A method and kit for analyzing the phenotype and function of CD303+ dendritic cell subsets

Abstract

The present application provides a method and kit for analyzing the phenotype and function of CD303+ dendritic cell subsets. The kit includes an antibody combination formula composed of specific antibodies for CD303, CD205, CD103, IL-12 and TGF-β, and its detection objects include CD303, CD205, CD103, IL-12 and TGF-β. The detection kit of the present application can be used to efficiently and quickly identify the phenotype of CD303+ dendritic cell subsets in peripheral blood and analyze their functions, while ensuring accuracy and reducing the economic cost caused by detecting a large number of surface antigen molecules, and the detection method is simple and easy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to immunoassay analysis of human peripheral blood dendritic cells by flow cytometry, and more particularly to a method for clinical immunoassay analysis of CD303 + Kits and applications for phenotype and function of dendritic cell subsets. Background Art

[0002] Dendritic cells (DCs) are one of the hot topics in immunology research as they play a major regulatory role in the body's immune system. At present, the detection of DCs mainly relies on flow cytometry analysis technology, but there are many different analysis schemes for measuring DCs, and there is no unified standard model. This is mainly because the research on DCs is changing with each passing day, and many different DC subtypes have been reported; while the flow cytometric analysis scheme for DCs is rather rough and can no longer meet the current clinical requirements for accurate analysis of DCs of different subtypes.

[0003] Flow cytometer 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 cytometer can quickly measure, store and display a series of important biophysical and biochemical characteristic parameters of dispersed cells suspended in liquid, and can sort out designated cell subpopulations according to the pre-selected parameter range. Most flow cytometers are zero-resolution instruments, which can only measure indicators such as total nucleic acid content and total protein content of a cell.

[0004] Flow cytometers can measure multiple parameters simultaneously, and the information mainly comes from specific fluorescence signals and non-fluorescence scattering signals. Its commonly used technical indicators include fluorescence resolution, fluorescence sensitivity, applicable sample concentration, sorting purity, and analyzable measurement parameters.

[0005] Cells that have not suffered any damage have characteristic scattering of light, so different scattered light signals can be used to analyze and sort unstained living cells. In actual use, the instrument must first measure the light scattering signal. When light scattering analysis is used in conjunction with fluorescent probes, stained and unstained cells in the sample can be identified. The most effective use of light scattering measurement is to identify certain subpopulations from non-uniform populations. Fluorescence signals mainly include two parts: ① autofluorescence, that is, the fluorescence emitted by fluorescent molecules inside cells after irradiation without fluorescent staining; ② characteristic fluorescence, that is, the fluorescence emitted by fluorescent dyes bound to cells after irradiation, with weak fluorescence intensity and a wavelength different from the irradiating laser. Autofluorescence signals are noise signals, which in most cases will interfere with the resolution and measurement of specific fluorescence signals.

[0006] In measurements such as immunocytochemistry, for fluorescent antibodies with low binding levels, how to improve the signal-to-noise ratio is the key. Generally speaking, the higher the content of molecules that can produce autofluorescence (such as riboflavin, cytochrome, etc.) in cell components, the stronger the autofluorescence; the higher the ratio of dead cells to living cells in cultured cells, the stronger the autofluorescence; the higher the proportion of bright cells in the cell sample, the stronger the autofluorescence. The main measures to reduce autofluorescence interference and improve the signal-to-noise ratio are: ① Try to use brighter fluorescent dyes; ② Choose appropriate laser and filter optical systems; ③ Use electronic compensation circuits to compensate for the background contribution of autofluorescence.

[0007] CD303 + Dendritic cells are distributed in human peripheral blood and are a newly discovered dendritic cell subset in recent years. Clinical and basic research shows that CD303 + Dendritic cell subsets play an important role in the occurrence of many diseases, such as certain malignancies 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 phenotypic and functional abnormalities. + The clinical data of phenotypic and functional determination of dendritic cells can become one of the auxiliary judgment indicators for clinicians to judge the development of the above-mentioned diseases and the clinical treatment effect, and it has very important clinical diagnostic significance.

