WO2023159343A1 - 一种用于检测cd141 +树突状细胞亚群表型和功能的方法及其应用试剂盒 - Google Patents

一种用于检测cd141 +树突状细胞亚群表型和功能的方法及其应用试剂盒 Download PDF

Info

Publication number
WO2023159343A1
WO2023159343A1 PCT/CN2022/077229 CN2022077229W WO2023159343A1 WO 2023159343 A1 WO2023159343 A1 WO 2023159343A1 CN 2022077229 W CN2022077229 W CN 2022077229W WO 2023159343 A1 WO2023159343 A1 WO 2023159343A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
phenotype
function
dendritic cell
dendritic cells
Prior art date
Application number
PCT/CN2022/077229
Other languages
English (en)
French (fr)
Inventor
周放
陈小平
秦莉
胡晓晶
高素素
容志恩
牛欢
刘玉梅
李海铭
吴智华
危元磊
Original Assignee
中科蓝华(广州)生物医药技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中科蓝华(广州)生物医药技术有限公司 filed Critical 中科蓝华(广州)生物医药技术有限公司
Priority to AU2022436042A priority Critical patent/AU2022436042A1/en
Priority to CN202280000607.2A priority patent/CN115004032A/zh
Priority to PCT/CN2022/077229 priority patent/WO2023159343A1/zh
Publication of WO2023159343A1 publication Critical patent/WO2023159343A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the application belongs to the field of biotechnology, and in particular relates to an antibody combination for detecting the phenotype and function of CD141 + dendritic cell subsets, and its analysis method and application.
  • CD141 + dendritic cells are distributed in human peripheral blood and are a newly discovered subpopulation of dendritic cells in recent years.
  • Clinical and basic studies have shown that CD141 + dendritic cell subsets play an important role in the occurrence of various diseases, such as malignant tumors (such as lung cancer, melanoma, prostate cancer and kidney cancer), dermatitis, viral infections (such as HIV- 1 infection), infectious diseases (such as malaria) and some autoimmune diseases (such as rheumatoid arthritis, etc.), clinical data show that in these diseases, CD141 + dendritic cells show abnormal phenotype and function. Therefore, the clinical data of CD141 + dendritic cell phenotype and functional determination can become an auxiliary judgment index for clinicians to judge the development of the above diseases and the clinical treatment effect, which has very important clinical diagnostic significance.
  • Dendritic cells as the main regulatory cells in the human immune system, are one of the hotspots in immunology research.
  • the detection of dendritic cells mainly relies on flow cytometry analysis technology, but there are various analysis schemes of flow cytometry for detecting dendritic cells, and there is no unified and standardized mode.
  • the research on dendritic cells is changing with each passing day, and a variety of different subtypes of dendritic cells have been reported and discovered.
  • the flow cytometry analysis scheme for the detection of dendritic cells is relatively extensive, which cannot meet the current clinical needs for different subtypes. The precise analysis of dendritic cell types is required.
  • CN105911292A discloses a kit for combined analysis of CD11c + , CD11b + dendritic cell subsets and their degree of differentiation and function
  • the kit contains the following 8 kinds of 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 + , CD11b + dendritic cell subsets and a full set of data on their differentiation degree and function at one time.
  • the morphology and immune function of dendritic cells are not the same, and there are a large number of surface antigen molecules. Different specific molecules need to be selected for detection of different dendritic cell subpopulations.
  • CD11c + , CD11b + dendritic cell subsets are completely different from those of CD141 + dendritic cell subsets, and they play roles in different diseases. Therefore, the kit for analyzing CD11c + , CD11b + dendritic cell subsets and their differentiation degree and function cannot meet the needs of researching CD141 + dendritic cell subsets. Therefore, it is of great significance to develop an immunoassay kit for detecting the phenotype and function of CD141 + dendritic cell subsets.
  • the present application provides an antibody combination for detecting the phenotype and function of CD141 + dendritic cell subsets, its analysis method and application.
  • Antigens detected by the antibody combination for detecting the phenotype and function of CD141 + dendritic cell subsets include CD141, Linage, HLA-DR, IL-10, IL-12, IL-23 and IL-27; the present application
  • a kit for detecting the phenotype and function of CD141 + dendritic cell subsets which can efficiently and rapidly analyze the phenotype and function of CD141 + dendritic cell subsets in peripheral blood, so
  • the above-mentioned kit has high accuracy, reduces the economic cost caused by detecting a large number of surface antigen molecules, and the detection and analysis methods are simple and easy, which has important application value.
  • the present application provides an antibody combination for detecting the phenotype and function of a CD141 + dendritic cell subgroup
  • the antibody combination for detecting the phenotype and function of a CD141 + dendritic cell subgroup includes CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody.
  • This application detects the phenotype and function of dendritic cell subsets through the combination of 7 specifically selected antibody molecules, including CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody, the formula design can detect the phenotype and function of CD141 + dendritic cell subsets with high specificity and high sensitivity, laying the foundation for related scientific research.
  • the antibodies in the antibody combination for detecting the phenotype and function of the CD141 + dendritic cell subpopulation are labeled with fluorescent dyes.
  • the fluorescent pigment includes any one or a combination of at least two of BV510, BV-786, Pacific blue, PE-Cy7, AF700, eFluor660 or PE.
  • the present application provides a kit for detecting the phenotype and function of a CD141 + dendritic cell subgroup
  • the kit for detecting the phenotype and function of a CD141 + dendritic cell subgroup includes the first aspect
  • kits for dendritic cell testing can only analyze overall dendritic cell data in general and do not include functional analysis.
  • multiple new subpopulations of dendritic cells in human peripheral blood have been discovered, such as CD141 + dendritic cells, which have different phenotypes and functions.
  • the kit for detecting the phenotype and function of CD141 + dendritic cell subsets provided by this application can analyze the proportion of CD141 + phenotype in dendritic cell subsets, and can control the differentiation and maturation of CD141 + dendritic cell subsets To judge the status, the function of CD141 + dendritic cell subsets can also be analyzed.
  • the present application provides an analysis method for detecting the phenotype and function of CD141 + dendritic cell subsets described in the second aspect, the analysis method comprising the following steps:
  • CD141 + dendritic cells were incubated and detected with 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; as well as
  • the analysis method uses human whole blood one-step method to measure the dendritic cell subpopulation and its function in human peripheral blood.
  • the analytical method described in the application is simple and easy to implement and saves cost. Isolation of dendritic cells using traditional PBMC separation techniques requires a large volume of blood collection (usually ranging from tens of milliliters) and takes a long time.
  • the present application adopts the one-step method of measuring human whole blood, and only one drop of blood (10-100 ⁇ L) of the patient can be detected to obtain the required complete set of information. It saves a lot of time for separating PBMCs, and can be measured simply, quickly and in one step. It is suitable for clinical testing of large batches of samples.
  • step (1) the step of pretreating peripheral blood to isolate CD141 + dendritic cells includes:
  • Peripheral blood was mixed with erythrocyte lysate, left standing in the dark, centrifuged, and CD141 + dendritic cells were separated, and the CD141 + dendritic cells were resuspended in cell staining solution, and leukocyte stimulating factor was added for incubation.
  • the volume of the peripheral blood is 10-100 ⁇ L, such as 10 ⁇ L, 20 ⁇ L, 30 ⁇ L, 40 ⁇ L, 50 ⁇ L, 60 ⁇ L, 70 ⁇ L, 80 ⁇ L, 90 ⁇ L or 100 ⁇ L.
  • the mixed volume ratio of the peripheral blood and the erythrocyte lysate is (1-10):200, for example, it can be 1:200, 3:200, 5:200, 7:200, 9:200 or 10: 200 etc.
  • the time for standing in the dark is 10 to 15 minutes, such as 10 min, 11 min, 12 min, 13 min, 14 min or 15 min, etc.
  • the temperature for standing in the dark is 20 to 25° C., for example, it can be 20 °C, 21°C, 22°C, 23°C, 24°C or 25°C etc.
  • the centrifugal force of the centrifugation is 300-350g, such as 300g, 310g, 320g, 330g, 340g or 350g, etc.
  • the centrifugation time is 5-10min, such as 5min, 6min, 7min, 8min , 9min or 10min, etc.
  • the cell staining solution includes PBS buffer containing fetal bovine serum.
  • the final concentration of the leukocyte-stimulating factor in the CD141 + dendritic cell suspension is 0.08-0.1 wt%, such as 0.08 wt%, 0.09 wt% or 0.1 wt%.
  • the incubation time is 4-6 hours, such as 4 hours, 5 hours or 6 hours, etc.
  • the incubation temperature is 35-38°C, such as 35°C, 36°C, 37°C or 38°C.
  • the CD141 antibody, Linage antibody, HLA-DR antibody, IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody labeled with different fluorochromes are effective against CD141 +
  • the steps for incubation and detection of dendritic cells include:
  • CD141 + dendritic cells were first incubated with CD141 antibody, Linage antibody and HLA-DR antibody labeled with different fluorochromes;
  • step (a) the step of first incubating CD141 + dendritic cells with CD141 antibodies, Linage antibodies and HLA-DR antibodies labeled with different fluorochromes includes:
  • step (1) Resuspend the CD141 + dendritic cells incubated in step (1), and incubate the CD141 + dendritic cell resuspension with different fluorochrome-labeled CD141 antibodies, Linage antibodies and HLA-DR antibodies for the first time, and use For CD141 + dendritic cells were fixed in Malin solution and incubated in the dark.
  • the mass fraction of the formalin solution is 1.5-2.5 vol%, such as 1.5 vol%, 1.8 vol%, 2.0 vol%, 2.2 vol% or 2.5 vol%.
  • the time for the first incubation is 25 to 35 minutes, such as 25 minutes, 28 minutes, 30 minutes, 32 minutes or 35 minutes, etc.
  • the temperature for the first incubation is 20 to 25° C., for example, 20° C., 21° C., 22° C. °C, 23°C, 24°C or 25°C etc.
  • the time of the shading incubation is 15-20 min, such as 15 min, 16 min, 17 min, 18 min, 19 min or 20 min, etc.
  • the temperature of the shading incubation is 20-25°C, such as 20°C, 21°C , 22°C, 23°C, 24°C or 25°C, etc.
  • step (b) the step of incubating CD141 + dendritic cells for a second time with IL-10 antibodies, IL-12 antibodies, IL-23 antibodies and IL-27 antibodies labeled with different fluorescent dyes includes:
  • step (a) Resuspend the CD141 + dendritic cells obtained in step (a) with cell penetration solution, centrifuge to remove the supernatant, resuspend the pellet with cell penetration solution, mix the CD141 + dendritic cell suspension with different fluorescent dyes Labeled IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody were incubated for the second time.
  • the centrifugal force of the centrifugation is 300-350g, such as 300g, 310g, 320g, 330g, 340g or 350g, etc.
  • the centrifugation time is 5-10min, such as 5min, 6min, 7min, 8min, 9min or 10min etc.
  • the time for the second incubation is 24-26h, for example, 24h, 25h or 26h, etc.
  • the temperature for the second incubation is 0-4°C, for example, it can be 0°C, 1°C, 2°C, 3°C or 4°C, etc.
  • the second incubation is carried out under light-shielding conditions.
  • step (3) the steps of detecting and analyzing with a flow cytometer include:
  • the CD141 + dendritic cells after the second incubation were resuspended with the cell penetration medium, centrifuged to remove the supernatant, the precipitated cells were resuspended with the cell staining solution, and analyzed and detected by flow cytometry.
  • the centrifugal force of the centrifugation is 300-350g, such as 300g, 310g, 320g, 330g, 340g or 350g, etc.
  • the centrifugation time is 5-10min, such as 5min, 6min, 7min, 8min, 9min or 10min etc.
  • the method of said analysis comprises:
  • CD141 antibody and Linage antibody According to the binding of CD141 antibody and Linage antibody to CD141 and Linage on the surface of CD141 + dendritic cells, the proportion of CD141 + dendritic cell subpopulation CD141 + phenotype was analyzed;
  • CD141 was analyzed based on the binding of IL-10 antibody, IL-12 antibody, IL-23 antibody, and IL-27 antibody to IL-10, IL-12, IL-23, and IL-27 on the surface of CD141 + dendritic cells + Function of dendritic cell subsets.
  • the analysis method for detecting the phenotype and function of the CD141 + dendritic cell subsets includes the following steps:
  • CD141 + dendritic cells were incubated with 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:
  • step (1) Resuspend the CD141 + dendritic cells incubated in step (1), and incubate the CD141 + dendritic cell resuspension with different fluorochrome-labeled CD141 antibodies, Linage antibodies and HLA-DR antibodies for the first time , the time for the first incubation is 25-35min, the temperature is 20-25°C, and the volume ratio of the CD141 + dendritic cell resuspension to CD141 antibody, Linage antibody and HLA-DR antibody is 100:(2 ⁇ 2 ⁇ 2.5): (2-2.5): (2-2.5), fix the CD141 + dendritic cells with a formalin solution with a mass fraction of 1.5-2.5 vol%, and incubate at 20-25°C for 15-20 minutes in the dark;
  • step (b) Resuspend the CD141 + dendritic cells obtained in step (a) with the cell penetrating medium, centrifuge at 300-350g for 5-10 minutes to remove the supernatant, resuspend the pellet with the cell penetrating medium, and dissolve the CD141 + dendritic cells
  • the resuspension of CD141+ dendritic cells was incubated with IL-10 antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody labeled with different fluorochromes, and the CD141 + dendritic cell suspension was mixed with IL-10
  • the volume ratio of antibody, IL-12 antibody, IL-23 antibody and IL-27 antibody is 100:(2 ⁇ 2.5):(2 ⁇ 2.5):(2 ⁇ 2.5):(2 ⁇ 2.5):(2 ⁇ 2.5), the secondary The incubation is carried out under light-shielding conditions, the time of the second incubation is 24-26 hours, and the temperature is 0-4°C.
  • CD141 antibody and Linage antibody According to the binding of CD141 antibody and Linage antibody to CD141 and Linage on the surface of CD141 + dendritic cells, the proportion of CD141 + dendritic cell subpopulation CD141 + phenotype was analyzed;
  • CD141 was analyzed based on the binding of IL-10 antibody, IL-12 antibody, IL-23 antibody, and IL-27 antibody to IL-10, IL-12, IL-23, and IL-27 on the surface of CD141 + dendritic cells + Function of dendritic cell subsets.
  • the present application provides a system for detecting the phenotype and function of a CD141 + dendritic cell subset, the system for detecting the phenotype and function of a CD141 + dendritic cell subset includes:
  • Sample processing module pretreat peripheral blood to isolate CD141 + dendritic cells
  • CD141 + dendritic cells were detected with 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. Incubation testing; and
  • Analysis module detection and analysis by flow cytometer.
  • the present application provides the antibody combination for detecting the phenotype and function of the CD141 + dendritic cell subpopulation described in the first aspect, and the antibody combination for detecting the phenotype and function of the CD141 + dendritic cell subpopulation described in the second aspect.
  • Any one or a combination of at least two of the functional kit or the system for detecting the phenotype and function of the CD141 + dendritic cell subpopulation described in the fourth aspect is useful in identifying the phenotype and function of the CD141 + dendritic cell subpopulation Functional and/or application in the preparation of products for identifying the phenotype and function of CD141 + dendritic cell subsets.
  • the product includes a kit and/or a detection reagent for detecting malignant tumors
  • the malignant tumor includes any one or a combination of at least two of lung cancer, melanoma, prostate cancer or kidney cancer.
  • the analysis method for detecting the phenotype and function of CD141 + dendritic cell subsets described in this application is simple, fast and easy to implement.
  • PBMC peripheral blood mononuclear cell
  • the present application adopts the one-step method of measuring human whole blood, and only one drop of blood (10-100 ⁇ L) of the patient can be detected to obtain the required complete set of information. It saves a lot of time for separating PBMCs, can be measured simply and quickly in one step, and is suitable for clinical testing of large batches of samples.
  • the analysis method for detecting the phenotype and function of the CD141 + dendritic cell subsets described in this application provides comprehensive results.
  • This application specifically selects 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.
  • Specifically and highly sensitively analyze the proportion of CD141 + phenotype in CD141 + dendritic cell subsets can judge the differentiation and maturation status of CD141 + dendritic cell subsets, and can also analyze CD141 + dendritic cell subsets group function.
  • the analytical method for detecting the phenotype and function of CD141 + dendritic cell subsets described in this application has high accuracy of the results.
  • the analysis scheme for detecting the phenotype and function of CD141 + dendritic cell subsets in this application has the advantages of high sensitivity and good specificity, and the detection results are more accurate and reliable.
  • the analysis scheme of the currently commercially available dendritic cell analysis kits can only test the data of overall dendritic cells, and does not include functional analysis.
  • the kit for detecting the phenotype and function of CD141 + dendritic cell subsets described in this application can precisely provide a complete set of data on the phenotype and function of the latest CD141 + dendritic cell subsets in human peripheral blood. Compared to previously developed protocols, this application is the first to test the combination of phenotype and function of CD141 + dendritic cell subsets.
  • FIG. 1 is a scatter diagram of FSC-SSC of healthy human peripheral blood cells in Example 3.
  • FIG. 2 is a flow cytometric analysis diagram of healthy human peripheral blood in Example 3.
  • FIG. 2 is a flow cytometric analysis diagram of healthy human peripheral blood in Example 3.
  • Example 3 is a flow cytometric analysis result of CD141 + dendritic cell subsets in the peripheral blood of healthy people/non-small cell lung cancer patients in Example 3.
  • Example 4 is a statistical graph of the expression of CD141 + dendritic cell subset HLA-DR in the peripheral blood of healthy people/non-small cell lung cancer patients in Example 3.
