CN110836966A - Detection nanoparticles, detection methods and kits for antigen-specific T cell content detection, etc. - Google Patents
Detection nanoparticles, detection methods and kits for antigen-specific T cell content detection, etc.Download PDFInfo
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Abstract
Description
Translated fromChinese技术领域technical field
本发明属于免疫检测领域，具体涉及一种能有效用于抗原特异性T细胞含量检测的检测纳米颗粒、其相应的制备方法、包括其的试剂盒以及利用其进行抗原特异性T细胞含量检测的检测方法。The invention belongs to the field of immune detection, and in particular relates to a detection nanoparticle that can be effectively used for detection of antigen-specific T cell content, a corresponding preparation method thereof, a kit including the same, and a detection nanoparticle using the same for detection of antigen-specific T cell content. Detection method.
背景技术Background technique
近些年来免疫技术的发展极其迅速，尤其是癌症的免疫治疗领域获得了突破性进展。随着对各类疾病认识的不断提高，人们发现免疫细胞在各类疾病发生和治疗中扮演着越来越重要的角色。In recent years, the development of immune technology has been extremely rapid, especially in the field of cancer immunotherapy, breakthroughs have been made. With the continuous improvement of understanding of various diseases, it has been found that immune cells play an increasingly important role in the occurrence and treatment of various diseases.
免疫细胞包括B细胞和T细胞等。T细胞在自身免疫反应和适应性免疫反应过程中都发挥了巨大的作用。抗原特异性T细胞是适应性免疫反应的主要组成部分之一。抗原特异性T细胞在某些疾病比如癌症和自身免疫性疾病的发生和发展过程中扮演着重要的角色。Immune cells include B cells and T cells. T cells play a huge role in both autoimmune and adaptive immune responses. Antigen-specific T cells are one of the main components of the adaptive immune response. Antigen-specific T cells play an important role in the occurrence and development of certain diseases such as cancer and autoimmune diseases.
因而如何能够有效地检测抗原特异性T细胞尤其是外周血中的抗原特异性T细胞就显得尤为重要，但是由于某些疾病如1型糖尿病的患者外周血中某些抗原特异性T细胞数量的稀少，目前尚无非常有效的技术方法用于检测一些疾病相关的抗原特异性T细胞的含量。Therefore, how to effectively detect antigen-specific T cells, especially antigen-specific T cells in peripheral blood, is particularly important. Rare, there is currently no very effective technical method for detecting the content of some disease-related antigen-specific T cells.
发明内容SUMMARY OF THE INVENTION
本发明提供一种能有效用于抗原特异性T细胞含量检测的检测纳米颗粒、其相应的制备方法、包括其的试剂盒以及利用其进行抗原特异性T细胞含量检测的检测方法。The present invention provides a detection nanoparticle which can be effectively used for antigen-specific T cell content detection, a corresponding preparation method thereof, a kit including the same, and a detection method for antigen-specific T cell content detection using the same.
为了实现上述目的，本发明采用了如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
本发明的一个目的是提供一种用于抗原特异性T细胞含量检测的检测纳米颗粒，其特征在于：在基于待测样品中的被激活的抗原特异性T细胞所分泌的细胞分泌物、被激活的抗原特异性T细胞的增殖状态或被激活的抗原特异性T细胞的细胞表面标志物中的任意一种或多种而进行该抗原特异性T细胞的含量的检测中用于激活抗原特异性T细胞，检测纳米颗粒包载有与疾病相关的抗原或包载有与疾病相关的抗原合成需要的遗传物质，其中，检测纳米颗粒的粒径大小为纳米级，检测纳米颗粒在处理待测样品时能被待测样品中的抗原提呈细胞吞噬以释放抗原或基于遗传物质生成抗原以让抗原提呈细胞将释放或生成的抗原的抗原表位提呈到该抗原提呈细胞的表面以让相应的抗原特异性T细胞识别并激活抗原特异性T细胞，抗原特异性T细胞为与疾病相关的抗原特异性T细胞。An object of the present invention is to provide a detection nanoparticle for antigen-specific T cell content detection, which is characterized in that: based on the cell secretion secreted by the activated antigen-specific T cells in the sample to be tested, the It is used to activate the antigen-specific T cells in the detection of the content of the antigen-specific T cells by the proliferation state of the activated antigen-specific T cells or any one or more of the cell surface markers of the activated antigen-specific T cells. Sexual T cells, the detection nanoparticles contain disease-related antigens or genetic material required for the synthesis of disease-related antigens, wherein the detection nanoparticles are nanoscale in size, and the detection nanoparticles are processed to be tested. The sample can be phagocytosed by the antigen-presenting cells in the sample to be tested to release antigens or generate antigens based on genetic material so that the antigen-presenting cells can present the released or generated antigen epitopes to the surface of the antigen-presenting cells for Let the corresponding antigen-specific T cells recognize and activate the antigen-specific T cells, and the antigen-specific T cells are disease-related antigen-specific T cells.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，检测纳米颗粒的粒径大小为10-2000nm。The detection nanoparticle provided by the present invention also has the feature that the particle size of the detection nanoparticle is 10-2000 nm.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，检测纳米颗粒的粒径大小为30-1000nm。The detection nanoparticle provided by the present invention also has the feature that the particle size of the detection nanoparticle is 30-1000 nm.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，检测纳米颗粒的粒径大小为100-500nm。The detection nanoparticle provided by the present invention also has the feature that the particle size of the detection nanoparticle is 100-500 nm.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，抗原包括与癌症疾病相关的抗原以及与自身免疫性疾病相关的抗原中的一种或多种。The detection nanoparticle provided by the present invention also has the feature that the antigens include one or more of antigens related to cancer diseases and antigens related to autoimmune diseases.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，自身免疫性疾病可为1型糖尿病。The detection nanoparticle provided by the present invention also has the feature that the autoimmune disease can be
本发明提供的检测纳米颗粒，还具有这样的特征，其中，检测纳米颗粒包载有与疾病相关的抗原，抗原为细胞裂解物、蛋白质、质粒、DNA、RNA、多肽、组织裂解物、外泌体裂解物、细胞分泌颗粒裂解物、糖类以及脂类中的任意一种或多种。The detection nanoparticle provided by the present invention also has the following characteristics, wherein the detection nanoparticle contains antigens related to diseases, and the antigens are cell lysates, proteins, plasmids, DNA, RNA, polypeptides, tissue lysates, exocytosis Any one or more of body lysate, cell secretory granule lysate, carbohydrates and lipids.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，检测纳米颗粒含有与疾病相关的抗原合成需要的遗传物质，遗传物质来于细胞裂解物、质粒、DNA、RNA、组织裂解物、外泌体裂解物、细胞分泌颗粒裂解物中的任意一种或多种。The detection nanoparticle provided by the present invention also has the feature that the detection nanoparticle contains genetic material required for the synthesis of disease-related antigens, and the genetic material is derived from cell lysate, plasmid, DNA, RNA, tissue lysate, external Any one or more of exosome lysate and cell secretory granule lysate.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，检测纳米颗粒采用包括医用高分子材料、制备脂质体所需的材料、制备纳米粒所需的金属材料以及制备纳米粒所需的非金属材料中的任意一种或任意几种制备而成。The detection nanoparticle provided by the present invention also has such a feature, wherein the detection nanoparticle adopts the medical polymer material, the material required for preparing the liposome, the metal material required for preparing the nanoparticle, and the material required for preparing the nanoparticle. It is prepared from any one or any of several non-metallic materials.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，检测纳米颗粒为纳米粒、脂质体、纳米乳、外泌体、外泌体类似物、病毒、细胞分泌颗粒及细胞分泌颗粒类似物中的任意一种或多种。The detection nanoparticle provided by the present invention also has such a feature, wherein the detection nanoparticle is nanoparticle, liposome, nanoemulsion, exosome, exosome analog, virus, cell secretory particle and cell secretory particle similar any one or more of these.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，抗原分布于检测纳米颗粒的内部以及检测纳米颗粒的表面这两个位置中的任意一个位置或两个位置。The detection nanoparticle provided by the present invention also has the feature that the antigen is distributed in any one or two positions of the inside of the detection nanoparticle and the surface of the detection nanoparticle.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，待测样品为血液、外周血单核细胞、病灶部位组织以及病灶部位细胞中的任意一种或多种。The detection nanoparticle provided by the present invention also has the feature that the sample to be tested is any one or more of blood, peripheral blood mononuclear cells, tissue at the lesion site, and cells at the lesion site.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，细胞分泌物包括干扰素-γ、白介素1、白介素2、转化生长因子-β、肿瘤坏死因子-α、白介素6、白介素7、白介素15、白介素21、IP-10、白介素17、穿孔素以及颗粒酶和白介素10中的一种或多种。The detection nanoparticle provided by the present invention also has the feature that the cell secretions include interferon-γ, interleukin-1, interleukin-2, transforming growth factor-β, tumor necrosis factor-α, interleukin-6, interleukin-7, interleukin 15. One or more of interleukin 21, IP-10, interleukin 17, perforin and granzyme and interleukin 10.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，检测纳米颗粒在处理待测样品时，加入白介素内毒素、内毒素、细胞因子内毒素、化学因子内毒素、趋化因子、二甲双胍、黄连素、青蒿素以及青蒿素类似物中的任意一种或多种。The detection nanoparticle provided by the present invention also has the feature that, when the detection nanoparticle processes the sample to be tested, interleukin endotoxin, endotoxin, cytokine endotoxin, chemical factor endotoxin, chemokine, metformin, Any one or more of berberine, artemisinin and artemisinin analogs.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，采用免疫学测定法、生物活性测定法以及分子生物学检测方法中的任意一种或多种细胞分泌物检测方法检测细胞分泌物。The detection nanoparticle provided by the present invention also has the feature that any one or more cell secretion detection methods among immunological assays, biological activity assays and molecular biology detection methods are used to detect cell secretions.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，细胞分泌物检测方法为酶联免疫斑点法、酶联免疫吸附法、胞内染色的流式细胞仪检测、Cytometric Bead Array、多聚体技术、多细胞因子检测法、高效液相法以及液质联用中的任意一种或多种。The detection nanoparticle provided by the present invention also has the following characteristics, wherein, the method for detecting cell secretions is enzyme-linked immunospot method, enzyme-linked immunosorbent assay, flow cytometry detection of intracellular staining, Cytometric Bead Array, polyplex Any one or more of in vivo technology, multi-cytokine detection method, high performance liquid phase method and liquid chromatography-mass spectrometry.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，基于的细胞分泌物的种类为一种或多种。The detection nanoparticle provided by the present invention also has the feature that the types of cell secretions based on it are one or more.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，抗原特异性T细胞激活后的增殖状态的检测采用基于外源标记的减少检测方法、基于分子标记的表达检测方法、基于代谢标记的增加检测方法、基于细胞群体生物量的变化检测方法以及基于细胞数量的变化检测方法中的任意一项或多项进行检测。The detection nanoparticle provided by the present invention also has the feature that the detection of the proliferation state of antigen-specific T cells after activation adopts the reduction detection method based on exogenous markers, the expression detection method based on molecular markers, and the detection method based on metabolic markers. Add any one or more of a detection method, a cell population biomass-based change detection method, and a cell number-based change detection method for detection.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，基于外源标记的减少检测方法为蛋白质结合染料CFDA-SE、CellTrace以及亲脂性染料PKH67和PKH26标记后采用流式细胞仪检测。The detection nanoparticle provided by the present invention also has the feature that the reduction detection method based on exogenous labeling is detection by flow cytometry after labeling with protein binding dyes CFDA-SE, CellTrace and lipophilic dyes PKH67 and PKH26.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，基于分子标记的表达检测方法中的基于的分子标记为Ki-67和PCNA。The detection nanoparticle provided by the present invention also has the feature that the molecular markers based on the molecular marker-based expression detection method are Ki-67 and PCNA.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，基于代谢标记的增加检测方法中的基于的代谢标记为氧化还原代谢标记和基于DNA代谢标记。The detection nanoparticle provided by the present invention also has such a feature, wherein the metabolic labeling based on the metabolic labeling increase detection method is a redox metabolic labeling and a DNA-based metabolic labeling.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，基于细胞群体生物量的变化检测方法中，基于的细胞群体生物量包括DNA含量和蛋白质含量中的一种或多种。The detection nanoparticle provided by the present invention also has the feature that, in the method for detecting changes based on the biomass of the cell population, the biomass based on the cell population includes one or more of DNA content and protein content.
