WO2022246764A1 - 一种分析cd303+树突状细胞亚群表型和功能的方法及试剂盒 - Google Patents

一种分析cd303+树突状细胞亚群表型和功能的方法及试剂盒 Download PDF

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WO2022246764A1
WO2022246764A1 PCT/CN2021/096525 CN2021096525W WO2022246764A1 WO 2022246764 A1 WO2022246764 A1 WO 2022246764A1 CN 2021096525 W CN2021096525 W CN 2021096525W WO 2022246764 A1 WO2022246764 A1 WO 2022246764A1
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antibody
cells
dendritic cell
function
antibodies
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French (fr)
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周放
陈小平
秦莉
胡晓晶
高素素
常旭
谢简明
黄烁珊
容志恩
牛欢
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中科蓝华(广州)生物医药技术有限公司
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Priority to PCT/CN2021/096525 priority Critical patent/WO2022246764A1/zh
Priority to AU2021447099A priority patent/AU2021447099A1/en
Priority to CN202180002895.0A priority patent/CN114391102A/zh
Publication of WO2022246764A1 publication Critical patent/WO2022246764A1/zh

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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5434—IL-12
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/7056—Selectin superfamily, e.g. LAM-1, GlyCAM, ELAM-1, PADGEM

Definitions

  • the application belongs to the field of biotechnology, and specifically relates to immunoassay analysis of human peripheral blood dendritic cells by flow cytometry, and in particular to a method for clinical immunoassay analysis of the phenotype and function of CD303 + dendritic cell subsets Kits and applications.
  • Dendritic cells as cells that play a major regulatory role in the immune system in vivo, are one of the hotspots in immunology research.
  • the detection of dendritic cells mainly relies on flow cytometry analysis technology, but there are various analysis schemes for the determination of dendritic cells, and there is no unified standard mode. This is mainly because the research on dendritic cells is changing with each passing day, and many different subtypes of dendritic cells have been reported; however, the flow cytometric analysis scheme for dendritic cells is too extensive, which cannot meet the current clinical needs for different subtypes. The precise analysis of dendritic cell types is required.
  • Flow cytometry is a device for automatic analysis and sorting of cells. It is mainly composed of four parts, including flow chamber and liquid flow system, laser source and optical system, photoelectric tube and detection system, and computer and analysis system. . Flow cytometry can quickly measure, store, and display a series of important biophysical and biochemical characteristic parameters of dispersed cells suspended in liquid, and can sort specified cell subgroups from them according to the pre-selected parameter range come out. Most flow cytometers are zero-resolution instruments, which can only measure indicators such as total nucleic acid and total protein in a cell.
  • Flow cytometry can measure multiple parameters at the same time, and the information mainly comes from specific fluorescent signals and non-fluorescent scattering signals.
  • the commonly used technical indicators include fluorescence resolution, fluorescence sensitivity, applicable sample concentration, sorting purity, analytical measurement parameters, etc.
  • Undamaged cells characteristically scatter light, so unstained live cells can be analyzed and sorted using the different scattered light signatures.
  • the instrument first measures the light scattering signal.
  • the fluorescent signal mainly includes two parts: 1 autofluorescence, that is, the fluorescence emitted by the fluorescent molecules inside the cell after being irradiated with light without fluorescent dyeing; Fluorescence, which has a weaker fluorescence intensity and a wavelength different from that of irradiating laser light.
  • Autofluorescence signals are noise signals that in most cases interfere with the resolution and measurement of specific fluorescence signals.
  • CD303 + dendritic cells are distributed in human peripheral blood and are a newly discovered subpopulation of dendritic cells in recent years.
  • Clinical and basic studies have shown that CD303 + dendritic cell subsets play an important role in the occurrence of various diseases, such as certain malignant tumors such as lung cancer, melanoma, prostate cancer and kidney cancer, dermatitis, certain viral infections such as HIV -1 infection, certain infectious diseases such as malaria infection, and some autoimmune diseases such as rheumatoid arthritis; in these diseases, CD303 + dendritic cells show abnormal phenotype and function. Therefore, the clinical data of CD303 + dendritic cell phenotype and function determination can become one of the auxiliary judgment indicators for clinicians to judge the development of the above diseases and the clinical treatment effect, which has very important clinical diagnostic significance.
  • dendritic cell subpopulations usually requires separation and extraction of peripheral blood mononuclear cells, which is complicated and cumbersome and takes a long time. If cell subpopulations are analyzed by means of cell molecular surface antigen detection, a large number of dendritic cells are usually selected.
  • the detection of dendritic cell surface antigen molecules can improve the accuracy and specificity of detection, but the detection and analysis of a large number of surface antigens takes a long time and increases the economic cost of detection, which is not conducive to the rapid and efficient analysis of dendritic cell subsets. .
  • CN105911292A discloses a kit for combined analysis of CD11c + CD11b + dendritic cell subsets and their degree of differentiation and function, including the following 8 kinds of antibodies: CD11c, CD80, CD86, CD11b, HLA-DR, IL-12 , IL-23 and IL-27.
  • the invention also provides a method for combined analysis of CD11c + CD11b + dendritic cell subsets and their degree of differentiation and function, which can detect CD11c + CD11b + dendritic cell subsets and their degree of differentiation and function at one time The full set of data for the function.
  • the morphology and immune function of dendritic cells are not the same, and the number of surface antigen molecules is large.
  • the present application provides a kit for analyzing the phenotype and function of CD303 + dendritic cell (CD303 + DC) subpopulation and its application.
  • the antibody combination formula design for the CD303 + DC subgroup used in the method can efficiently and quickly analyze the phenotype and function of the CD303 + DC subgroup in peripheral blood, while ensuring the accuracy, it can also reduce the risk of detection of a large number of surface antigens. Molecule-induced cost, simple, high accuracy.
