CN115004032A - For detecting CD141 + Method for phenotype and function of dendritic cell subgroup and application kit thereof - Google Patents
For detecting CD141 + Method for phenotype and function of dendritic cell subgroup and application kit thereof Download PDFInfo
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Abstract
The present application provides a method for detecting CD141 + Antibody combination for phenotype and function of dendritic cell subpopulation and analysis method and application thereof, and CD141 detection method + Combinations of antibodies to the phenotype and function of a dendritic cell subpopulation include CD141 antibodies, Linage antibodies, HLA-DR antibodies, IL-10 antibodies, IL-12 antibodies, IL-23 antibodies, and IL-27 antibodies. The application also provides a method for detecting CD141 + Kit for the phenotypic and functional analysis of dendritic cell subpopulations for the efficient and rapid analysis of CD141 in peripheral blood + The dendritic cell subgroup phenotype and the function thereof, the kit has high accuracy, can reduce the economic cost generated by detecting a large amount of surface antigen molecules, and has simple and easy detection and analysis methods and important application value.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a method for detecting CD141 + Antibody combinations for phenotype and function of dendritic cell subpopulations and methods of analysis and use thereof.
Background
CD141 + Dendritic cells are distributed in human peripheral blood, and are a subgroup of dendritic cells newly found in recent years. Clinical and basic studies have shown that CD141 + The dendritic cell subpopulation plays an important role in the development of various diseases, such as malignancies (e.g., lung cancer, melanoma, prostate cancer, and renal cancer), dermatitis, viral infections (e.g., HIV-1 infection), infectious diseases (e.g., malaria), and some autoimmune diseases (e.g., rheumatoid arthritis), among which CD141 has been shown by clinical data + Dendritic cells all exhibit phenotypic and functional abnormalities. Thus CD141 + Clinical data for phenotypic and functional assays of dendritic cells can become clinicalThe bed doctor judges the development status of the disease and the auxiliary judgment index of the clinical treatment effect, and has very important clinical diagnosis significance.
The flow cytometry analysis technology has been widely used in clinical and scientific research works as the main technology of immunology. Dendritic cells are one of the hot spots in immunological research as cells playing a major role in regulating the immune system in human body. At present, the detection of dendritic cells mainly depends on a flow cytometry analysis technology, but the flow cytometry for detecting the dendritic cells has various analysis schemes, and a unified standardized mode is not provided. The research on dendritic cells is in the future, and a plurality of different dendritic cell subtypes are continuously reported to be discovered, however, the analysis scheme of a flow cytometer aiming at dendritic cell detection is relatively extensive, and the requirement of accurate analysis aiming at the different dendritic cell subtypes in clinic at present cannot be met.
CN105911292A discloses a method for combined analysis of CD11c + 、CD11b + A kit for subpopulations of dendritic cells and their degree of differentiation and function, said kit comprising the following 8 antibodies: CD11c antibody, CD80 antibody, CD86 antibody, CD11b antibody, HLA-DR antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody. The kit can detect CD11c at one time + 、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 a large number of surface antigen molecules, so that different specific molecules need to be selected for detection aiming at different dendritic cell subsets.
The study showed that CD11c + 、CD11b + Function of dendritic cell subsets and CD141 + The function of dendritic cell subsets is quite different and plays a role in different diseases. Thus, the assay CD11c + 、CD11b + The kit for researching the dendritic cell subgroup and the differentiation degree and function thereof cannot meet the requirement of researching CD141 + Requirement of dendritic cell subpopulation. Therefore, it was developed to provide a method for detecting CD141 + The immunoassay kit for the phenotype and function of the dendritic cell subpopulation has important significance.
Disclosure of Invention
The present application provides a method for detecting CD141 + Antibody combinations for phenotype and function of dendritic cell subpopulations and methods of analysis and use thereof. The detection of CD141 + Antigens detected by the combination of antibodies specific for the phenotype and function of the dendritic cell subpopulation include CD141, Linage, HLA-DR, IL-10, IL-12, IL-23, and IL-27; the present application further provides a method of detecting CD141 + Kit for the phenotyping and the function of a subpopulation of dendritic cells, which allows an efficient and rapid analysis of CD141 in peripheral blood + The dendritic cell subgroup phenotype and the function thereof, the kit has high accuracy, reduces the economic cost generated by detecting a large amount of surface antigen molecules, and has simple and easy detection and analysis methods and important application value.
In a first aspect, the present application provides a method for detecting CD141 + Combination of antibodies for the phenotype and function of a subpopulation of dendritic cells, said detection of CD141 + Antibody combinations of phenotype and function of dendritic cell subsets include CD141 antibodies, Linage antibodies, HLA-DR antibodies, IL-10 antibodies, IL-12 antibodies, IL-23 antibodies, and IL-27 antibodies.
In the prior art, peripheral blood mononuclear cells are usually required to be separated and extracted for identifying the dendritic cell subsets through a flow cytometer, the process is complex and tedious, the time period is long, if the cell subsets are analyzed through a cell molecule surface antigen detection mode, a large number of dendritic cell surface antigen molecules are usually required to be selected for detection, so that the accuracy and the specificity of detection can be improved, but the detection and analysis of a large number of surface antigens require long time, the economic cost of detection is high, and the rapid and efficient analysis and research on the dendritic cell subsets are not facilitated. The phenotype and the function of the dendritic cell subpopulation are detected by specifically selecting a combination of 7 antibody molecules, including a CD141 antibody, a Linage antibody, an HLA-DR antibody, an IL-10 antibody, an IL-12 antibody, an IL-23 antibody and an IL-27 antibody, and the formula can be designed to detect the CD141 with high specificity and high sensitivity + The phenotype and the function of the dendritic cell subgroup lay a foundation for related scientific research.