本发明提供的检测纳米颗粒，还具有这样的特征，其中，抗原特异性T细胞激活的细胞表面标志物为CD137、CD69、CD38、CD44、CD62L或CD25中的一种或多种。The detection nanoparticle provided by the present invention also has the feature that the cell surface marker of antigen-specific T cell activation is one or more of CD137, CD69, CD38, CD44, CD62L or CD25.
本发明的另一个目的是提供一种用于抗原特异性T细胞含量检测的检测纳米颗粒的制备方法，其特征在于，检测纳米颗粒为纳米粒，制备过程包括以下步骤：步骤1，将第一预定体积的含有第一预定浓度的抗原或抗原合成需要的遗传物质加入第二预定体积的含有第二预定浓度医用高分子材料的有机相中；步骤2，将步骤1得到的抗混合物进行大于3秒的超声处理；步骤3，将步骤2处理后得到的混合物加入第三预定体积的第三预定浓度的乳化剂水溶液中并进行大于5秒的超声处理；步骤4，将步骤3处理后得到的混合物加入第四预定体积的第四预定浓度的乳化剂水溶液中，并进行搅拌直至满足预定搅拌条件；步骤5，将步骤4处理满足预定搅拌条件的混悬液在以大于8000RPM的转速进行大于7分钟的离心后，去除上清液，并将剩下的沉淀物重新混悬于水中，如此反复多次，最后得到去除上清液的最终沉淀物；步骤6，将步骤5最后得到的最终沉淀物混悬于第五预定体积的第五预定浓度的冻干保护剂中得到混悬液；步骤7，将步骤6得到的混悬液进行冷冻干燥处理后得到冻干粉末，将含有检测纳米颗粒的冻干粉末用于检测抗原特异性T细胞的含量。Another object of the present invention is to provide a method for preparing detection nanoparticles for antigen-specific T cell content detection, characterized in that the detection nanoparticles are nanoparticles, and the preparation process includes the following steps:
本发明提供的制备方法，还具有这样的特征：第一预定体积与第二预定体积比例范围为1:3-1:50，第二预定体积与第三预定体积比例范围为1:1.5-1:100，第三预定体积与第四预定体积之比范围为1:3-1:50。The preparation method provided by the present invention also has the following characteristics: the ratio of the first predetermined volume to the second predetermined volume is in the range of 1:3-1:50, and the ratio of the second predetermined volume to the third predetermined volume is in the range of 1:1.5-1 : 100, and the ratio of the third predetermined volume to the fourth predetermined volume ranges from 1:3 to 1:50.
本发明提供的制备方法，还具有这样的特征：其中，有机相中的有机溶剂为二氯甲烷。The preparation method provided by the present invention also has the following characteristics: wherein, the organic solvent in the organic phase is dichloromethane.
本发明提供的制备方法，还具有这样的特征：其中，医用高分子材料为聚乳酸-羟基乙酸共聚物。The preparation method provided by the present invention also has the following characteristics: wherein, the medical polymer material is a polylactic acid-glycolic acid copolymer.
本发明提供的制备方法，还具有这样的特征：其中，乳化剂水溶液为聚乙烯醇水溶液。The preparation method provided by the present invention also has the following characteristics: wherein, the aqueous emulsifier solution is an aqueous solution of polyvinyl alcohol.
本发明提供的制备方法，还具有这样的特征，其中，第一预定浓度为大于0.01ng/mL，第二预定浓度范围为0.5mg/mL-500mg/mL，第三预定浓度范围为0.5mg/mL-500mg/mL，第四预定浓度范围为0.5mg/mL-500mg/mL。The preparation method provided by the present invention also has the feature that the first predetermined concentration is greater than 0.01ng/mL, the second predetermined concentration range is 0.5mg/mL-500mg/mL, and the third predetermined concentration range is 0.5mg/mL mL-500mg/mL, and the fourth predetermined concentration range is 0.5mg/mL-500mg/mL.
本发明提供的制备方法，还具有这样的特征：其中，预定搅拌条件为直至有机溶剂挥发完全。The preparation method provided by the present invention also has the following characteristics: wherein, the predetermined stirring condition is until the organic solvent is completely volatilized.
本发明提供的制备方法，还具有这样的特征：其中，冻干保护剂为糖类，如海藻糖，冻干保护剂的含量百分比为大于1％。The preparation method provided by the present invention also has the following characteristics: wherein, the freeze-drying protective agent is saccharide, such as trehalose, and the content percentage of the freeze-drying protective agent is greater than 1%.
本发明提供的制备方法，还具有这样的特征：其中，所冷冻干燥处理具体为：将步骤5得到的混悬液在-80℃的温度下冷冻8小时以上，再使用冷冻干燥机冷冻干燥12小时以上。The preparation method provided by the present invention also has the following characteristics: wherein, the freeze-drying treatment specifically includes: freezing the suspension obtained in
本发明的再一个目的是提供一种抗原特异性T细胞检测用试剂盒，其特征在于，包括：检测纳米颗粒，其中，检测纳米颗粒为上述的检测纳米颗粒。Another object of the present invention is to provide an antigen-specific T cell detection kit, which is characterized by comprising: detection nanoparticles, wherein the detection nanoparticles are the above detection nanoparticles.
本发明的又一个目的是提供一种抗原特异性T细胞含量的检测方法，其特征在于，包括以下步骤：采用含有与疾病相关的抗原或含有与疾病相关的抗原合成需要的遗传物质的检测纳米颗粒处理含有抗原提呈细胞和T细胞的待测样品以将释放或者利用遗传物质合成的抗原的抗原表位提呈到抗原提呈细胞表面让相应的抗原特异性T细胞识别并激活抗原特异性T细胞；基于激活的抗原特异性T细胞所分泌的细胞分泌物、被激活的抗原特异性T细胞的增殖状态或被激活的抗原特异性T细胞的细胞表面标志物检测待测样品的抗原特异性T细胞含量，其中，检测纳米颗粒为上述的检测纳米颗粒。Another object of the present invention is to provide a method for detecting the content of antigen-specific T cells, which is characterized by comprising the following steps: using a detection nanometer containing a disease-related antigen or a genetic material required for the synthesis of a disease-related antigen. Particles process the test sample containing antigen-presenting cells and T cells to present antigen epitopes of antigens released or synthesized using genetic material to the surface of antigen-presenting cells for the corresponding antigen-specific T cells to recognize and activate antigen-specific T cells; detect the antigen specificity of the test sample based on the cell secretions secreted by the activated antigen-specific T cells, the proliferation state of the activated antigen-specific T cells, or the cell surface markers of the activated antigen-specific T cells The content of sexual T cells, wherein the detection nanoparticles are the above detection nanoparticles.
发明作用与效果Invention action and effect
根据本发明所提供的用于抗原特异性T细胞含量检测的检测纳米颗粒、抗原特异性T细胞检测用试剂盒以及相应的抗原特异性T细胞含量的检测方法中，通过含有与疾病相关的抗原或含有合成该抗原所需的遗传物质的检测纳米颗粒，并且该检测纳米颗粒的尺寸大小为纳米级，就可以使得能被待测样品中的抗原提呈细胞吞噬，并释放抗原或抗原合成所需的遗传物质以合成相应的抗原，进一步对被释放的抗原或新合成的抗原进行提呈，就可以激活相应的与疾病相关的抗原特异性T细胞，通过对被激活的抗原特异性T细胞分泌的细胞分泌物、激活的抗原特异性T细胞的增殖状态或被激活的抗原特异性T细胞的细胞表面标志物中的任意一种或多种的检测，并基于细胞分泌物、状态或细胞表面标志物与相应抗原特异性T细胞之间的关系，就能测定相应抗原特异性T细胞的含量，所以我们可以用于监测疾病的进展、预测和监测疾病治疗方案的治疗效果或者在治疗过程中评估治疗方案的治疗效果，这样就为我们实施精准治疗避免医疗资源的浪费提供了条件，并为做相关研究选择适宜的实验对象以取得良好的实验结果和避免不必要的浪费提供了条件，并且，该检测方法成本较低，而且操作相对简单，使用方便。According to the detection nanoparticle for antigen-specific T cell content detection, the antigen-specific T cell detection kit and the corresponding antigen-specific T cell content detection method provided by the present invention, the antigen-specific T cell content is detected by containing disease-related antigens. Or a detection nanoparticle containing the genetic material required for synthesizing the antigen, and the size of the detection nanoparticle is nanoscale, so that it can be phagocytosed by the antigen-presenting cells in the sample to be tested, and release the antigen or antigen synthesis components. The required genetic material is used to synthesize the corresponding antigen, and the released antigen or newly synthesized antigen can be further presented, and the corresponding disease-related antigen-specific T cells can be activated. Detection of any one or more of secreted cellular secretions, proliferative status of activated antigen-specific T cells, or cell surface markers of activated antigen-specific T cells, based on cellular secretions, status or cells The relationship between the surface markers and the corresponding antigen-specific T cells can determine the content of the corresponding antigen-specific T cells, so we can use it to monitor the progress of the disease, predict and monitor the therapeutic effect of the disease treatment plan or in the treatment process. It provides conditions for us to implement precise treatment to avoid waste of medical resources, and to select appropriate experimental subjects for relevant research to obtain good experimental results and avoid unnecessary waste. In addition, the detection method has low cost, relatively simple operation and convenient use.
附图说明Description of drawings
图1为检测纳米颗粒包载抗原时检测抗原特异性T细胞含量的检测方法的检测机理示意图。图1中：1，抗原提呈细胞；2，抗原特异性T细胞；3，包载有抗原的检测纳米颗粒；4，抗原分子；5：抗原表位；6，主要组织相容性复合体；7，T细胞受体；8，细胞分泌物，如干扰素-γ。FIG. 1 is a schematic diagram of the detection mechanism of the detection method for detecting the content of antigen-specific T cells when the nanoparticle-encapsulated antigen is detected. In Figure 1: 1, antigen presenting cells; 2, antigen-specific T cells; 3, detection nanoparticles loaded with antigen; 4, antigen molecule; 5: antigenic epitope; 6, major histocompatibility complex ; 7, T cell receptors; 8, cellular secretions, such as interferon-gamma.
图2为检测纳米颗粒包载能生成抗原的遗传物质时检测抗原特异性T细胞含量的检测方法的检测机理示意图。图2中：1，抗原提呈细胞；2，抗原特异性T细胞；3，包载有能生成抗原的遗传物质的检测纳米颗粒；4，能生成抗原的遗传物质；5：遗传物质生成的抗原；6：抗原表位；7，主要组织相容性复合体；8，T细胞受体；9，细胞分泌物，如干扰素-γFIG. 2 is a schematic diagram of the detection mechanism of the detection method for detecting the content of antigen-specific T cells when the genetic material capable of generating antigen is detected when the nanoparticles are encapsulated. In Figure 2: 1, antigen-presenting cells; 2, antigen-specific T cells; 3, detection nanoparticles loaded with genetic material capable of producing antigen; 4, genetic material capable of producing antigen; 5: generated by genetic material antigen; 6: epitope; 7, major histocompatibility complex; 8, T cell receptor; 9, cellular secretions, such as interferon-γ
具体实施方式Detailed ways
基于被激活的抗原特异性T细胞所分泌的细胞分泌物的检测原理：Based on the detection principle of cell secretions secreted by activated antigen-specific T cells:
图1为抗原特异性T细胞含量的检测方法的检测机理示意图。FIG. 1 is a schematic diagram of the detection mechanism of the method for detecting the content of antigen-specific T cells.
图1中，1，抗原提呈细胞；2，抗原特异性T细胞；3，包载有抗原的检测纳米颗粒；4，抗原分子；5：抗原表位；6，主要组织相容性复合体；7，T细胞受体；8，细胞分泌物，如干扰素-γ。In Figure 1, 1, antigen-presenting cells; 2, antigen-specific T cells; 3, detection nanoparticles loaded with antigen; 4, antigen molecule; 5: antigenic epitope; 6, major histocompatibility complex ; 7, T cell receptors; 8, cellular secretions, such as interferon-gamma.