  • the present application provides an antibody combination formula for analyzing the phenotype and function of CD303 + dendritic cell subsets, the molecular combination formula includes: CD303 antibody, CD205 antibody, CD103 antibody, IL-12 antibody and Antibody to TGF-beta.
  • the present application provides an application of an antibody combination formulation in the analysis and/or preparation of products for analyzing the phenotype and function of CD303 + dendritic cell subsets.
  • the antibody combination formula can be used to analyze the phenotype and function of the CD303 + dendritic cell subgroup; the antibody combination formula can be used to prepare products for analyzing the phenotype and function of the CD303 + dendritic cell subgroup.
  • the product comprises kits and/or detection reagents.
  • the antibody combination formula described in this application can be used to analyze the phenotype and function of CD303 + dendritic cell subsets, while identifying the phenotype of CD303 + dendritic cell subsets, it can also perform functional analysis, and the obtained product
  • the kit can obtain comprehensive information on dendritic cells based on a small amount of samples (including blood samples from healthy people), providing convenient and rapid detection methods for cell subtype identification, drug development, and disease prevention.
  • the present application also provides a kit for analyzing the phenotype and function of CD303 + dendritic cell subsets, the kit includes the antibody combination formula as described in the first aspect; and the antibody combination formula consists of Labeled with five different fluorochromes.
  • the fluorochrome label is selected from any five of BV421, BV711, PerCP-Cy5.5, Pacific blue, PE-CF594, FITC, PE-Cy7, Amcyan, APC-Cy7 or Q-Dot, preferably BV421, BV711, PerCP-Cy5.5, Pacific blue and PE-CF594.
  • the kit described in this application includes antibodies to CD303, CD205, CD103, IL-12 and TGF- ⁇ , which are labeled with 5 different fluorescent pigments.
  • Dendritic cell test kits currently on the market can only analyze data on overall dendritic cells in general and do not include functional analysis.
  • the CD303 + DC phenotype and function analysis kit and method of the present application are aimed at the recently reported human peripheral blood CD303 + dendritic cell subsets, adding function-related cytokines (IL-12 and TGF- ⁇ ), which can Provide a full set of data on the latest CD303 + DC subsets and their functions that have been reported in human peripheral blood.
  • the present application provides a method for identifying the phenotype and function of CD303 + dendritic cell subsets, the method is carried out by using the antibody combination formula as described in the first aspect or the kit as described in the third aspect Detect, said method comprises:
  • the resulting cells were permeabilized, mixed with IL-12 antibodies and TGF- ⁇ antibodies labeled with different fluorochromes, and incubated again.
  • the method includes the following steps:
  • step (2) staining the cells obtained in step (1), adding different fluorochrome-labeled CD205 antibodies, CD103 antibodies and CD303 antibodies for first incubation, staining again, and then fixing the obtained cells with formalin solution;
  • step (3) Resuspend the cells obtained in step (2) in the cell penetration solution, and after centrifugation, resuspend the precipitated cells in the cell penetration solution again, add IL-12 antibodies and TGF- ⁇ antibodies labeled with different fluorescent dyes, carry out re-incubation;
  • step (3) Resuspend the cells obtained in step (3) in the cell penetration solution, centrifuge and resuspend the precipitated cells in the cell staining solution, analyze and detect with flow cytometry, and obtain the CD303 + dendritic cell subpopulation Phenotype and function.
  • the method described in this application uses whole blood to determine human peripheral blood dendritic cell subsets and their functions in one step, which is simpler and easier than the previous cumbersome steps of using isolated peripheral blood mononuclear cells (PBMC) to measure DC Much, save a lot of manpower, material resources and financial resources.
  • Isolation of dendritic cells using the traditional PBMC method requires a large blood collection volume, usually ranging from tens of milliliters, and takes a long time.
  • only one drop of blood (10-100 ⁇ L) is used for the determination of the patient.
  • the full set of information we need can be obtained, which saves a lot of time for separating PBMCs. It is simple and can be quickly determined in one step. It is suitable for clinical testing of large batches of samples.
  • the volume of peripheral blood in step (1) is 10-100 ⁇ L, for example, 10 ⁇ L, 20 ⁇ L, 30 ⁇ L, 40 ⁇ L, 50 ⁇ L, 60 ⁇ L, 70 ⁇ L, 80 ⁇ L, 90 ⁇ L or 100 ⁇ L.
  • the volume concentration of the leukocyte stimulating factor in step (1) is 0.08-0.1%, such as 0.08%, 0.082%, 0.084%, 0.085%, 0.086%, 0.088% or 0.1%.
  • the first incubation time in step (2) is 20-60 min, for example, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min, etc., preferably 25-35 min.
  • the first incubation temperature in step (2) is 16-28°C, such as 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C °C, 26°C, 27°C or 28°C etc.
  • the mass fraction of the formalin solution in step (2) is 2 to 4%, such as 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, 3.2%, 3.5%, 3.6%, 3.8% or 4%, etc.
  • the condition for re-incubation in step (3) is to incubate at 4°C in the dark for 10-15h (such as 10h, 11h, 12h, 13h, 14h or 15h, etc.) or 16-28°C (such as 16°C, 18°C, 20°C, 22°C, 24°C, 25°C or 28°C, etc.) and incubate in the dark for 20-40min (eg 20min, 22min, 25min, 28min, 30min, 35min, 38min or 40min, etc.).
  • 10-15h such as 10h, 11h, 12h, 13h, 14h or 15h, etc.
  • 16-28°C such as 16°C, 18°C, 20°C, 22°C, 24°C, 25°C or 28°C, etc.
  • 20-40min eg 20min, 22min, 25min, 28min, 30min, 35min, 38min or 40min, etc.