  • Fig. 5 is a graph showing the ratio of expression of IL-10 on CD141 + dendritic cells in peripheral blood of isotype control/healthy person/lung cancer patient in Example 3.
  • FIG. 6 is a statistical graph of the expression of IL-10 on CD141 + dendritic cells in the peripheral blood of healthy people/lung cancer patients in Example 3.
  • FIG. 6 is a statistical graph of the expression of IL-10 on CD141 + dendritic cells in the peripheral blood of healthy people/lung cancer patients in Example 3.
  • Fig. 7 is a graph showing the ratio of expression of IL-12 on CD141 + dendritic cells in peripheral blood of isotype control/healthy person/lung cancer patient in Example 3.
  • FIG. 8 is a statistical graph of the expression of IL-12 on CD141 + dendritic cells in the peripheral blood of healthy people/lung cancer patients in Example 3.
  • FIG. 8 is a statistical graph of the expression of IL-12 on CD141 + dendritic cells in the peripheral blood of healthy people/lung cancer patients in Example 3.
  • Fig. 9 is a graph showing the ratio of expression of IL-23 on CD141 + dendritic cells in peripheral blood of isotype control/healthy person/lung cancer patient in Example 3.
  • FIG. 10 is a statistical graph of the expression of IL-23 on CD141 + dendritic cells in the peripheral blood of healthy people/lung cancer patients in Example 3.
  • Fig. 11 is a graph showing the ratio of expression of IL-27 on CD141 + dendritic cells in peripheral blood of isotype control/healthy person/lung cancer patient in Example 3.
  • Fig. 12 is a statistical graph of the expression of IL-27 on CD141 + dendritic cells in the peripheral blood of healthy people/lung cancer patients in Example 3.
  • This embodiment provides an antibody combination for detecting the phenotype and function of CD141 + dendritic cell subsets
  • the antibody combination for detecting the phenotype and function of CD141 + dendritic cell subsets includes CD141 antibody (Biolegend), Linage antibody (Biolegend), HLA-DR antibody (eBioscience), IL-10 antibody (BD), IL-12 antibody (Biolegend), IL-23 antibody (eBioscience) and IL-27 antibody (Biolegend).
  • the antibodies in the antibody combination for detecting the phenotype and function of CD141 + dendritic cell subsets are labeled with fluorescent pigments.
  • the fluorochrome of the CD141 antibody is BV510; the fluorochrome of the Linage antibody is BV786; the fluorochrome of the HLA-DR antibody is Pacific blue; the fluorochrome of the IL-10 antibody is PE-Cy7;
  • the fluorochrome of the -12 antibody is AF700; the fluorochrome of the IL-23 antibody is eFluor660; the fluorochrome of the IL-27 antibody is PE.
  • This embodiment provides a kit for detecting the phenotype and function of CD141 + dendritic cell subgroups
  • the kit for detecting the phenotype and function of CD141 + dendritic cell subgroups includes the Antibody combinations for the detection of phenotype and function of CD141 + dendritic cell subsets as described.
  • the kit for detecting the phenotype and function of CD141 + dendritic cell subsets described in Example 2 was used to detect CD141 + dendritic cell subsets in the peripheral blood of non-small cell lung cancer patients (experimental group) and healthy people (control group). CD141 + dendritic cell subsets were analyzed for differentiation and function, while isotype controls were analyzed.
  • CD141 + dendritic cells were incubated with 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:
  • step (1) Resuspend the CD141 + dendritic cells obtained in step (1) in 100 ⁇ L cell staining solution, mix the CD141 + dendritic cell suspension with 2 ⁇ L BV510-labeled CD141 antibody, 2 ⁇ L BV786-labeled Linage antibody Incubate with 2 ⁇ L of Pacific blue labeled HLA-DR antibody for the first time, the time of the first incubation is 30min, the temperature is 25°C, add 2mL of cell staining solution, centrifuge at 350g for 5min, repeat the centrifugation twice, and use 2mL with a mass fraction of 2% CD141 + dendritic cells were fixed with formalin solution, and incubated at 25°C for 20 minutes in the dark;
  • step (b) Resuspend the CD141 + dendritic cells obtained in step (a) with 2 mL of cell penetration solution (Biolegend), centrifuge at 350 g for 5 min, repeat the centrifugation twice, resuspend the pellet with 100 ⁇ L of cell penetration solution, and dissolve CD141 + Dendritic cell suspension was incubated with 2 ⁇ L PE-Cy7-labeled IL-10 antibody, 2 ⁇ L AF700-labeled IL-12 antibody, 2 ⁇ L eFluor660-labeled IL-23 antibody and 2 ⁇ L PE-labeled IL-27 antibody , the second incubation is carried out under the condition of shading, the time of the second incubation is 30min, and the temperature is 25°C.
  • cell penetration solution Biolegend
  • CD141 + dendritic cells were resuspended with 2 mL of cell penetration medium, centrifuged at 350 g for 5 min to remove the supernatant, and the precipitated cells were resuspended with 0.5 mL of cell staining liquid, and detected and analyzed by flow cytometry.
  • the dendritic cell subsets (characterized by the expression of CD141 and Linage), the degree of differentiation (characterized by the expression of HLA-DR) and cell function (characterized by the expression of IL-10) were obtained at one time using flow cytometry. , IL-12, IL-23 and IL-27 expression characterization), and through the comparison of these information, the differences between non-small cell lung cancer patients and healthy people can be studied, so as to provide a basis for the study of non-small cell lung cancer patients The immune regulation mechanism provides the basis.
  • Figure 5 is a graph showing the expression ratio of IL-10 on CD141 + dendritic cells in the peripheral blood of isotype controls/healthy people/lung cancer patients
  • Figure 6 is the expression ratio of IL-10 in the peripheral blood of healthy people/lung cancer patients CD141
  • Statistical diagram of the expression of + dendritic cells; IL-10 has the effect of inhibiting immune function in vivo.
  • the CD141 + dendritic cells of patients with non-small cell lung cancer are more than those of healthy people.
  • CD141 + dendritic cells secreted more IL-10, and the results showed that CD141 + dendritic cells in patients with non-small cell lung cancer suppressed the anti-tumor immune response in vivo by secreting more IL-10.
  • Figure 7 is a graph showing the expression ratio of IL-12 on CD141 + dendritic cells in the peripheral blood of isotype controls/healthy people/lung cancer patients
  • Figure 8 is the expression ratio of IL-12 in the peripheral blood of healthy people/lung cancer patients CD141 + Statistical graph of expression on dendritic cells.
  • IL-12 is a cytokine that promotes immune response.
  • CD141 + dendritic cells in patients with non-small cell lung cancer secrete less IL-12 than CD141 + dendritic cells in healthy people. 12.
  • the results showed that CD141 + dendritic cells in patients with non-small cell lung cancer secrete IL-12 insufficiently, which may affect the anti-tumor immune response mediated by IL-12.
  • Figure 9 is a graph showing the expression ratio of IL-23 on the CD141 + dendritic cells in the peripheral blood of the same type control/healthy person/lung cancer patient
  • Figure 10 is the CD141 expression of IL-23 in the peripheral blood of the healthy person/lung cancer patient +
  • Figure 11 is the expression ratio diagram of IL-27 on CD141 + dendritic cells in the peripheral blood of isotype control/healthy person/lung cancer patient
  • Figure 12 is the expression ratio diagram of IL-27 Statistical graph of the expression of 27 on CD141 + dendritic cells in the peripheral blood of healthy people/lung cancer patients. It can be seen from the figure that the expression ratios of IL - 23 and IL-27 on CD141 + dendritic cells of healthy people are 17.7% and 36.6% respectively; The expression ratios on the cells were 22.2% and 38.8%.
  • IL-23 is a cytokine that promotes immune response. If dendritic cells can secrete more IL-23, it indicates that dendritic cells can promote immune response by secreting more IL-23.
  • Figure 9 and Figure 10 The results showed that the IL-23 secreted by CD141 + dendritic cells of normal healthy people was similar to the IL-23 secreted by CD141 + dendritic cells of lung cancer patients, which indicated that CD141 + dendritic cells in lung cancer patients were not regulated by IL-23 to affect the immune response mediated by CD141 + DC.
  • IL-27 is a cytokine that can suppress immune function. If dendritic cells secrete more IL-27, it indicates that dendritic cells have immunosuppressive effect. Dendritic cells can secrete more IL-27 to Suppresses the immune response.
  • the detection results in Figure 11 and Figure 12 show that there is no significant difference in the amount of IL-27 secreted by CD141 + dendritic cells in lung cancer patients and CD141 + dendritic cells in normal healthy people, which indicates that CD141 in lung cancer patients + dendritic cells do not suppress immune function by secreting more IL-27, and CD141 + dendritic cells in lung cancer patients are a kind of immunosuppressive cells compared with CD141 + dendritic cells in healthy people. Dendritic Cells.
  • this 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.
  • the formula design can analyze the proportion of CD141 + phenotype in CD141 + dendritic cell subpopulation with high specificity and high sensitivity, can judge the differentiation and maturity status of CD141 + dendritic cell subpopulation, and can also analyze CD141 + dendritic cell subpopulation Functions of dendritic cell subsets.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Secondary Cells (AREA)