如图1，本发明的原理是用包载有抗原或包载有合成抗原所需的遗传物质的粒径大小为纳米粒的检测纳米颗粒，去处理含有抗原提呈细胞(antigen-presenting cells,APC)和T细胞的待测样品。利用抗原提呈细胞具有吞噬纳米级别的颗粒的特性，抗原或抗原合成所需遗传物质能够通过纳米大小的检测纳米颗粒进入抗原提呈细胞，并被抗原提呈细胞处理后与主要组织相容性复合体(major histocompatibility complex，MHC)结合，而会在抗原提呈细胞内释放出包载在检测纳米颗粒中的抗原，或释放遗传物质以在抗原提呈细胞中生成抗原。被释放或生成的抗原被抗原提呈细胞处理和提呈到抗原提呈细胞表面。被提呈到抗原提呈细胞表面的抗原表位即可被相应的抗原特异性T细胞识别并激活抗原特异性T细胞。抗原特异性T细胞被激活后会分泌一些细胞分泌物，通过一定的检测手段检测相关细胞分泌物的含量即可知道抗原特异性T细胞的含量。As shown in Fig. 1, the principle of the present invention is to use detection nanoparticles with a particle size of nanoparticles containing antigens or genetic materials required for synthesizing antigens to process antigen-presenting cells (antigen-presenting cells, APC) and T cells to be tested. Taking advantage of the ability of antigen-presenting cells to phagocytose nano-sized particles, the genetic material required for antigen or antigen synthesis can enter antigen-presenting cells through nano-sized detection nanoparticles, and be processed by antigen-presenting cells for major histocompatibility When the major histocompatibility complex (MHC) binds, the antigens encapsulated in the detection nanoparticles are released in the antigen-presenting cells, or the genetic material is released to generate the antigens in the antigen-presenting cells. The released or generated antigen is processed and presented to the surface of the antigen-presenting cell by the antigen-presenting cell. The antigenic epitopes presented on the surface of the antigen-presenting cells can be recognized by the corresponding antigen-specific T cells and activate the antigen-specific T cells. Antigen-specific T cells will secrete some cell secretions after being activated, and the content of antigen-specific T cells can be known by detecting the content of relevant cell secretions by certain detection methods.
以下结合附图来说明本发明的具体实施方式，以下实施例中的检测均基于被激活的抗原特异性T细胞所分泌的细胞分泌物。对于实施例中所用到的具体方法或材料，本领域技术人员可以在本发明技术思路的基础上，根据已有的技术进行常规的替换选择，而不仅限于本发明实施例的具体记载。The specific embodiments of the present invention are described below with reference to the accompanying drawings. The detections in the following examples are all based on the cell secretions secreted by activated antigen-specific T cells. For the specific methods or materials used in the embodiments, those skilled in the art can make conventional replacement selections based on the technical ideas of the present invention and existing technologies, and are not limited to the specific descriptions of the embodiments of the present invention.
实施例中所使用的方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。The methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources.
实施例1癌症患者中癌细胞抗原特异性T细胞检测Example 1 Detection of cancer cell antigen-specific T cells in cancer patients
本实施例以对癌细胞中的抗原特异性T细胞检测为例进行说明。In this example, the detection of antigen-specific T cells in cancer cells is taken as an example for description.
癌症的免疫治疗最近几年取得了巨大的进步，如抗PD-1抗体、抗PD-L1抗体和CAR-T细胞疗法的抗癌效果良好，并已有多个相关抗癌药物获得美国食品药品监督管理局(FDA)的批准投入市场使用。癌症免疫疗法的治疗机理是通过自身免疫系统的癌症相关抗原特异性T细胞去识别和杀灭癌细胞。因为癌症抗原特异性T细胞具有特异性和记忆性的特性，所以一旦癌症抗原特异性T细胞能够识别和攻击癌细胞，副作用很小而且作用持久。但是当前癌症免疫治疗的总体响应率较低，只有20％-40％左右。这其中很重要的一个原因是由于部分患者体内没有足够的癌细胞抗原特异性T细胞。一般体内癌症抗原特异性T细胞含量多的癌症患者癌症免疫治疗的效果都较好，而体内癌症抗原特异性T细胞含量少的患者癌症免疫治疗的效果都较差。所以在癌症患者开始癌症免疫治疗之前，测定癌症患者体内癌细胞抗原特异性T细胞的含量。如果患者体内癌细胞抗原特异性T细胞的数量较多，则说明免疫治疗的治疗效果会较好；如果患者体内癌细胞抗原特异性T细胞的数量较少，则说明进行免疫疗法的预后可能较差。Cancer immunotherapy has made great progress in recent years, such as anti-PD-1 antibody, anti-PD-L1 antibody and CAR-T cell therapy have good anti-cancer effects, and many related anti-cancer drugs have been approved by the US Food and Drug Administration FDA approval for market use. The therapeutic mechanism of cancer immunotherapy is to recognize and kill cancer cells through cancer-associated antigen-specific T cells of the autoimmune system. Because of the specificity and memory properties of cancer antigen-specific T cells, once cancer antigen-specific T cells are able to recognize and attack cancer cells, side effects are minimal and long-lasting. However, the overall response rate of current cancer immunotherapy is low, only about 20%-40%. An important reason for this is that some patients do not have enough cancer cell antigen-specific T cells. Generally, cancer patients with a high content of cancer antigen-specific T cells in the body have better cancer immunotherapy effects, while patients with a low content of cancer antigen-specific T cells in the body have poor cancer immunotherapy effects. Therefore, before cancer patients start cancer immunotherapy, the content of cancer cell antigen-specific T cells in cancer patients is measured. If the number of cancer cell antigen-specific T cells in the patient is high, the therapeutic effect of immunotherapy will be better; if the number of cancer cell antigen-specific T cells in the patient is low, the prognosis of immunotherapy may be better. Difference.
为此，本实施例对癌细胞中抗原特异性T细胞含量进行了检测。To this end, the present example detects the content of antigen-specific T cells in cancer cells.
本实施例中，先对检测纳米颗粒进行制备，再采用制备得到的检测纳米颗粒对癌细胞中的抗原特异性T细胞含量进行检测。In this embodiment, the detection nanoparticles are prepared first, and then the content of antigen-specific T cells in cancer cells is detected by using the prepared detection nanoparticles.
1.检测纳米颗粒的制备1. Preparation of Assay Nanoparticles
本实施例中采用的检测纳米颗粒为纳米粒，其制备方法具体包括以下步骤：The detection nanoparticles used in this embodiment are nanoparticles, and the preparation method thereof specifically includes the following steps:
步骤1，将第一预定体积的含有第一预定浓度抗原的水相溶液加入第二预定体积的含有第二预定浓度医用高分子材料的有机相中；
本实施例中，抗原存在于癌细胞裂解物中。In this example, the antigen was present in the cancer cell lysate.
本实施例中，将含有抗原的癌细胞裂解物(生物样本)，溶解到水或任何含有水的对后续检测不产生影响的水溶液中，制备得到水相的抗原溶液，并且得到的该抗原溶液的浓度，也即第一预定浓度要求抗原浓度含量大于0.1mg/mL，之所以选择这样的浓度，是发明人经大量试验发现，一般所用抗原浓度越大，制备的纳米粒中包载的抗原越多，当制备得到的抗原浓度大于0.1mg/mL，也即第一预定浓度大于0.1mg/ml时，才有足够抗原进行后续的检测。另外，本实施例中第一预定体积为200μL。In this example, the antigen-containing cancer cell lysate (biological sample) is dissolved in water or any aqueous solution containing water that does not affect subsequent detection to prepare an aqueous antigen solution, and the obtained antigen solution The concentration of the antigen, that is, the first predetermined concentration requires the antigen concentration to be greater than 0.1 mg/mL. The reason for choosing such a concentration is that the inventors have found through a large number of experiments that the larger the concentration of the antigen used, the more likely the antigen contained in the prepared nanoparticles will be. More, when the prepared antigen concentration is greater than 0.1 mg/mL, that is, when the first predetermined concentration is greater than 0.1 mg/ml, there will be enough antigen for subsequent detection. In addition, in this embodiment, the first predetermined volume is 200 μL.
将医用高分子材料溶解于有机溶剂中，得到第二预定体积的含有第二预定浓度医用高分子材料的有机相，本实施例中，医用高分子材料为聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid)，PLGA)，有机溶剂选用二氯甲烷，得到的有机相的体积，也即第二预定体积为2mL。另外，本实施例中，医用高分子材料的第二预定浓度的范围为0.5mg/mL-500mg/mL，优选为100mg/mL。The medical polymer material is dissolved in an organic solvent to obtain a second predetermined volume of an organic phase containing a second predetermined concentration of the medical polymer material. In this embodiment, the medical polymer material is a polylactic acid-glycolic acid copolymer (poly(( lactic-co-glycolic acid), PLGA), dichloromethane was selected as the organic solvent, and the volume of the obtained organic phase, that is, the second predetermined volume, was 2 mL. In addition, in this embodiment, the range of the second predetermined concentration of the medical polymer material is 0.5 mg/mL-500 mg/mL, preferably 100 mg/mL.
本实施例中之所以选择PLGA，是由于本发明人发现，该材料属于pH敏感材料，在生理中性pH条件下不会释放抗原，但是一旦被吞噬进入抗原细胞后，首先进入endosome,endosome是酸性的，在酸性条件下PLGA会被快速降解因而抗原可以快速被释放。The reason why PLGA was selected in this example is because the inventors found that this material is a pH-sensitive material and will not release antigens under physiological neutral pH conditions, but once it is phagocytosed into antigen cells, it first enters the endosome, and the endosome is a Acidic, PLGA is rapidly degraded under acidic conditions and the antigen can be released rapidly.
实际中，有机相的第二预定体积根据其和水相的第一预定体积的比例进行设定，本实施例中，水相的第一预定体积和有机相的第二预定体积之比的范围为1:2-1:50，优先地为1:10。在具体实施过程中可根据需要对第一预定体积、第二预定体积和第一预定体积与第二预定体积之比进行调整以调整制备的纳米粒的尺寸大小。In practice, the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase. In this embodiment, the range of the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is 1:2-1:50, preferably 1:10. In the specific implementation process, the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as required to adjust the size of the prepared nanoparticles.
步骤2，将步骤1得到的抗原高分子混合液进行大于2秒的超声处理。In
该步骤是为了进行纳米化，超声时间长短能控制制备的纳米粒子大小，过长或过短都会带来粒径大小的变化，为此，需要选择合适的超声时间，本实施例中超声时间大于2秒。The purpose of this step is to carry out nanometerization. The length of the ultrasonic time can control the size of the prepared nanoparticles. Too long or too short will bring about changes in the particle size. Therefore, it is necessary to select an appropriate ultrasonic time. In this embodiment, the ultrasonic time is longer than 2 seconds.
步骤3，将步骤2处理后得到的混合物加入第三预定体积的含有第三预定浓度乳化剂的水溶液中并进行大于5秒的超声处理。In
该步骤将步骤2得到的混合物加入到乳化剂水溶液中继续超声纳米化。In this step, the mixture obtained in
本实施例中，乳化剂水溶液为聚乙烯醇(PVA)水溶液，第三预定体积为5mL，第三预定浓度为20mg/mL。第三预定体积根据其与第二预定体积的比例进行调整，本实施例中，第二预定体积与第三预定体积之的范围为1:1.5-1:100进行设定，优先地可以为2:5。在具体实施过程中为了控制纳米粒子的尺寸，可以对第二预定体积和第三预定体积之比进行调整。In this embodiment, the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution, the third predetermined volume is 5 mL, and the third predetermined concentration is 20 mg/mL. The third predetermined volume is adjusted according to its ratio to the second predetermined volume. In this embodiment, the range between the second predetermined volume and the third predetermined volume is set at 1:1.5-1:100, preferably 2 :5. In the specific implementation process, in order to control the size of the nanoparticles, the ratio of the second predetermined volume to the third predetermined volume can be adjusted.
同样地，本步骤的超声时间、乳化剂水溶液的体积以及浓度的取值根据，均为了得到尺寸大小合适的纳米粒。Similarly, the ultrasonic time, the volume and concentration of the emulsifier aqueous solution in this step are all based on obtaining nanoparticles of suitable size.
