CN114391102A - Method and kit for analyzing phenotype and function of CD303+ dendritic cell subset - Google Patents
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Abstract
The application provides a method and a kit for analyzing phenotype and function of CD303+ dendritic cell subset. The kit comprises an antibody combination formula consisting of specific antibodies of CD303, CD205, CD103, IL-12 and TGF-beta, and detection objects of the antibody combination formula comprise CD303, CD205, CD103, IL-12 and TGF-beta. The detection kit can be used for efficiently and quickly identifying the phenotype of the CD303+ dendritic cell subset in peripheral blood and analyzing the function of the phenotype, the economic cost caused by detecting a large number of surface antigen molecules is reduced while the accuracy is ensured, and the detection method is simple and easy to implement.
Description
Technical Field
The application belongs to the technical field of biology, particularly relates to a method for analyzing human peripheral blood dendritic cells by immunoassay with flow cytometry, and particularly relates to a method for analyzing CD303 by clinical immunoassay+A kit for the phenotype and function of dendritic cell subgroups and application thereof.
Background
Dendritic Cells (DCs), which are cells that play a major regulatory role in the immune system in vivo, are one of the hot spots in immunological research. At present, the detection of dendritic cells mainly depends on a flow cytometry analysis technology, but the analysis scheme for measuring the dendritic cells is various and has no uniform standard mode. This is mainly due to the fact that the research on dendritic cells is on the rise, and a plurality of different dendritic cell subtypes have been reported to be discovered; however, the flow cytometry analysis scheme aiming at the dendritic cells is not extensive, and the current clinical accurate analysis requirement aiming at the dendritic cells of different subtypes can not be met.
Flow cytometry (Flow cytometry) is a device for automatically analyzing and sorting cells, and mainly comprises four parts, including a Flow chamber and a liquid Flow system, a laser source and an optical system, a photoelectric tube and a detection system, and a computer and an analysis system. Flow cytometers can rapidly measure, store and display a series of important biophysical and biochemical characteristic parameters of dispersed cells suspended in a liquid, and can sort out specified cell subsets according to preselected parameter ranges. Most flow cytometers are zero resolution instruments that can only measure one cell for such things as total nucleic acid, total protein, etc.
The flow cytometer can measure multiple parameters simultaneously, and the information mainly comes from specific fluorescent signals and non-fluorescent scattering signals. The common technical indexes include fluorescence resolution, fluorescence sensitivity, applicable sample concentration, sorting purity, analyzable measurement parameters and the like.
Cells that have not suffered any damage have a characteristic scatter of light, and therefore live cells that have not been stained can be analyzed and sorted using different scattered light signals. In actual use, the instrument first measures the light scattering signal. When light scattering analysis is used in conjunction with a fluorescent probe, stained and unstained cells in the sample can be identified. The most efficient use of light scattering measurements is to identify certain subpopulations from heterogeneous populations. The fluorescence signal mainly comprises two parts: autofluorescence, namely fluorescence emitted by fluorescent molecules in cells after being irradiated by light without fluorescent staining; ② characteristic fluorescence, namely fluorescence emitted by fluorescent dye on dyed and combined cells under illumination, the fluorescence intensity is weaker, and the wavelength is different from that of the illuminating laser. The autofluorescence signal is a noise signal that in most cases interferes with the resolution and measurement of specific fluorescence signals.
In the measurement of immunocytochemistry and the like, how to improve the signal-to-noise ratio is crucial for fluorescent antibodies with low binding levels. In general, the higher the content of autofluorescent molecules (e.g., riboflavin, cytochromes, etc.) that can be produced in the cell components, the stronger the autofluorescence; the higher the ratio of dead cells/live cells in the cultured cells, the stronger the autofluorescence; the higher the proportion of bright cells contained in the cell sample, the stronger the autofluorescence. The main measures for reducing the autofluorescence interference and improving the signal-to-noise ratio are as follows: selecting brighter fluorescent dye as much as possible; selecting proper laser and filter optical system; and thirdly, compensating the background contribution of the autofluorescence by adopting an electronic compensation circuit.