[0008] Identification of dendritic cell subsets by flow cytometry usually requires separation and extraction of peripheral blood mononuclear cells, a complex and time-consuming process. If cell subsets are analyzed by cell molecule surface antigen detection, a large number of dendritic cell surface antigen molecules are usually selected for detection to improve the accuracy and specificity of the detection. However, the detection and analysis of a large number of surface antigens takes a long time and increases the economic cost of the detection, which is not conducive to rapid and efficient dendritic cell subset analysis research.

[0009] CN105911292A discloses a method for combined analysis of CD11c + CD11b + A kit for dendritic cell subsets and their differentiation degree and function, comprising the following 8 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 differentiation and function can be detected by one-time detection of CD11c + CD11b + A complete set of data on dendritic cell subsets and their differentiation degree and function. However, the morphology and immune function of dendritic cells are different, and there are many surface antigen molecules. Different specific detection molecules need to be selected for different dendritic cell subsets. For example, studies have shown that CD11c + CD11b + Function of DC subsets and CD303 + DC subsets have completely different functions and are involved in different diseases. + CD11b + DC subset detection kit cannot meet the needs of studying CD303 + The requirement for DC subsets.

[0010] In view of this, a method for identifying CD303 is provided. + Immunoassay kits for detecting the phenotype and function of dendritic cell subsets are of great significance. Summary of the invention

[0011] The present application provides a method for analyzing CD303 + Dendritic cells (CD303 + Kit for detecting the phenotype and function of DC subsets and its application. + Antibody combination formulation for DC subsets is designed to efficiently and quickly analyze CD303 in peripheral blood +The DC subset phenotype and its function can ensure accuracy while reducing the cost caused by detecting a large number of surface antigen molecules. It is simple, easy and highly accurate.

[0012] In a first aspect, the present application provides a method for analyzing CD303 + An antibody combination formula for the phenotype and function of dendritic cell subsets, the molecular combination formula comprising: CD303 antibody, CD205 antibody, CD103 antibody, IL-12 antibody and TGF-β antibody.

[0013] Since there are many types of dendritic cell subpopulations in human peripheral blood, with different phenotypes and functions, in order to fully understand different dendritic cells, researchers need to list each subtype separately and study them one by one, which will consume a lot of manpower and material resources. In this application, by selecting specific antibodies for 5 molecules, namely, specific antibodies for CD303, CD205, CD103, IL-12 and TGF-β, this antibody combination formula targets the reported human peripheral blood CD303+ dendritic cell subpopulation, and also adds antibodies for function-related cytokines (IL-12 and TGF-β), which can detect CD303 with high specificity and high sensitivity. + DC subsets, analyze cell functions, and lay the foundation for related scientific research.

[0014] In a second aspect, the present application provides an antibody combination formulation for analyzing and / or preparing an analysis of CD303 + Application of products to dendritic cell subset phenotype and function.

[0015] The antibody combination formulation can be used to analyze CD303 + Dendritic cell subset phenotype and function; the antibody combination formula can be used to prepare and analyze CD303 + Products of dendritic cell subset phenotype and function.

[0016] Preferably, the product comprises a kit and / or a detection reagent.

[0017] The antibody combination formulation described in this application can be used to analyze CD303 + Dendritic cell subset phenotype and function, in the identification of CD303 + In addition to analyzing the phenotype of dendritic cell subsets, their functions can also be analyzed. The resulting products, such as test kits, can obtain comprehensive information on dendritic cells based on a small amount of samples (including blood samples from healthy people), providing a convenient and rapid detection method for cell subtype identification, drug development, and disease prevention.

[0018] In a third aspect, the present application also provides a method for analyzing CD303 +A kit for detecting the phenotype and function of dendritic cell subsets, the kit comprising the antibody combination formula as described in the first aspect; and the antibody combination formula is labeled with five different fluorescent pigments.