步骤4，将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中，并进行搅拌直至满足预定搅拌条件。In
本步骤中，乳化剂水溶液依然为PVA，第四预定体积为大于50mL，第四预定浓度为5mg/mL，第四预定浓度的选择，以得到尺寸大小合适的纳米粒为依据。第四预定体积的选择依据第三预定体积与第四预定体积之比决定。在本实施例中，第三预定体积与第三预定体积之比为范围为1:3-1:50，优先地为1:10。在具体实施过程中为了控制纳米粒子的尺寸可以对第三预定体积和第四预定体积之比进行调整。In this step, the emulsifier aqueous solution is still PVA, the fourth predetermined volume is greater than 50 mL, and the fourth predetermined concentration is 5 mg/mL. The selection of the fourth predetermined concentration is based on obtaining nanoparticles of suitable size. The selection of the fourth predetermined volume is determined according to the ratio of the third predetermined volume to the fourth predetermined volume. In this embodiment, the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:3-1:50, preferably 1:10. In the specific implementation process, the ratio of the third predetermined volume to the fourth predetermined volume may be adjusted in order to control the size of the nanoparticles.
本实施例中，本步骤的预定搅拌条件为直至有机溶剂挥发完成，也即步骤1中的二氯甲烷挥发完成。In this embodiment, the predetermined stirring condition in this step is until the volatilization of the organic solvent is completed, that is, the volatilization of the dichloromethane in
步骤5，将步骤4处理满足预定搅拌条件的混合液在以大于8000RPM的转速进行大于7分钟的离心后，去除上清液，并将剩下的沉淀物重新混悬于水中，如此反复多次，最后得到去除上清液的最终沉淀物。Step 5: After centrifugation for more than 7 minutes at a speed of more than 8000 RPM, the mixture that meets the predetermined stirring conditions in
本步骤的目的去除残留的一些聚合物和未包裹的抗原，本实施例中，本步骤具体为：The purpose of this step is to remove some residual polymers and unwrapped antigens. In this embodiment, this step is specifically:
步骤5.1，将步骤4处理满足预定搅拌条件的混合液在超高速离心机上以大于8000RPM离心大于7分钟，弃去上清液；In step 5.1, the mixed solution that meets the predetermined stirring conditions in
步骤5.2，将步骤5.1中留下的沉淀物重新混悬于水中；Step 5.2, resuspend the sediment left in step 5.1 in water;
步骤5.3，重复步骤5.1；Step 5.3, repeat step 5.1;
步骤5.4，重复步骤5.2；Step 5.4, repeat step 5.2;
步骤5.5，重复步骤5.1，得到最终沉淀物。Step 5.5, repeat step 5.1 to obtain the final pellet.
实际中，根据实际情况，可以反复进行步骤5.1-步骤5.5的循环，以能得到需要的最终沉淀物。In practice, according to the actual situation, the cycle of step 5.1-step 5.5 can be repeated to obtain the desired final precipitate.
步骤6，将步骤5最后得到的最终沉淀物混悬于第三预定体积的第三预定浓度的冻干保护剂中得到混悬液。In
本实施例中的冻干保护剂选用海藻糖(trehalose)。The freeze-drying protective agent in this example was selected from trehalose.
本实施例中，该步骤的冻干保护剂的第五预定体积为20mL，第五预定浓度为质量百分比4％，之所以如此设定，是为了在后续进行冷冻干燥中不影响冻干效果。In this embodiment, the fifth predetermined volume of the freeze-drying protective agent in this step is 20 mL, and the fifth predetermined concentration is 4% by mass. The reason for this setting is to not affect the freeze-drying effect in subsequent freeze-drying.
步骤7，将步骤6得到的混悬液进行冷冻干燥处理后，将冻干物质用于检测抗原特异性T细胞的含量的检测纳米颗粒。In
本实施例中，该步骤具体为：In this embodiment, this step is specifically:
步骤7.1：将步骤6得到的混悬液在-80℃冷冻8小时以上得到冷冻产物；Step 7.1: Freeze the suspension obtained in
步骤步骤7.2：使用冷冻干燥机冷冻干燥步骤7.1所得的冷冻产物得到冻干的检测纳米颗粒；Step 7.2: use a freeze dryer to freeze dry the frozen product obtained in step 7.1 to obtain freeze-dried detection nanoparticles;
步骤7.3：分析步骤7.2所得冻干颗粒的粒径、表面电荷和抗原包载量。根据这些分析结果，选择合适的检测纳米颗粒用于检测抗原特异性T细胞的含量。Step 7.3: Analyze the particle size, surface charge and antigen loading capacity of the lyophilized particles obtained in step 7.2. Based on these analysis results, suitable detection nanoparticles were selected for detecting the content of antigen-specific T cells.
检测纳米颗粒的粒径大小为纳米级，这样能在用于抗原特异性T细胞的检测时被抗原提呈细胞吞噬，而为了提高吞噬效率，粒径大小要在适宜的范围内，本实施例中，检测纳米颗粒的粒径大小为10-2000nm，更优选地，粒径大小为30-1000nm，最优选地，粒径大小为100-500nm。The particle size of the detection nanoparticles is nanoscale, so that they can be phagocytosed by antigen-presenting cells when used for the detection of antigen-specific T cells, and in order to improve the phagocytosis efficiency, the particle size should be within an appropriate range. Among them, the particle size of the detected nanoparticles is 10-2000 nm, more preferably, the particle size is 30-1000 nm, and most preferably, the particle size is 100-500 nm.
另外，本实施例检测纳米颗粒的制备过程中，直接采用抗原进行包载，最终得到含有抗原的纳米粒，且该抗原为细胞裂解物，实际中，当直接包载抗原时，抗原还可以为为细胞裂解物、蛋白质、质粒、DNA、RNA、多肽、组织裂解物、外泌体裂解物、细胞分泌颗粒裂解物、糖类以及脂类中的任意一种或多种。In addition, in the preparation process of the detection nanoparticles in this embodiment, antigens are directly used for encapsulation, and finally nanoparticles containing antigens are obtained, and the antigens are cell lysates. In practice, when the antigens are directly encapsulated, the antigens can also be It is any one or more of cell lysate, protein, plasmid, DNA, RNA, polypeptide, tissue lysate, exosome lysate, cell secretory granule lysate, carbohydrate and lipid.
另外，在检测纳米颗粒的制备过程中，也可以对与疾病相关的抗原合成需要的遗传物质进行包载，这样最终得到包载了这种遗传物质的检测纳米颗粒，遗传物质来于细胞裂解物、质粒、DNA、RNA、组织裂解物、外泌体裂解物、细胞分泌颗粒裂解物中的任意一种或多种In addition, in the preparation process of detection nanoparticles, the genetic material required for the synthesis of disease-related antigens can also be encapsulated, so that detection nanoparticles encapsulated with this genetic material are finally obtained. The genetic material comes from cell lysates. , plasmid, DNA, RNA, tissue lysate, exosome lysate, any one or more of cell secretory granule lysate
另外，制备得到的检测纳米颗粒，抗原可以分布在检测纳米颗粒的内部或者表面，或者内部和表面这两个位置都有分布。In addition, in the prepared detection nanoparticle, the antigen can be distributed in the interior or the surface of the detection nanoparticle, or both the interior and the surface are distributed.
另外，在实施例中采用的检测纳米颗粒为纳米粒，在实际中也可以采用脂质体、纳米乳、外泌体、外泌体类似物、病毒、细胞分泌颗粒及细胞分泌颗粒类似物中的任意一种或多种。In addition, the detection nanoparticles used in the examples are nanoparticles, and in practice, liposomes, nanoemulsions, exosomes, exosome analogs, viruses, cell secretion particles and cell secretion particle analogs can also be used. any one or more of them.
2.癌细胞中抗原特异性T细胞的含量检测2. Detection of the content of antigen-specific T cells in cancer cells
该检测的过程通过对激活的T细胞分泌的细胞分泌物的含量进行检测，而间接得到T细胞的含量，包括以下步骤：采用上述制备方法得到的检测纳米颗粒处理含有抗原提呈细胞和T细胞的待测样品以将释放或者利用遗传物质合成的抗原的抗原表位提呈到抗原提呈细胞表面让抗原特异性T细胞识别并激活抗原特异性T细胞；基于激活的抗原特异性T细胞分泌的细胞分泌物检测待测样品中的抗原特异性T细胞的含量。The detection process indirectly obtains the content of T cells by detecting the content of cell secretions secreted by activated T cells, including the following steps: using the detection nanoparticles obtained by the above preparation method to treat antigen-presenting cells and T cells The test sample is used to present the antigen epitope of the antigen released or synthesized by using genetic material to the surface of the antigen-presenting cell for the antigen-specific T cell to recognize and activate the antigen-specific T cell; based on the activated antigen-specific T cell secretion The cell secretions were used to detect the content of antigen-specific T cells in the sample to be tested.
在本实施例中采用ELISPOT检测技术检测细胞分泌物的方法作为检测抗原特异性T细胞数量的方法，细胞分泌物可选用干扰素-γ，白介素1(IL-1),白介素2(IL-2)，转化生长因子(TGF)-β，白介素6(IL-6)，白介素7(IL-7)、白介素15(IL-15)、白介素21(IL-21)、IP-10(Interferon gamma-induced protein 10，CXCL10)、白介素17(IL-17)和白介素10(IL-10)中的一种或多种。本实施例中T细胞的分泌物以干扰素-γ为例进行说明。In this example, the method of detecting cell secretions using ELISPOT detection technology is used as the method for detecting the number of antigen-specific T cells, and the cell secretions can be selected from interferon-γ, interleukin-1 (IL-1), interleukin-2 (IL-2) ), transforming growth factor (TGF)-β, interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 21 (IL-21), IP-10 (Interferon gamma- One or more of induced protein 10, CXCL10), interleukin 17 (IL-17) and interleukin 10 (IL-10). In this example, the secretion of T cells is described by taking interferon-γ as an example.
本实施例中的细胞分泌物为干扰素-γ。在具体实践中检测过程具体如下：The cellular secretion in this example is interferon-gamma. In specific practice, the detection process is as follows:
步骤1，采样
抽取患者外周血若干毫升，在抽血过程中加入肝素抗凝血。Several milliliters of peripheral blood were drawn from the patient, and heparin was added for anticoagulation during the blood drawing.
步骤2，待测样品制备——提取分离PBMCs(外周血单核细胞)
本实施能力中的待测样品为PBMCs。The samples to be tested in this implementation capability are PBMCs.
PBMCs提取分离过程采用实验室常规采用的提取分离方法，如Ficoll密度梯度离心法和percoll分层液法。提取分离得到的含有抗原提呈细胞以及T细胞的PBMCs加入AIM V细胞培养基中。The extraction and separation process of PBMCs adopts the extraction and separation methods routinely used in the laboratory, such as Ficoll density gradient centrifugation and percoll layered liquid method. The isolated PBMCs containing antigen-presenting cells and T cells were added to AIM V cell culture medium.
步骤3，将制备得到的检测纳米颗粒混悬于AIM V细胞培养基中，也即复溶于单纯的AIM V培养基。Step 3: Suspend the prepared detection nanoparticles in AIM V cell culture medium, that is, redissolve in pure AIM V medium.
步骤4，将在AIM V培养基中的PBMCs和AIM V培养基中的检测纳米颗粒混合加入预先包被有干扰素-γ抗体a(capture antibody，吸附抗体)并经过AIM V培养基封闭处理1小时以上的96孔PVDF板中。Step 4: Mix the PBMCs in the AIM V medium and the detection nanoparticles in the AIM V medium, add the pre-coated interferon-γ antibody a (capture antibody, adsorption antibody), and block the AIM V medium 1 hours in a 96-well PVDF plate.
在混合加入PBMCs和检测纳米颗粒前，96孔板中未结合的干扰素-γ抗体a已被洗去，在96孔板中加入AIM V培养基于37℃(5％CO2)培养箱中放置作用超过1个小时进行封闭以避免非特异性吸附。Before mixing PBMCs and detecting nanoparticles, the unbound interferon-γ antibody a in the 96-well plate was washed away, and AIM V was added to the 96-well plate and placed in a 37°C (5% CO2 ) incubator. Blocking was performed over 1 hour to avoid nonspecific adsorption.
步骤5，将步骤4得到的PBMCs和步骤3中复溶检测纳米颗粒得到的产物进行混合得到的混合物在37℃(5％CO2)培养箱中培养24到96个小时。In
步骤6：弃去PBMCs和检测纳米颗粒的混合物，洗涤96孔板。Step 6: Discard the mixture of PBMCs and detection nanoparticles and wash the 96-well plate.