Preferably, the detection of CD141 + The antibodies in the combination of phenotypic and functional antibodies of the dendritic cell subpopulation are labeled with a fluorescent dye.
Preferably, the fluorescent pigment comprises any one of BV510, BV-786, Pacific blue, PE-Cy7, AF700, eFluor660 or PE or a combination of at least two of the above.
In a second aspect, the present application provides a method for detecting CD141 + Kit for the detection of the phenotype and function of a subpopulation of dendritic cells, said kit detecting CD141 + A kit for the phenotype and function of a subpopulation of dendritic cells comprising the detection of CD141 according to the first aspect + A combination of antibodies to the phenotype and function of a subpopulation of dendritic cells.
The currently marketed kits for dendritic cell testing can only analyze data of total dendritic cells in general and do not include functional analysis. With the development of scientific research, several new sub-populations of dendritic cells in human peripheral blood have been discovered, such as CD141 + Dendritic cells, which differ in their phenotype and function. The present application provides for detecting CD141 + Kits for the phenotypic and functional analysis of dendritic cell subsets for CD141 + The ratio of the phenotype can be to CD141 + Judging the differentiation and maturation status of dendritic cell subpopulation, and analyzing CD141 + Function of the dendritic cell subpopulation.
In a third aspect, the present application provides a method of detecting CD141 of the second aspect + A method for the analysis of the phenotype and function of a subpopulation of dendritic cells, said analysis comprising the steps of:
(1) pretreatment of peripheral blood to isolate CD141 + A dendritic cell;
(2) CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody labeled with different fluorochromes for binding to CD141 + Incubating and detecting the dendritic cells; and
(3) detection and analysis were performed using a flow cytometer.
In the application, the analysis method adopts a one-step method of human whole blood to measure the dendritic cell subset and the functions thereof in human peripheral blood, and compared with the method of measuring the dendritic cells by using a Peripheral Blood Mononuclear Cell (PBMC) separation technology, the analysis method is simple, convenient and feasible and saves the cost. The conventional PBMC separation technology is used for separating dendritic cells, and a large amount of blood is required (usually, tens of milliliters or so) and the time is long. The method adopts a one-step method for measuring the whole blood of the human body, and only one drop of blood (10-100 mu L) of the patient is needed to detect and obtain the required complete set of information. Saves a great deal of time for separating PBMC, and can simply, quickly and one-step determine in place. Is suitable for testing clinical large-batch samples.
Preferably, in step (1), the pretreatment of peripheral blood separates CD141 + The step of dendritic cells comprises:
mixing peripheral blood and erythrocyte lysate, standing in dark place, centrifuging, and separating out CD141 + Dendritic cells, the CD141 + The dendritic cells are resuspended in a cell staining solution and incubated with leukocyte stimulating factors.
In the present application, the volume of the peripheral blood is 10 to 100. mu.L, and may be, 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.
In the present application, the volume ratio of the peripheral blood to the erythrocyte lysate is (1 to 10):200, and may be, for example, 1:200, 3:200, 5:200, 7:200, 9:200, or 10: 200.
Preferably, the light-shielding standing time is 10-15 min, such as 10min, 11min, 12min, 13min, 14min or 15min, and the light-shielding standing temperature is 20-25 ℃, such as 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃ or 25 ℃.
In the present application, the centrifugal force of the centrifugation is 300-350 g, for example, 300g, 310g, 320g, 330g, 340g, or 350g, and the time of the centrifugation is 5-10 min, for example, 5min, 6min, 7min, 8min, 9min, or 10 min.
Preferably, the cell staining solution comprises PBS buffer containing fetal bovine serum.
Preferably, the leukocyte stimulating factor is CD141 + The final concentration in the dendritic cell resuspension was 0.08 to 0.1 wt%, for example, 0.08 wt%, 0.09 wt%, or 0.1 wt%, etc.
Preferably, the incubation time is 4-6 h, for example, 4h, 5h or 6h, and the incubation temperature is 35-38 ℃, for example, 35 ℃, 36 ℃, 37 ℃ or 38 ℃.
Preferably, in step (2), the CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody labeled with different fluorescent dyes are used for the CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody and IL-27 antibody + The step of incubating and detecting the dendritic cells comprises the following steps:
(a) CD141 antibody, Linage antibody and HLA-DR antibody labeled with different fluorochromes + Incubating the dendritic cells for the first time;
(b) IL-10, IL-12, IL-23 and IL-27 antibodies labeled with different fluorochromes on CD141 + The dendritic cells were incubated a second time.
Preferably, in the step (a), the CD141 antibody, the Linage antibody and the HLA-DR antibody labeled with different fluorescent dyes are used for the CD141 antibody + The step of incubating the dendritic cells for the first time comprises:
the CD141 incubated in the step (1) is added + Resuspending the dendritic cells and resuspending CD141 + Performing primary incubation on dendritic cell resuspension different fluorescent pigment labeled CD141 antibody, Linage antibody and HLA-DR antibody, and fixing CD141 with formalin solution + Dendritic cells, light-shielded incubation.