CD303+Dendritic cells are distributed in human peripheral blood, and are a subset of dendritic cells that have been newly discovered in recent years. Clinical and basic studies have shown that CD303+The dendritic cell subpopulation plays an important role in the development of various diseases, such as certain malignancies such as lung cancer, melanoma, prostate 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, etc.; among these diseases, CD303+Dendritic cells all exhibit phenotypic and functional abnormalities. Thus, CD303+The phenotype and function measurement clinical data of the dendritic cells can become one of auxiliary judgment indexes for a clinician to judge the development condition and the clinical treatment effect of the diseases, and have very important clinical diagnosis significance.
The identification of the dendritic cell subsets by the flow cytometry usually requires the separation and extraction of peripheral blood mononuclear cells, the process is complex and tedious, and the time period is long, if the cell subsets are analyzed by a cell molecule surface antigen detection mode, a large number of dendritic cell surface antigen molecules are usually selected for detection, so that the detection accuracy and specificity are improved, but the detection and analysis of a large number of surface antigens require a long time and improve the economic cost of detection, and the rapid and efficient analysis and research on the dendritic cell subsets are not facilitated.
CN105911292A discloses a method for combined analysis of CD11c+CD11b+A kit of dendritic cell subpopulations and their degree of differentiation 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+Method for detecting dendritic cell subgroup and its differentiation degree and function by one-time detection of CD11c+CD11b+A complete set of data on the subpopulations of dendritic cells and their degree of differentiation and function. However, dendritic cells have different morphologies and immune functions, and many surface antigen molecules are present, so that different specific detection molecules need to be selected for different dendritic cell subsets. For example, studies have shown CD11c+CD11b+Function of DC subsets and CD303+The DC subsets function quite differently and function in different diseases. Therefore, the CD11c+CD11b+DC subgroup detection kit cannot meet the requirement of researching CD303+The requirement of DC subpopulation.
In view of this, a method for identifying CD303 is provided+The immunoassay kit for the phenotype and the function of the dendritic cell subgroup has important significance.
Disclosure of Invention
The present application provides a method for analyzing CD303+Dendritic cells (CD 303)+DC) subgroup phenotype and function and application thereof. For CD303 used in the method+The antibody combination formula design of the DC subgroup can efficiently and quickly analyze CD303 in peripheral blood+The DC subgroup phenotype and the function thereof can ensure the accuracy and reduce the cost caused by detecting a large amount of surface antigen molecules, and is simple and easy to implement and high in accuracy.
In a first aspect, the present application provides a method for analyzing CD303+A combination formulation of antibodies phenotypic and functional for a subpopulation of dendritic cells, said combination formulation of molecules comprising: CD303 antibodies, CD205 antibodies, CD103 antibodies, IL-12 antibodies, and TGF- β antibodies.
Because the dendritic cell subsets in human peripheral blood are of various types and have different phenotypes and functions, researchers need to respectively list and research each subtype one by one in order to comprehensively know different dendritic cells, and the process consumes a large amount of manpower and material resources. In the present application, antibodies against the CD303+ dendritic cell subpopulation of human peripheral blood that have been reported are formulated by selecting 5 molecules of antibodies specific for CD303, CD205, CD103, IL-12 and TGF-beta, and adding antibodies to functionally related cytokines (IL-12 and TGF-beta)Can detect CD303 with high specificity and high sensitivity+The DC subgroup analyzes the cell function and lays a foundation for related scientific research.
In a second aspect, the present application provides an antibody combinatorial formulation for analysis and/or preparation of CD303+Use in products for the phenotype and function of a subpopulation of dendritic cells.
The antibody combination formula can be used for analyzing CD303+Dendritic cell subpopulation phenotype and function; the antibody combination formula can be used for preparing and analyzing CD303+Products of dendritic cell subpopulation phenotype and function.