  • the expression of CD303 molecules is obtained by CD303 antibody, and the proportion of dendritic cell subpopulation phenotype CD303 + is analyzed;
  • CD103 molecule and CD205 molecule were obtained by CD103 antibody and CD205 antibody, and the differentiation and maturation of CD303 + dendritic cell subsets were analyzed;
  • the secretion and expression of IL-12 molecules and TGF- ⁇ molecules were obtained by IL-12 antibody and TGF- ⁇ antibody, and the function of CD303 + dendritic cell subsets was analyzed.
  • the method specifically includes the following steps:
  • step (2) Incubate the cells obtained in step (1) with CD103, CD205 and CD303 antibodies labeled with different fluorochromes, incubate at 16-28°C for 25-35 min, and stain again. Fix with 4% formalin solution, and incubate at 16-28°C in the dark for 15-20 minutes;
  • step (3) Resuspend the cells obtained in step (2) in the cell penetration solution, centrifuge and discard the supernatant, resuspend the precipitated cells in the cell penetration solution, add IL-12 and TGF- ⁇ labeled with different fluorescent dyes Antibody, incubate at 4°C for 10-15 hours in the dark;
  • step (3) Resuspend the cells incubated in step (3) in the cell penetration solution, centrifuge to remove the supernatant, resuspend the precipitated cells in the cell staining solution, and analyze and detect with a flow cytometer;
  • analytical testing includes:
  • CD303 molecules The expression of CD303 molecules was obtained by CD303 antibody, and the proportion of dendritic cell subpopulation phenotype CD303 + was analyzed;
  • CD103 molecule and CD205 molecule by CD103 antibody and CD205 antibody, and analyze the differentiation and maturation status of CD303 + dendritic cell subsets;
  • the secretion and expression of IL-12 molecules and TGF- ⁇ molecules were obtained by IL-12 antibody and TGF- ⁇ antibody, and the function of CD303 + dendritic cell subsets was analyzed.
  • the present application also provides a kit as described in the third aspect or a method as described in the fourth aspect in identifying CD303 + dendritic cell subsets and/or analyzing the function of CD303 + dendritic cells Applications for non-disease diagnosis and treatment purposes.
  • Figure 1 is a graph showing the detection results of CD205 and CD103 expression levels in peripheral blood CD303 + DC subsets of healthy people and non-small cell lung cancer patients.
  • Figure 2 is a comparison chart of the expression levels of CD205 + and CD103 + in the peripheral blood CD303 + DC subsets of healthy people and patients with non-small cell lung cancer, in which the figure I is the expression level of CD205 + , and the figure II is the expression level of CD103 + .
  • Fig. 3 is a graph showing the detection results of CD303 + DC IL-12 expression in healthy people and non-small cell lung cancer patients.
  • Fig. 4 is a graph comparing the expression level of IL-12 + in peripheral blood CD303 + DC of healthy people and patients with non-small cell lung cancer.
  • Fig. 5 is a graph showing the detection results of CD303 + DC TGF- ⁇ expression in healthy people and non-small cell lung cancer patients.
  • Fig. 6 is a graph comparing the expression level of TGF- ⁇ + in peripheral blood CD303 + DCs of healthy people and patients with non-small cell lung cancer.
  • This embodiment provides a kit for identifying the phenotype and function of CD303+ dendritic cell subsets.
  • the kit includes: CD303 antibody, CD205 antibody, CD103 antibody, IL-12 antibody and TGF- ⁇ antibody; the kit also includes: red blood cell lysate, cell staining solution, cell fixation solution, and cell penetration solution.
  • the CD303 antibody was labeled with BV421
  • the CD205 antibody was labeled with BV711
  • the CD103 antibody was labeled with PerCP-Cy5.5
  • the IL-12 antibody was labeled with Pacific blue
  • the TGF- ⁇ antibody was labeled with PE-CF594.
  • This example provides a method for identifying the phenotype and function of CD303 + dendritic cell subsets.
  • BD leukocyte-stimulating factor
  • the incubated cells were suspended in 2 mL of cell permeabilization buffer, centrifuged (350 g, 5 min) twice, the supernatant was poured off, the pelleted cells were resuspended in 0.5 mL of cell staining buffer, and analyzed by flow cytometry (Cytek) test.
  • Example 2 the method provided in Example 2 was used to conduct phenotype identification and functional analysis of CD303 + dendritic cells in the peripheral blood of non-small cell lung cancer patients and healthy adults.
  • Flow cytometry was used to detect the expression of costimulatory molecules CD205 and CD103 on human peripheral blood CD303 + DC subsets (expressed as % ratio), and to evaluate the differentiation and maturation status of human peripheral blood CD303 + DC subsets.
  • the CD205 expression (mean value) on the surface of dendritic cells of healthy people is significantly less than that of patients with non-small cell lung cancer ( Figure I), and the expression of CD103 is also less than that of patients with non-small cell lung cancer, but the difference is not significant (II figure);
  • Flow cytometry was used to detect the secretion and expression of cytokines IL-12 and TGF- ⁇ in CD303 + DC (expressed as a percentage %), and to analyze the function of human peripheral blood CD303 + DC.
  • IL-12 is a cytokine that promotes immune response. If dendritic cells can secrete more IL-12, it indicates that dendritic cells can promote immune response by secreting more IL-12; the above results show that normal The IL-12 secreted by healthy CD303 + DC was significantly less than the IL-12 secreted by CD303 + dendritic cells of lung cancer patients (as shown in Figure 4);
  • CD303 + DCs in NSCLC patients have the ability to enhance the immune response by secreting IL-12, which is a sign that the immune function mediated by CD303 + DCs in NSCLC patients is different from that in healthy people.
  • TGF- ⁇ is a cytokine that can suppress immune function. If DC secretes more TGF- ⁇ , it indicates that DC has immunosuppressive effect and can suppress immune response by secreting more TGF- ⁇ .
  • CD303 + DC in patients with non-small cell lung cancer inhibits immune function by secreting more TGF- ⁇ than CD303 + DC in healthy people
  • CD303 + DC in patients with non-small cell lung cancer + DC is a kind of dendritic cell with immunosuppressive function, which may be realized by regulating the secretion of TGF- ⁇ .