Abstract

提供了一种检测CD141+树突状细胞亚群的表型和功能的抗体组合及其分析方法和应用，所述检测CD141+树突状细胞亚群的表型和功能的抗体组合包括CD141抗体、Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体。本申请还提供了一种检测CD141+树突状细胞亚群的表型和功能的试剂盒。

Description

一种用于检测CD141 +树突状细胞亚群表型和功能的方法及其应用试剂盒 技术领域
本申请属于生物技术领域，具体涉及一种检测CD141 +树突状细胞亚群的表型和功能的抗体组合及其分析方法和应用。
背景技术
CD141 +树突状细胞分布于人外周血中，是近年来新发现的一个树突状细胞亚群。临床和基础研究表明，CD141 +树突状细胞亚群在多种疾病的发生中起重要作用，例如恶性肿瘤(如肺癌、黑色素瘤、***癌和肾癌)、皮炎、病毒感染(如HIV-1感染)、传染病(如疟疾)以及一些自身免疫病(如类风湿关节炎等)，临床数据表明在这些疾病中CD141 +树突状细胞都显现出表型和功能的异常。因此CD141 +树突状细胞的表型和功能测定的临床数据可成为临床医生判断上述疾病发展状况和临床治疗效果的辅助判断指标，具有十分重要的临床诊断意义。
流式细胞仪分析技术已作为免疫学的主要技术，广泛应用于临床和科研工作。树突状细胞作为人体内免疫***中起主要调控作用的细胞，是免疫学研究热点之一。目前树突状细胞的检测主要依靠流式细胞仪分析技术，但检测树突状细胞的流式细胞仪的分析方案多种多样，没有一个统一标准化的模式。树突状细胞的研究日新月异，多种不同的树突状细胞亚型不断被报导发现，然而，针对树突状细胞检测的流式细胞仪的分析方案较为粗放，已不能满足目前临床针对不同亚型的树突状细胞的精确分析要求。
CN105911292A公开了一种用于组合分析CD11c +、CD11b +树突状细胞亚群及其分化程度和功能的试剂盒，所述试剂盒包含以下8种抗体：CD11c抗体、CD80抗体、CD86抗体、CD11b抗体、HLA-DR抗体、IL-12抗体、IL-23抗体和IL-27抗体。所述试剂盒可一次性检测CD11c +、CD11b +树突状细胞亚群及其分化程度和功能的全套数据。但树突状细胞的形态、免疫功能不尽相同，其表面抗原分子数量众多，针对不同树突状细胞亚群需要选择不同的特异性分子进行检测。
研究表明CD11c +、CD11b +树突状细胞亚群的功能与CD141 +树突状细胞亚 群的功能完全不同，并且在不同的疾病中起用。因此，所述分析CD11c +、CD11b +树突状细胞亚群及其分化程度和功能的试剂盒并不能满足研究CD141 +树突状细胞亚群的需要。因此，研发提供一种用于检测CD141 +树突状细胞亚群的表型和功能的免疫检测试剂盒具有重要意义。
发明内容
本申请提供了一种检测CD141 +树突状细胞亚群的表型和功能的抗体组合及其分析方法和应用。所述检测CD141 +树突状细胞亚群的表型和功能的抗体组合所检测的抗原包括CD141、Linage、HLA-DR、IL-10、IL-12、IL-23和IL-27；本申请还提供一种检测CD141 +树突状细胞亚群的表型和功能的试剂盒，所述试剂盒可高效快速地分析外周血中的CD141 +树突状细胞亚群表型及其功能，所述试剂盒的准确率高，同时降低因检测大量表面抗原分子产生的经济成本，且检测、分析方法简单易行，具有重要的应用价值。
第一方面，本申请提供一种检测CD141 +树突状细胞亚群的表型和功能的抗体组合，所述检测CD141 +树突状细胞亚群的表型和功能的抗体组合包括CD141抗体、Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体。
在现有技术中通过流式细胞仪鉴定树突状细胞亚群通常需要分离提取外周血单核细胞，该过程复杂繁琐，时间周期长，若通过细胞分子表面抗原检测的方式分析细胞亚群则通常需要选择大量树突状细胞表面抗原分子进行检测，才能提高检测的准确性和特异性，但大量表面抗原的检测分析需要较长时间，且检测的经济成本较高，不利于快速高效地进行树突状细胞亚群分析研究。本申请通过特异性选择的7个抗体分子的组合对树突状细胞亚群的表型和功能进行检测，包括CD141抗体、Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体，该配方设计可以高特异性和高灵敏度地检测CD141 +树突状细胞亚群的表型和功能，为相关科学研究奠定基础。
优选地，所述的检测CD141 +树突状细胞亚群的表型和功能的抗体组合中的抗体带有荧光色素标记。
优选地，所述荧光色素包括BV510、BV-786、Pacific blue、PE-Cy7、AF700、eFluor660或PE中任意一种或至少两种的组合。
第二方面，本申请提供一种检测CD141 +树突状细胞亚群的表型和功能的试剂盒，所述检测CD141 +树突状细胞亚群的表型和功能的试剂盒包括第一方面所述的检测CD141 +树突状细胞亚群的表型和功能的抗体组合。
目前市售的树突状细胞测试的试剂盒只能笼统地分析总体树突状细胞的数据，且不包括功能分析。随着科学研究的发展，人外周血中多个树突状细胞新亚群已经被发现，如CD141 +树突状细胞，它们的表型和功能各不相同。本申请提供的检测CD141 +树突状细胞亚群的表型和功能的试剂盒能分析树突状细胞亚群中CD141 +表型的比例，能对CD141 +树突状细胞亚群的分化成熟状况进行判断，还可以分析CD141 +树突状细胞亚群的功能。
第三方面，本申请提供一种第二方面所述的检测CD141 +树突状细胞亚群的表型和功能的分析方法，所述分析方法包括如下步骤：
(1)对外周血进行预处理分离出CD141 +树突状细胞；
(2)用不同荧光色素标记的CD141抗体、Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体对CD141 +树突状细胞进行孵育检测；以及
(3)用流式细胞仪进行检测和分析。
本申请中，所述分析方法采用人体全血一步法测定人外周血中树突状细胞亚群及其功能，与使用外周血单核细胞(PBMC)分离技术测定树突状细胞相比，本申请中所述的分析方法简便易行、节约成本。用传统的PBMC分离技术分离树突状细胞，需要较大的采血量(通常几十毫升不等)，消耗时间长。而本申请采用人体全血一步法测定，只需患者一滴血(10～100μL)即可检测得到所需的全套信息。省去了分离PBMC的大量时间，能够简单快速、一步测定到位。适合临床大批量样品的测试。
优选地，步骤(1)中，所述对外周血进行预处理分离出CD141 +树突状细胞的步骤包括：
将外周血与红细胞裂解液混合，避光静置，离心，分离出CD141 +树突状细胞，将所述CD141 +树突状细胞重悬于细胞染色液中，加入白细胞刺激因子孵育。
本申请中，所述外周血的体积为10～100μL，例如可以是10μL、20μL、30μL、40μL、50μL、60μL、70μL、80μL、90μL或100μL等。
本申请中，所述外周血与红细胞裂解液混合的体积比为(1～10):200，例如可 以是1:200、3:200、5:200、7:200、9:200或10:200等。
优选地，所述避光静置的时间为10～15min，例如可以是10min、11min、12min、13min、14min或15min等，所述避光静置的温度为20～25℃，例如可以是20℃、21℃、22℃、23℃、24℃或25℃等。