[0019] Preferably, the fluorescent pigment 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.

[0020] The kit described in the present application includes antibodies to CD303, CD205, CD103, IL-12 and TGF-β, and the antibodies are labeled with 5 different fluorescent pigments.

[0021] The kits for dendritic cell testing currently sold on the market can only analyze the data of overall dendritic cells in general and do not include functional analysis. + DC phenotype and function analysis kit and method are targeted at the recently reported human peripheral blood CD303 + Dendritic cell subsets, with the addition of functionally relevant cytokines (IL-12 and TGF-β), can provide the latest CD303 reported in human peripheral blood. + Comprehensive set of data on DC subsets and their functions.

[0022] In a fourth aspect, the present application provides a method for identifying CD303 + A method for detecting the phenotype and function of a dendritic cell subset, the method using the antibody combination formulation as described in the first aspect or the kit as described in the third aspect for detection, the method comprising:

[0023] Mixing the cells to be tested with CD205 antibodies, CD103 antibodies and CD303 antibodies labeled with different fluorescent pigments, performing a first incubation, staining and fixing; and

[0024] The obtained cells were permeabilized, mixed with IL-12 antibodies and TGF-β antibodies labeled with different fluorescent dyes, and incubated again.

[0025] As a preferred technical solution of the present application, the method comprises the following steps:

[0026] (1) Pre-treating peripheral blood, isolating dendritic cells, and then adding leukocyte stimulating factors;

[0027] (2) staining the cells obtained in step (1), adding CD205 antibodies, CD103 antibodies and CD303 antibodies labeled with different fluorescent pigments for a first incubation, staining again, and then fixing the obtained cells with a formalin solution;

[0028] (3) resuspending the cells obtained in step (2) in a cell penetration solution, centrifuging and resuspending the precipitated cells in the cell penetration solution again, adding IL-12 antibodies and TGF-β antibodies labeled with different fluorescent dyes, and incubating again;

[0029] (4) The cells obtained in step (3) were resuspended in a cell permeabilization solution, and after centrifugation, the precipitated cells were resuspended in a cell staining solution, and analyzed and detected by flow cytometry to obtain CD303 + Dendritic cell subset phenotype and function.

[0030] The method described in this application uses a whole blood one-step method to determine human peripheral blood dendritic cell subsets and their functions. Compared with the cumbersome steps of separating peripheral blood mononuclear cells (PBMC) to determine DC, it is much simpler and easier, saving a lot of manpower, material and financial resources. Using the traditional PBMC method to separate dendritic cells requires a large amount of blood, usually ranging from tens of milliliters, and takes a long time. In this application, whole blood determination is used, and only a drop of blood (10 to 100 μL) from the patient is needed to obtain the full set of information we need, saving a lot of time for separating PBMC, and achieving simple, fast one-step determination, which is suitable for the testing of large quantities of clinical samples.

[0031] Preferably, the volume of the peripheral blood in step (1) is 10 to 100 μL, for example, 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL or 100 μL.

[0032] Preferably, the volume concentration of the leukocyte stimulating factor in step (1) is 0.08-0.1%, for example, it can be 0.08%, 0.082%, 0.084%, 0.085%, 0.086%, 0.088% or 0.1%, etc.

[0033] Preferably, the first incubation time in step (2) is 20 to 60 min, for example, it can be 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min, etc., preferably 25 to 35 min.

[0034] Preferably, the temperature of the first incubation in step (2) is 16-28°C, for example, it can be 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C or 28°C, etc.

[0035] Preferably, the mass fraction of the formalin solution in step (2) is 2-4%, for example, it can be 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, 3.2%, 3.5%, 3.6%, 3.8% or 4%, etc.