  • the molecular composition and identification method of the present application can effectively identify CD303 + DC subsets in peripheral blood, and analyze their differentiation, maturation and function, and can efficiently and quickly compare the peripheral blood of patients with non-small cell lung cancer and healthy people. Developmental differentiation and functional differences of CD303 + DCs.
  • this method adopts human whole blood one-step method to determine human peripheral blood dendritic cell subsets and their functions, which is much simpler and easier than the traditional method of separating PBMCs, saves a lot of manpower, material and financial resources, and only needs a drop of blood from the patient. (10-100 ⁇ L) can get the full set of information required, saving a lot of time for separating PBMCs, simple, fast one-step determination, suitable for clinical testing of large quantities of samples.

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Abstract

本申请提供了一种分析CD303+树突状细胞亚群表型和功能的方法及试剂盒。所述试剂盒包括CD303、CD205、CD103、IL-12和TGF-β的特异性抗体组成的抗体组合配方，其检测对象包括CD303、CD205、CD103、IL-12和TGF-β。本申请的检测试剂盒可用于高效快速地鉴别外周血中CD303+树突状细胞亚群表型同时分析其功能，保证准确率的同时降低因检测大量表面抗原分子导致的经济成本，且检测方法简单易行。

Description

一种分析CD303 +树突状细胞亚群表型和功能的方法及试剂盒 技术领域
本申请属于生物技术领域，具体涉及用流式细胞光度术进行免疫检测分析人外周血树突状细胞，尤其涉及一种用于临床免疫检测分析CD303 +树突状细胞亚群表型和功能的试剂盒及应用。
背景技术
树突状细胞(Dendritic cell，DC)作为体内免疫系统中起主要调控作用的细胞，是免疫学研究热点之一。目前，树突状细胞的检测主要依靠流式细胞分析技术，但测定树突状细胞的分析方案多种多样，没有统一的标准模式。这主要是由于树突状细胞的研究日新月异，多个不同的树突状细胞亚型已被报导发现；而针对树突状细胞的流式细胞分析方案未免粗放，已不能满足目前临床针对不同亚型的树突状细胞的精确分析要求。
流式细胞仪(Flow cytometry)是对细胞进行自动分析和分选的装置，主要由四部分组成，包括流动室和液流系统、激光源和光学系统、光电管和检测系统以及计算机和分析系统。流式细胞仪可以快速测量、存贮、显示悬浮在液体中的分散细胞的一系列重要的生物物理、生物化学方面的特征参量，并可以根据预选的参量范围把指定的细胞亚群从中分选出来。多数流式细胞仪是一种零分辨率的仪器，它只能测量一个细胞的诸如总核酸量，总蛋白量等指标。
流式细胞仪可同时进行多参数测量，信息主要来自特异性荧光信号及非荧光散射信号。其常用的技术指标有荧光分辨率、荧光灵敏度、适用样品浓度、分选纯度、可分析测量参数等。
未遭受任何损坏的细胞对光线都具有特征性的散射，因此可利用不同的散射光信号对不经染色活细胞进行分析和分选。在实际使用中，仪器首先要对光散射信号进行测量。当光散射分析与荧光探针联合使用时，可鉴别出样品中被染色和未被染色细胞。光散射测量最有效的用途是从非均一的群体中鉴别出某些亚群。荧光信号主要包括两部分：①自发荧光，即不经荧光染色，细胞内部的荧光分子经光照射后所发出的荧光；②特征荧光，即由细胞经染色结合上的荧光染料受光照而发出的荧光，其荧光强度较弱，波长也与照射激光不同。自 发荧光信号为噪声信号，在多数情况下会干扰对特异荧光信号的分辨和测量。
在免疫细胞化学等测量中，对于结合水平不高的荧光抗体来说，如何提高信噪比是个关键。一般说来，细胞成分中能够产生的自发荧光的分子(例如核黄素、细胞色素等)的含量越高，自发荧光越强；培养细胞中死细胞/活细胞比例越高，自发荧光越强；细胞样品中所含亮细胞的比例越高，自发荧光越强。减少自发荧光干扰、提高信噪比的主要措施是：①尽量选用较亮的荧光染料；②选用适宜的激光和滤片光学系统；③采用电子补偿电路，将自发荧光的本底贡献予以补偿。
CD303 +树突状细胞分布于人外周血中，是近年来新发现的一个树突状细胞亚群。临床和基础研究表明，CD303 +树突状细胞亚群在多种疾病的发生中起重要作用，例如某些恶性肿瘤如肺癌，黑色素瘤，前列腺癌和肾癌，皮炎，某些病毒感染如HIV-1感染，某些传染病如疟疾感染以及一些自身免疫病如类风湿关节炎等；在这些疾病中，CD303 +树突状细胞都显现出表型和功能的异常。因此，CD303 +树突状细胞的表型和功能测定临床数据可成为临床医生判断上述疾病发展状况和临床治疗效果的辅助判断指标之一，具有十分重要的临床诊断意义。