Preferably, the formalin solution has a mass fraction of 1.5 to 2.5 vol%, and may be, for example, 1.5 vol%, 1.8 vol%, 2.0 vol%, 2.2 vol%, 2.5 vol%, or the like.
Preferably, the time of the first incubation is 25-35 min, such as 25min, 28min, 30min, 32min or 35min, and the temperature of the first incubation is 20-25 ℃, such as 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃ or 25 ℃.
Preferably, the time of the light-shielding incubation is 15-20 min, for example, 15min, 16min, 17min, 18min, 19min or 20min, and the temperature of the light-shielding incubation is 20-25 ℃, for example, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃ or 25 ℃.
Preferably, in step (b), the IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody labeled with different fluorescent dyes are used for the CD141 antibody + The step of performing secondary incubation on the dendritic cells comprises:
subjecting the CD141 obtained in step (a) to + Resuspending dendritic cells with cell-penetrating fluid, centrifuging to remove supernatant, resuspending the precipitate with cell-penetrating fluid, and resuspending CD141 + The dendritic cell resuspension was incubated twice with different fluorochrome-labeled IL-10, IL-12, IL-23 and IL-27 antibodies.
Preferably, the centrifugal force of the centrifugation is 300-350 g, such as 300g, 310g, 320g, 330g, 340g or 350g, and the time of the centrifugation is 5-10 min, such as 5min, 6min, 7min, 8min, 9min or 10 min.
Preferably, the time of the secondary incubation is 24-26 h, for example, 24h, 25h or 26h, and the temperature of the secondary incubation is 0-4 ℃, for example, 0 ℃, 1 ℃, 2 ℃, 3 ℃ or 4 ℃.
Preferably, the second incubation is performed under light-shielding conditions.
Preferably, in the step (3), the step of detecting and analyzing by a flow cytometer comprises:
subjecting the twice incubated CD141 + The dendritic cells are resuspended with a cell-penetrating fluid, the supernatant is centrifuged off, the precipitated cells are resuspended with a cell staining fluid, and the cells are analyzed and detected by a flow cytometer.
Preferably, the centrifugal force of the centrifugation is 300-350 g, such as 300g, 310g, 320g, 330g, 340g or 350g, and the time of the centrifugation is 5-10 min, such as 5min, 6min, 7min, 8min, 9min or 10 min.
Preferably, the method of analysis comprises:
according to the CD141 antibody and Linage antibody with CD141 + Binding of CD141 and Linage on the surface of dendritic cells, CD141 was analyzed + Dendritic cell subgroup CD141 + The ratio of phenotypes;
according to HLA-DR antibody and CD141 + Binding of HLA-DR to the surface of dendritic cells, CD141 was analyzed + The differentiation maturation status of the subpopulation of dendritic cells; and
based on IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody and CD141 + Binding of IL-10, IL-12, IL-23 and IL-27 on the surface of dendritic cells, CD141 was analyzed + Function of the dendritic cell subpopulation.
As a preferred technical scheme of the application, the CD141 is detected + The method for analyzing the phenotype and function of the dendritic cell subpopulation comprises the following steps:
(1) pretreatment of peripheral blood to isolate CD141 + Dendritic cell:
mixing 10-100 μ L of peripheral blood with erythrocyte lysate, standing in dark at 20-25 deg.C for 10-15 min, centrifuging at 300-350 g for 5-10 min, and separating CD141 + Dendritic cells, the CD141 + The dendritic cells are resuspended in cell staining solution, and leukocyte stimulating factors are added to the cell staining solution to incubate for 4 to 6 hours at the temperature of 35 to 38 ℃, wherein the leukocyte stimulating factors are CD141 + The final concentration of the dendritic cell resuspension is 0.08-0.1%.
(2) CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody labeled with different fluorochromes for binding to CD141 + Incubation detection of dendritic cells:
(a) the CD141 incubated in the step (1) is added + Resuspending the dendritic cells and resuspending CD141 + The dendritic cell resuspension is incubated with CD141 antibodies, Linage antibodies and HLA-DR antibodies marked by different fluorescent pigments for the first time, wherein the time of incubation for the first time is 25-35 min, the temperature is 20-25 ℃, and the CD141 is incubated + The volume ratio of the dendritic cell resuspension to the CD141 antibody, the Linage antibody and the HLA-DR antibody is 100 (2-2.5): 2-2.5), and the CD141 is fixed by formalin solution with the mass fraction of 1.5-2.5 vol% + Incubating the dendritic cells in a shading mode at the temperature of 20-25 ℃ for 15-20 min;
(b) subjecting the CD141 obtained in step (a) to + Cell puncture for dendritic cellsResuspending the solution, centrifuging at 300-350 g for 5-10 min to remove supernatant, resuspending the precipitate with cell penetrating solution, and resuspending CD141 + Performing secondary incubation on the dendritic cell resuspension solution and different fluorochrome-labeled IL-10 antibodies, IL-12 antibodies, IL-23 antibodies and IL-27 antibodies to obtain CD141 + The volume ratio of the dendritic cell resuspension to the IL-10 antibody, the IL-12 antibody, the IL-23 antibody and the IL-27 antibody is 100 (2-2.5): (2-2.5): 2-2.5), the secondary incubation is carried out under the condition of shading, the time of the secondary incubation is 24-26 hours, and the temperature is 0-4 ℃.