本申请中，所述离心的离心力为300～350g，例如可以是300g、310g、320g、330g、340g或350g等，所述离心的时间为5～10min，例如可以是5min、6min、7min、8min、9min或10min等。
优选地，所述细胞染色液包括含胎牛血清的PBS缓冲液。
优选地，所述白细胞刺激因子在CD141 +树突状细胞重悬液中的终浓度为0.08～0.1wt％，例如可以是0.08wt％、0.09wt％或0.1wt％等。
优选地，所述孵育的时间为4～6h，例如可以是4h、5h或6h等，所述孵育的温度为35～38℃，例如可以是35℃、36℃、37℃或38℃等。
优选地，步骤(2)中，所述用不同荧光色素标记的CD141抗体、Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体对CD141 +树突状细胞进行孵育检测的步骤包括：
(a)用不同荧光色素标记的CD141抗体、Linage抗体和HLA-DR抗体对CD141 +树突状细胞进行首次孵育；
(b)用不同荧光色素标记的IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体对CD141 +树突状细胞进行二次孵育。
优选地，步骤(a)中，所述用不同荧光色素标记的CD141抗体、Linage抗体和HLA-DR抗体对CD141 +树突状细胞进行首次孵育的步骤包括：
将步骤(1)中孵育后的CD141 +树突状细胞重悬，将CD141 +树突状细胞重悬液不同荧光色素标记的CD141抗体、Linage抗体和HLA-DR抗体进行首次孵育，用***溶液固定CD141 +树突状细胞，遮光孵育。
优选地，所述***溶液的质量分数为1.5～2.5vol％，例如可以是1.5vol％、1.8vol％、2.0vol％、2.2vol％或2.5vol％等。
优选地，所述首次孵育的时间为25～35min，例如可以是25min、28min、30min、32min或35min等，所述首次孵育的温度为20～25℃，例如可以是20℃、21℃、22℃、23℃、24℃或25℃等。
优选地，所述遮光孵育的时间为15～20min，例如可以是15min、16min、 17min、18min、19min或20min等，所述遮光孵育的温度为20～25℃，例如可以是20℃、21℃、22℃、23℃、24℃或25℃等。
优选地，步骤(b)中，用不同荧光色素标记的IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体对CD141 +树突状细胞进行二次孵育的步骤包括：
将步骤(a)所得的CD141 +树突状细胞用细胞穿透液重悬，离心去上清，将沉淀用细胞穿透液重悬，将CD141 +树突状细胞重悬液与不同荧光色素标记的IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体进行二次孵育。
优选地，所述离心的离心力为300～350g，例如可以是300g、310g、320g、330g、340g或350g等，所述离心的时间为5～10min，例如可以是5min、6min、7min、8min、9min或10min等。
优选地，所述二次孵育的时间为24～26h，例如可以是24h、25h或26h等，所述二次孵育的温度为0～4℃，例如可以是0℃、1℃、2℃、3℃或4℃等。
优选地，所述二次孵育在遮光的条件下进行。
优选地，步骤(3)中，用流式细胞仪进行检测和分析的步骤包括：
将二次孵育后的CD141 +树突状细胞用细胞穿透液重悬，离心去上清，将沉淀细胞用细胞染色液重悬，用流式细胞仪进行分析检测。
优选地，所述离心的离心力为300～350g，例如可以是300g、310g、320g、330g、340g或350g等，所述离心的时间为5～10min，例如可以是5min、6min、7min、8min、9min或10min等。
优选地，所述分析的方法包括：
根据CD141抗体和Linage抗体与CD141 +树突状细胞表面的CD141和Linage的结合情况，分析CD141 +树突状细胞亚群CD141 +表型的比例；
根据HLA-DR抗体与CD141 +树突状细胞表面的HLA-DR的结合情况，分析CD141 +树突状细胞亚群的分化成熟状况；以及
根据IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体与CD141 +树突状细胞表面的IL-10、IL-12、IL-23和IL-27的结合情况，分析CD141 +树突状细胞亚群的功能。
作为本申请的优选技术方案，所述检测CD141 +树突状细胞亚群的表型和功能的分析方法包括如下步骤：
(1)对外周血进行预处理分离出CD141 +树突状细胞：
将10～100μL外周血与红细胞裂解液混合，20～25℃避光静置10～15min，300～350g离心5～10min，分离出CD141 +树突状细胞，将所述CD141 +树突状细胞重悬于细胞染色液中，加入白细胞刺激因子35～38℃孵育4～6h，所述白细胞刺激因子在CD141 +树突状细胞重悬液中的终浓度为0.08～0.1％。
(2)用不同荧光色素标记的CD141抗体、Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体对CD141 +树突状细胞进行孵育检测：
(a)将步骤(1)中孵育后的CD141 +树突状细胞重悬，将CD141 +树突状细胞重悬液与不同荧光色素标记的CD141抗体、Linage抗体和HLA-DR抗体进行首次孵育，所述首次孵育的时间为25～35min、温度为20～25℃，所述CD141 +树突状细胞重悬液与CD141抗体、Linage抗体和HLA-DR抗体的体积比为100:(2～2.5):(2～2.5):(2～2.5)，用质量分数为1.5～2.5vol％***溶液固定CD141 +树突状细胞，20～25℃遮光孵育15～20min；
(b)将步骤(a)所得的CD141 +树突状细胞用细胞穿透液重悬，300～350g离心5～10min去上清，将沉淀用细胞穿透液重悬，将CD141 +树突状细胞重悬液与不同荧光色素标记的IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体进行二次孵育，所述CD141 +树突状细胞重悬液与IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体的体积比为100:(2～2.5):(2～2.5):(2～2.5):(2～2.5)，所述二次孵育在遮光的条件下进行，所述二次孵育的时间为24～26h、温度为0～4℃。
(3)用流式细胞仪进行检测和分析：
将二次孵育后的CD141 +树突状细胞用细胞穿透液重悬，300～350g离心5～10min去上清，将沉淀细胞用细胞染色液重悬，用流式细胞仪进行检测和分析；
根据CD141抗体和Linage抗体与CD141 +树突状细胞表面的CD141和Linage的结合情况，分析CD141 +树突状细胞亚群CD141 +表型的比例；
根据HLA-DR抗体与CD141 +树突状细胞表面的HLA-DR的结合情况，分析CD141 +树突状细胞亚群的分化成熟状况；以及
根据IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体与CD141 +树突状细胞表面的IL-10、IL-12、IL-23和IL-27的结合情况，分析CD141 +树突状细胞亚群的功能。
第四方面，本申请提供一种检测CD141 +树突状细胞亚群的表型和功能的系 统，所述检测CD141 +树突状细胞亚群的表型和功能的***包括：