步骤7：加入干扰素-γ抗体b(detection antibody，检测抗体),并在37℃(5％CO2)培养箱中培养2小时以上。Step 7: Add interferon-γ antibody b (detection antibody, detection antibody), and incubate at 37° C. (5% CO2 ) in an incubator for more than 2 hours.
步骤8：弃去含有干扰素-γ抗体b的溶液，洗涤96孔板并采用相应的方法显色以在96孔板表面形成斑点。Step 8: Discard the solution containing the interferon-γ antibody b, wash the 96-well plate and develop color using a corresponding method to form spots on the surface of the 96-well plate.
步骤9：采用ELISPOT分析仪读取数据并分析实验结果。Step 9: Use the ELISPOT analyzer to read the data and analyze the experimental results.
在ELISPOT检测方法中，抗原特异性T细胞被激活后分泌的细胞分泌物如干扰素-γ会被96孔板包载的抗体a吸附，在加入抗体b后，会形成双抗体夹心结构。当采用一定的显色方法对双抗体夹心结构处理后，细胞所在的位置会形成斑点。一个斑点的形成就代表着一个被激活的抗原特异性T细胞，所以通过测量96孔板中每个孔中显色形成的斑点的数量，我们可以得知被检测样品中抗原特异性T细胞的数量。这样，根据被检测对象体内癌细胞抗原特异性T细胞含量的多少，就从以下方面评估免疫治疗效果：In the ELISPOT detection method, the cell secretions such as interferon-γ secreted by the activated antigen-specific T cells will be adsorbed by the antibody a contained in the 96-well plate, and after adding the antibody b, a double-antibody sandwich structure will be formed. When the double-antibody sandwich structure is processed by a certain color development method, spots will be formed where the cells are located. The formation of a spot represents an activated antigen-specific T cell, so by measuring the number of spots formed in each well of the 96-well plate, we can know the number of antigen-specific T cells in the tested sample. In this way, according to the content of antigen-specific T cells of cancer cells in the test object, the effect of immunotherapy can be evaluated from the following aspects:
1.可以作为患者是否选择免疫治疗的一种参考指标，以避免患者由于不适宜免疫治疗而引起的不必要的财务负担、宝贵的治疗时间的浪费和不必要的副作用，并且可以有效防止不必要的医疗资源的浪费；1. It can be used as a reference indicator for whether patients choose immunotherapy, so as to avoid unnecessary financial burden, waste of precious treatment time and unnecessary side effects caused by unsuitable immunotherapy for patients, and can effectively prevent unnecessary waste of medical resources;
2.作为肿瘤防治研究中选择研究对象的一种参考指标，以让肿瘤防治研究能有效地选择研究对象进行免疫治疗的研究，从而得到科学合理地研究结果，更好地推动肿瘤防治的科学进步，并且避免了不必要的研究经费、人力以及物力的浪费，有效防治公共资源的浪费。2. As a reference index for selecting research objects in tumor prevention and treatment research, so that tumor prevention and treatment research can effectively select research objects for immunotherapy research, so as to obtain scientific and reasonable research results, and better promote the scientific progress of tumor prevention and treatment. , and avoid unnecessary waste of research funds, manpower and material resources, and effectively prevent the waste of public resources.
实施例2Example 2
以下是对实施例2的说明。本实施例中与实施例1相同的检测原理以及各个制备过程中的涉及的物质以及参数选择依据相同的部分，省略相同的说明。The following is a description of Example 2. In this embodiment, the same detection principle as in
本实施例以对1型糖尿病相关的抗原特异性T细胞检测为例进行说明。In this example, the detection of antigen-specific T cells related to
1型糖尿病是由于β细胞特异性T细胞异常识别和攻击胰腺内的β细胞所造成的。一旦β细胞被完全杀灭后，人体自身将无法合成和分泌能降低血糖的胰岛素，进而血糖就会升高，只能依靠外来途径补充胰岛素来维持血糖的平衡。由此可以看出β细胞特异性T细胞在1型糖尿病的发生和发展过程中扮演了关键角色。
为此，本实施例对1型糖尿病相关的β细胞特异性T细胞含量进行了检测。To this end, in this example, the content of β-cell-specific T cells related to
本实施例中，先对检测纳米颗粒进行制备，再采用制备得到的检测纳米颗粒对癌细胞中的抗原特异性T细胞含量进行检测。In this embodiment, the detection nanoparticles are prepared first, and then the content of antigen-specific T cells in cancer cells is detected by using the prepared detection nanoparticles.
1.检测纳米颗粒的制备1. Preparation of Assay Nanoparticles
本实施例中采用的检测纳米颗粒为纳米粒，其包载1型糖尿病相关抗原GAD65和preproinsulin，其制备方法具体包括以下步骤：The detection nanoparticles used in this embodiment are nanoparticles, which encapsulate the
步骤1，将预定第一体积含有第一预定浓度的抗原蛋白的抗原溶液加入到预定第二体积的预定第二预定浓度医用高分子材料的有机相中。
本实施例中，抗原来自1型糖尿病相关抗原蛋白质GAD65和preproinsulin。第一预定体积为200μL。第一预定浓度为GAD65和preproinsulin的浓度分别大于0.01mg/mL。In this example, the antigens were derived from
本实施例中，将1型糖尿病相关抗原蛋白质GAD65和preproinsulin，溶解到水或任何含有水的对后续检测不产生影响的水溶液中，制备得到抗原溶液。In this example,
将医用高分子材料溶解于预定第二体积的有机溶剂中，得到有机溶液，本实施例中，医用高分子材料为聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid)，PLGA)，有机溶剂选用二氯甲烷，预定有机体积为2mL。The medical polymer material is dissolved in a predetermined second volume of organic solvent to obtain an organic solution. In this embodiment, the medical polymer material is poly(lactic-co-glycolic acid) (PLGA). , the organic solvent is dichloromethane, and the predetermined organic volume is 2mL.
同样地，水相的第一预定体积和有机相的第二预定体积之比为1:10。在具体实施过程中可根据需要对第一预定体积、第二预定体积和第一预定体积与第二预定体积之比进行调整。Likewise, the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is 1:10. In the specific implementation process, the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as required.
步骤2，将步骤1得到的混合液进行大于2秒的超声处理。In
步骤3，将步骤2处理后得到的液体加入第三预定体积的第三预定浓度的乳化剂水溶液中并进行大于5秒的超声处理。In
本实施例中，乳化剂水溶液为聚乙烯醇(PVA)水溶液，第三预定体积为5mL，第三预定浓度为20mg/mL。本实施例中，第二预定体积与第三预定体积之比为2:5。在具体实施过程中为了控制纳米粒子的尺寸可以对第二预定体积和第三预定体积之比进行调整。In this embodiment, the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution, the third predetermined volume is 5 mL, and the third predetermined concentration is 20 mg/mL. In this embodiment, the ratio of the second predetermined volume to the third predetermined volume is 2:5. In the specific implementation process, in order to control the size of the nanoparticles, the ratio of the second predetermined volume to the third predetermined volume can be adjusted.
同样地，本步骤的超声时间、乳化剂水溶液的体积以及浓度的取值根据，均为了得到尺寸大小合适的纳米粒。Similarly, the ultrasonic time, the volume and concentration of the emulsifier aqueous solution in this step are all based on obtaining nanoparticles of suitable size.
步骤4，将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中，并进行搅拌直至满足预定搅拌条件。In
本步骤中，乳化剂水溶液依然为PVA，第四预定体积为大于50mL，第四预定浓度为5mg/mL，第四预定体积的选择依据第三预定体积与第四预定体积之比决定。在本实施例中，第三预定体积与第四预定体积之比为1:10。在具体实施过程中为了控制纳米粒子的尺寸可以对第三预定体积和第四预定体积之比进行调整。In this step, the emulsifier aqueous solution is still PVA, the fourth predetermined volume is greater than 50 mL, the fourth predetermined concentration is 5 mg/mL, and the selection of the fourth predetermined volume is determined according to the ratio of the third predetermined volume to the fourth predetermined volume. In this embodiment, the ratio of the third predetermined volume to the fourth predetermined volume is 1:10. In the specific implementation process, the ratio of the third predetermined volume to the fourth predetermined volume may be adjusted in order to control the size of the nanoparticles.
本实施例中，本步骤的预定搅拌条件为直至有机溶剂挥发完全，也即步骤1中的二氯甲烷挥发完全。In this embodiment, the predetermined stirring condition in this step is until the organic solvent is completely volatilized, that is, the dichloromethane in
步骤5，将步骤4处理满足预定搅拌条件的液体在以大于8000RPM的转速进行大于7分钟的离心后，去除上清液，并将剩下的沉淀物重新混悬于水中，如此反复多次，最后得到去除上清液的最终沉淀物。Step 5: After centrifuging the liquid that meets the predetermined stirring conditions in
本步骤的目的去除残留的一些聚合物和未包裹的抗原，本实施例中，本步骤具体为：The purpose of this step is to remove some residual polymers and unwrapped antigens. In this embodiment, this step is specifically:
步骤5.1，将步骤4处理满足预定搅拌条件的液体在超高速离心机上以大于8000RPM离心大于7分钟，弃去上清液；Step 5.1, centrifuge the liquid that meets the predetermined stirring conditions in
步骤5.2，将步骤5.1中留下的沉淀物重新混悬于水中；Step 5.2, resuspend the sediment left in step 5.1 in water;
步骤5.3，重复步骤5.1；Step 5.3, repeat step 5.1;
步骤5.4，重复步骤5.2；Step 5.4, repeat step 5.2;
步骤5.5，重复步骤5.1，得到最终沉淀物。Step 5.5, repeat step 5.1 to obtain the final pellet.
实际中，根据实际情况，可以反复进行步骤5.1-步骤5.5的循环，以能得到需要的最终沉淀物。In practice, according to the actual situation, the cycle of step 5.1-step 5.5 can be repeated to obtain the desired final precipitate.
步骤6，将步骤5最后得到的最终沉淀物混悬于第五预定体积的第五预定浓度的冻干保护剂中得到混悬液。In
本实施例中的冻干保护剂选用海藻糖(trehalose)。The freeze-drying protective agent in this example was selected from trehalose.
本实施例中，该步骤的冻干保护剂的第五预定体积为20mL，第五预定浓度为4％，之所以如此设定，是为了在后续进行冷冻干燥中不影响冻干效果。In this embodiment, the fifth predetermined volume of the freeze-drying protective agent in this step is 20 mL, and the fifth predetermined concentration is 4%. The reason for this setting is to not affect the freeze-drying effect in the subsequent freeze-drying.
步骤7，将步骤6得到的混悬液进行冷冻干燥处理后得到冻干粉末，含有检测纳米颗粒的冻干粉末可以被用于检测抗原特异性T细胞的含量。In
本实施例中，该步骤具体为：In this embodiment, this step is specifically:
步骤7.1：将步骤6得到的混悬液在-80℃冷冻8小时以上得到冷冻产物；Step 7.1: Freeze the suspension obtained in
步骤步骤7.2：使用冷冻干燥机冷冻干燥步骤7.1所得的冷冻产物，冷冻干燥时间大于12小时，得到检测纳米颗粒；Step 7.2: use a freeze dryer to freeze dry the frozen product obtained in step 7.1, and the freeze drying time is greater than 12 hours to obtain detection nanoparticles;
步骤7.3：分析步骤7.2所得检测纳米颗粒的粒径、表面电荷和抗原包载量。Step 7.3: Analyze the particle size, surface charge and antigen loading capacity of the detection nanoparticles obtained in step 7.2.
本实施例中，检测纳米颗粒的粒径大小为10-2000nm，更优选地，粒径大小为30-1000nm，最优选地，粒径大小为100-500nm。In this embodiment, the particle size of the detected nanoparticles is 10-2000 nm, more preferably, the particle size is 30-1000 nm, and most preferably, the particle size is 100-500 nm.
2.1型糖尿病病人抗原特异性T细胞的含量检测2. Detection of antigen-specific T cells in patients with
在本实施例中依然采用酶联免疫斑点法(ELISPOT)检测技术检测细胞分泌物的方法作为检测抗原特异性T细胞数量的方法。在实际中还可以采用酶联免疫吸附法(ELISA)、细胞内细胞因子的流式细胞仪检测、多聚体技术、Luminex多细胞因子检测法、高效液相法以及液质联用中的任意一种或多种。In this example, the enzyme-linked immunospot assay (ELISPOT) detection technique is still used to detect cell secretions as the method for detecting the number of antigen-specific T cells. In practice, any of enzyme-linked immunosorbent assay (ELISA), flow cytometry detection of intracellular cytokines, multimer technology, Luminex multi-cytokine detection method, high performance liquid method and liquid chromatography-mass spectrometry can also be used. one or more.