Preferably, the product comprises a kit and/or a detection reagent.
The antibody combinatorial formulations described herein can be used to analyze CD303+Phenotype and function of dendritic cell subsets in the identification of CD303+The dendritic cell subpopulation can be subjected to functional analysis while having phenotype, and the obtained product such as a kit can obtain comprehensive information of the dendritic cells according to a small amount of samples (including blood samples of healthy people), so that a convenient and rapid detection means is provided for the fields of cell subtype identification, drug research and development, disease prevention and the like.
In a third aspect, the present application also provides an assay for CD303+A kit for the phenotype and function of a subpopulation of dendritic cells, said kit comprising an antibody combination formulation according to the first aspect; and the antibody combination formula is marked by five different fluorescent pigments.
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-CF 594.
The kit of the present application comprises antibodies to CD303, CD205, CD103, IL-12 and TGF-. beta.which are labeled with 5 different fluorescent dyes.
The kits for dendritic cell assay currently on the market can analyze only data of total dendritic cells in general and do not include functional analysis. CD303 of the present application+DC phenotypic and functional assay kits and methods for newly reported human peripheral blood CD303+The dendritic cell subpopulation, added with functionally related cytokines (IL-12 and TGF-beta), can provide the latest CD303 reported from human peripheral blood+Complete set of data for DC subsets and their functions.
In a fourth aspect, the present application provides a method for authenticating a CD303+A method of detecting the phenotype and function of a subpopulation of dendritic cells using an antibody combination formulation according to the first aspect or a kit according to the third aspect, the method comprising:
mixing the cells to be detected with CD205 antibodies, CD103 antibodies and CD303 antibodies marked by different fluorescent pigments, performing primary incubation, dyeing and fixing; and
the cells obtained were permeabilized, mixed with IL-12 antibody and TGF-beta antibody labeled with different fluorescent dyes, and incubated again.
As a preferred technical solution of the present application, the method comprises the steps of:
(1) pretreating peripheral blood, separating to obtain dendritic cells, and adding leukocyte stimulating factors;
(2) staining the cells obtained in the step (1), adding CD205 antibodies, CD103 antibodies and CD303 antibodies marked by different fluorescent pigments for primary incubation, staining again, and then fixing the obtained cells by formalin solution;
(3) resuspending the cells obtained in the step (2) in a cell penetration solution, centrifuging, then resuspending the precipitated cells in the cell penetration solution again, adding IL-12 antibodies and TGF-beta antibodies labeled by different fluorescent pigments, and incubating again;
(4) resuspending the cells obtained in the step (3) in a cell penetration solution, centrifuging, then resuspending the precipitated cells in a cell staining solution, and analyzing and detecting by using a flow cytometer to obtain CD303+Dendritic cell subpopulations are phenotypic and functional.
Compared with the prior complicated steps of measuring DC by separating Peripheral Blood Mononuclear Cells (PBMC), the method has the advantages of simplicity, convenience, practicability and great saving of manpower, material resources and financial resources. The traditional PBMC method is used for separating dendritic cells, a large blood collection amount is needed, generally dozens of milliliters of blood are different, the consumption time is long, in the application, whole blood measurement is adopted, a patient only needs one drop of blood (10-100 mu L) to obtain a whole set of information needed by the patient, a large amount of time for separating PBMC is saved, the method is simple, rapid and one-step measurement is in place, and the method is suitable for testing clinical large-batch samples.
Preferably, the volume of the peripheral blood in the step (1) is 10 to 100. mu.L, for example, 10. mu.L, 20. mu.L, 30. mu.L, 40. mu.L, 50. mu.L, 60. mu.L, 70. mu.L, 80. mu.L, 90. mu.L or 100. mu.L.
Preferably, the volume concentration of the leukocyte stimulating factor in step (1) is 0.08-0.1%, for example, 0.08%, 0.082%, 0.084%, 0.085%, 0.086%, 0.088%, or 0.1%.