(3) Detection and analysis with flow cytometry:
subjecting the secondarily incubated CD141 + Resuspending the dendritic cells by using a cell penetration solution, centrifuging for 5-10 min at 300-350 g to remove supernatant, resuspending the precipitated cells by using a cell staining solution, and detecting and analyzing by using a flow cytometer;
according to the CD141 antibody and Linage antibody with CD141 + Binding of CD141 and Linage on the surface of dendritic cells, CD141 was analyzed + Dendritic cell subgroup CD141 + The ratio of phenotypes;
according to HLA-DR antibody and CD141 + Binding of HLA-DR to the surface of dendritic cells, CD141 was analyzed + The differentiation maturation status of the subpopulation of dendritic cells; and
based on IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody and CD141 + Binding of IL-10, IL-12, IL-23 and IL-27 on the surface of dendritic cells, CD141 was analyzed + Function of the dendritic cell subpopulation.
In a fourth aspect, the present application provides a method for detecting CD141 + System for the detection of the phenotype and function of a subpopulation of dendritic cells, said system detecting CD141 + A system of phenotype and function of a subpopulation of dendritic cells comprising:
(1) a sample processing module: pretreatment of peripheral blood to isolate CD141 + A dendritic cell;
(2) a detection module: CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody labeled with different fluorochromes for binding to CD141 + Incubating and detecting the dendritic cells; and
(3) an analysis module: detection and analysis were performed using a flow cytometer.
In a fifth aspect, the present application provides the method for detecting CD141 of the first aspect + Combination of antibodies for the phenotype and function of a subpopulation of dendritic cells, detection of CD141 according to the second aspect + Kit for detecting the phenotype and function of a subpopulation of dendritic cells or detecting CD141 according to the fourth aspect + Characterization of CD141 by any one of, or a combination of at least two of, a system for the phenotype and function of a subpopulation of dendritic cells + Phenotypic and functional and/or preparative identification of CD141 from dendritic cell subsets + Use in products for the phenotype and function of a subpopulation of dendritic cells.
Preferably, the product comprises a kit and/or a detection reagent for detecting malignancy;
preferably, the malignancy comprises any one of, or a combination of at least two of, lung cancer, melanoma, prostate cancer, or renal cancer.
The recitation herein of ranges of values herein includes not only the recited values, but also any values within any range of values not recited above, and for the sake of brevity and clarity, this application is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the method has the following beneficial effects:
(1) detecting CD141 as described herein + The method for analyzing the phenotype and the function of the dendritic cell subgroup is simple, rapid and easy to implement. The method for measuring the dendritic cell subpopulation in the human peripheral blood by adopting the human whole blood one-step method and the function thereof is simple, convenient and feasible, and saves the cost compared with the method for measuring the dendritic cell by using the Peripheral Blood Mononuclear Cell (PBMC) separation technology. The conventional PBMC separation technology is used for separating dendritic cells, and a large amount of blood is required (usually, tens of milliliters or so) and the time is long. The method adopts a one-step method for measuring the whole blood of the human body, and only one drop of blood (10-100 mu L) of the patient is needed to detect and obtain the required complete set of information. Saves a great deal of time for separating PBMC, and can be simply, quickly and one-step measuredThe test strip is suitable for testing large-batch samples clinically.
(2) Detecting CD141 as described herein + The results of the analysis of the phenotype and function of the dendritic cell subpopulation are comprehensive. The present application is designed to analyze CD141 with high specificity and high sensitivity by specifically selecting 7 antibody molecule combinations including CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody + CD141 in dendritic cell subpopulation + The ratio of the phenotype can be to CD141 + Judging the differentiation and maturation status of dendritic cell subpopulation, and analyzing CD141 + Function of the dendritic cell subpopulation.
(3) Detecting CD141 as described herein + The accuracy of the result of the analysis method of the phenotype and the function of the dendritic cell subgroup is high. Detecting CD141 in the present application + Compared with other technologies, the analysis scheme for the phenotype and the function of the dendritic cell subgroup has the advantages of high flexibility and good specificity, and the detection result is more accurate and reliable.
(4) The assay protocol of the currently marketed dendritic cell assay kit can only test data of total dendritic cells and does not include functional analysis. Detecting CD141 as described herein + A kit for the phenotype and function of a subpopulation of dendritic cells can finely provide the most recent CD141 in human peripheral blood + Complete data set of phenotype and function of dendritic cell subpopulations, compared to previously developed protocols, this application presents for the first time CD141 + The phenotype of the dendritic cell subpopulation is tested in combination with function.
Drawings
FIG. 1 is a FSC-SSC scattergram of healthy human peripheral blood cells obtained in example 3.
FIG. 2 is a diagram of flow cytometry analysis of peripheral blood of a healthy human in example 3.
FIG. 3 is a graph showing the results of flow cytometry analysis of CD141+ dendritic cell subsets in peripheral blood of healthy human/non-small cell lung cancer patients in example 3.
FIG. 4 is a statistical graph showing HLA-DR expression of CD141+ dendritic cell subpopulation in peripheral blood of healthy human/non-small cell lung cancer patients in example 3.
FIG. 5 is a graph showing the expression ratio of IL-10 in example 3 on CD141+ dendritic cells in peripheral blood of isotype control/healthy human/lung cancer patients.
FIG. 6 is a statistical graph of IL-10 expression in peripheral blood of CD141+ dendritic cells in healthy human/lung cancer patients in example 3.
