CN115078732A - 结核分枝杆菌特异性刺激抗原、试剂盒及其应用 - Google Patents
结核分枝杆菌特异性刺激抗原、试剂盒及其应用 Download PDFInfo
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Abstract
本发明涉及一种结核分枝杆菌特异性刺激抗原、试剂盒及其应用，本发明首先提供了TB27.4抗原作为检测结核分枝杆菌感染的刺激抗原的应用，本发明还提供了包括TB27.4抗原的刺激抗原组合，其还包括ESAT‑6抗原和CFP‑10抗原。本发明提供的刺激抗原组合可在不降低特异性的情况下显著降低结核感染的漏检率。本发明还提供了包括该刺激抗原组合的试剂盒，其具有性能好，检出率高等优点，对传染病防控具有实际和广泛的正向作用。
Description
技术领域
本发明涉及免疫检测技术领域，具体涉及一种结核分枝杆菌特异性刺激抗原、试剂盒及其应用。
背景技术
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。结核分枝杆菌侵入呼吸道后，原肺泡中未活化的树突状细胞(Dendriticcells,DC)抗菌活性弱，不能防止所吞噬的结核分枝杆菌生长，但可将结核分枝杆菌吞噬并带到他处，提呈抗原，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(INF-γ、IL-2、IL-6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。根据这一免疫机制，通过对已感染结核分枝杆菌的患者及健康人血液中的细胞因子进行分析对比，发现感染结核分枝杆菌的患者其血液中INF-γ、IL-2、IL-6、TNF-α的浓度明显升高。
γ-干扰素，又称IFN-γ或Ⅱ型干扰素，是由有丝分裂原刺激T淋巴细胞产生。干扰素是一种高效的抗病毒生物活性物质，又是一种具有广泛免疫调节作用的淋巴因子。该产品广泛运用于干扰素释放实验(IGRA)试剂盒。刺激抗原作为刺激T细胞产生细胞因子的重要组分，在此类试剂盒中起到了至关重要的作用。
然而市场现售的结核特异性细胞因子检测试剂盒使用的ESAT-6、CFP-10联用的方案均存在漏检的问题，鉴于结核病是传染性较强的疾病，因此存在降低其漏检率的需求。
发明内容
为了克服上述现有技术的不足，本发明的目的在于提供一种结核分枝杆菌特异性的刺激抗原、试剂盒及其应用。
为此，第一方面，本发明提供TB27.4抗原作为检测结核分枝杆菌感染的刺激抗原的应用。
本发明的第二方面，提供一种用于检测结核分枝杆菌感染的刺激抗原组合，所述刺激抗原组合包括TB27.4抗原。
进一步，所述刺激抗原组合还包括ESAT-6抗原和CFP-10抗原。
本发明通过混合TB27.4、ESAT-6、CFP-10三种结核特异性抗原作为刺激抗原，发现其对T淋巴细胞的刺激效果显著高于仅混合ESAT-6与CFP-10的对照组，因此，添加TB27.4可以有效提高刺激抗原的刺激效果，添加了TB27.4的刺激抗原可以作为更优的试剂盒组分以提高试剂盒性能，降低漏检率。
进一步，所述刺激抗原组合中，TB27.4抗原、ESAT-6抗原和CFP-10抗原的质量之比为1:1-5:1-5；例如1:1:1、1:2:2、1:5:5等。
本发明的第三方面，提供一种用于检测结核分枝杆菌感染的反应体系，所述反应体系包括本发明第二方面所述的刺激抗原组合。
进一步，所述反应体系中，所述TB27.4抗原的浓度为2-50μg/ml；例如2μg/ml、5μg/ml、10μg/ml、20μg/ml、30μg/ml、40μg/ml、50μg/ml等。
在一些实施方式中，所述刺激抗原组合包括TB27.4抗原、ESAT-6抗原和CFP-10抗原；所述反应体系中，所述ESAT-6抗原和CFP-10抗原的浓度各自独立地选自2-50μg/ml；例如2μg/ml、5μg/ml、10μg/ml、20μg/ml、30μg/ml、40μg/ml、50μg/ml等。