[0036] Preferably, the conditions for the second incubation in step (3) are incubation in the dark at 4°C for 10 to 15 h (for example, 10 h, 11 h, 12 h, 13 h, 14 h or 15 h, etc.) or incubation in the dark at 16 to 28°C (for example, 16°C, 18°C, 20°C, 22°C, 24°C, 25°C or 28°C, etc.) for 20 to 40 min (for example, 20 min, 22 min, 25 min, 28 min, 30 min, 35 min, 38 min or 40 min, etc.).

[0037] Preferably, the expression of CD303 molecules is obtained by using CD303 antibodies to analyze the phenotype of dendritic cell subsets. + proportion;

[0038] The expression of CD103 and CD205 molecules was obtained by using CD103 and CD205 antibodies, and CD303 + Differentiation and maturation status of dendritic cell subsets;

[0039] The secretion and expression of IL-12 and TGF-β molecules were obtained by using IL-12 and TGF-β antibodies, and CD303 was analyzed. + Functions of dendritic cell subsets.

[0040] As a preferred technical solution of the present application, the method specifically comprises the following steps:

[0041] (1) 10-100 μL of peripheral blood was anticoagulated, the whole peripheral blood was mixed with red blood cell lysis buffer, vortexed and shaken, and then allowed to stand for 10-15 min. The supernatant was discarded after centrifugation, and the precipitated cells were resuspended in cell staining solution, and leukocyte stimulating factor was added at a volume concentration of 0.08-0.1%, and incubated at 30-37°C for 4-6 h;

[0042] (2) mixing the cells obtained in step (1) with CD103, CD205 and CD303 antibodies labeled with different fluorescent pigments and incubating them at 16 to 28° C. for 25 to 35 minutes, staining them again, fixing the obtained cells with a formalin solution having a mass fraction of 2 to 4%, and incubating them at 16 to 28° C. in the dark for 15 to 20 minutes;

[0043] (3) resuspending the cells obtained in step (2) in a cell permeabilization buffer, centrifuging and discarding the supernatant, resuspending the precipitated cells in a cell permeabilization buffer, adding IL-12 and TGF-β antibodies labeled with different fluorescent dyes, and incubating at 4° C. in the dark for 10 to 15 hours;

[0044] (4) resuspending the cells incubated in step (3) in a cell permeabilization solution, centrifuging to remove the supernatant, resuspending the precipitated cells in a cell staining solution, and analyzing and detecting using a flow cytometer;

[0045] The analysis and testing include:

[0046] The expression of CD303 molecules was obtained by CD303 antibody, and the phenotype of dendritic cell subsets was analyzed. + proportion;

[0047] The expression of CD103 and CD205 molecules was obtained by using CD103 and CD205 antibodies, and CD303 + Differentiation and maturation status of dendritic cell subsets; and

[0048] The secretion and expression of IL-12 and TGF-β molecules were obtained by using IL-12 and TGF-β antibodies, and CD303 was analyzed. + Functions of dendritic cell subsets.

[0049] The fifth application also provides a kit as described in the third aspect or a method as described in the fourth aspect for identifying CD303 + Analysis of dendritic cell subsets and / or CD303 + Application of dendritic cell functions, which is for non-disease diagnosis and treatment purposes.

[0050] The numerical range described in this application includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to limited space and for the sake of brevity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0051] Compared with the prior art, the beneficial effects of this application are:

[0052] (1) Comprehensive information: The kits for dendritic cell testing sold on the market can only analyze the data of overall dendritic cells in general and do not include functional analysis. The CD303 designed in this application + The dendritic cell phenotype and function analysis program targets the recently reported human peripheral blood CD303 + Dendritic cell subsets, and the addition of functionally relevant cytokines (IL-12 and TGF-β), can be measured at one time CD303 +Dendritic cell phenotype and its function;

[0053] (2) Rapid, simple, easy and highly accurate: In this application, a one-step method using whole human blood is used to determine human peripheral blood dendritic cell subsets and their functions. Compared with the method of separating PBMC to determine DC, the method is much simpler and easier to implement, saving a lot of manpower, material resources and financial resources. The traditional PBMC method for separating DC requires a large amount of blood and takes a long time, while the whole blood method only requires 10-100 μL of blood to obtain the required full set of information, saving the time of separating PBMC, and achieving a simple, rapid and one-step determination, which is suitable for the testing of large quantities of clinical samples; at the same time, the analysis scheme described in this application is based on flow cytometer analysis technology, and the results obtained are more accurate and reliable, with high sensitivity and good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 CD303 in peripheral blood of healthy subjects and patients with non-small cell lung cancer + Detection results of CD205 and CD103 expression in DC subsets.