FIG. 7 is a graph showing the expression ratio of IL-12 in example 3 on CD141+ dendritic cells in peripheral blood of isotype-controlled/healthy/lung cancer patients.
FIG. 8 is a statistical graph of the expression of IL-12 in example 3 on CD141+ dendritic cells in peripheral blood of healthy human/lung cancer patients.
FIG. 9 is a graph showing the expression ratio of IL-23 in example 3 on CD141+ dendritic cells in peripheral blood of isotype-controlled/healthy human/lung cancer patients.
FIG. 10 is a statistical graph of the expression of IL-23 in example 3 on CD141+ dendritic cells in peripheral blood of healthy human/lung cancer patients.
FIG. 11 is a graph showing the expression ratio of IL-27 in example 3 on CD141+ dendritic cells in peripheral blood of an isotype-controlled/healthy human/lung cancer patient.
FIG. 12 is a statistical graph of the expression of IL-27 on CD141+ dendritic cells in peripheral blood of healthy human/lung cancer patients in example 3.
Detailed Description
The technical solution of the present application is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present application and should not be construed as a specific limitation of the present application.
The examples do not specify particular techniques or conditions, and are to be construed in accordance with the description of the art in the literature or with the specification of the product. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
The experimental materials used in the following specific embodiments are as follows:
flow cytometry (Cytek); a line antibody (Biolegend); CD141 antibody (Biolegend): HLA-DR antibody (eBioscience); an IL-10 antibody (BD); IL-12 antibody (Biolegend); IL-23 antibody (eBioscience); and IL-27 antibody (Biolegend).
Example 1
The present embodiment provides a method for detecting CD141 + Combination of antibodies for the phenotype and function of a subpopulation of dendritic cells, said detection of CD141 + Antibody combinations for phenotype and function of dendritic cell subsets include CD141 antibody (Biolegged), Link antibody (Biolegged), HLA-DR antibody (eBioscience), IL-10 antibody (BD), IL-12 antibody (Biolegged), IL-23 antibody (eBioscience), and IL-27 antibody (Biolegged).
The detection CD141 + The antibodies in the combination of phenotypic and functional antibodies of the dendritic cell subpopulation are labeled with a fluorescent dye. The fluorescent pigment of the CD141 antibody is BV 510; the fluorescent pigment of the Linage antibody is BV 786; the fluorescent pigment of the HLA-DR antibody is Pacific blue; the fluorescent dye of the IL-10 antibody is PE-Cy 7; the fluorescent dye of the IL-12 antibody is AF 700; the fluorescent pigment of the IL-23 antibody is eFluor 660; the fluorescent dye of the IL-27 antibody is PE.
Example 2
The present embodiment provides a method for detecting CD141 + Kit for the detection of the phenotype and function of a subpopulation of dendritic cells, said kit detecting CD141 + The kit for the phenotype and function of a subpopulation of dendritic cells comprises the detection of CD141 as described in example 1 + A combination of antibodies to the phenotype and function of a subpopulation of dendritic cells.
Example 3
This example used the detection of CD141 described in example 2 + Phenotypic and functional kits for dendritic cell subsets CD141 in peripheral blood of non-small cell lung cancer patients (experimental group) and healthy persons (control group) + The dendritic cell subpopulations were analyzed for differentiation and function, while isotype controls were analyzed.
(1) Pretreatment of peripheral blood to isolate CD141 + Dendritic cell:
respectively collecting non-small cell lung cancer patients and their medicinal materials100 mu L of venous peripheral blood of healthy adults and carrying out anticoagulation treatment; mixing peripheral blood whole blood with 2mL of 1 × erythrocyte lysate (Biolegend), rotating and shaking for 10s, standing for 15min at 25 ℃ in a dark place, centrifuging by using a centrifuge (350g and centrifuging for 5min), pouring supernatant, suspending precipitated cells in 2mL of cell staining solution (PBS solution containing 2.5% fetal calf serum), adding leukocyte stimulation factor (BD) to incubate cells for 6h at 37 ℃ for later use, wherein the leukocyte stimulation factor is CD141 for incubation for 6h + The final concentration in the dendritic cell resuspension was 0.1%.
(2) CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody labeled with different fluorochromes for binding to CD141 + Incubation detection of dendritic cells:
(a) the CD141 obtained in the step (1) + The dendritic cells were resuspended in 100. mu.L of cell staining solution and CD141 was added + Performing primary incubation on the dendritic cell resuspension solution, 2 mu L of BV 510-labeled CD141 antibody, 2 mu L of BV 786-labeled Link antibody and 2 mu L of Pacific blue-labeled HLA-DR antibody, wherein the primary incubation time is 30min, the temperature is 25 ℃, 2mL of cell staining solution is added, 350g of cell staining solution is centrifuged for 5min, the centrifugation is repeated twice, and 2mL of formalin solution with the mass fraction of 2% is used for fixing CD141 + Incubating the dendritic cells at 25 ℃ in shade for 20 min;
(b) subjecting the CD141 obtained in step (a) to + The dendritic cells were resuspended in 2mL of cell-permeabilizing fluid (Biolegend), centrifuged at 350g for 5min, centrifuged twice, the pellet resuspended in 100. mu.L of cell-permeabilizing fluid, CD141 + And (3) carrying out secondary incubation on the dendritic cell resuspension and 2 mu L of PE-Cy7 labeled IL-10 antibody, 2 mu L of AF700 labeled IL-12 antibody, 2 mu L of eFluor660 labeled IL-23 antibody and 2 mu L of PE labeled IL-27 antibody, wherein the secondary incubation is carried out under the condition of shading, and the time of the secondary incubation is 30min and the temperature is 25 ℃.