在某实施方式中，所述反应体系中，所述TB27.4抗原的浓度为10μg/ml，ESAT-6抗原的浓度为10μg/ml，所述CFP-10抗原的浓度为10μg/ml。
在另一实施方式中，所述反应体系中，所述TB27.4抗原的浓度为2μg/ml，ESAT-6抗原的浓度为10μg/ml，所述CFP-10抗原的浓度为10μg/ml。
在又一实施方式中，所述反应体系中，所述TB27.4抗原的浓度为5μg/ml，ESAT-6抗原的浓度为10μg/ml，所述CFP-10抗原的浓度为10μg/ml。
在再一实施方式中，所述反应体系中，所述TB27.4抗原的浓度为50μg/ml，ESAT-6抗原的浓度为50μg/ml，所述CFP-10抗原的浓度为50μg/ml。
进一步，所述反应体系还包括PBS缓冲液。
本发明的第四方面，提供一种用于检测结核分枝杆菌感染的试剂盒，所述试剂盒包括本发明第二方面所述的刺激抗原组合。
本发明的第五方面，提供一种结核分枝杆菌感染的检测方法，所述检测方法包括应用本发明第三方面所述的反应体系或者本发明第四方面所述的试剂盒，对待测样本进行检测。
根据本发明所述的检测方法，其具有非疾病诊断治疗目的。
进一步，所述待测样本包括受试者的T细胞。
进一步，所述T细胞来源于受试者的外周血液、肺泡灌洗液、脑脊液、淋巴液等。
进一步，所述检测方法包括：使所述刺激抗原组合与受试者的T细胞相接触，检测T细胞分泌的细胞因子。
进一步，所述细胞因子为INF-γ、IL-2、IL-6或TNF-α。
进一步，所述细胞因子的检测方法包括：酶联免疫吸附测定(ELISA)、酶联免疫斑点法(ELISPOT)、免疫印迹法、细胞内细胞因子染色法等。
与现有技术相比，本发明具有以下有益效果：
本发明提供了TB27.4抗原作为检测结核分枝杆菌感染的刺激抗原的应用，在此基础上，还提供了包括TB27.4抗原在内的刺激抗原组合，该刺激抗原组合还包括ESAT-6抗原和CFP-10抗原，可在不降低特异性的情况下显著降低结核感染的漏检率。将该刺激抗原组合用于检测结核分枝杆菌感染的试剂盒，能得到性能更好，检出率更高的试剂盒，对传染病防控具有实际和广泛的正向作用。
具体实施方式
下面将更详细地描述本公开的示例性实施方式。应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
下述实验例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
以下实施方式中，用于测定γ-干扰素含量的方法是本领域技术人员已知的，刺激抗原中是否含有TB27.4对测定γ-干扰素含量的方法步骤没有任何影响。
实施例1
取3mg/ml的TB27.4、ESAT-6、CFP-10各500μl，混合均匀配制得到刺激抗原组1，其三种抗原的终浓度分别为1mg/ml；取3mg/ml的ESAT-6、CFP-10各500μl混合均匀，并加入500μl PBS缓冲液配制成刺激抗原组2，其两种抗原的终浓度分别为1mg/ml。
使用肝素锂抗凝采血管分别采集50例结核确诊患者(结核核酸阳性)、50例无结核病人群(结核核酸阴性)的外周血各3ml，每份样本分装为A、B、C三组，每组为1ml/支。向A组外周血中加入10μl PBS缓冲液，B组加入10μl刺激抗原组1，C组加入10μl刺激抗原组2。混匀后37℃孵育16小时，离心取血浆，进行γ-干扰素含量测定。
γ-干扰素含量测定：
使用ELISA法进行血浆样本中γ-干扰素含量的测定。使用γ-干扰素国际标准品配制标准曲线测定样本中的γ-干扰素浓度。结果显示于表1。
使用统计学分析软件对实验产生的数据进行对比分析，针对检测得到的γ-干扰素浓度值，研究了不同刺激抗原组之间的统计学差异。数据显示，组B与组C结核确诊患者血浆γ-干扰素含量显著高于仅添加PBS的组A。说明刺激抗原组1与刺激抗原组2均具有诊断结核病的潜力。将组B、组C的检测结果分别进行ROC曲线分析，组B的曲线下面积为0.9640，较组C的0.9184明显更大；与临床诊断进行一致性分析，组B的kappa值0.8600明显高于组C的0.7800；说明组B的诊断效能优于组C。因此，刺激抗原组1较刺激抗原组2的效果更好。