通过流式细胞仪鉴定树突状细胞亚群通常需要分离提取外周血单核细胞，该过程复杂繁琐，时间周期长，若通过细胞分子表面抗原检测的方式分析细胞亚群则通常选择大量树突状细胞表面抗原分子进行检测，提高检测的准确性和特异性，但大量表面抗原的检测分析需要较长时间并提高了检测的经济成本，不利于快速高效地进行树突状细胞亚群分析研究。
CN105911292A公开了一种用于组合分析CD11c +CD11b +树突状细胞亚群及其分化程度和功能的试剂盒，包含以下8种抗体：CD11c、CD80、CD86、CD11b、HLA-DR、IL-12、IL-23和IL-27。该发明还提供了一种用于组合分析CD11c +CD11b +树突状细胞亚群及其分化程度和功能的方法，可一次性地检测CD11c +CD11b +树突状细胞亚群及其分化程度和功能的全套数据。但树突状细胞的形态、免疫功能不尽相同，其表面抗原分子数量众多，针对不同树突状细胞亚群需要选择不同的特异性检测分子。例如，研究表明CD11c +CD11b +DC亚群的功能与CD303 +DC亚群的功能完全不同，并且在不同的疾病中起用。因此，上述CD11c +CD11b +DC亚群检测试剂盒不能满足研究CD303 +DC亚群的需要。
有鉴于此，研发提供一种用于鉴定CD303 +树突状细胞亚群表型和功能的免疫检测试剂盒具有重要意义。
发明内容
本申请提供了一种用于分析CD303 +树突状细胞(CD303 +DC)亚群表型和功能的试剂盒及其应用。所述方法中使用的针对CD303 +DC亚群的抗体组合配方设计，可高效快速地分析外周血中CD303 +DC亚群表型及其功能，保证准确率的同时也可降低因检测大量表面抗原分子导致的成本，简单易行，准确度高。
第一方面，本申请提供一种用于分析CD303 +树突状细胞亚群表型和功能的抗体组合配方，所述分子组合配方包括：CD303抗体、CD205抗体、CD103抗体、IL-12抗体和TGF-β抗体。
由于人外周血中树突状细胞亚群种类较多，表型和功能各不相同，为了全方位的了解不同的树突状细胞，研究人员需要将各个亚型分别列出并逐一加以研究，该过程会耗费大量的人力物力。本申请中，通过选择5个分子的特异性抗体，即CD303、CD205、CD103、IL-12和TGF-β的特异性抗体，该抗体组合配方针对已被报道的人外周血CD303+树突状细胞亚群，还加入功能相关性细胞因子(IL-12和TGF-β)的抗体，可以高特异性和高灵敏度检测CD303 +DC亚群，分析细胞功能，为相关科学研究奠定基础。
第二方面，本申请提供一种抗体组合配方在分析和/或制备分析CD303 +树突状细胞亚群表型和功能的产品中的应用。
所述抗体组合配方能够用于分析CD303 +树突状细胞亚群表型和功能；所述抗体组合配方能够用于制备分析CD303 +树突状细胞亚群表型和功能的产品。
优选地，所述产品包括试剂盒和/或检测试剂。
本申请所述的抗体组合配方能够用于分析CD303 +树突状细胞亚群表型和功能，在鉴定CD303 +树突状细胞亚群表型的同时，还能对其进行功能分析，所得产品如试剂盒可以根据少量的样本(包括健康人血液样本)获取树突状细胞的全方位信息，为细胞亚型鉴定、药物研发以及疾病的预防等领域提供了方便快速的检测手段。
第三方面，本申请还提供一种分析CD303 +树突状细胞亚群表型和功能的试剂盒，所述试剂盒包括如第一方面所述的抗体组合配方；且所述抗体组合配方 由五种不同的荧光色素标记。
优选地，所述荧光色素标记选自BV421、BV711、PerCP-Cy5.5、Pacific blue、PE-CF594、FITC、PE-Cy7、Amcyan、APC-Cy7或Q-Dot中的任意五种，优选为BV421、BV711、PerCP-Cy5.5、Pacific blue和PE-CF594。
本申请所述试剂盒包括CD303、CD205、CD103、IL-12和TGF-β的抗体，所述抗体由5种不同的荧光色素标记。
目前市场上出售的关于树突状细胞测试的试剂盒只能笼统地分析总体树突状细胞的数据，且不包括功能分析。本申请的CD303 +DC表型及功能分析试剂盒和方法针对新近已被报道的人外周血CD303 +树突状细胞亚群，加入功能相关性细胞因子(IL-12和TGF-β)，可以精细地提供人外周血已被报道的最新的CD303 +DC亚群及其功能的全套数据。
第四方面，本申请提供一种鉴定CD303 +树突状细胞亚群表型和功能的方法，所述方法采用如第一方面所述的抗体组合配方或如第三方面所述的试剂盒进行检测，所述方法包括：
将待测细胞与不同荧光色素标记的CD205抗体、CD103抗体和CD303抗体混合，进行首次孵育，染色，固定；以及
将所得细胞进行透化处理，再与不同荧光色素标记的IL-12抗体和TGF-β抗体混合，进行再次孵育。
作为本申请优选的技术方案，所述方法包括如下步骤：
(1)预处理外周血，分离得到树突状细胞后加入白细胞刺激因子；
(2)将步骤(1)得到的细胞染色，加入不同荧光色素标记的CD205抗体、CD103抗体和CD303抗体进行首次孵育，再次染色，而后将得到的细胞用福尔马林溶液固定；
(3)将步骤(2)所得的细胞重悬于细胞穿透液，离心后将沉淀的细胞再次重悬于细胞穿透液，加入不同荧光色素标记的IL-12抗体和TGF-β抗体，进行再次孵育；
(4)将步骤(3)所得的细胞重悬于细胞穿透液中，离心后将沉淀细胞重悬于细胞染色液，用流式细胞仪进行分析检测，得到CD303 +树突状细胞亚群表型和功能。