(3) Detection and analysis were performed using a flow cytometer.
Subjecting the twice incubated CD141 + The dendritic cells were resuspended in 2mL of cell-penetrating fluid, centrifuged at 350g for 5min to remove supernatant, the precipitated cells were resuspended in 0.5mL of cell staining fluid, and examined by flow cytometryAnd (6) measuring and analyzing.
Through the method, all information about dendritic cell subsets (characterized by the expression conditions of CD141 and Linage), differentiation degree (characterized by the expression conditions of HLA-DR) and cell functions (characterized by the expression conditions of IL-10, IL-12, IL-23 and IL-27) is obtained at one time by using a flow cytometer, and the difference between a non-small cell lung cancer patient and a healthy person can be researched through the comparison of the information, so that a basis is provided for researching the immune regulation mechanism of the non-small cell lung cancer patient.
And (3) analysis results:
(1) by CD141 antibody and Linage (Lin) antibody with CD141 + The binding condition of CD141 and Linage on the surface of the dendritic cells is analyzed, and the CD141 of the dendritic cells is analyzed + The ratio of the phenotypes. The detection results are shown in FIG. 1, in which FIG. 1 is a FSC-SSC scattergram of peripheral blood cells of a healthy person, FSC represents the size of the cells, SSC represents the granularity of the cells; FIG. 2 is a diagram showing flow cytometry analysis of healthy human peripheral blood, and the left diagram is a diagram showing the expression ratio of Linage in dendritic cells in healthy human peripheral blood - (Lin - ) 12.9% of the total cell count, and the right graph is a graph showing the expression ratio of CD141 in dendritic cells in peripheral blood of a healthy person, CD141 + Accounting for 41.9 percent of the total cell number.
(2) Detection of CD141 of co-signal stimulating molecule HLA-DR in human peripheral blood by flow cytometry + Expression status on a subpopulation of dendritic cells, this data being used to assess CD141 in human peripheral blood + Differentiation maturation of dendritic cell subpopulations. FIG. 3 is CD141 in peripheral blood of healthy/non-small cell lung cancer patients + FIG. 4 is a graph showing the result of flow cytometry analysis of dendritic cell subsets, and FIG. 4 is CD141 in peripheral blood of healthy human/non-small cell lung cancer patients + Statistical analysis of HLA-DR expression of dendritic cell subsets, wherein the bold line in FIG. 3 indicates healthy individuals and the dashed line indicates non-small cell lung cancer patients, revealed that CD141 + The higher the HLA-DR co-expression on the surface of dendritic cells, the higher CD141 + The higher the degree of immune maturation of dendritic cells, the more healthy human CD141 can be seen from the results of FIGS. 3 and 4 + HLA-DR on dendritic cell surfaceCo-expression is significantly greater in lung cancer patients; indicating CD141 in lung cancer patients + Dendritic cells are significantly less immune mature than healthy humans.
(3) Determination of CD141 by IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody + Secretion expression of IL-10, IL-12, IL-23 and IL-27 on the surface of dendritic cells, CD141 was analyzed + Function of the dendritic cell subpopulation.
FIG. 5 is a graph of IL-10 CD141 in peripheral blood of isotype control/healthy human/lung cancer patients + FIG. 6 is a graph showing an expression ratio on dendritic cells, and CD141 of IL-10 in peripheral blood of healthy human/lung cancer patients + A statistical map of expression on dendritic cells; IL-10 has an inhibitory effect on immune function in vivo, and the results of FIGS. 5 and 6 show that CD141 is present in patients with non-small cell lung cancer + Dendritic cells are more than CD141 of healthy people + The dendritic cells secreted more IL-10, and the results indicated that CD141 was present in patients with non-small cell lung cancer + Dendritic cells suppress the anti-tumor immune response in vivo by secreting more IL-10.
FIG. 7 is a graph of IL-12 CD141 in peripheral blood of isotype control/healthy human/lung cancer patients + FIG. 8 is a graph of the expression ratio on dendritic cells, showing CD141 of IL-12 in peripheral blood of healthy human/lung cancer patients + Statistical map of expression on dendritic cells. IL-12 is a cytokine that promotes immune response, and the results in FIGS. 7 and 8 show that CD141 is present in patients with non-small cell lung cancer + Dendritic cells are more than CD141 of healthy people + The dendritic cells secreted less IL-12, and the results indicated that CD141 was present in patients with non-small cell lung cancer + Dendritic cells secrete insufficient amounts of IL-12, which likely affects IL-12-mediated anti-tumor immune responses.
FIG. 9 is a photograph of the CD141 of IL-23 in the peripheral blood of isotype control/healthy person/lung cancer patients + FIG. 10 is a graph showing an expression ratio on dendritic cells, and CD141 of IL-23 in peripheral blood of healthy human/lung cancer patients + Statistical plot of expression on dendritic cells, FIG. 11 is CD141 of IL-27 in peripheral blood of isotype controlled/healthy/lung cancer patients + FIG. 12 is a graph of the expression ratio of IL-27 on dendritic cells, CD141 in peripheral blood of healthy human/lung cancer patients + Statistical profiles of expression on dendritic cells. From the figure, it can be seen that IL-23 and IL-27 are present in CD141 of healthy persons + The expression ratio on dendritic cells was 17.7% and 36.6%, respectively; while IL-23 and IL-27 are found in CD141 of patients with non-small cell lung cancer + The expression ratio on dendritic cells was 22.2% and 38.8%, respectively.