[0055] Figure 2 CD303 in peripheral blood of healthy subjects and patients with non-small cell lung cancer + CD205 in DC subsets + and CD103 + The expression level comparison chart of CD205 + Expression level, Figure II shows CD103 + Expression amount.

[0056] Figure 3 CD303 in healthy people and patients with non-small cell lung cancer + Figure 2 shows the detection results of DC IL-12 expression.

[0057] Figure 4 CD303 in peripheral blood of healthy subjects and patients with non-small cell lung cancer + IL-12 in DCs + Expression level comparison chart.

[0058] Figure 5 CD303 in healthy people and patients with non-small cell lung cancer + Figure 2 shows the detection results of DC TGF-β expression.

[0059] Figure 6 CD303 in peripheral blood of healthy subjects and patients with non-small cell lung cancer + TGF-β in DCs + Expression level comparison chart. DETAILED DESCRIPTION

[0060] The technical solution of the present application is further explained below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are merely simple examples of the present application and do not represent or limit the scope of protection of the present application. The scope of protection of the present application shall be subject to the claims.

[0061] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0062] Example 1

[0063] This embodiment provides a kit for identifying the phenotype and function of CD303+ dendritic cell subsets.

[0064] The kit comprises: CD303 antibody, CD205 antibody, CD103 antibody, IL-12 antibody and TGF-β antibody; the kit also comprises: red blood cell lysis solution, cell staining solution, cell fixative solution and cell penetration solution.

[0065] Among them, the CD303 antibody is labeled with BV421, the CD205 antibody is labeled with BV711, the CD103 antibody is labeled with PerCP-Cy5.5, the IL-12 antibody is labeled with Pacific blue, and the TGF-β antibody is labeled with PE-CF594.

[0066] It should be noted that the combination of antibodies and fluorescent markers described in this application is not fixed, and other combinations are also applicable. It is only necessary to use five different fluorescent pigments to label CD303 antibody, CD205 antibody, CD103 antibody, IL-12 antibody and TGF-β antibody.

[0067] Example 2

[0068] This embodiment provides a method for identifying CD303 + Methods for phenotyping and function of dendritic cell subsets.

[0069] (1) Pretreatment of human peripheral blood

[0070] One drop of human venous and peripheral blood was collected and anticoagulated, and then the peripheral whole blood was mixed in 2 mL of 1× red blood cell lysis solution (Biolegend), rotated and shaken for 10 s, and then allowed to stand at room temperature (25°C) in the dark for 15 min;

[0071] Centrifuge (350 g, 5 min), pour off the supernatant, and then suspend the precipitated cells in 2 mL of cell staining solution (PBS solution containing 2.5% fetal bovine serum by volume);

[0072] Leukocyte stimulating factor (BD) was added at a volume concentration of 0.1%, and the cells were incubated at 37°C for 6 h;

[0073] (2) First incubation

[0074] The blood cells were centrifuged (350 g, 5 min), and after the supernatant was poured off, the blood cells were suspended in 100 μL of cell staining solution;

[0075] 2 μL of anti-human CD303 antibody, CD103 antibody (Biolegend), and CD205 antibody (BD) were added and incubated at room temperature for 30 min;

[0076] Add 2 mL of cell staining solution, repeat centrifugation (350 g, 5 min) twice, pour off the supernatant, fix the cells with 2 mL of 2% formalin solution, and incubate at room temperature (25°C) in the dark for 20 min;