本实施例中T细胞的分泌物依然选用干扰素-γ。在实际中，细胞分泌物可根据需要选用干扰素-γ、白介素1(IL-1)、白介素2(IL-2)、转化生长因子(TGF)-β、白介素6(IL-6)、白介素7(IL-7)、白介素15(IL-15)、白介素21(IL-21)、IP-10(Interferon gamma-induced protein 10，CXCL10)、白介素17(IL-17)和白介素10(IL-10)中的一种或几种。待测样品依然为外周血。In this example, the secretion of T cells still uses interferon-γ. In practice, cell secretions can be selected according to the needs of interferon-γ, interleukin-1 (IL-1), interleukin-2 (IL-2), transforming growth factor (TGF)-β, interleukin-6 (IL-6), interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 21 (IL-21), IP-10 (Interferon gamma-induced protein 10, CXCL10), interleukin 17 (IL-17) and interleukin 10 (IL- 10) one or more of them. The sample to be tested is still peripheral blood.
另外，在实施例中采用的检测纳米颗粒为纳米粒，在实际中也可以采用脂质体、纳米乳、外泌体、外泌体类似物、病毒、细胞分泌颗粒及细胞分泌颗粒类似物中的任意一种或多种。In addition, the detection nanoparticles used in the examples are nanoparticles, and in practice, liposomes, nanoemulsions, exosomes, exosome analogs, viruses, cell secretion particles and cell secretion particle analogs can also be used. any one or more of them.
本实施例的待测样本以及具体检测过程具体与实施例1中的一样。The sample to be tested and the specific detection process in this embodiment are the same as those in
在ELISPOT检测方法中，一个斑点的形成就代表着一个被激活的抗原特异性T细胞，所以通过测量96孔板中每个孔中显色形成的斑点的数量，我们可以得知被检测样品中抗原特异性T细胞的数量。这样，根据被检测对象体内1型糖尿病相关抗原特异性T细胞含量的多少，就能监测1型糖尿病的进展或者对于1型糖尿病的不同治疗方法的治疗效果进行评价：In the ELISPOT assay, the formation of a spot represents an activated antigen-specific T cell, so by measuring the number of spots formed in each well of the 96-well plate, we can know the antigen-specific T cells in the tested sample. number of sex T cells. In this way, according to the content of
1、抗原特异性T细胞在1型糖尿病患者发病前已存在于患者体内，因而检测外周血中抗原特异性T细胞的数量可以知道1型糖尿病患者的病情进展，患者体内抗原特异性T细胞数量越多说明对患者β细胞的杀伤和破坏越大，这样能让患者提前对自己的状况有所了解，所以能提前做一定的预防措施；1. Antigen-specific T cells already exist in patients with
2、在对1型糖尿病患者进行治疗时，对抗原特异性T细胞的检测可以用于对治疗效果进行评价：如果某一治疗手段能显著的降低抗原特异性T细胞的数量，则说明该治疗手段治疗效果良好，从而能作为患者后续是否采用这一治疗手段的评价依据，或者也能作为药企或药物开发机构对不同治疗手段或治疗方法的治疗效果进行评价的依据，能提高精准医疗，节省治疗资源或实验资源。2. When treating patients with
实施例3Example 3
以下是对实施例3的说明。本实施例中与实施例1相同的检测原理以及各个制备过程中的涉及的物质以及参数选择依据相同的部分，省略相同的说明。The following is a description of Example 3. In this embodiment, the same detection principle as in
本实施例与实施例2的检测目的和意义一样，也是以对1型糖尿病相关的抗原特异性T细胞检测为例进行说明，也是为了检测胰岛素依赖性糖尿病(1型糖尿病)病人外周血中β细胞抗原特异性T细胞的含量。本实施例与实施例2的不同在于，制备检测纳米颗粒时，不是直接包载与1型糖尿病相关的抗原，而是包载合成这种抗原所需的遗传物质DNA。The purpose and significance of this example is the same as that of Example 2, and the detection of antigen-specific T cells related to
本实施例中，也先对检测纳米颗粒进行制备，再采用制备得到的检测纳米颗粒对癌细胞中的抗原特异性T细胞含量进行检测。In this embodiment, the detection nanoparticles are also prepared first, and then the content of antigen-specific T cells in cancer cells is detected by using the prepared detection nanoparticles.
1.检测纳米颗粒的制备1. Preparation of Assay Nanoparticles
本实施例中采用的检测纳米颗粒为纳米粒，其包载1型糖尿病相关抗原GAD65的合成所需的遗传物质DNA，其制备方法具体包括以下步骤：The detection nanoparticles used in this embodiment are nanoparticles, which encapsulate the genetic material DNA required for the synthesis of the
步骤1，将第一预定体积的含有第一预定浓度抗原合成所需DNA的抗原溶液加入到第二预定体积的含有第二预定浓度的医用高分子材料有机溶液中。
本实施例中，抗原合成所需的DNA为抗原GAD合成所需的DNA。In this example, the DNA required for antigen synthesis is the DNA required for antigen GAD synthesis.
本实施例中，将合成抗原所需的DNA，溶解到水或任何含有水的对后续检测不产生影响的水溶液中，制备得到水相溶液，第一预定体积为200μL,第一预定浓度为大于0.01ng/mL。In this embodiment, the DNA required for synthesizing the antigen is dissolved in water or any aqueous solution containing water that does not affect subsequent detection to prepare an aqueous phase solution. The first predetermined volume is 200 μL, and the first predetermined concentration is greater than or equal to 200 μL. 0.01ng/mL.
将医用高分子材料溶解于预定有机溶剂中，得到高分子有机溶液，本实施例中，医用高分子材料为聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid)，PLGA)，有机溶剂选用二氯甲烷，预定有机体积为2mL。The medical polymer material is dissolved in a predetermined organic solvent to obtain a polymer organic solution. In this embodiment, the medical polymer material is poly(lactic-co-glycolic acid) (PLGA), an organic Dichloromethane was selected as the solvent, and the predetermined organic volume was 2 mL.
水相的第一预定体积和有机相的第二预定体积之比为1:10。在具体实施过程中可根据需要对第一预定体积、第二预定体积和第一预定体积与第二预定体积之比进行调整。The ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is 1:10. In the specific implementation process, the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as required.
步骤2，将步骤1得到的混合物进行大于3秒的超声处理。In
步骤3，将步骤2处理后得到的液体加入第三预定体积的第三预定浓度的乳化剂水溶液中并进行大于5秒的超声处理。In
本实施例中，乳化剂水溶液为聚乙烯醇(PVA)水溶液，第三预定体积为5mL，第三预定浓度为20mg/mL。In this embodiment, the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution, the third predetermined volume is 5 mL, and the third predetermined concentration is 20 mg/mL.
同样地，本步骤的超声时间、乳化剂水溶液的体积以及浓度的取值根据，均为了得到尺寸大小合适的纳米粒。第三预定体积的选择依据第二预定体积与第三预定体积之比决定。在本实施例中，第二预定体积与第三预定体积之比为2:5。在具体实施过程中为了控制纳米粒子的尺寸可以对第二预定体积和第三预定体积之比进行调整。Similarly, the ultrasonic time, the volume and concentration of the emulsifier aqueous solution in this step are all based on obtaining nanoparticles of suitable size. The selection of the third predetermined volume is determined according to the ratio of the second predetermined volume to the third predetermined volume. In this embodiment, the ratio of the second predetermined volume to the third predetermined volume is 2:5. In the specific implementation process, in order to control the size of the nanoparticles, the ratio of the second predetermined volume to the third predetermined volume can be adjusted.
步骤4，将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中，并进行搅拌直至满足预定搅拌条件。In
本步骤中，乳化剂水溶液依然为PVA，第四预定体积为大于50mL，In this step, the emulsifier aqueous solution is still PVA, and the fourth predetermined volume is greater than 50mL,
第四预定浓度为5mg/mL，第四预定体积以及第四预定浓度的选择，依然以得到尺寸大小合适的纳米粒为依据。第四预定体积的选择依据第三预定体积与第四预定体积之比决定。在本实施例中，第三预定体积与第四预定体积之比为1:10。在具体实施过程中为了控制纳米粒子的尺寸可以对第三预定体积和第四预定体积之比进行调整。The fourth predetermined concentration is 5 mg/mL, and the selection of the fourth predetermined volume and the fourth predetermined concentration is still based on obtaining nanoparticles of suitable size. The selection of the fourth predetermined volume is determined according to the ratio of the third predetermined volume to the fourth predetermined volume. In this embodiment, the ratio of the third predetermined volume to the fourth predetermined volume is 1:10. In the specific implementation process, the ratio of the third predetermined volume to the fourth predetermined volume may be adjusted in order to control the size of the nanoparticles.
本实施例中，本步骤的预定搅拌条件为直至有机溶剂挥发完全，也即步骤1中的二氯甲烷挥发完全。In this embodiment, the predetermined stirring condition in this step is until the organic solvent is completely volatilized, that is, the dichloromethane in
步骤5，将步骤4处理满足预定搅拌条件的混合液在以大于8000RPM的转速进行大于7分钟的离心后，去除上清液，并将剩下的沉淀物重新混悬于水中，如此反复多次，最后得到去除上清液的最终沉淀物。Step 5: After centrifugation for more than 7 minutes at a speed of more than 8000 RPM, the mixture that meets the predetermined stirring conditions in
本步骤的目的去除残留的一些聚合物和未包裹的DNA，本实施例中，本步骤具体为：The purpose of this step is to remove some residual polymers and unwrapped DNA. In this embodiment, this step is specifically:
步骤5.1，将步骤4处理满足预定搅拌条件的液体在超高速离心机上以大于8000RPM离心大于7分钟，弃去上清液；Step 5.1, centrifuge the liquid that meets the predetermined stirring conditions in
步骤5.2，将步骤5.1中留下的沉淀物重新混悬于水中；Step 5.2, resuspend the sediment left in step 5.1 in water;
步骤5.3，重复步骤5.1；Step 5.3, repeat step 5.1;
步骤5.4，重复步骤5.2；Step 5.4, repeat step 5.2;
步骤5.5，重复步骤5.1，得到最终沉淀物。Step 5.5, repeat step 5.1 to obtain the final pellet.
实际中，根据实际情况，可以反复进行步骤5.1-步骤5.5的循环，以能得到需要的最终沉淀物。In practice, according to the actual situation, the cycle of step 5.1-step 5.5 can be repeated to obtain the desired final precipitate.
步骤6，将步骤5最后得到的最终沉淀物混悬于第五预定体积的第五预定浓度的冻干保护剂中得到混悬液。In
本实施例中的冻干保护剂选用海藻糖(trehalose)。The freeze-drying protective agent in this example was selected from trehalose.
本实施例中，该步骤的冻干保护剂的第五预定体积为20mL，第五预定浓度为4％，之所以如此设定，是为了在后续进行冷冻干燥中不影响冻干效果。In this embodiment, the fifth predetermined volume of the freeze-drying protective agent in this step is 20 mL, and the fifth predetermined concentration is 4%. The reason for this setting is to not affect the freeze-drying effect in the subsequent freeze-drying.
步骤7，将步骤6得到的混悬液进行冷冻干燥处理后得到冻干粉末。含有检测纳米颗粒的冻干粉末可以被用于检测抗原特异性T细胞的含量。
本实施例中，该步骤具体为：In this embodiment, this step is specifically:
步骤7.1：将步骤6得到的混悬液在-80℃冷冻8小时以上得到冷冻产物；Step 7.1: Freeze the suspension obtained in
步骤步骤7.2：使用冷冻干燥机冷冻干燥步骤7.1所得的冷冻产物，冷冻干燥时间大于12小时，得到冻干的检测纳米颗粒；Step 7.2: use a freeze dryer to freeze dry the frozen product obtained in step 7.1, and the freeze drying time is greater than 12 hours to obtain freeze-dried detection nanoparticles;
步骤7.3：分析步骤7.2所得检测纳米颗粒的粒径、表面电荷和DNA包载量。Step 7.3: Analyze the particle size, surface charge and DNA package capacity of the detection nanoparticles obtained in step 7.2.
