ和IL-2与病人的疾病状态有密切的联系，通过检测这两个因子的分泌量可对患者是否为活动性结核进行准确判断，更有利于临床医生对病人的疾病状态及时做出诊断。
本发明所采取的技术方案是：
定量检测结核相关因子IFN-γ和IL-2的试剂在体外检测结核中的应用。
优选的，定量检测试剂包括酶联免疫吸附法、酶联免疫斑点法、化学发光法、液态芯片法所用的试剂。
优选的，结核为活动性结核。
优选的，活动性结核包括肺结核、活动性淋巴结核、活动性肠结核、活动性骨结核、活动性肾结核等。
一种体外检测活动性结核的方法，使用特异性刺激抗原体外诱导外周血单个核细胞，使其产生结核相关因子，通过检测其中IFN-γ和IL-2的分泌量，判断样本是否为活动性结核，该方法不用于疾病诊断。
优选的，特异性抗原包括ESAT-6、CFP-10和Rv1985c三种蛋白的至少一种，形式选自这三种蛋白的融合蛋白、蛋白混合物或肽段。
优选的，检测所用的方法包括酶联免疫吸附法、酶联免疫斑点法、化学发光法、液态芯片法。
本发明的有益效果是：
1)本发明提供了一种有效判断活动性结核的方法，该方法通过刺激外周血单个核细胞产生结核相关细胞因子，检测其中IFN-γ和IL-2的分泌量并进行分析，得出结论。与现有的活动性结核检测方法相比，这一方法在活动性结核的判断结果上更准确、特异性更高。
2)本发明提供的活动性结核检测方法与现有的金标准痰培养相比，检测时间大大缩短，为临床医生及时诊断病人、指导用药提供了依据，对结核病的控制具有非常积极的意义。
3)本发明中所述试剂用于检测的结核病是针对所有结核病，包括肺结核及肺外结核(如淋巴结核、肠结核、骨结核、肾结核等)，覆盖面和实用性更广。
附图说明
图1为ELISA检测IFN-γ和IL-2的分泌水平；
图2为ELISPOT检测IFN-γ和IL-2的分泌水平；
图3为ELISA检测IFN-γ、IL-2和IL-10的分泌水平。
具体实施方式
活动性结核：是指患有结核病，证明有结核分枝杆菌排出，病灶属于活动期，胸片上常有斑片状阴影或是结核空洞，或者播散病灶，说明结核分枝杆菌繁殖活跃，毒力强。活动性结核患者是结核病的主要传染源，如不能及时诊断治疗，会造成结核病的大范围传播，快速准确的检出活动性结核患者是降低结核病发病率及病死率的关键。
活动性结核包括但不限于活动性肺结核、活动性淋巴结核、活动性肠结核、活动性骨结核、活动性肾结核。
下面结合具体实施例对本发明作进一步的说明，但并不局限于此。
实施例1：密度梯度离心法分离人外周血单个核细胞(PBMC)
使用新鲜肝素锂或肝素钠的采血管无菌抽取待检人员外周静脉血4-5ml，颠倒使抗凝剂和血液混匀待用。准备2个15ml离心管，分别加入与采血量等体积的注射用生理盐水和淋巴细胞分离液。将新鲜肝素抗凝血与生理盐水混匀后，匀速缓慢加入到分离液中，加入时需保持分离液与血液分层，然后于22℃、1800g离心20min，离心后可见PBMCs细胞存在于云雾状层中。吸取PBMCs细胞层至新的15ml离心管内，用RPMI-1640培养液补齐至12ml，于22℃，600g离心10min。倒掉上清，用RPMI-1640培养液补齐至5ml，轻轻吹打沉淀重悬后，于22℃，350g离心10min。弃上清，加入0.5ml无血清培养基重悬细胞。采用台盼蓝染色手工计数法或使用合适的设备自动计数。根据计数结果，用无血清培养基将其稀释为所需浓度的PBMC细胞悬液。
实施例2酶联免疫吸附法(ELISA)检测临床诊断活动性结核患者和健康人的诱导后PBMC分泌IFN-γ和IL-2的水平
1)选取10例临床诊断为活动性结核的患者和10例健康人的肝素钠抗凝外周静脉血样本，按实施例1中的密度分层法分离PBMC，经过细胞计数后使用无血清培养基将PBMC稀释到2.5×106个/ml的浓度，将细胞接种至96孔板中，每孔加入2.5×105个细胞，再加入用无血清培养基稀释的特异性刺激抗原(融合蛋白CFP-10-ESAT-6-Rv1985c，浓度2μg/ml)，37℃、5％CO2条件下孵育18小时，取细胞培养液进行IFN-γ和IL-2细胞因子的检测。