[0077] (3) Re-incubation

[0078] The fixed cells were suspended in 2 mL of cell permeabilization buffer (Biolegend) and centrifuged (350 g, 10 min) twice;

[0079] The pelleted cells after centrifugation were resuspended in 100 μL of cell permeabilization buffer, and 2 μL of IL-12 antibody (Biolegend) and TGF-β antibody (BD) were added, respectively, and incubated at room temperature (25°C) in the dark for 30 min;

[0080] The incubated cells were suspended in 2 mL of cell penetration solution, centrifuged (350 g, 5 min) twice, the supernatant was poured out, the precipitated cells were resuspended in 0.5 mL of cell staining solution, and analyzed using a flow cytometer (Cytek).

[0081] Example 3

[0082] This example uses the method provided in Example 2 to detect CD303 in the peripheral blood of patients with non-small cell lung cancer and healthy adults. + Phenotypic characterization and functional analysis of dendritic cells.

[0083] (1) Human peripheral blood CD303 + Dendritic cells (CD303 + Analysis of differentiation degree of DC subpopulations:

[0084] Flow cytometry was used to detect the expression of co-signaling molecules CD205 and CD103 in human peripheral blood CD303 + Expression on DC subsets (expressed as %), evaluation of human peripheral blood CD303 + Differentiation and maturation status of DC subsets.

[0085] Among them, CD303 in healthy people and non-small cell lung cancer patients + The expression of CD205 and CD103 in DC is shown in Figure 1 and Figure 2 :

[0086] Depend on Figure 1 It can be seen that CD205 and CD103 are in healthy people CD303 + The expression ratios of CD205 and CD103 on DCs were 2.23% and 4.93%, respectively, while CD303 in patients with non-small cell lung cancer was significantly higher than that in patients with non-small cell lung cancer. + The expression ratios on DC were 7.96% and 5.19%, respectively;

[0087] like Figure 2 As shown, the expression level of CD205 (mean) on the surface of dendritic cells of healthy people is significantly lower than that of patients with non-small cell lung cancer (Figure I), and the expression level of CD103 is also lower than that of patients with non-small cell lung cancer, but the difference is not significant (Figure II);

[0088] The higher the co-expression of CD205 and CD103 on the surface of dendritic cells, the higher the immune tolerance of dendritic cells. The above results show that CD303 in patients with non-small cell lung cancer + The immune tolerance of DC is significantly higher than that of CD303 in healthy humans. + DC.

[0089] (2) Functional analysis of human peripheral blood CD303+ dendritic cells

[0090] Flow cytometry was used to detect the expression of cytokines IL-12 and TGF-β in CD303 + Secretory expression in DC (expressed as percentage %), analysis of human peripheral blood CD303 + Functions of DC.

[0091] Among them, CD303 in healthy people and non-small cell lung cancer patients + The secretion and expression of IL-12 and TGF-β in DCs are shown in Figure 3 to Figure 6 :

[0092] Depend on Figure 3 It can be seen that IL-12 is expressed in healthy human CD303 + The expression ratio of IL-12 on DC was 4.31%, while the expression ratio of IL-12 on DC in patients with non-small cell lung cancer was 4.31%. + The expression ratio on DC was 26.4%;

[0093] IL-12 is a cytokine that promotes immune response. If dendritic cells can secrete more IL-12, it means that dendritic cells can promote immune response by secreting more IL-12. The above results show that normal healthy human CD303 + The IL-12 secreted by DCs is significantly less than that secreted by CD303+ dendritic cells in lung cancer patients (e.g. Figure 4 );

[0094] This suggests that CD303 in NSCLC patients + DCs can enhance immune response by secreting IL-12, which indicates that CD303 in NSCLC patients + DC-mediated immune function is different from that of healthy people.

[0095] Depend on Figure 5 It is known that TGF-β in healthy human CD303 + The expression ratio of TGF-β on DC was 6.18%, while the expression ratio of TGF-β on DC in patients with non-small cell lung cancer was 6.18%. + The expression ratio on DC was 23.4%.