(1)样品处理模块：对外周血进行预处理分离出CD141 +树突状细胞；
(2)检测模块：用不同荧光色素标记的CD141抗体、Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体对CD141 +树突状细胞进行孵育检测；以及
(3)分析模块：用流式细胞仪进行检测和分析。
第五方面，本申请提供第一方面所述的检测CD141 +树突状细胞亚群的表型和功能的抗体组合、第二方面所述的检测CD141 +树突状细胞亚群的表型和功能的试剂盒或第四方面所述的检测CD141 +树突状细胞亚群的表型和功能的***中任意一种或至少两种的组合在鉴定CD141 +树突状细胞亚群表型和功能和/或制备鉴定CD141 +树突状细胞亚群表型和功能的产品中的应用。
优选地，所述产品包括检测恶性肿瘤的试剂盒和/或检测试剂；
优选地，所述恶性肿瘤包括肺癌、黑色素瘤、***癌或肾癌中的任意一种或至少两种的组合。
本申请所述的数值范围不仅包括上述列举的点值，还包括没有列举出的上述数值范围之间的任意的点值，限于篇幅及出于简明的考虑，本申请不再穷尽列举所述范围包括的具体点值。
相对于现有技术，本申请具有以下有益效果：
(1)本申请所述的检测CD141 +树突状细胞亚群的表型和功能的分析方法简单、快速易行。采用人体全血一步法测定人外周血中树突状细胞亚群及其功能，与使用外周血单核细胞(PBMC)分离技术测定树突状细胞相比，本申请中所述分析方法简便易行、节约成本。用传统的PBMC分离技术分离树突状细胞，需要较大的采血量(通常几十毫升不等)，消耗时间长。而本申请采用人体全血一步法测定，只需患者一滴血(10～100μL)即可检测得到所需的全套信息。省去了分离PBMC的大量时间，能够简单快速、一步测定到位，适合临床的大批量样品的测试。
(2)本申请所述的检测CD141 +树突状细胞亚群的表型和功能的分析方发的结果信息全面。本申请通过特异性地选择7个抗体分子组合，包括CD141抗体、Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体，该配方设计可以高特异性和高灵敏度地分析CD141 +树突状细胞亚群中 CD141 +表型的比例，能对CD141 +树突状细胞亚群的分化成熟状况进行判断，还可以分析CD141 +树突状细胞亚群的功能。
(3)本申请所述的检测CD141 +树突状细胞亚群的表型和功能的分析方发的结果准确性高。本申请中检测CD141 +树突状细胞亚群的表型和功能的分析方案，相较其它技术具有灵明度高，特异性好的优点，检测的结果更加准确可靠。
(4)目前市售的树突状细胞分析试剂盒的分析方案只能测试总体树突状细胞的数据，且不包括功能分析。本申请所述的检测CD141 +树突状细胞亚群的表型和功能的试剂盒可以精细地提供人外周血中最新的CD141 +树突状细胞亚群的表型和功能的全套数据，相较于先前已开发的方案，本申请首次把CD141 +树突状细胞亚群的表型与功能相结合来进行测试。
附图说明
图1为实施例3中健康人外周血细胞FSC-SSC散点图。
图2为实施例3中健康人外周血的流式细胞分析图。
图3为实施例3中健康人/非小细胞肺癌病人的外周血中的CD141 +树突状细胞亚群的流式细胞仪分析结果图。
图4为实施例3中健康人/非小细胞肺癌病人的外周血中的CD141 +树突状细胞亚群HLA-DR的表达情况的统计图。
图5为实施例3中IL-10在同型对照/健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达比例图。
图6为实施例3中IL-10在健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达情况的统计图。
图7为实施例3中IL-12在同型对照/健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达比例图。
图8为实施例3中IL-12在健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达情况的统计图。
图9为实施例3中IL-23在同型对照/健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达比例图。
图10为实施例3中IL-23在健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达情况的统计图。
图11为实施例3中IL-27在同型对照/健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达比例图。
图12为实施例3中IL-27在健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达情况的统计图。
具体实施方式
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
以下具体实施方式中所使用的实验材料如下：
流式细胞仪(Cytek)；linage抗体(Biolegend)；CD141抗体(Biolegend)：HLA-DR抗体(eBioscience)；IL-10抗体(BD)；IL-12抗体(Biolegend)；IL-23抗体(eBioscience)；和IL-27抗体(Biolegend)。
实施例1
本实施例提供一种检测CD141 +树突状细胞亚群的表型和功能的抗体组合，所述检测CD141 +树突状细胞亚群的表型和功能的抗体组合包括CD141抗体(Biolegend)、Linage抗体(Biolegend)、HLA-DR抗体(eBioscience)、IL-10抗体(BD)、IL-12抗体(Biolegend)、IL-23抗体(eBioscience)和IL-27抗体(Biolegend)。
所述的检测CD141 +树突状细胞亚群的表型和功能的抗体组合中的抗体带有荧光色素标记。所述CD141抗体的荧光色素为BV510；所述Linage抗体的荧光色素为BV786；所述HLA-DR抗体的荧光色素为Pacific blue；所述IL-10抗体的荧光色素为PE-Cy7；所述IL-12抗体的荧光色素为AF700；所述IL-23抗体的荧光色素为eFluor660；所述IL-27抗体的荧光色素为PE。
实施例2
本实施例提供一种检测CD141 +树突状细胞亚群的表型和功能的试剂盒，所述检测CD141 +树突状细胞亚群的表型和功能的试剂盒中包括实施例1中所述的检测CD141 +树突状细胞亚群的表型和功能的抗体组合。
实施例3
本实施例使用实施例2中所述的检测CD141 +树突状细胞亚群的表型和功能的试剂盒对非小细胞肺癌病人(实验组)与健康人(对照组)的外周血中的CD141 +树突状细胞亚群进行分化和功能分析，同时对同型对照进行分析。
(1)对外周血进行预处理分离出CD141 +树突状细胞：
分别取非小细胞肺癌病人和健康成年人的静脉外周血100μL，并进行抗凝处理；将外周血全血混合于2mL 1×红细胞裂解液(Biolegend)中，旋转震荡10s后，25℃避光静置15min，用离心机离心(350g离心5min)，倾倒上清液，将沉淀细胞悬浮于2mL细胞染色液(含2.5％胎牛血清的PBS溶液)中，加入白细胞刺激因子(BD)在37℃恒温孵育细胞6h备用，所述白细胞刺激因子在CD141 +树突状细胞重悬液中的终浓度为0.1％。
(2)用不同荧光色素标记的CD141抗体、Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体对CD141 +树突状细胞进行孵育检测：