本实施例中，检测纳米颗粒的粒径大小为10-2000nm，更优选地，粒径大小为30-1000nm，最优选地，粒径大小为100-500nm。In this embodiment, the particle size of the detected nanoparticles is 10-2000 nm, more preferably, the particle size is 30-1000 nm, and most preferably, the particle size is 100-500 nm.
另外，在实施例中采用的检测纳米颗粒为纳米粒，在实际中也可以采用脂质体、纳米乳、外泌体、外泌体类似物、病毒、细胞分泌颗粒及细胞分泌颗粒类似物中的任意一种或多种。In addition, the detection nanoparticles used in the examples are nanoparticles, and in practice, liposomes, nanoemulsions, exosomes, exosome analogs, viruses, cell secretion particles and cell secretion particle analogs can also be used. any one or more of them.
2 1型糖尿病病人抗原特异性T细胞的含量检测Detection of the content of antigen-specific T cells in patients with
在本实施例中采用常规的酶联免疫吸附法(ELISA)检测技术检测细胞分泌物的方法作为检测抗原特异性T细胞数量的方法。在具体应用中可以根据不同ELISA试剂和检测试剂盒的实际条件进行调整。In this example, a conventional enzyme-linked immunosorbent assay (ELISA) detection technique is used to detect cell secretions as a method for detecting the number of antigen-specific T cells. In specific applications, it can be adjusted according to the actual conditions of different ELISA reagents and detection kits.
步骤1，采样
抽取患者外周血若干毫升，在抽血过程中加入肝素抗凝血。本实验采用新鲜采集的外周血进行。Several milliliters of peripheral blood were drawn from the patient, and heparin was added for anticoagulation during the blood drawing. The experiment was performed with freshly collected peripheral blood.
步骤2，将制备得到的检测纳米颗粒混悬于AIM V细胞培养基中。
步骤3，外周血和AIM V培养基中的检测纳米颗粒混合在37℃(5％CO2)培养箱中培养24到96个小时。In
步骤4，离心去除血细胞并收集上清液。
步骤5，将上清液直接或稀释后采用干扰素-γ的ELISA检测方法检测上清液中干扰素-γ的含量。具体检测步骤与ELISA常规检测相同。简述如下：In
上清液直接或经PBS稀释后加入包被有抗干扰素-γ抗体a并经0.5％BSA封闭处理的96孔板中。作用至少2个小时以后洗涤并加入抗干扰素-γ抗体b。抗体b上连接有能够进行显色反应或者荧光检测的物质。在与抗体b作用一段时间后采用显色法、化学发光法或荧光法检测上清液中干扰素-γ的含量。The supernatant was added directly or diluted with PBS to a 96-well plate coated with anti-interferon-γ antibody a and blocked with 0.5% BSA. After at least 2 hours of action, wash and add anti-interferon-gamma antibody b. A substance capable of performing a color reaction or fluorescent detection is linked to the antibody b. After reacting with antibody b for a period of time, the content of interferon-γ in the supernatant was detected by chromogenic method, chemiluminescence method or fluorescence method.
在本实施例中酶联免疫吸附法(ELISA)检测技术检测细胞分泌物的方法作为检测抗原特异性T细胞数量的方法，在实际中还可以采用酶联免疫斑点法(ELISPOT)、酶联免疫吸附法(ELISA)、细胞内细胞因子的流式细胞仪检测、多聚体技术、Luminex多细胞因子检测法、高效液相法以及液质联用中的任意一种或多种。细胞分泌物可选用干扰素-γ，白介素1(IL-1)、白介素2(IL-2)、转化生长因子(TGF)-β、白介素6(IL-6)、白介素7(IL-7)、白介素15(IL-15)、白介素21(IL-21)、IP-10(Interferon gamma-induced protein 10，CXCL10)、白介素17(IL-17)和白介素10(IL-10)中的一种或几种。本实施例中T细胞的分泌物依然选用干扰素-γ，待测样品为外周血。In this example, the method for detecting cell secretions by enzyme-linked immunosorbent assay (ELISA) detection technology is used as a method for detecting the number of antigen-specific T cells. Any one or more of adsorption method (ELISA), flow cytometry detection of intracellular cytokines, multimer technology, Luminex multi-cytokine detection method, high performance liquid chromatography and liquid chromatography-mass spectrometry. Cell secretions can be selected from interferon-γ, interleukin 1 (IL-1), interleukin 2 (IL-2), transforming growth factor (TGF)-β, interleukin 6 (IL-6), interleukin 7 (IL-7) , one of interleukin 15 (IL-15), interleukin 21 (IL-21), IP-10 (Interferon gamma-induced protein 10, CXCL10), interleukin 17 (IL-17) and interleukin 10 (IL-10) or several. In this example, the secretion of T cells still uses interferon-γ, and the sample to be tested is peripheral blood.
本实施例的待测样本以及具体检测过程与实施例1与实施例2中的一样，不一样的是，本这个检测过程中，本实施例中，检测纳米颗粒中含有的合成抗原需要的遗传物质DNA进入抗原提呈细胞被释放后，在抗原提呈细胞内先合成了相应的抗原，再进行后续的提呈与激活。The sample to be tested and the specific detection process in this example are the same as those in Example 1 and Example 2. The difference is that in this detection process, in this example, the genetic code required for the detection of synthetic antigen contained in nanoparticles After the substance DNA enters the antigen-presenting cells and is released, the corresponding antigens are first synthesized in the antigen-presenting cells, and then the subsequent presentation and activation are carried out.
图2为检测纳米颗粒包载能生成抗原的遗传物质时检测抗原特异性T细胞含量的检测方法的检测机理示意图。FIG. 2 is a schematic diagram of the detection mechanism of the detection method for detecting the content of antigen-specific T cells when the genetic material capable of generating antigen is detected when the nanoparticles are encapsulated.
图2中，1，抗原提呈细胞；2，抗原特异性T细胞；3，包载有能生成抗原的遗传物质的检测纳米颗粒；4，能生成抗原的遗传物质；5：遗传物质生成的抗原；6：抗原表位；7，主要组织相容性复合体；8，T细胞受体；9，细胞分泌物，如干扰素-γ。In Fig. 2, 1, antigen-presenting cells; 2, antigen-specific T cells; 3, detection nanoparticles loaded with genetic material capable of producing antigen; 4, genetic material capable of producing antigen; 5: generated by genetic material antigen; 6: epitope; 7, major histocompatibility complex; 8, T cell receptor; 9, cellular secretions, such as interferon-gamma.
从图2中可以看出，本实施例的检测机理也是基于细胞分泌物，与实施例1中的不同的是，由于包载的是遗传物质，所以，包载物释放进入细胞后，要先在细胞内生成抗原，然后再进行抗原的处理和提呈。It can be seen from Figure 2 that the detection mechanism of this example is also based on cell secretions. The difference from Example 1 is that since the encapsulated material is genetic material, after the encapsulated material is released into the cell, it must be Intracellular production of antigens followed by processing and presentation of the antigens.
在ELISA检测方法中，细胞分泌物含量不同相应的显色或荧光读数会不同，通过最终读数的高低就能判断细胞分泌物含量的高低进而确定抗原特异性T细胞含量的高低。这样，根据被检测对象体内1型糖尿病相关抗原特异性T细胞含量的多少，就能监测1型糖尿病的疾病进展和评价1型糖尿病不同治疗方法的治疗效果。In the ELISA detection method, the corresponding color or fluorescence readings will be different depending on the content of cell secretions. The level of the final readings can determine the level of cell secretions and then determine the level of antigen-specific T cells. In this way, according to the content of
实施例作用与效果Example function and effect
根据实施例1-3所提供的用于抗原特异性T细胞含量检测的检测纳米颗粒以及抗原特异性T细胞含量的检测方法中，通过制备含有与疾病相关的抗原或含有合成该抗原所需的遗传物质的检测纳米颗粒，并且该检测纳米颗粒的尺寸大小为纳米级，就可以使得能被待测样品中的抗原提呈细胞吞噬，并释放抗原或抗原合成所需的遗传物质以合成相应的抗原，进一步对被释放的抗原或新合成的抗原进行提呈，就可以激活相应的与疾病相关的抗原特异性T细胞，通过激活的抗原特异性T细胞分泌的细胞分泌物的检测，并基于细胞分泌物与相应抗原特异性T细胞之间的关系，就能测定相应抗原特异性T细胞的含量，所以我们可以用于监测疾病的进展或者评价比较疾病的治疗方案，这样就为我们实施精准治疗提供了条件，并为做相关研究选择适宜的实验对象以取得良好的实验结果和避免不必要的浪费提供了条件，并且，该检测方法成本较低，而且操作相对简单，使用方便。In the detection nanoparticles for the detection of antigen-specific T cell content and the detection method for antigen-specific T cell content provided according to Examples 1-3, by preparing a disease-related antigen or containing the antigen required for synthesis of the antigen Detection of genetic material nanoparticles, and the size of the detection nanoparticles is nanoscale, so that they can be phagocytosed by antigen-presenting cells in the sample to be tested, and release the antigen or the genetic material required for antigen synthesis to synthesize the corresponding Antigen, further presentation of the released antigen or newly synthesized antigen can activate the corresponding disease-related antigen-specific T cells, through the detection of cell secretions secreted by the activated antigen-specific T cells, and based on The relationship between cell secretions and the corresponding antigen-specific T cells can determine the content of the corresponding antigen-specific T cells, so we can use it to monitor the progress of the disease or evaluate the treatment plan for the comparison of the disease, so that we can accurately implement the The treatment provides conditions, and provides conditions for selecting suitable experimental objects for relevant research to obtain good experimental results and avoid unnecessary waste, and the detection method has low cost, relatively simple operation and convenient use.
另外，实施例1-3中，抗原特异性T细胞含量的检测基于被激活的抗原特异性T细胞所分泌的细胞分泌物，作为本发明，抗原特异性T细胞含量的检测还可以基于被激活的抗原特异性T细胞的增殖状态或被激活的抗原特异性T细胞的细胞表面标志物：In addition, in Examples 1-3, the detection of the antigen-specific T cell content was based on the cell secretions secreted by the activated antigen-specific T cells. As the present invention, the detection of the antigen-specific T cell content can also be based on the activated antigen-specific T cell content. Proliferation status of antigen-specific T cells or cell surface markers of activated antigen-specific T cells:
当基于被激活的抗原特异性T细胞的增殖状态进行检测时，检测原理是抗原特异性T细胞被激活后会增殖，其增殖速度快于未被激活时，所以通过检测抗原特异性T细胞增值速度和状态我们就能确定抗原特异性T细胞是否被激活了并测定其含量。When the detection is based on the proliferation state of the activated antigen-specific T cells, the detection principle is that the antigen-specific T cells will proliferate after being activated, and their proliferation rate is faster than that when they are not activated. Therefore, by detecting the proliferation of antigen-specific T cells Speed and status we can determine whether antigen-specific T cells are activated and measure their content.
当基于被激活的抗原特异性T细胞的细胞表面标志物进行检测时，检测原理是抗原特异性T细胞被激活后细胞表面会表达一些特异性的分子表明该抗原特异性T细胞为激活状态的抗原特异性T细胞，通过检测这些细胞表面的特异性的分子的有无和其含量我们就能确定抗原特异性T细胞是否被激活了及其含量。When the detection is based on the cell surface markers of activated antigen-specific T cells, the detection principle is that after the antigen-specific T cells are activated, some specific molecules will be expressed on the cell surface, indicating that the antigen-specific T cells are activated. For antigen-specific T cells, we can determine whether antigen-specific T cells are activated and their content by detecting the presence and content of specific molecules on the surface of these cells.