本申请所述的方法采用全血一步法测定人外周血树突状细胞亚群及其功 能，较之先前用分离外周血单核细胞(PBMC)测定DC的繁琐步骤来说，要简便易行得多，节约大量人力、物力和财力。用传统的PBMC法分离树突状细胞，需要较大的采血量，通常几十毫升不等，消耗时间长，而本申请中，采用全血测定，只需患者一滴血(10～100μL)即可得到我们所需的全套信息，省却了分离PBMC的大量时间，做到简单，快速一步测定到位，适合临床大批量样品的测试。
优选地，步骤(1)所述外周血的体积为10～100μL，例如可以是10μL、20μL、30μL、40μL、50μL、60μL、70μL、80μL、90μL或100μL。
优选地，步骤(1)所述白细胞刺激因子的体积浓度为0.08～0.1％，例如可以是0.08％、0.082％、0.084％、0.085％、0.086％、0.088％或0.1％等。
优选地，步骤(2)所述首次孵育的时间为20～60min，例如可以是20min、25min、30min、35min、40min、45min、50min、55min或60min等，优选为25～35min。
优选地，步骤(2)所述首次孵育的温度为16～28℃，例如可以是16℃、17℃、18℃、19℃、20℃、21℃、22℃、23℃、24℃、25℃、26℃、27℃或28℃等。
优选地，步骤(2)所述福尔马林溶液的质量分数为2～4％，例如可以是2％、2.2％、2.4％、2.5％、2.6％、2.8％、3％、3.2％、3.5％、3.6％、3.8％或4％等。
优选地，步骤(3)所述再次孵育的条件为4℃避光孵育10～15h(例如10h、11h、12h、13h、14h或15h等)或16～28℃(例如16℃、18℃、20℃、22℃、24℃、25℃或28℃等)避光孵育20～40min(例如20min、22min、25min、28min、30min、35min、38min或40min等)。
优选地，通过CD303抗体得到CD303分子的表达情况，分析树突状细胞亚群表型CD303 +的比例；
通过CD103抗体和CD205抗体得到CD103分子和CD205分子的表达情况，分析CD303 +树突状细胞亚群的分化成熟状况；
通过IL-12抗体和TGF-β抗体得到IL-12分子和TGF-β分子的分泌表达情况，分析CD303 +树突状细胞亚群的功能。
作为本申请的优选技术方案，所述方法具体包括如下步骤：
(1)取10～100μL外周血进行抗凝处理，外周血全血混合红细胞裂解液中，旋转震荡后，静置10～15min，离心弃上清，将沉淀细胞重悬于细胞染色液中， 按照体积浓度0.08-0.1％加入白细胞刺激因子，并在30～37℃下孵育4～6h；
(2)将步骤(1)得到的细胞与不同荧光色素标记的CD103、CD205和CD303抗体混合进行孵育，16～28℃下孵育25～35min，再次染色，将得到的细胞用质量分数为2～4％的福尔马林溶液固定，16～28℃下避光孵育15～20min；
(3)将步骤(2)所得的细胞重悬于细胞穿透液中，离心弃上清，将沉淀的细胞重悬于细胞穿透液，加入不同荧光色素标记的IL-12和TGF-β抗体，在4℃避光条件下孵育10～15h；
(4)将步骤(3)孵育好的细胞重悬于细胞穿透液，离心去上清，将沉淀细胞重悬于细胞染色液，用流式细胞仪进行分析检测；
其中，分析检测包括：
通过CD303抗体得到CD303分子的表达情况，分析树突状细胞亚群表型CD303 +的比例；
通过CD103抗体和CD205抗体得到CD103分子和CD205分子的表达情况，分析CD303 +树突状细胞亚群的分化成熟状况；和
通过IL-12抗体和TGF-β抗体得到IL-12分子和TGF-β分子的分泌表达情况，分析CD303 +树突状细胞亚群的功能。
第五申请，本申请还提供一种如第三方面所述的试剂盒或如第四方面所述的方法在鉴定CD303 +树突状细胞亚群和/或分析CD303 +树突状细胞功能中的应用，所述应用为非疾病的诊断和治疗目的的应用。
本申请所述的数值范围不仅包括上述列举的点值，还包括没有列举出的上述数值范围之间的任意的点值，限于篇幅及出于简明的考虑，本申请不再穷尽列举所述范围包括的具体点值。
与现有技术相比，本申请的有益效果为：
(1)信息全面：市场上出售的关于树突状细胞测试的试剂盒只能笼统地分析总体树突状细胞的数据，且不包括功能分析，本申请设计的CD303 +树突状细胞表型及功能分析方案针对新近已被报道的人外周血CD303 +树突状细胞亚群，并加入功能相关性细胞因子(IL-12和TGF-β)，能够一次性测定CD303 +树突状细胞表型及其功能；
(2)快速简便易行且准确度高：本申请中，采用人体全血一步法测定人外周血树突状细胞亚群及其功能，相较于使用分离PBMC法测定DC，所述方法 简便易行得多，节约大量人力、物力和财力。用传统的PBMC法分离DC，需要较大的采血量，消耗时间长，而采用全血测定，只需10-100μL的血量即可得到所需的全套信息，省却了分离PBMC的时间，做到简单、快速一步测定到位，适合临床大批量样品的测试；同时，本申请所述的分析方案建立在流式细胞仪分析技术的基础之上，所得结果更加准确可靠，且灵敏度高，准确性好。
附图说明
图1为健康人与非小细胞肺癌病人外周血CD303 +DC亚群中CD205和CD103表达量的检测结果图。
图2为健康人与非小细胞肺癌病人外周血CD303 +DC亚群中CD205 +和CD103 +的表达量对比图，其中I图为CD205 +表达量，II图为CD103 +表达量。
图3为健康人与非小细胞肺癌病人CD303 +DC IL-12表达量的检测结果图。
图4为健康人与非小细胞肺癌病人外周血CD303 +DC中IL-12 +的表达量对比图。
图5为健康人与非小细胞肺癌病人CD303 +DC TGF-β表达量的检测结果图。
图6为健康人与非小细胞肺癌病人外周血CD303 +DC中TGF-β +的表达量对比图。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本申请的技术方案，但下述的实例仅仅是本申请的简易例子，并不代表或限制本申请的权利保护范围，本申请的保护范围以权利要求书为准。