(a)将步骤(1)中所得的CD141 +树突状细胞重悬于100μL细胞染色液中，将CD141 +树突状细胞重悬液与2μL BV510标记的CD141抗体、2μL BV786标记的Linage抗体和2μL Pacific blue标记的HLA-DR抗体进行首次孵育，所述首次孵育的时间为30min、温度为25℃，加2mL细胞染色液，350g离心5min，重复离心两次，用2mL质量分数为2％***溶液固定CD141 +树突状细胞，25℃遮光孵育20min；
(b)将步骤(a)所得的CD141 +树突状细胞用2mL细胞穿透液(Biolegend)重悬，350g离心5min，重复离心两次，将沉淀用100μL细胞穿透液重悬，将CD141 +树突状细胞重悬液与2μL PE-Cy7标记的IL-10抗体、2μL AF700标记的IL-12抗体、2μL eFluor660标记的IL-23抗体和2μL PE标记的IL-27抗体进行二次孵育，所述二次孵育在遮光的条件下进行，所述二次孵育的时间为30min、温度为25℃。
(3)用流式细胞仪进行检测和分析。
将二次孵育后的CD141 +树突状细胞用2mL细胞穿透液重悬，350g离心5min去上清，将沉淀细胞用0.5mL细胞染色液重悬，用流式细胞仪进行检测和分析。
通过以上方法，使用流式细胞仪一次性得到了关于树突状细胞亚群(通过 CD141和Linage的表达情况表征)、分化程度(HLA-DR的表达情况表征)以及细胞功能(通过IL-10、IL-12、IL-23和IL-27的表达情况表征)的所有信息，并可通过这些信息的比较对非小细胞肺癌病人与健康人的差异进行研究，从而为研究非小细胞肺癌病人的免疫调节机制提供依据。
分析结果：
(1)通过CD141抗体和Linage(Lin)抗体与CD141 +树突状细胞表面的CD141和Linage的结合情况，分析树突状细胞CD141 +表型的比例。检测结果如图1所示，图1为健康人外周血细胞FSC-SSC散点图，FSC代表细胞的大小，SSC代表细胞的颗粒度；图2为健康人外周血的流式细胞分析图，左图为Linage在健康人外周血中的树突状细胞的表达比例图，Linage -(Lin -)占总细胞数的12.9％，右图为CD141在健康人外周血中的树突状细胞的表达比例图，CD141 +占总细胞数的41.9％。
(2)采用流式细胞仪检测共信号刺激分子HLA-DR在人外周血中的CD141 +树突状细胞亚群上的表达状况，此数据用于评估人外周血中的CD141 +树突状细胞亚群的分化成熟状况。图3为健康人/非小细胞肺癌病人的外周血中的CD141 +树突状细胞亚群的流式细胞仪分析结果图，图4为健康人/非小细胞肺癌病人的外周血中的CD141 +树突状细胞亚群HLA-DR的表达情况的统计图，其中图3中粗线表示健康人，虚线表示非小细胞肺癌病人，结果表明，CD141 +树突状细胞表面HLA-DR共表达越高，CD141 +树突状细胞的免疫成熟度越高，从图3和图4的结果也可以看出健康人的CD141 +树突状细胞表面的HLA-DR共表达明显多于肺癌病人；表明肺癌病人体内的CD141 +树突状细胞的免疫成熟程度显著低于健康人。
(3)通过IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体测定CD141 +树突状细胞表面的IL-10、IL-12、IL-23和IL-27的分泌表达情况，分析CD141 +树突状细胞亚群的功能。
图5为IL-10在同型对照/健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达比例图，图6为IL-10在健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达情况的统计图；IL-10有抑制体内免疫功能的作用，通过图5和图6的结果可知，非小细胞肺癌病人的CD141 +树突状细胞比健康人的CD141 +树突状细胞分泌更多的IL-10，结果表明非小细胞肺癌病人体内的CD141 +树突状细 胞通过分泌较多的IL-10来抑制体内的抗肿瘤免疫反应。
图7为IL-12在同型对照/健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达比例图，图8为IL-12在健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达情况的统计图。IL-12是促进免疫反应的细胞因子，通过图7和图8的结果可知，非小细胞肺癌病人体内的CD141 +树突状细胞比健康人的CD141 +树突状细胞分泌较少的IL-12，结果表明非小细胞肺癌病人体内的CD141 +树突状细胞分泌IL-12的数量不足，这很可能影响IL-12介导的抗肿瘤免疫反应。
图9为IL-23在同型对照/健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达比例图，图10为IL-23在健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达情况的统计图，图11为IL-27在同型对照/健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达比例图，图12为IL-27在健康人/肺癌病人的外周血中的CD141 +树突状细胞上的表达情况的统计图。从图中可知IL-23和IL-27在健康人CD141 +树突状细胞上的表达比例分别为17.7％和36.6％；而IL-23和IL-27在非小细胞肺癌病人CD141 +树突状细胞上的表达比例分别为22.2％和38.8％。
IL-23是一个促进免疫反应的细胞因子，如果树突状细胞能分泌较多的IL-23，表明树突状细胞能够通过分泌较多的IL-23来促进免疫反应，图9和图10的结果表明，正常健康人的CD141 +树突状细胞分泌的IL-23与肺癌病人CD141 +树突状细胞分泌的IL-23相似，这表明肺癌病人体内的CD141 +树突状细胞不是通过调控IL-23来影响CD141 +DC介导的免疫反应。
IL-27是一个能抑制免疫功能的细胞因子，如果树突状细胞分泌较多的IL-27，表明树突状细胞具有免疫抑制功效，树突状细胞能够通过分泌较多的IL-27来抑制免疫反应。图11和图12的检测结果表明，肺癌病人体内的CD141 +树突状细胞与正常健康人的CD141 +树突状细胞分泌的IL-27数量相比无明显区别，这表明肺癌病人体内的CD141 +树突状细胞不是通过分泌更多的IL-27来抑制免疫功能，肺癌病人体内的CD141 +树突状细胞相较于健康人体内的CD141 +树突状细胞是一种具有免疫抑制功能的树突状细胞。
上述结果表明，本申请的抗体分子组合以及分析方法可以有效鉴别外周血中的CD141 +树突状细胞亚群，并分析其分化成熟情况以及功能。
综上所述，本申请特异性地选择7个抗体分子组合，包括CD141抗体、Linage 抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体，该配方设计可以高特异性和高灵敏度地分析CD141 +树突状细胞亚群中CD141 +表型的比例，能对CD141 +树突状细胞亚群的分化成熟状况进行判断，还可以分析CD141 +树突状细胞亚群的功能。
申请人声明，以上所述仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到的变化或替换，均落在本申请的保护范围和公开范围之内。