Preferably, the time of the first incubation in the step (2) is 20-60 min, for example, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min or 60min, and the like, and preferably 25-35 min.
Preferably, the temperature of the first incubation in step (2) is 16-28 deg.C, such as 16 deg.C, 17 deg.C, 18 deg.C, 19 deg.C, 20 deg.C, 21 deg.C, 22 deg.C, 23 deg.C, 24 deg.C, 25 deg.C, 26 deg.C, 27 deg.C or 28 deg.C.
Preferably, the formalin solution in the step (2) has a mass fraction of 2-4%, for example, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, 3.2%, 3.5%, 3.6%, 3.8%, or 4%.
Preferably, the conditions of the re-incubation in step (3) are 4 ℃ for 10-15 h (e.g., 10h, 11h, 12h, 13h, 14h or 15h, etc.) or 16-28 ℃ (e.g., 16 ℃, 18 ℃, 20 ℃, 22 ℃, 24 ℃, 25 ℃ or 28 ℃, etc.) for 20-40 min (e.g., 20min, 22min, 25min, 28min, 30min, 35min, 38min or 40min, etc.) in the absence of light.
Preferably, the expression of CD303 molecules is obtained by CD303 antibodies, and the phenotype CD303 of the dendritic cell subpopulation is analyzed+The ratio of (A) to (B);
by CD103 and CD205 antibodiesObtaining the expression of CD103 molecule and CD205 molecule, and analyzing CD303+The differentiation maturation status of the subpopulation of dendritic cells;
obtaining the secretory expression of IL-12 molecule and TGF-beta molecule by IL-12 antibody and TGF-beta antibody, analyzing CD303+Function of the dendritic cell subpopulation.
As a preferred technical solution of the present application, the method specifically comprises the following steps:
(1) taking 10-100 mu L of peripheral blood for anticoagulation treatment, mixing the peripheral blood and whole blood into erythrocyte lysate, rotating and shaking, standing for 10-15 min, centrifuging and discarding supernatant, suspending precipitated cells into cell staining solution, adding leukocyte stimulation factor according to the volume concentration of 0.08-0.1%, and incubating for 4-6 h at 30-37 ℃;
(2) mixing the cells obtained in the step (1) with CD103, CD205 and CD303 antibodies marked by different fluorescent pigments for incubation, incubating for 25-35 min at 16-28 ℃, dyeing again, fixing the obtained cells with a formalin solution with the mass fraction of 2-4%, and incubating for 15-20 min at 16-28 ℃ in a dark place;
(3) resuspending the cells obtained in the step (2) in a cell penetration solution, centrifuging and discarding supernatant, resuspending the precipitated cells in the cell penetration solution, adding IL-12 and TGF-beta antibodies labeled by different fluorescent pigments, and incubating for 10-15 h at 4 ℃ in a dark place;
(4) resuspending the cells incubated in the step (3) in a cell penetration solution, centrifuging to remove supernatant, resuspending the precipitated cells in a cell staining solution, and analyzing and detecting by using a flow cytometer;
wherein, the analysis and detection comprises:
obtaining the expression of CD303 molecule by CD303 antibody, analyzing the phenotype CD303 of the dendritic cell subgroup+The ratio of (A) to (B);
CD103 and CD205 molecules expression status obtained by CD103 and CD205 antibodies, and CD303 is analyzed+The differentiation maturation status of the subpopulation of dendritic cells; and
obtaining the secretory expression of IL-12 molecule and TGF-beta molecule by IL-12 antibody and TGF-beta antibody, analyzing CD303+Dendritic cellsThe function of the cell subpopulation.
In a fifth application, the present application also provides a kit according to the third aspect or a method according to the fourth aspect for identifying CD303+Dendritic cell subpopulation and/or analysis of CD303+Use in dendritic cell function for non-disease diagnostic and therapeutic purposes.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, this application is not intended to be exhaustive of the specific values subsumed within the range.