表1三组样本血浆中γ-干扰素含量测定结果
实施例2
分别配制刺激抗原组A：0.6mg/ml的TB27.4、3mg/ml的ESAT-6与3mg/ml的CFP-10各500μl，混合均匀，配制得到刺激抗原组A；
刺激抗原组B：1.5mg/ml的TB27.4、3mg/ml的ESAT-6与3mg/ml的CFP-10各500μl，混合均匀，配制得到刺激抗原组B；
刺激抗原组C：3mg/ml的TB27.4、3mg/ml的ESAT-6与3mg/ml的CFP-10各500μl，混合均匀，配制得到刺激抗原组C；
使用肝素锂抗凝采血管分别采集20例结核确诊患者(结核核酸阳性)、20例无结核病人群(结核核酸阴性)的外周血各4ml，每份样本分装为A、B、C、D三组，每组为1ml/支。向A组外周血中加入10μl刺激抗原组A，B组加入10μl刺激抗原组B，C组加入10μl刺激抗原组C，D组加入50μl刺激抗原组C。混匀后37℃孵育16小时，离心取血浆，进行γ-干扰素含量测定。
γ-干扰素含量测定：
使用ELISA法进行血浆样本中γ-干扰素含量的测定。使用γ-干扰素国际标准品配制标准曲线测定样本中的γ-干扰素浓度。结果显示于表2。
使用统计学分析软件对实验产生的数据进行对比分析，针对检测得到的γ-干扰素浓度值，研究了不同刺激抗原组之间的统计学差异。数据显示，A、B、C、D四组的γ-干扰素含量没有显著性差异，因此TB27.4、ESAT-6、CFP-10的工作浓度为2-50μg/ml；且在2-50μg/ml浓度范围内刺激抗原对T淋巴细胞的刺激效果没有显著差异。
表2四组样本血浆中γ-干扰素含量测定结果
| 样本 | 组A | 组B | 组C | 组D | 样本 | 组A | 组B | 组C | 组D |
| 确诊1 | 74.442 | 67.294 | 62.115 | 63.019 | 无结核1 | 5.980 | 2.955 | 2.876 | 5.194 |
| 确诊2 | 430.638 | 416.554 | 424.217 | 434.326 | 无结核2 | 7.119 | 9.833 | 8.213 | 7.047 |
| 确诊3 | 303.773 | 332.113 | 340.805 | 345.543 | 无结核3 | 9.605 | 8.670 | 7.498 | 4.774 |
| 确诊4 | 321.014 | 313.497 | 344.248 | 360.458 | 无结核4 | 9.117 | 9.160 | 6.566 | 7.132 |
| 确诊5 | 82.164 | 85.042 | 87.007 | 85.116 | 无结核5 | 8.457 | 3.330 | 0.768 | 3.622 |
| 确诊6 | 363.010 | 377.291 | 379.360 | 345.425 | 无结核6 | 9.268 | 2.810 | 5.565 | 2.726 |
| 确诊7 | 179.827 | 175.946 | 159.582 | 151.148 | 无结核7 | 1.887 | 1.549 | 1.841 | 5.619 |
| 确诊8 | 448.526 | 411.125 | 407.218 | 390.690 | 无结核8 | 0.306 | 1.644 | 1.730 | 8.174 |
| 确诊9 | 118.993 | 129.970 | 117.931 | 114.486 | 无结核9 | 6.155 | 7.314 | 7.862 | 1.661 |
| 确诊10 | 501.226 | 526.971 | 534.607 | 525.857 | 无结核10 | 7.449 | 0.239 | 9.410 | 4.680 |