[0096] On the contrary, TGF-β is a cytokine that can suppress immune function. If DC secretes more TGF-β, it indicates that DC has immunosuppressive effect and can suppress immune response by secreting more TGF-β. + DC and CD303 of normal healthy people + The amount of TGF-β secreted by DC was significantly increased compared with that in Figure 6 ).

[0097] This suggests that CD303 in NSCLC patients + DCs are higher than CD303 in healthy people + DCs suppress immune function by secreting more TGF-β, and CD303 in patients with non-small cell lung cancer + DCs compared to CD303 in healthy humans + DC is a dendritic cell with immunosuppressive function, which may be achieved by regulating the secretion of TGF-β.

[0098] In summary, the molecular composition and identification method of the present application can effectively identify CD303 in peripheral blood + DC subsets, and analyze their differentiation, maturation and function, can efficiently and quickly compare the peripheral blood CD303 of non-small cell lung cancer patients and healthy people + Differences in development, differentiation and function of DCs.

[0099] In summary, this method uses human whole blood in a one-step method to determine human peripheral blood dendritic cell subsets and their functions. It is much simpler and easier than the traditional PBMC isolation method, saving a lot of manpower, material and financial resources. Only one drop of blood (10-100 μL) from the patient is needed to obtain the required full set of information, saving a lot of time for isolating PBMCs, and achieving simple and rapid one-step determination, which is suitable for testing large quantities of clinical samples.

[0100] The applicant declares that the above is only a specific implementation method of the present application, but the protection scope of the present application is not limited thereto. Technical personnel in the relevant technical field should understand that any changes or substitutions that can be easily thought of by technical personnel in the relevant technical field within the technical scope disclosed in the present application are within the protection scope and disclosure scope of the present application.

Claims

1. A method for analyzing CD303 + Antibody combination formulation for phenotype and function of dendritic cell subsets in the analysis of CD303 in peripheral blood of patients with non-small cell lung cancer + Analysis of phenotype and function of dendritic cell subsets or preparation of CD303 in peripheral blood of patients with non-small cell lung cancer + Application in products for the phenotype and function of dendritic cell subsets, wherein the antibody combination formula is: CD303 antibody, CD205 antibody, CD103 antibody, IL-12 antibody and TGF-β antibody; The analysis of CD303 + The phenotypes and functions of dendritic cell subsets are: The expression of CD303 molecules was obtained by CD303 antibody, and the phenotype of dendritic cell subsets was analyzed. + proportion; The expression of CD103 and CD205 molecules was obtained by using CD103 and CD205 antibodies, and CD303 + Differentiation and maturation status of dendritic cell subsets; The secretion and expression of IL-12 and TGF-β molecules were obtained by using IL-12 and TGF-β antibodies, and CD303 was analyzed. + Functions of dendritic cell subsets.

2. The use according to claim 1, wherein: The product includes a kit and / or a detection reagent.

3. The use according to claim 1, wherein: The antibody combination formula is labeled with five different fluorescent pigments; the fluorescent pigments are selected from any five of BV421, BV711, PerCP-Cy5.5, Pacific blue, PE-CF594, FITC, PE-Cy7, Amcyan, APC-Cy7 or Q-Dot.

4. The use according to claim 3, wherein: The fluorescent pigments are BV421, BV711, PerCP-Cy5.5, Pacific blue and PE-CF594.