Compared with the prior art, the beneficial effect of this application is:
(1) the information is comprehensive: the commercially available kit for testing dendritic cells can only analyze data of general dendritic cells in general and does not include functional analysis, and the CD303 designed by the application+Dendritic cell phenotypic and functional analysis protocol for newly reported human peripheral blood CD303+Dendritic cell subpopulation and addition of functionally relevant cytokines (IL-12 and TGF-beta) enable one-time determination of CD303+Dendritic cell phenotype and its function;
(2) quick simple accurate and high degree of accuracy: in the application, the human peripheral blood dendritic cell subset and the functions thereof are measured by adopting a human whole blood one-step method, compared with the method for measuring DC by using a PBMC separation method, the method is simple, convenient and feasible, and saves a large amount of manpower, material resources and financial resources. The traditional PBMC method is used for separating the DC, so that a large amount of blood is required, the consumption time is long, the whole set of required information can be obtained only by 10-100 mu L of blood by adopting whole blood measurement, the time for separating the PBMC is saved, the one-step measurement is simply and quickly carried out, and the method is suitable for testing clinical large-batch samples; meanwhile, the analysis scheme is established on the basis of the flow cytometer analysis technology, and the obtained result is more accurate and reliable, and the sensitivity is high and the accuracy is good.
Drawings
FIG. 1 shows the peripheral blood CD303 of healthy and non-small cell lung cancer patients+DC subsetsThe results of the detection of the expression levels of CD205 and CD 103.
FIG. 2 is the peripheral blood CD303 of healthy human and non-small cell lung cancer patients+CD205 in DC subpopulation+And CD103+Wherein the I picture is CD205+Expression level, FIG. II is CD103+The expression level.
FIG. 3 shows CD303 of healthy and non-small cell lung cancer patients+DC IL-12 expression level test results.
FIG. 4 is the peripheral blood CD303 of healthy human and non-small cell lung cancer patients+IL-12 in DC+The expression level of (2) is shown in the graph.
FIG. 5 shows CD303 of healthy and non-small cell lung cancer patients+Test results of the expression level of DC TGF-. beta.s.
FIG. 6 is the peripheral blood CD303 of healthy human and non-small cell lung cancer patients+TGF-beta in DC+The expression level of (2) is shown in the graph.
Detailed Description
The technical solutions of the present application are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present application and do not represent or limit the scope of the claims of the present application, and the scope of the present application is controlled by the claims.
In the following examples, reagents and consumables used were obtained from conventional reagent manufacturers in the field unless otherwise specified; unless otherwise indicated, all experimental methods and technical means are conventional in the art.
Example 1
This example provides a kit for identifying the phenotype and function of a subpopulation of CD303+ dendritic cells.
The kit comprises: CD303 antibodies, CD205 antibodies, CD103 antibodies, IL-12 antibodies, and TGF- β antibodies; the kit further comprises: erythrocyte lysate, cell staining solution, cell fixing solution and cell penetrating solution.
Wherein the CD303 antibody is marked by BV421, the CD205 antibody is marked by BV711, the CD103 antibody is marked by PerCP-Cy5.5, the IL-12 antibody is marked by Pacific blue, and the TGF-beta antibody is marked by PE-CF 594.
It should be noted that the combination of the antibody and the fluorescent label described in the present application is not fixed, and other combinations are also applicable, and it is only necessary to label the CD303 antibody, the CD205 antibody, the CD103 antibody, the IL-12 antibody, and the TGF- β antibody with five different fluorescent dyes.
Example 2
This example provides an authentication CD303+Methods for the phenotype and function of a subpopulation of dendritic cells.