IL-23 is a cytokine that promotes immune response, if dendritic cells secrete more IL-23, indicating that dendritic cells can promote immune response by secreting more IL-23, the results in FIGS. 9 and 10 indicate that CD141 is a normal healthy human + IL-23 secreted by dendritic cells and CD141 of lung cancer patients + Similar IL-23 secretion by dendritic cells indicates that CD141 is present in lung cancer patients + Dendritic cells do not affect CD141 by modulating IL-23 + DC-mediated immune responses.
IL-27 is a cytokine that can suppress immune function, and if dendritic cells secrete more IL-27, it indicates that dendritic cells have immunosuppressive effects, and dendritic cells can suppress immune response by secreting more IL-27. The results of the tests in FIGS. 11 and 12 indicate that CD141 is present in lung cancer patients + Dendritic cell and CD141 of normal healthy human + There was no significant difference in the amount of IL-27 secreted by dendritic cells, indicating that CD141 was present in lung cancer patients + Dendritic cells do not suppress immune function by secreting more IL-27, CD141 in lung cancer patients + Dendritic cells compared to CD141 in healthy humans + The dendritic cell is a dendritic cell with an immunosuppressive function.
The above results indicate that the antibody molecule combination and analysis method of the present application can effectively identify CD141 in peripheral blood + Dendritic cell subpopulations and analyzed for differentiation maturation and function.
In summary, the present application specifically selects 7 combinations of antibody molecules, including CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody, which can be used for analyzing CD141 with high specificity and high sensitivity + CD141 in dendritic cell subpopulation + Ratio of phenotype to CD141 + Judging the differentiation and maturation status of dendritic cell subpopulation, and analyzing CD141 + Function of the dendritic cell subpopulation.
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. Detect CD141 + Combinations of antibodies to the phenotype and function of a dendritic cell subpopulation comprising a CD141 antibody, a Linage antibody, an HLA-DR antibody, an IL-10 antibody, an IL-12 antibody, an IL-23 antibody, and an IL-27 antibody.
2. Detecting CD141 according to claim 1 + Combination of antibodies for the phenotype and function of a subpopulation of dendritic cells, wherein said detection of CD141 + The antibodies in the combination of antibodies for the phenotype and function of the dendritic cell subpopulation are labeled with a fluorescent dye.
3. Detecting CD141 according to claim 2 + A combination of antibodies to the phenotype and function of a subpopulation of dendritic cells, wherein said fluorescent pigment comprises any one or a combination of at least two of BV510, BV-786, Pacific blue, PE-Cy7, AF700, eFluor660 or PE.
4. Detect CD141 + Kit for the detection of the phenotype and function of a subpopulation of dendritic cells comprising the detection of CD141 according to any one of claims 1 to 3 + A combination of antibodies to the phenotype and function of a subpopulation of dendritic cells.
5. Detect CD141 + DendriteA method for analyzing phenotype and function of a sub-population of somatic cells, comprising the steps of:
(1) pretreatment of peripheral blood to isolate CD141 + A dendritic cell;
(2) CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody labeled with different fluorochromes for binding to CD141 + Incubating and detecting the dendritic cells; and
(3) detection and analysis were performed using a flow cytometer.
6. Detecting CD141 as claimed in claim 5 + Method for analyzing phenotype and function of dendritic cell subpopulation, wherein in step (1), CD141 is separated by pre-treating peripheral blood + The step of dendritic cells comprises:
mixing peripheral blood and erythrocyte lysate, standing in dark place, centrifuging, and separating out CD141 + Dendritic cells, the CD141 + Resuspending the dendritic cells in a cell staining solution, and adding a leukocyte stimulating factor for incubation;
preferably, the light-proof standing time is 10-15 min, and the light-proof standing temperature is 20-25 ℃;
preferably, the cell staining solution comprises PBS buffer containing fetal bovine serum;
preferably, the leukocyte stimulating factor is in CD141 + The final concentration of the dendritic cell resuspension is 0.08-0.1 wt%;
preferably, the incubation time is 4-6 h, and the incubation temperature is 35-38 ℃.
7. Detecting CD141 according to claim 5 or6 + A method for analyzing the phenotype and function of a dendritic cell subpopulation, wherein in step (2), the CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody are labeled with different fluorescent dyes to bind to CD141 + The step of incubating and detecting the dendritic cells comprises the following steps:
(a) labelling with different fluorescent dyesThe CD141 antibody, Linage antibody and HLA-DR antibody of (1) + Incubating the dendritic cells for the first time;
(b) IL-10, IL-12, IL-23 and IL-27 antibodies labeled with different fluorochromes on CD141 + The dendritic cells were incubated a second time.