以下实施例中，若无特殊说明，所用试剂及耗材均购自本领域常规试剂厂商；若无特殊说明，所用实验方法和技术手段均为本领域常规的方法和手段。
实施例1
本实施例提供一种鉴定CD303+树突状细胞亚群表型和功能的试剂盒。
所述试剂盒包括：CD303抗体、CD205抗体、CD103抗体、IL-12抗体和TGF-β抗体；所述试剂盒还包括：红细胞裂解液、细胞染色液、细胞固定液、细胞穿透液。
其中，所述CD303抗体采用BV421进行标记，CD205抗体采用BV711、CD103抗体采用PerCP-Cy5.5进行标记，IL-12抗体采用Pacific blue进行标记， TGF-β抗体采用PE-CF594进行标记。
需要注意的是，本申请中所述抗体与荧光标记的组合并不是固定的，其余组合也适用，只需CD303抗体、CD205抗体、CD103抗体、IL-12抗体和TGF-β抗体采取五种不同的荧光色素进行标记即可。
实施例2
本实施例提供一种鉴定CD303 +树突状细胞亚群表型和功能的方法。
(1)人外周血的预处理
取人静脉外周血各1滴，并进行抗凝处理，再将外周血全血混合于2mL 1×红细胞裂解液(Biolegend)中，旋转震荡10s后，室温(25℃)避光静置15min；
用离心机离心(350g，5min)，倾倒上清液，然后将沉淀细胞悬浮于2mL细胞染色液(含体积分数为2.5％胎牛血清的PBS溶液)中；
以体积浓度0.1％的比例加入白细胞刺激因子(BD)，在37℃恒温条件下孵育细胞6小时；
(2)首次孵育
血细胞离心(350g，5min)，倾倒上清液后，将血细胞悬浮于100μL细胞染色液中；
加入2μL抗人CD303抗体、CD103抗体(Biolegend)和CD205抗体(BD)，室温孵育30min；
加2mL细胞染色液，重复离心(350g，5min)两次，倾倒上清液后，用2mL 2％福尔马林溶液固定细胞，室温(25℃)避光孵育20min；
(3)再次孵育
将固定好的细胞悬浮于2mL细胞穿透液(Biolegend)中，重复离心(350g，10min)两次；
将离心后的沉淀细胞重新悬浮于100μL细胞穿透液中，各加2μL IL-12抗体(Biolegend)和TGF-β抗体(BD)，室温(25℃)避光孵育30min；
孵育好的细胞悬浮于2mL细胞穿透液中，重复离心(350g，5min)两次，倾倒上清液，将沉淀细胞重新悬浮于0.5mL细胞染色液中，用流式细胞仪(Cytek)分析测试。
实施例3
本实施例采用实施例2提供的方法对非小细胞肺癌病人和健康成年人外周血中的CD303 +树突状细胞进行表型鉴定和功能分析。
(1)人外周血CD303 +树突状细胞(CD303 +DC)亚群的分化程度分析：
采用流式细胞仪检测共信号刺激分子CD205和CD103在人外周血CD303 +DC亚群上的表达情况(用％比表示)，评估人外周血CD303 +DC亚群的分化成熟状况。
其中，健康人和非小细胞肺癌病人的CD303 +DC中CD205和CD103的表达情况见图1和图2：
由图1可知，CD205、CD103在健康人CD303 +DC上的表达比例分别为2.23％和4.93％，而CD205和CD103在非小细胞肺癌病人CD303 +DC上的表达比例分别为7.96％和5.19％；
如图2所示，健康人的树突状细胞表面CD205表达量(均值)明显少于非小细胞肺癌病人(I图)，CD103表达量也少于非小细胞肺癌病人，但是差异并不显著(II图)；
树突状细胞表面CD205和CD103共表达越高，则表明树突状细胞的免疫耐受度越高；上述结果表明，非小细胞肺癌病人体内的CD303 +DC的免疫耐受程度显著高于健康人体内的CD303 +DC。
(2)人外周血CD303+树突状细胞功能分析
采用流式细胞仪检测细胞因子IL-12和TGF-β在CD303 +DC中的分泌表达情况(用百分比％表示)，分析人外周血CD303 +DC的功能。
其中，健康人和非小细胞肺癌病人的CD303 +DC中IL-12和TGF-β的分泌表达情况见图3～图6：
由图3可知，IL-12在健康人CD303 +DC上的表达比例为4.31％，而IL-12在非小细胞肺癌病人CD303 +DC上的表达比例为26.4％；
IL-12是一个促进免疫反应的细胞因子，若树突状细胞能分泌较多的IL-12，表明树突状细胞能够通过分泌较多的IL-12来促进免疫反应；上述结果显示，正常健康人CD303 +DC分泌的IL-12显著少于肺癌病人CD303+树突状细胞分泌的IL-12(如图4)；
这表明非小细胞肺癌病人体内的CD303 +DC通过分泌IL-12来加强免疫反应的能力，这是提示非小细胞肺癌病人体内CD303 +DC介导的免疫功能有异于 健康人的一个标志。
由图5可知，TGF-β在健康人CD303 +DC上的表达比例为6.18％，而TGF-β在非小细胞肺癌病人CD303 +DC上的表达比例为23.4％，
而与此相反，TGF-β是一个能抑制免疫功能的细胞因子，DC如果分泌较多的TGF-β，表明DC具有免疫抑制功效，能够通过分泌较多的TGF-β来抑制免疫反应。上述实验证明，非小细胞肺癌病人体内的CD303 +DC与正常健康人的CD303 +DC分泌的TGF-β数量相比显著升高(如图6)。
这表明非小细胞肺癌病人体内的CD303 +DC比健康人的CD303 +DC通过分泌更多的TGF-β来抑制免疫功能，非小细胞肺癌病人体内的CD303 +DC相较于健康人体内的CD303 +DC是一种具有免疫抑制功能的树突状细胞，可能是通过调控TGF-β的分泌量来实现的。
综上所述，本申请的分子组合物以及鉴定方法可以有效鉴别外周血中的CD303 +DC亚群，并分析其分化成熟情况以及功能，能够高效快速比较非小细胞肺癌病人与健康人外周血CD303 +DC的发育分化差异以及功能差异。