2)采用预先包被了抗人IFN-γ单克隆抗体的ELISA板，加入上步所提细胞培养上清液，室温孵育2小时，用洗涤缓冲液清洗ELISA板3-5次，往检测孔中加入抗IFN-γ抗体，室温孵育1小时，然后去除抗体，用洗涤缓冲液清洗ELISA板3-5次，再加入HRP标记的二抗，37℃孵育1小时，然后去除二抗，用洗涤缓冲液清洗ELISA板3-5次，加入配制好的TMB显色底物，室温下避光显色20分钟，最后加入终止液终止显色，30分钟内用酶标仪进行结 果分析。
3)采用预先包被了抗人IL-2单克隆抗体的ELISA板，加入上步所提细胞培养上清液，室温孵育2小时，用洗涤缓冲液清洗ELISA板3-5次，往检测孔中加入抗IL-2抗体，室温孵育1小时，然后去除抗体，用洗涤缓冲液清洗ELISA板3-5次，再加入HRP标记的二抗，37℃孵育1小时，然后去除二抗，用洗涤缓冲液清洗ELISA板3-5次，加入配制好的TMB显色底物，室温下避光显色20分钟，最后加入终止液终止显色，30分钟内用酶标仪进行结果分析。
检测结果如图1所示，从中可以看出与健康人相比，活动性结核患者的PBMC在经过特异性刺激抗原诱导后其IFN-γ和IL-2这两种细胞因子的分泌量都有明显提高。以IFN-γ分泌量＞20、IL-2分泌量＞15为标准，两个细胞因子检测结果均呈阳性的样本可判断为活动性结核。根据这一判定方案，用该方法检测的10例活动性结核患者样本都判定为活动性结核，10例健康人样本均判定为非活动性结核。
实施例3酶联免疫斑点法(ELISPOT)检测临床诊断活动性结核患者和健康人的诱导后PBMC分泌IFN-γ和IL-2的水平
1)选取10例活动性结核患者和10例健康人的肝素钠抗凝外周静脉血样本，按实施例1中的密度分层法分离PBMC，经过细胞计数后使用无血清培养基将其稀释到2.5×106个/ml的浓度备用。
2)选择PVDF膜检测板(分别预先使用抗人IFN-γ单克隆抗体和抗人IL-2单克隆抗体包被过夜，4℃条件保存)作为反应板，每孔加入2.5×105个上步制备的PBMC，每个样本每种细胞因子设置3个检测孔，分别是空白对照(加入无血清培养基)、实验孔(加入特异性刺激抗原进行刺激，浓度为2μg/ml)和阳性对照孔(加入植物凝集素PHA进行刺激，浓度为5μg/ml)，37℃、5％CO2培养箱中孵育16-20小时。
3)取出检测板，弃培养上清液，用PBS冲洗检测板3-4遍，再用PBS洗板1-2次，每次来回轻轻晃动5下，最后一次洗板后拍干。在每个孔中加入相应细胞因子的标记抗体工作液，置37℃孵育1小时。弃液体，用PBS洗板3-5次，每次来回轻轻晃动5下，最后一次洗板后拍干，每孔加入酶结合物工作液，置37℃孵育1小时。弃液体，用PBS洗板5次，每次来回轻轻晃动5下，最后一次洗板后拍干，每孔加入100μl的显色液，置37℃避光显色5-10分钟至斑点大小肉眼清晰可见，弃液体，用去离子水或自来水洗涤检测板正反面及底座3-5遍，扣干孔里的水，终止显色，将板放入37℃烘箱干燥4小时或者室温干燥过夜。显色结果用CTL ImmunoSpot S6 ELISpot分析仪进行自动分析。
实验分析结如图2所示，从中可以看出活动性结核患者的PBMC在经过特异性刺激抗原 诱导后其IFN-γ和IL-2的分泌量都有明显提高。
实施例4分别对活动性结核患者、潜伏感染及健康人的PBMC进行诱导刺激，并对其中IFN-γ、IL-2和IL-10的分泌量进行对比
1)选取10例活动性结核患者、10例潜伏感染及10例健康人的肝素钠抗凝外周静脉血样本，按实施例1中的密度分层法分离PBMC，经过细胞计数后使用无血清培养基将PBMC稀释到2.5×106个/ml的浓度，将细胞接种至96孔板中，每孔加入2.5×105个细胞，再加入用无血清培养基稀释的特异性刺激抗原(浓度2μg/ml)，37℃、5％CO2条件下孵育18小时，取细胞培养液进行IFN-γ、IL-2和TNF-α细胞因子的检测。