| 确诊11 | 145.236 | 153.251 | 165.633 | 164.463 | 无结核11 | 2.108 | 6.451 | 3.848 | 7.160 |
| 确诊12 | 477.222 | 488.743 | 536.514 | 574.883 | 无结核12 | 0.157 | 1.408 | 9.902 | 3.028 |
| 确诊13 | 247.049 | 261.577 | 247.043 | 240.230 | 无结核13 | 1.395 | 9.290 | 8.461 | 5.504 |
| 确诊14 | 301.898 | 329.165 | 327.519 | 337.923 | 无结核14 | 3.755 | 9.310 | 8.810 | 8.579 |
| 确诊15 | 440.558 | 469.230 | 489.690 | 497.345 | 无结核15 | 8.430 | 6.402 | 1.884 | 4.691 |
| 确诊16 | 218.151 | 210.774 | 191.991 | 198.427 | 无结核16 | 9.995 | 0.967 | 5.447 | 7.491 |
| 确诊17 | 455.826 | 424.647 | 422.875 | 391.575 | 无结核17 | 8.229 | 5.313 | 5.050 | 4.910 |
| 确诊18 | 383.127 | 405.368 | 426.326 | 385.078 | 无结核18 | 5.197 | 0.239 | 4.009 | 3.455 |
| 确诊19 | 368.089 | 404.735 | 373.899 | 398.826 | 无结核19 | 3.664 | 1.920 | 2.666 | 9.119 |
| 确诊20 | 450.816 | 446.464 | 455.689 | 493.557 | 无结核20 | 9.557 | 6.634 | 1.324 | 9.148 |
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
Claims (10)
1.TB27.4抗原作为检测结核分枝杆菌感染的刺激抗原的应用。
2.一种用于检测结核分枝杆菌感染的刺激抗原组合，其特征在于，所述刺激抗原组合包括TB27.4抗原。
3.如权利要求2所述的刺激抗原组合，其特征在于，所述刺激抗原组合还包括ESAT-6抗原和CFP-10抗原；
优选地，所述刺激抗原组合中，TB27.4抗原、ESAT-6抗原和CFP-10抗原的质量之比为1:1-5:1-5。
4.一种用于检测结核分枝杆菌感染的反应体系，其特征在于，所述反应体系包括权利要求2-3任一项所述的刺激抗原组合。
5.如权利要求4所述的反应体系，其特征在于，所述反应体系中，所述TB27.4抗原的浓度为2-50μg/ml。
6.如权利要求4所述的反应体系，其特征在于，所述反应体系还包括PBS缓冲液。
7.一种用于检测结核分枝杆菌感染的试剂盒，其特征在于，所述试剂盒包括权利要求2-3任一项所述的刺激抗原组合。
8.一种结核分枝杆菌感染的检测方法，其特征在于，所述检测方法包括应用权利要求2-3任一项所述的反应体系或者权利要求7所述的试剂盒，对待测样本进行检测。
9.如权利要求8所述的检测方法，其特征在于，所述待测样本包括受试者的T细胞；
优选地，所述T细胞来源于受试者的外周血液、肺泡灌洗液、脑脊液或淋巴液。
10.如权利要求9所述的检测方法，其特征在于，所述检测方法包括：使所述刺激抗原组合与受试者的T细胞相接触，检测T细胞分泌的细胞因子；
优选地，所述细胞因子为INF-γ、IL-2、IL-6或TNF-α；
优选地，所述细胞因子的检测方法包括：酶联免疫吸附测定、酶联免疫斑点法、免疫印迹法、细胞内细胞因子染色法。
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