(1) Pretreatment of human peripheral blood
Taking 1 drop of human venous peripheral blood, performing anticoagulation treatment, mixing the peripheral blood whole blood in 2mL of 1 x erythrocyte lysate (Biolegend), rotating and shaking for 10s, and standing for 15min at room temperature (25 ℃) in a dark place;
centrifuging (350g, 5min) with a centrifuge, pouring the supernatant, and suspending the precipitated cells in 2mL of cell staining solution (PBS solution containing 2.5% by volume of fetal bovine serum);
adding leukocyte stimulating factor (BD) at a volume concentration of 0.1%, and incubating the cells at a constant temperature of 37 deg.C for 6 hr;
(2) first incubation
Blood cells were centrifuged (350g, 5min), and after the supernatant was poured, the blood cells were suspended in 100. mu.L of a cell staining solution;
add 2. mu.L anti-human CD303 antibody, CD103 antibody (Biolegend) and CD205 antibody (BD) and incubate for 30min at room temperature;
adding 2mL of cell staining solution, repeatedly centrifuging (350g, 5min) twice, pouring out supernatant, fixing cells with 2mL of 2% formalin solution, and incubating at room temperature (25 ℃) in the dark for 20 min;
(3) incubating again
The fixed cells were suspended in 2mL of cell-permeabilized solution (Biolegend) and centrifuged (350g, 10min) twice;
resuspending the centrifuged precipitated cells in 100. mu.L of cell-penetrating fluid, adding 2. mu.L each of IL-12 antibody (Biolegend) and TGF-beta antibody (BD), and incubating in the dark at room temperature (25 ℃) for 30 min;
the incubated cells were suspended in 2mL of cell-permeabilized solution, centrifuged (350g, 5min) twice, the supernatant decanted, and the pelleted cells resuspended in 0.5mL of cell staining solution and assayed by flow cytometry (Cytek).
Example 3
This example used the method provided in example 2 to treat CD303 in peripheral blood of non-small cell lung cancer patients and healthy adults+The dendritic cells were phenotypically identified and functionally analyzed.
(1) Human peripheral blood CD303+Dendritic cells (CD 303)+DC) analysis of degree of differentiation of subpopulation:
flow cytometry is adopted to detect co-signal stimulating molecules CD205 and CD103 in human peripheral blood CD303+Expression (in%) on DC subsets, human peripheral blood CD303 was evaluated+Differentiation maturation status of DC subsets.
Among them, CD303 of healthy human and non-small cell lung cancer patient+Expression of CD205 and CD103 in DCs is shown in figures 1 and 2:
as can be seen from FIG. 1, CD205 and CD103 are found in CD303 of healthy people+The expression ratio on DC is 2.23% and 4.93%, respectively, while CD205 and CD103 are expressed on CD303 of non-small cell lung cancer patients+The expression ratios on DC were 7.96% and 5.19%, respectively;
as shown in FIG. 2, the expression level (mean) of CD205 on the surface of dendritic cells of healthy people is obviously less than that of non-small cell lung cancer patients (graph I), and the expression level of CD103 is also less than that of non-small cell lung cancer patients, but the difference is not significant (graph II);
the higher the co-expression of CD205 and CD103 on the surface of the dendritic cells, the higher the immune tolerance of the dendritic cells is; the above results indicate that CD303 in patients with non-small cell lung cancer+The degree of immune tolerance of DC is significantly higher than that of CD303 in healthy human body+DC。
(2) Functional analysis of human peripheral blood CD303+ dendritic cells
Detection of cytokines IL-12 and TGF-beta in CD303 by flow cytometry+Secretion expression in DCs (expressed as a percentage) human peripheral blood CD303 was analyzed+The function of DC.
Among them, CD303 of healthy human and non-small cell lung cancer patient+The secretory expression of IL-12 and TGF-. beta.in DC is shown in FIGS. 3 to 6:
as can be seen from FIG. 3, IL-12 is found in CD303 of healthy persons+The expression ratio on DC is 4.31%, while IL-12 is expressed on CD303 of non-small cell lung cancer patients+The expression ratio on DC was 26.4%;
IL-12 is a cytokine for promoting the immune response, if dendritic cells can secrete more IL-12, the dendritic cells can promote the immune response by secreting more IL-12; the above results show that normal healthy human CD303+IL-12 secreted by DCs was significantly less than IL-12 secreted by CD303+ dendritic cells in lung cancer patients (see FIG. 4);
this indicates CD303 in non-small cell lung cancer patients+The ability of DCs to boost the immune response by secreting IL-12 suggests CD303 in patients with non-small cell lung cancer+DC-mediated immune function has a sign that is different from that of healthy people.