2)采用预先包被了抗人IFN-γ单克隆抗体的ELISA板，加入上步所提细胞培养上清液，室温孵育2小时，用洗涤缓冲液清洗ELISA板3-5次，往检测孔中加入抗IFN-γ抗体，室温孵育1小时，然后去除抗体，用洗涤缓冲液清洗ELISA板3-5次，再加入HRP标记的二抗，37℃孵育1小时，然后去除二抗，用洗涤缓冲液清洗ELISA板3-5次，加入配制好的TMB显色底物，室温下避光显色20分钟，最后加入终止液终止显色，30分钟内用酶标仪进行结果分析。
3)采用预先包被了抗人IL-2单克隆抗体的ELISA板，加入上步所提细胞培养上清液，室温孵育2小时，用洗涤缓冲液清洗ELISA板3-5次，往检测孔中加入抗IL-2抗体，室温孵育1小时，然后去除抗体，用洗涤缓冲液清洗ELISA板3-5次，再加入HRP标记的二抗，37℃孵育1小时，然后去除二抗，用洗涤缓冲液清洗ELISA板3-5次，加入配制好的TMB显色底物，室温下避光显色20分钟，最后加入终止液终止显色，30分钟内用酶标仪进行结果分析。
4)采用预先包被了抗人IL-10单克隆抗体的ELISA板，加入上步所提细胞培养上清液，室温孵育2小时，用洗涤缓冲液清洗ELISA板3-5次，往检测孔中加入抗IL-2抗体，室温孵育1小时，然后去除抗体，用洗涤缓冲液清洗ELISA板3-5次，再加入HRP标记的二抗，37℃孵育1小时，然后去除二抗，用洗涤缓冲液清洗ELISA板3-5次，加入配制好的TMB显色底物，室温下避光显色20分钟，最后加入终止液终止显色，30分钟内用酶标仪进行结果分析。
实验结果如图3所示，按照IFN-γ分泌量＞20、IL-2分泌量＞15和IL-10分泌量＞25分别作为三个细胞因子检测阳性的判断标准：
1)10例活动性结核样本中，9例判断为IFN-γ阳性，9例判断为IL-2阳性，4例判断为IL-10阳性，这三个因子判定的交叉样本集如表1显示。
表1
Figure PCTCN2016105469-appb-000001
根据表1可以看出，IFN-γ与IL-2这两个因子结合即可判定8例样本为阳性，而增加了IL-10因子进行判断，对提高检测的灵敏度没有帮助。
2)10例潜伏感染样本中，6例判断为IFN-γ阳性，3例判断为IL-2阳性，IL-10全部为阴性。没有一例样本的IFN-γ和IL-2因子检测同时判定为阳性。
3)10例健康人样本中，IFN-γ和IL-2因子全部显示为阴性，有2例判断为IL-10阳性。
根据以上结果，结合IFN-γ和IL-2这两个细胞因子进行检测分析即能够判断出活动性结核样本，灵敏度高、特异性好，如果再加入其他的因子辅助判断也不能有效提高检出率，同时也会增加误判的风险。
目前，由于没有专门针对活动性结核的检测方法，基本都是临床医生依靠多个检查指标进行综合判断来诊断，因此本发明中的检测方法准确性高，可以帮助临床医生快速判断活动性结核(也可以结合其他少量指标综合判断)，而本发明方法中判定为阴性的样本的也可以进一步进行其他方法的检测而最终确定结果。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (6)

  1. 定量检测结核相关因子IFN-γ和IL-2的试剂在体外检测结核中的应用。
  2. 根据权利要求1所述的应用，其特征在于：定量检测试剂包括酶联免疫吸附法、酶联免疫斑点法、化学发光法、液态芯片法所用的试剂。
  3. 根据权利要求1所述的应用，其特征在于：结核为活动性结核。
  4. 一种体外检测活动性结核的方法，其特征在于：使用特异性刺激抗原体外诱导外周血单个核细胞，使其产生结核相关因子，通过检测其中IFN-γ和IL-2的分泌量，判断样本是否为活动性结核，该方法不用于疾病诊断。
  5. 根据权利要求4所述的方法，其特征在于：特异性抗原包括ESAT-6、CFP-10和Rv1985c三种蛋白的至少一种，形式选自这三种蛋白的融合蛋白、蛋白混合物或肽段。
  6. 根据权利要求4所述的方法，其特征在于：检测所用的方法包括酶联免疫吸附法、酶联免疫斑点法、化学发光法、液态芯片法。
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