8. Detecting CD141 as claimed in claim 7 + Method for analyzing the phenotype and function of a dendritic cell subpopulation, wherein, in step (a), the CD141 antibody, Linage antibody and HLA-DR antibody labeled with different fluorochromes are used for the CD141 antibody + The step of incubating the dendritic cells for the first time comprises:
the CD141 incubated in the step (1) is added + Resuspending the dendritic cells and resuspending CD141 + The dendritic cell heavy suspension is incubated with CD141 antibody, Linage antibody and HLA-DR antibody marked by different fluorescent pigments for the first time, and the CD141 is fixed by formalin solution + Dendritic cells, and shading and incubating;
preferably, the mass fraction of the formalin solution is 1.5-2.5 vol%;
preferably, the first incubation time is 25-35 min, and the first incubation temperature is 20-25 ℃;
preferably, the shading incubation time is 15-20 min, and the shading incubation temperature is 20-25 ℃;
preferably, in step (b), the IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody labeled with different fluorescent dyes are directed against CD141 + The step of performing secondary incubation on the dendritic cells comprises the following steps:
subjecting the CD141 obtained in step (a) to + Resuspending dendritic cells with cell-penetrating fluid, centrifuging to remove supernatant, resuspending the precipitate with cell-penetrating fluid, and resuspending CD141 + Performing secondary incubation on the dendritic cell resuspension and different fluorescent pigment labeled IL-10 antibodies, IL-12 antibodies, IL-23 antibodies and IL-27 antibodies;
preferably, the centrifugal force of the centrifugation is 300-350 g, and the time of the centrifugation is 5-10 min;
preferably, the time of the secondary incubation is 24-26 h, and the temperature of the secondary incubation is 0-4 ℃;
preferably, the second incubation is performed under light-shielding conditions.
9. Detecting CD141 according to any of claims 5-8 + A method for analyzing the phenotype and function of a subpopulation of dendritic cells, wherein in step (3), said step of detecting and analyzing by flow cytometry comprises:
subjecting the secondarily incubated CD141 + Resuspending the dendritic cells by using a cell penetration solution, centrifuging to remove supernatant, resuspending the precipitated cells by using a cell staining solution, and detecting and analyzing by using a flow cytometer;
preferably, the centrifugal force of the centrifugation is 300-350 g, and the time of the centrifugation is 5-10 min;
preferably, the method of analysis comprises:
according to CD141 antibody and Linage antibody and CD141 + Binding of CD141 and Linage on the surface of dendritic cells, CD141 was analyzed + CD141 in dendritic cell subpopulation + The ratio of phenotypes;
according to HLA-DR antibody and CD141 + Binding of HLA-DR to the surface of dendritic cells, CD141 was analyzed + The differentiation maturation status of the dendritic cell subpopulation; and
based on IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody and CD141 + Binding of IL-10, IL-12, IL-23 and IL-27 on the surface of dendritic cells, CD141 was analyzed + Function of the dendritic cell subpopulation.
10. Detect CD141 + A system for the phenotype and function of a subpopulation of dendritic cells comprising:
(1) a sample processing module: pretreatment of peripheral blood to isolate CD141 + A dendritic cell;
(2) a detection module: CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody labeled with different fluorochromes for binding to CD141 + Dendritic shapeCarrying out incubation detection on the cells; and
(3) an analysis module: detection and analysis was performed using a flow cytometer.
11. The method of detecting CD141 of any of claims 1-3 + Combination of antibodies for the detection of CD141 according to claim 4, for the phenotype and function of a subpopulation of dendritic cells + Kit for the detection of the phenotype and function of a subpopulation of dendritic cells or of CD141 according to claim 10 + Characterization of CD141 by any one of, or a combination of at least two of, a system for the phenotype and function of a subpopulation of dendritic cells + Phenotypic and functional and/or preparative identification of CD141 from dendritic cell subsets + Use in products of dendritic cell subpopulation phenotype and function;
preferably, the product comprises a kit and/or a detection reagent for detecting malignancy;
preferably, the malignancy comprises any one of, or a combination of at least two of, lung cancer, melanoma, prostate cancer, or renal cancer.
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| PCT/CN2022/077229 WO2023159343A1 (en) | 2022-02-22 | 2022-02-22 | Method for detecting phenotype and function of cd141+ dendritic cell subsets, and use kit thereof |
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| CN117624367A (en) * | 2023-11-29 | 2024-03-01 | 武汉爱博泰克生物科技有限公司 | Anti-human CD141 protein rabbit monoclonal antibody and application thereof |
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| US20040219612A1 (en) * | 2003-05-02 | 2004-11-04 | Beckman Coulter, Inc. | Methods for detecting intracellular defensins in various leukocyte subpopulations |
| CN104360053B (en) * | 2014-09-22 | 2016-08-17 | 重庆医科大学附属儿童医院 | A kind of method of bone-marrow-derived lymphocyte immunophenotyping and test kit |
| CN110389221B (en) * | 2019-08-30 | 2021-03-16 | 广州中科蓝华生物科技有限公司 | Combined formula kit for analyzing phenotype and function of CD1c + dendritic cell subset and application thereof |
| CN112147326B (en) * | 2020-09-04 | 2022-04-08 | 北京大学 | Accurate detection kit for tumor immune cell subset typing |
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| CN117624367A (en) * | 2023-11-29 | 2024-03-01 | 武汉爱博泰克生物科技有限公司 | Anti-human CD141 protein rabbit monoclonal antibody and application thereof |
| CN117624367B (en) * | 2023-11-29 | 2024-06-25 | 武汉爱博泰克生物科技有限公司 | Anti-human CD141 protein rabbit monoclonal antibody and application thereof |
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