As can be seen from FIG. 5, TGF-. beta.is found in CD303 of healthy human+The expression ratio on DC is 6.18%, while TGF-beta is expressed on CD303 of non-small cell lung cancer patient+The expression ratio on DC was 23.4%,
in contrast, TGF-beta is a cytokine that can suppress immune function, and if DC secretes a large amount of TGF-beta, DC has immunosuppressive effect and can suppress immune response by secreting a large amount of TGF-beta. The above experiments demonstrate that CD303 in patients with non-small cell lung cancer+DC and CD303 of normal healthy people+The amount of TGF-beta secreted by DC was significantly increased compared to figure 6.
This indicates CD303 in non-small cell lung cancer patients+DC vs. CD303 of healthy person+DCs suppress immune function by secreting more TGF-beta, CD303 in non-small cell lung cancer patients+DC vs CD303 in healthy humans+DC is a kind of dendritic cell with immune suppression function, and may be realized by regulating the secretion of TGF-beta.
In summary, the molecular compositions and methods of identification of the present application canEfficient discrimination of CD303 in peripheral blood+The DC subgroup is analyzed, and the differentiation and maturation conditions and functions of the DC subgroup are analyzed, so that the peripheral blood CD303 of the non-small cell lung cancer patient and the healthy human can be efficiently and quickly compared+Developmental differentiation and functional differences of DCs.
In a word, the method adopts a one-step method of human whole blood to measure the human peripheral blood dendritic cell subgroup and the functions thereof, is more simple and feasible compared with the traditional PBMC separation method, saves a large amount of manpower, material resources and financial resources, can obtain the required complete set of information only by one drop of blood (10-100 mu L) of a patient, saves a large amount of time for PBMC separation, achieves simple and rapid one-step measurement, and is suitable for testing clinical large-scale samples.
The applicant declares that the above description is only a specific embodiment of the present application, but the scope of the present application is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present disclosure fall within the scope and disclosure of the present application.
Claims (11)
1. For analyzing CD303+A combination formulation of antibodies phenotypic and functional for a subpopulation of dendritic cells comprising: CD303 antibodies, CD205 antibodies, CD103 antibodies, IL-12 antibodies, and TGF- β antibodies.
2. The antibody combinatorial formulation of claim 1 for analysis of CD303+Phenotypic and functional or preparative analysis of dendritic cell subsets CD303+Use in products for the phenotype and function of a subpopulation of dendritic cells.
3. Use according to claim 2, wherein the product comprises a kit and/or a detection reagent.
4. Analysis CD303+A kit for the phenotype and function of a subpopulation of dendritic cells comprising an antibody combinatorial formulation according to claim 1;
wherein the antibody combination formula is labeled by five different fluorescent pigments.
5. The kit according to claim 4, wherein the fluorescent pigment 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-CF 594.
6. Identification of CD303+A method of detecting a phenotype and function of a subpopulation of dendritic cells using the antibody combination formulation of claim 1 or the kit of claim 4 or 5, said method comprising:
mixing the cells to be detected with CD205 antibodies, CD103 antibodies and CD303 antibodies marked by different fluorescent pigments, performing primary incubation, dyeing and fixing; and
the cells obtained were permeabilized, mixed with IL-12 antibody and TGF-beta antibody labeled with different fluorescent dyes, and incubated again.