总之，本方法采用人体全血一步法测定人外周血树突状细胞亚群及其功能，较传统的分离PBMC法简便易行得多，节约了大量人力、物力和财力，只需患者一滴血(10-100μL)即可得到所需的全套信息，省却了分离PBMC的大量时间，做到简单，快速一步测定到位，适合临床大批量样品的测试。
申请人声明，以上所述仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到的变化或替换，均落在本申请的保护范围和公开范围之内。

Claims (11)

  1. 一种用于分析CD303 +树突状细胞亚群表型和功能的抗体组合配方，其包括：CD303抗体、CD205抗体、CD103抗体、IL-12抗体和TGF-β抗体。
  2. 如权利要求1所述的抗体组合配方在分析CD303 +树突状细胞亚群表型和功能或制备分析CD303 +树突状细胞亚群表型和功能的产品中的应用。
  3. 根据权利要求2所述的应用，其中，所述产品包括试剂盒和/或检测试剂。
  4. 一种分析CD303 +树突状细胞亚群表型和功能的试剂盒，其包括如权利要求1所述的抗体组合配方；
    其中，所述抗体组合配方由五种不同的荧光色素进行标记。
  5. 根据权利要求4所述的试剂盒，其中，所述荧光色素选自BV421、BV711、PerCP-Cy5.5、Pacific blue、PE-CF594、FITC、PE-Cy7、Amcyan、APC-Cy7或Q-Dot中的任意五种，优选为BV421、BV711、PerCP-Cy5.5、Pacific blue和PE-CF594。
  6. 一种鉴定CD303 +树突状细胞亚群表型和功能的方法，其采用如权利要求1所述的抗体组合配方或如权利要求4或5所述的试剂盒进行检测，所述方法包括：
    将待测细胞与不同荧光色素标记的CD205抗体、CD103抗体和CD303抗体混合，进行首次孵育，染色，固定；以及
    将所得细胞进行透化处理，再与不同荧光色素标记的IL-12抗体和TGF-β抗体混合，进行再次孵育。
  7. 根据权利要求6所述的方法，其中，所述方法包括如下步骤：
    (1)预处理外周血，分离得到树突状细胞后加入白细胞刺激因子；
    (2)将步骤(1)得到的细胞染色，加入不同荧光色素标记的CD205抗体、CD103抗体和CD303抗体进行首次孵育，再次染色，而后将得到的细胞用福尔马林溶液固定；
    (3)将步骤(2)所得的细胞重悬于细胞穿透液，离心后将沉淀的细胞再次重悬于细胞穿透液，加入不同荧光色素标记的IL-12抗体和TGF-β抗体，进行再次孵育；
    (4)将步骤(3)所得的细胞重悬于细胞穿透液中，离心后将沉淀细胞重悬于细胞染色液，用流式细胞仪进行分析检测，得到CD303 +树突状细胞亚群表型和功能。
  8. 根据权利要求7所述的方法，其中，步骤(1)所述外周血的体积为10～100μL；
    优选地，步骤(1)所述白细胞刺激因子的体积浓度为0.08～0.1％；
    优选地，步骤(2)所述首次孵育的时间为20～60min，优选为25～35min；
    优选地，步骤(2)所述首次孵育的温度为16～28℃；
    优选地，步骤(2)所述福尔马林溶液的质量分数为2～4％；
    优选地，步骤(3)所述再次孵育的条件为4℃避光孵育10～15h或16～28℃避光孵育20～40min。
  9. 根据权利要求7或8所述的方法，其中，所述分析检测包括如下步骤：
    通过CD303抗体得到CD303分子的表达情况，分析树突状细胞亚群表型CD303 +的比例；
    通过CD103抗体和CD205抗体得到CD103分子和CD205分子的表达情况，分析CD303 +树突状细胞亚群的分化成熟状况；和
    通过IL-12抗体和TGF-β抗体得到IL-12分子和TGF-β分子的分泌表达情况，分析CD303 +树突状细胞亚群的功能。
  10. 根据权利要求6～9任一项所述的方法，其中，所述方法具体包括如下步骤：
    (1)取10～100μL外周血进行抗凝处理，外周血全血混合红细胞裂解液中，旋转震荡后，静置10～15min，离心弃上清，将沉淀细胞重悬于细胞染色液中，按照体积浓度0.08～0.1％加入白细胞刺激因子，并在30～37℃下孵育4～6h；
    (2)将步骤(1)得到的细胞与不同荧光色素标记的CD103、CD205和CD303抗体混合进行孵育，16～28℃下孵育25～35min，再次染色，将得到的细胞用质量分数为2～4％的福尔马林溶液固定，16～28℃下避光孵育15～20min；
    (3)将步骤(2)所得的细胞重悬于细胞穿透液中，离心弃上清，将沉淀的细胞重悬于细胞穿透液，加入不同荧光色素标记的IL-12和TGF-β抗体，在4℃避光条件下孵育10～15h；
    (4)将步骤(3)孵育好的细胞重悬于细胞穿透液，离心去上清，将沉淀细胞重悬于细胞染色液，用流式细胞仪进行分析检测；
    其中，分析检测包括：
    通过CD303抗体得到CD303分子的表达情况，分析树突状细胞亚群表型 CD303 +的比例；
    通过CD103抗体和CD205抗体得到CD103分子和CD205分子的表达情况，分析CD303 +树突状细胞亚群的分化成熟状况；和
    通过IL-12抗体和TGF-β抗体得到IL-12分子和TGF-β分子的分泌表达情况，分析CD303 +树突状细胞亚群的功能。
  11. 如权利要求4或5所述的试剂盒或如权利要求6～10任一项所述的方法在鉴定CD303 +树突状细胞亚群和/或分析CD303 +树突状细胞功能中的应用。
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