5. A method for identifying CD303 in peripheral blood of patients with non-small cell lung cancer + A method for detecting the phenotype and function of dendritic cell subsets, which is performed using the antibody combination formulation in the application of claim 1, the method comprising: (1) Pre-treat peripheral blood, separate dendritic cells, and then add leukocyte stimulating factors; (2) staining the cells obtained in step (1), adding CD205 antibodies, CD103 antibodies and CD303 antibodies labeled with different fluorescent pigments for a first incubation, staining again, and then fixing the obtained cells with a formalin solution; (3) resuspending the cells obtained in step (2) in a cell permeabilization solution, centrifuging and resuspending the precipitated cells in the cell permeabilization solution again, adding IL-12 antibodies and TGF-β antibodies labeled with different fluorescent dyes, and incubating again; (4) The cells obtained in step (3) were resuspended in a cell permeabilization solution, and after centrifugation, the precipitated cells were resuspended in a cell staining solution, and analyzed and detected by flow cytometry to obtain CD303 + Dendritic cell subset phenotype and function; The analysis and testing include: The expression of CD303 molecules was obtained by CD303 antibody, and the phenotype of dendritic cell subsets was analyzed. + proportion; The expression of CD103 and CD205 molecules was obtained by using CD103 and CD205 antibodies, and CD303 + Differentiation and maturation status of dendritic cell subsets; and The secretion and expression of IL-12 and TGF-β molecules were obtained by using IL-12 and TGF-β antibodies, and CD303 was analyzed. + Functions of dendritic cell subsets.

6. The method according to claim 5, wherein: The volume of the peripheral blood in step (1) is 10-100 μL.

7. The method according to claim 5, wherein: The volume concentration of the leukocyte stimulating factor in step (1) is 0.08-0.1%.

8. The method according to claim 5, wherein: The first incubation time in step (2) is 20 to 60 min.

9. The method according to claim 8, wherein: The first incubation time in step (2) is 25 to 35 minutes.

10. The method according to claim 5, wherein: The temperature of the first incubation in step (2) is 16-28°C.

11. The method according to claim 5, wherein: The mass fraction of the formalin solution in step (2) is 2-4%.

12. The method according to claim 5, wherein: The conditions for the second incubation in step (3) are incubation at 4°C in the dark for 10-15 h or incubation at 16-28°C in the dark for 20-40 min.

13. The method according to claim 5, wherein: The method specifically comprises the following steps: (1) Take 10-100 μL of peripheral blood for anticoagulation treatment, mix the peripheral blood with red blood cell lysis buffer, rotate and shake, let it stand for 10-15 minutes, centrifuge and discard the supernatant, resuspend the precipitated cells in cell staining solution, add leukocyte stimulating factor at a volume concentration of 0.08-0.1%, and incubate at 30-37°C for 4-6 hours; (2) The cells obtained in step (1) were mixed with CD103, CD205 and CD303 antibodies labeled with different fluorescent pigments and incubated at 16-28°C for 25-35 min, and then stained again. The obtained cells were fixed with a 2-4% formalin solution and incubated at 16-28°C in the dark for 15-20 min; (3) Resuspend the cells obtained in step (2) in cell permeabilization buffer, centrifuge and discard the supernatant, resuspend the precipitated cells in cell permeabilization buffer, add IL-12 and TGF-β antibodies labeled with different fluorescent dyes, and incubate at 4°C in the dark for 10 to 15 h; (4) resuspending the cells incubated in step (3) in a cell permeabilization solution, centrifuging to remove the supernatant, resuspending the precipitated cells in a cell staining solution, and analyzing and detecting using a flow cytometer; The analysis and testing include: The expression of CD303 molecules was obtained by CD303 antibody, and the phenotype of dendritic cell subsets was analyzed. + proportion; The expression of CD103 and CD205 molecules was obtained by using CD103 and CD205 antibodies, and CD303 + Differentiation and maturation status of dendritic cell subsets; and The secretion and expression of IL-12 and TGF-β molecules were obtained by using IL-12 and TGF-β antibodies, and CD303 was analyzed. + Functions of dendritic cell subsets.

14. The method according to any one of claims 5 to 13 for identifying CD303 in the peripheral blood of patients with non-small cell lung cancer + Analysis of dendritic cell subsets and / or CD303 in peripheral blood of patients with non-small cell lung cancer + Applications in dendritic cell function.

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