7. The method according to claim 6, wherein the method comprises the steps of:
(1) pretreating peripheral blood, separating to obtain dendritic cells, and adding leukocyte stimulating factors;
(2) staining the cells obtained in the step (1), adding CD205 antibodies, CD103 antibodies and CD303 antibodies marked by different fluorescent pigments for primary incubation, staining again, and then fixing the obtained cells by formalin solution;
(3) resuspending the cells obtained in the step (2) in a cell penetration solution, centrifuging, then resuspending the precipitated cells in the cell penetration solution again, adding IL-12 antibodies and TGF-beta antibodies labeled by different fluorescent pigments, and incubating again;
(4) resuspending the cells obtained in the step (3) in a cell penetration solution, centrifuging, then resuspending the precipitated cells in a cell staining solution, and analyzing and detecting by using a flow cytometer to obtain CD303+Dendritic cell subpopulations are phenotypic and functional.
8. The method according to claim 7, wherein the volume of the peripheral blood in step (1) is 10-100 μ L;
preferably, the volume concentration of the leukocyte stimulating factor in the step (1) is 0.08-0.1%;
preferably, the time for the first incubation in the step (2) is 20-60 min, preferably 25-35 min;
preferably, the temperature of the first incubation in the step (2) is 16-28 ℃;
preferably, the mass fraction of the formalin solution in the step (2) is 2-4%;
preferably, the condition of the secondary incubation in the step (3) is that the incubation is carried out for 10-15 h at 4 ℃ in the dark or for 20-40 min at 16-28 ℃ in the dark.
9. The method according to claim 7 or 8, wherein the analytical detection comprises the steps of:
obtaining the expression of CD303 molecule by CD303 antibody, analyzing the phenotype CD303 of the dendritic cell subgroup+The ratio of (A) to (B);
CD103 and CD205 molecules expression status obtained by CD103 and CD205 antibodies, and CD303 is analyzed+The differentiation maturation status of the subpopulation of dendritic cells; and
obtaining the secretory expression of IL-12 molecule and TGF-beta molecule by IL-12 antibody and TGF-beta antibody, analyzing CD303+Function of the dendritic cell subpopulation.
10. The method according to any one of claims 6 to 9, wherein the method comprises the following steps:
(1) taking 10-100 mu L of peripheral blood for anticoagulation treatment, mixing the peripheral blood and whole blood into erythrocyte lysate, rotating and shaking, standing for 10-15 min, centrifuging and discarding supernatant, suspending precipitated cells into cell staining solution, adding leukocyte stimulation factor according to the volume concentration of 0.08-0.1%, and incubating for 4-6 h at 30-37 ℃;
(2) mixing the cells obtained in the step (1) with CD103, CD205 and CD303 antibodies marked by different fluorescent pigments for incubation, incubating for 25-35 min at 16-28 ℃, dyeing again, fixing the obtained cells with a formalin solution with the mass fraction of 2-4%, and incubating for 15-20 min at 16-28 ℃ in a dark place;
(3) resuspending the cells obtained in the step (2) in a cell penetration solution, centrifuging and discarding supernatant, resuspending the precipitated cells in the cell penetration solution, adding IL-12 and TGF-beta antibodies labeled by different fluorescent pigments, and incubating for 10-15 h at 4 ℃ in a dark place;
(4) resuspending the cells incubated in the step (3) in a cell penetration solution, centrifuging to remove supernatant, resuspending the precipitated cells in a cell staining solution, and analyzing and detecting by using a flow cytometer;
wherein, the analysis and detection comprises:
obtaining the expression of CD303 molecule by CD303 antibody, analyzing the phenotype CD303 of the dendritic cell subgroup+The ratio of (A) to (B);
CD103 and CD205 molecules expression status obtained by CD103 and CD205 antibodies, and CD303 is analyzed+The differentiation maturation status of the subpopulation of dendritic cells; and
obtaining the secretory expression of IL-12 molecule and TGF-beta molecule by IL-12 antibody and TGF-beta antibody, analyzing CD303+Function of the dendritic cell subpopulation.
11. Use of a kit according to claim 4 or 5 or a method according to any one of claims 6 to 10 for identifying CD303+Dendritic cell subpopulation and/or analysis of CD303+Use in dendritic cell function.
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