并且，抗原特异性T细胞被激活后的增殖状态的检测采用基于外源标记的减少检测方法、基于分子标记的表达检测方法、基于代谢标记的增加检测方法、基于细胞群体生物量的变化检测方法以及基于细胞数量的变化检测方法中的任意一项进行检测；其中，基于外源标记的减少检测方法为蛋白质结合染料CFDA-SE、CellTrace以及亲脂性染料PKH67和PKH26标记后采用流式细胞仪检测，并且，基于的分子标记可以为Ki-67和PCNA；基于代谢标记的增加检测方法中的基于的代谢标记可以为氧化还原代谢标记和基于DNA代谢标记；基于细胞群体生物量的变化检测方法中，基于的细胞群体生物量可以包括DNA含量和蛋白质含量中的一种或多种。In addition, the detection of the proliferation state of antigen-specific T cells after activation adopts the detection method of decrease based on exogenous markers, the detection method of expression based on molecular markers, the detection method of increase based on metabolic markers, and the detection method based on the change of cell population biomass. And any one of the detection methods based on changes in the number of cells is detected; wherein, the reduction detection method based on exogenous labeling is protein-binding dyes CFDA-SE, CellTrace and lipophilic dyes PKH67 and PKH26 are labeled with flow cytometry detection. , and the molecular markers based on can be Ki-67 and PCNA; the metabolic markers based on the increase detection method based on metabolic markers can be redox metabolic markers and DNA-based metabolic markers; in the detection method based on changes in cell population biomass , the cell population based biomass can include one or more of DNA content and protein content.
基于被激活的抗原特异性T细胞的细胞表面标志物进行检测时，抗原特异性T细胞激活的细胞表面标志物可以为CD137、CD69、CD38、CD44、CD62L或CD25中的一种或多种。When the detection is based on cell surface markers of activated antigen-specific T cells, the cell surface markers of antigen-specific T cell activation can be one or more of CD137, CD69, CD38, CD44, CD62L or CD25.
另外，在实施例1和2中，所选用的抗原为癌细胞抗原和1型糖尿病相关抗原，作为本发明，无论是基于被激活的抗原特异性T细胞所分泌的细胞分泌物、被激活的抗原特异性T细胞的增殖状态或被激活的抗原特异性T细胞的细胞表面标志物中的哪一种进行检测，抗原都可以根据需要使用与任何疾病的相关抗原，例如与自身免疫性疾病的相关抗原。In addition, in Examples 1 and 2, the selected antigens were cancer cell antigens and
另外，实施例1中，抗原为癌细胞裂解物，实施例2中，抗原为1型糖尿病相关的抗原蛋白质，作为本发明，无论是基于被激活的抗原特异性T细胞所分泌的细胞分泌物、被激活的抗原特异性T细胞的增殖状态或被激活的抗原特异性T细胞的细胞表面标志物中的哪一种进行检测，抗原都可以为癌细胞裂解物、抗原蛋白质、多肽、细胞裂解物、组织裂解物、DNA、RNA、外泌体(exosome)裂解物、细胞分泌颗粒裂解物、糖类以及脂类中的任意一种或多种。In addition, in Example 1, the antigen is a cancer cell lysate, and in Example 2, the antigen is a
另外，在实施例1-3中，所选用的制备纳米粒子的材料为PLGA，作为本发明，无论是基于被激活的抗原特异性T细胞所分泌的细胞分泌物、被激活的抗原特异性T细胞的增殖状态或被激活的抗原特异性T细胞的细胞表面标志物中的哪一种进行检测，所选用的制备纳米粒子的材料除了PLGA，还可以根据使用需要任意选择其他用于制备纳米粒子的材料。In addition, in Examples 1-3, the selected material for preparing nanoparticles is PLGA. As the present invention, whether it is based on cell secretions secreted by activated antigen-specific T cells, activated antigen-specific T cells Which of the cell proliferation status or activated antigen-specific T cell cell surface markers is to be detected, the materials selected for the preparation of nanoparticles, in addition to PLGA, can also be arbitrarily selected for the preparation of nanoparticles according to the needs of use s material.
另外，实施例1和实施例2检测纳米颗粒的制备过程中，直接采用抗原进行包载，最终得到含有抗原的纳米粒，且在实施例1中该抗原为细胞裂解物，实施例2中抗原为蛋白质，实际中，无论是基于被激活的抗原特异性T细胞所分泌的细胞分泌物、被激活的抗原特异性T细胞的增殖状态或被激活的抗原特异性T细胞的细胞表面标志物中的哪一种进行检测，当直接包载抗原时，抗原可以为细胞裂解物、蛋白质、质粒、多肽、DNA、RNA、组织裂解物、外泌体裂解物、细胞分泌颗粒裂解物以、糖类以及脂类中的任意一种或多种。In addition, in the preparation process of the detection nanoparticles in Examples 1 and 2, the antigen is directly used for encapsulation, and finally the nanoparticles containing the antigen are obtained. For proteins, in practice, whether based on cellular secretions secreted by activated antigen-specific T cells, the proliferative state of activated antigen-specific T cells, or cell surface markers of activated antigen-specific T cells Which one is used for detection, when the antigen is directly encapsulated, the antigen can be cell lysate, protein, plasmid, polypeptide, DNA, RNA, tissue lysate, exosome lysate, cell secretory granule lysate, carbohydrates and any one or more of lipids.
另外，在如同实施例3的检测纳米颗粒的制备过程中，无论是基于被激活的抗原特异性T细胞所分泌的细胞分泌物、被激活的抗原特异性T细胞的增殖状态或被激活的抗原特异性T细胞的细胞表面标志物中的哪一种进行检测，对与疾病相关的抗原合成需要的遗传物质进行包载，这样最终得到包载了这种遗传物质的检测纳米颗粒，遗传物质可以为来于细胞裂解物、质粒、DNA、RNA、组织裂解物、外泌体裂解物、细胞分泌颗粒裂解物中的任意一种或多种。In addition, in the preparation process of the detection nanoparticles as in Example 3, whether it is based on the cell secretion secreted by the activated antigen-specific T cells, the proliferative state of the activated antigen-specific T cells or the activated antigen Which of the cell surface markers of specific T cells is detected, and the genetic material required for the synthesis of disease-related antigens is encapsulated, so that the detection nanoparticles encapsulated with this genetic material are finally obtained. For any one or more of cell lysate, plasmid, DNA, RNA, tissue lysate, exosome lysate, cell secretory granule lysate.
另外，在实施例1-3中，抗原特异性T细胞的检测过程中未在纳米粒和待测样品混合物中加入其他物质以增强检测，在实际使用时，无论是基于被激活的抗原特异性T细胞所分泌的细胞分泌物、被激活的抗原特异性T细胞的增殖状态或被激活的抗原特异性T细胞的细胞表面标志物中的哪一种进行检测，都可根据需要在纳米粒子处理待测样品时加入白介素、内毒素、细胞因子、化学因子，趋化因子、二甲双胍、黄连素、青蒿素及其类似物等中的任意一种或多种，以增强检测技术的敏感性。In addition, in Examples 1-3, no other substances were added to the mixture of nanoparticles and the sample to be tested to enhance the detection during the detection of antigen-specific T cells. Which of the cell secretions secreted by T cells, the proliferation state of activated antigen-specific T cells, or the cell surface markers of activated antigen-specific T cells is detected can be treated with nanoparticles as needed. Add any one or more of interleukins, endotoxins, cytokines, chemical factors, chemokines, metformin, berberine, artemisinin and its analogs to the samples to be tested to enhance the sensitivity of the detection technology.
另外，在实施例1-3中，检测的细胞分泌物为一种，实际中，还可以根据需要选择多种进行检测，实施例1-3中的细胞分泌物均为干扰素-γ，实际中还可以为白介素1、白介素2、转化生长因子-β、肿瘤坏死因子-α、白介素6、白介素7、白介素15、白介素21、IP-10、白介素17、穿孔素以及颗粒酶和白介素10中的任意一种或多种；In addition, in Example 1-3, only one type of cell secretion was detected. In practice, multiple types of secretions can be selected for detection according to needs. The cell secretions in Example 1-3 are all interferon-γ. It can also be
另外，在实施例1-3中，检测T细胞分泌物的细胞分泌物检测方法选用免疫学检测方法，实际中，无论是基于被激活的抗原特异性T细胞所分泌的细胞分泌物、被激活的抗原特异性T细胞的增殖状态或被激活的抗原特异性T细胞的细胞表面标志物中的哪一种进行检测，都可以选用免疫学检测方法、生物活性测定法以及分子生物学检测方法中的任意一种；实施例1-3中，选用的免疫学检测方法为酶联免疫斑点(ELISPOT)，实际中还可以选择酶联免疫吸附法、胞内染色的流式细胞仪检测、Cytometric Bead Array、多聚体技术、多细胞因子检测法、高效液相法以及液质联用中的任意一种或多种。In addition, in Examples 1-3, the immunological detection method was used as the method for detecting the secretion of T cell secretions. The proliferation state of the activated antigen-specific T cells or the cell surface markers of the activated antigen-specific T cells can be detected by immunological detection methods, biological activity assays and molecular biology detection methods. In embodiment 1-3, the selected immunological detection method is enzyme-linked immunospot (ELISPOT), in practice, ELISA, intracellular staining flow cytometry detection, Cytometric Bead can also be selected Any one or more of Array, multimer technology, multi-cytokine detection method, high performance liquid phase method and liquid chromatography-mass spectrometry.
另外，实施例1-3中，待测样品为外周血单核细胞(peripheral bloodmononuclear cells,PBMCs)，实际中，无论是基于被激活的抗原特异性T细胞所分泌的细胞分泌物、被激活的抗原特异性T细胞的增殖状态或被激活的抗原特异性T细胞的细胞表面标志物中的哪一种进行检测，待测样品可以为外周血单核细胞、血液、血浆、血清、病灶部位组织以及病灶部位细胞中的任意一种或多种。In addition, in Examples 1-3, the samples to be tested are peripheral blood mononuclear cells (PBMCs). Which one of the proliferation state of antigen-specific T cells or the cell surface markers of activated antigen-specific T cells is detected. The samples to be tested can be peripheral blood mononuclear cells, blood, plasma, serum, and tissue at the lesion site. and any one or more of the cells at the lesion site.
另外，在实施例2和3中，涉及的疾病相关抗原为谷氨酸脱羧酶(GAD)和前胰岛素原(preproinsulin)，实际中还可以为胰岛素原(proinsulin)、胰岛素(insulin)、胰岛细胞抗体(ICA)、β细胞裂解物、ZnT8、chromgranin A、islet-specific glucose-6-phosphatasecatalytic subunit-related protein(IGRP)、CD38以及酪氨酸磷酸酶(IA-2)等1型糖尿病相关抗原作为检测1型糖尿病的抗原。In addition, in Examples 2 and 3, the disease-related antigens involved are glutamate decarboxylase (GAD) and preproinsulin, but in practice they can also be proinsulin, insulin, pancreatic islet cells Antibody (ICA), β-cell lysate, ZnT8, chromgranin A, islet-specific glucose-6-phosphatasecatalytic subunit-related protein (IGRP), CD38, and tyrosine phosphatase (IA-2) and
另外，实施例1-3的检测纳米颗粒均为纳米粒，作为本发明，外周血单核细胞，检测纳米颗粒还可以为任何含有疾病相关抗原或含有合成该抗原所需遗传物质的尺寸大小为纳米级的物质，例如为脂质体、纳米乳、外泌体、外泌体类似物、病毒、细胞分泌颗粒及细胞分泌颗粒类似物等中的任意一种或多种。In addition, the detection nanoparticles in Examples 1-3 are all nanoparticles, as the present invention, peripheral blood mononuclear cells, detection nanoparticles can also be any containing disease-related antigen or containing the size of the genetic material required for synthesizing the antigen. Nanoscale substances, for example, are any one or more of liposomes, nanoemulsions, exosomes, exosome analogs, viruses, cell secretory particles, and cell secretory particle analogs.
另外，实施例1-3中，制备检测纳米颗粒的材料包括医用高分子材料，实际中，外周血单核细胞，制备检测纳米颗粒的材料包括医用高分子材料、制备脂质体所需的材料、制备纳米粒所需的金属材料以及制备纳米粒所需的非金属材料中的任意一种或多种。In addition, in Examples 1-3, the materials for preparing detection nanoparticles include medical polymer materials. In practice, peripheral blood mononuclear cells, and the materials for preparing detection nanoparticles include medical polymer materials and materials required for preparing liposomes. , any one or more of the metal materials required for the preparation of nanoparticles and the non-metallic materials required for the preparation of nanoparticles.
另外，本发明还提供一种抗原特异性T细胞检测用试剂盒，其包括上述的检测纳米颗粒，所以采用本发明的抗原特异性T细胞检测方法，也能检测出抗原特异性T细胞含量。In addition, the present invention also provides an antigen-specific T cell detection kit, which includes the above-mentioned detection nanoparticles, so the antigen-specific T cell detection method of the present invention can also detect the antigen-specific T cell content.
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