Claims (11)

  1. 一种检测CD141 +树突状细胞亚群的表型和功能的抗体组合，其包括CD141抗体、Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体。
  2. 根据权利要求1所述的检测CD141 +树突状细胞亚群的表型和功能的抗体组合，其中，所述的检测CD141 +树突状细胞亚群的表型和功能的抗体组合中的抗体带有荧光色素标记。
  3. 根据权利要求2所述的检测CD141 +树突状细胞亚群的表型和功能的抗体组合，其中，所述荧光色素包括BV510、BV-786、Pacific blue、PE-Cy7、AF700、eFluor660或PE中任意一种或至少两种的组合。
  4. 一种检测CD141 +树突状细胞亚群的表型和功能的试剂盒，其包括权利要求1-3任一项所述的检测CD141 +树突状细胞亚群的表型和功能的抗体组合。
  5. 一种检测CD141 +树突状细胞亚群的表型和功能的分析方法，其包括如下步骤：
    (1)对外周血进行预处理分离出CD141 +树突状细胞；
    (2)用不同荧光色素标记的CD141抗体、Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体对CD141 +树突状细胞进行孵育检测；以及
    (3)用流式细胞仪进行检测和分析。
  6. 根据权利要求5所述的检测CD141 +树突状细胞亚群的表型和功能的分析方法，其中，步骤(1)中，所述对外周血进行预处理分离出CD141 +树突状细胞的步骤包括：
    将外周血与红细胞裂解液混合，避光静置，离心，分离出CD141 +树突状细胞，将所述CD141 +树突状细胞重悬于细胞染色液中，加入白细胞刺激因子孵育；
    优选地，所述避光静置的时间为10～15min，所述避光静置的温度为20～25℃；
    优选地，所述细胞染色液包括含胎牛血清的PBS缓冲液；
    优选地，所述白细胞刺激因子在CD141 +树突状细胞重悬液中的终浓度为0.08～0.1wt％；
    优选地，所述孵育的时间为4～6h，所述孵育的温度为35～38℃。
  7. 根据权利要求5或6所述的检测CD141 +树突状细胞亚群的表型和功能的分析方法，其中，步骤(2)中，所述用不同荧光色素标记的CD141抗体、 Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体对CD141 +树突状细胞进行孵育检测的步骤包括：
    (a)用不同荧光色素标记的CD141抗体、Linage抗体和HLA-DR抗体对CD141 +树突状细胞进行首次孵育；
    (b)用不同荧光色素标记的IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体对CD141 +树突状细胞进行二次孵育。
  8. 根据权利要求7所述的检测CD141 +树突状细胞亚群的表型和功能的分析方法，其中，步骤(a)中，所述用不同荧光色素标记的CD141抗体、Linage抗体和HLA-DR抗体对CD141 +树突状细胞进行首次孵育的步骤包括：
    将步骤(1)中孵育后的CD141 +树突状细胞重悬，将CD141 +树突状细胞重悬液与不同荧光色素标记的CD141抗体、Linage抗体和HLA-DR抗体进行首次孵育，用***溶液固定CD141 +树突状细胞，遮光孵育；
    优选地，所述***溶液的质量分数为1.5～2.5vol％；
    优选地，所述首次孵育的时间为25～35min，所述首次孵育的温度为20～25℃；
    优选地，所述遮光孵育的时间为15～20min，所述遮光孵育的温度为20～25℃；
    优选地，步骤(b)中，所述用不同荧光色素标记的IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体对CD141 +树突状细胞进行二次孵育的步骤包括：
    将步骤(a)所得的CD141 +树突状细胞用细胞穿透液重悬，离心去上清，将沉淀用细胞穿透液重悬，将CD141 +树突状细胞重悬液与不同荧光色素标记的IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体进行二次孵育；
    优选地，所述离心的离心力为300～350g，所述离心的时间为5～10min；
    优选地，所述二次孵育的时间为24～26h，所述二次孵育的温度为0～4℃；
    优选地，所述二次孵育在遮光的条件下进行。
  9. 根据权利要求5-8中任一项所述的检测CD141 +树突状细胞亚群的表型和功能的分析方法，其中，步骤(3)中，所述用流式细胞仪进行检测和分析的步骤包括：
    将二次孵育后的CD141 +树突状细胞用细胞穿透液重悬，离心去上清，将沉淀细胞用细胞染色液重悬，用流式细胞仪进行检测和分析；
    优选地，所述离心的离心力为300～350g，所述离心的时间为5～10min；
    优选地，所述分析的方法包括：
    根据CD141抗体和Linage抗体与CD141 +树突状细胞表面的CD141和Linage的结合情况，分析CD141 +树突状细胞亚群中CD141 +表型的比例；
    根据HLA-DR抗体与CD141 +树突状细胞表面的HLA-DR的结合情况，分析CD141 +树突状细胞亚群的分化成熟状况；以及
    根据IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体与CD141 +树突状细胞表面的IL-10、IL-12、IL-23和IL-27的结合情况，分析CD141 +树突状细胞亚群的功能。
  10. 一种检测CD141 +树突状细胞亚群的表型和功能的***，其包括：
    (1)样品处理模块：对外周血进行预处理分离出CD141 +树突状细胞；
    (2)检测模块：用不同荧光色素标记的CD141抗体、Linage抗体、HLA-DR抗体、IL-10抗体、IL-12抗体、IL-23抗体和IL-27抗体对CD141 +树突状细胞进行孵育检测；以及
    (3)分析模块：用流式细胞仪进行检测和分析。
  11. 权利要求1-3任一项所述的检测CD141 +树突状细胞亚群的表型和功能的抗体组合、权利要求4所述的检测CD141 +树突状细胞亚群的表型和功能的试剂盒或权利要求10所述的检测CD141 +树突状细胞亚群的表型和功能的***中任意一种或至少两种的组合在鉴定CD141 +树突状细胞亚群表型和功能和/或制备鉴定CD141 +树突状细胞亚群表型和功能的产品中的应用；
    优选地，所述产品包括检测恶性肿瘤的试剂盒和/或检测试剂；
    优选地，所述恶性肿瘤包括肺癌、黑色素瘤、***癌或肾癌中的任意一种或至少两种的组合。
PCT/CN2022/077229 2022-02-22 2022-02-22 一种用于检测cd141 +树突状细胞亚群表型和功能的方法及其应用试剂盒 WO2023159343A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU2022436042A AU2022436042A1 (en) 2022-02-22 2022-02-22 Method for detecting a phenotype and a function of a cd141+ dendritic cell subset and kit for use
CN202280000607.2A CN115004032A (zh) 2022-02-22 2022-02-22 一种用于检测cd141+树突状细胞亚群表型和功能的方法及其应用试剂盒
PCT/CN2022/077229 WO2023159343A1 (zh) 2022-02-22 2022-02-22 一种用于检测cd141 +树突状细胞亚群表型和功能的方法及其应用试剂盒

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/077229 WO2023159343A1 (zh) 2022-02-22 2022-02-22 一种用于检测cd141 +树突状细胞亚群表型和功能的方法及其应用试剂盒

Publications (1)

Publication Number Publication Date
WO2023159343A1 true WO2023159343A1 (zh) 2023-08-31

Family

ID=83022832

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/077229 WO2023159343A1 (zh) 2022-02-22 2022-02-22 一种用于检测cd141 +树突状细胞亚群表型和功能的方法及其应用试剂盒

Country Status (3)

Country Link
CN (1) CN115004032A (zh)
AU (1) AU2022436042A1 (zh)
WO (1) WO2023159343A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117624367A (zh) * 2023-11-29 2024-03-01 武汉爱博泰克生物科技有限公司 抗人cd141蛋白的兔单克隆抗体及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4987086A (en) * 1987-11-30 1991-01-22 Becton, Dickinson And Company Method for analysis of subpopulations of cells
US20040219612A1 (en) * 2003-05-02 2004-11-04 Beckman Coulter, Inc. Methods for detecting intracellular defensins in various leukocyte subpopulations
CN104360053A (zh) * 2014-09-22 2015-02-18 重庆医科大学附属儿童医院 一种b淋巴细胞免疫分型的方法和试剂盒
CN110389221A (zh) * 2019-08-30 2019-10-29 广州中科蓝华生物科技有限公司 一种用于分析CD1c+树突状细胞亚群表型和功能的组合配方试剂盒及其应用
CN112147326A (zh) * 2020-09-04 2020-12-29 北京大学 一种肿瘤免疫细胞亚群分型精准检测试剂盒

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4987086A (en) * 1987-11-30 1991-01-22 Becton, Dickinson And Company Method for analysis of subpopulations of cells
US20040219612A1 (en) * 2003-05-02 2004-11-04 Beckman Coulter, Inc. Methods for detecting intracellular defensins in various leukocyte subpopulations
CN104360053A (zh) * 2014-09-22 2015-02-18 重庆医科大学附属儿童医院 一种b淋巴细胞免疫分型的方法和试剂盒
CN110389221A (zh) * 2019-08-30 2019-10-29 广州中科蓝华生物科技有限公司 一种用于分析CD1c+树突状细胞亚群表型和功能的组合配方试剂盒及其应用
CN112147326A (zh) * 2020-09-04 2020-12-29 北京大学 一种肿瘤免疫细胞亚群分型精准检测试剂盒

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117624367A (zh) * 2023-11-29 2024-03-01 武汉爱博泰克生物科技有限公司 抗人cd141蛋白的兔单克隆抗体及其应用

Also Published As

Publication number Publication date
CN115004032A (zh) 2022-09-02
AU2022436042A1 (en) 2023-09-07

Similar Documents

Publication Publication Date Title
Shu et al. Purity and yield of melanoma exosomes are dependent on isolation method
Menard et al. Immunohematological reference ranges for adults from the Central African Republic
CN106635995B (zh) 一种循环肿瘤细胞阴性富集方法
Kimoto et al. Further development of the rat Pig‐a mutation assay: Measuring rat Pig‐a mutant bone marrow erythroids and a high throughput assay for mutant peripheral blood reticulocytes
WO2021036105A1 (zh) 一种用于分析CD1c+树突状细胞亚群表型和功能的组合配方试剂盒及其应用
Lopresti et al. Sensitive and easy screening for circulating tumor cells by flow cytometry
Ivison et al. A standardized immune phenotyping and automated data analysis platform for multicenter biomarker studies
WO2020107495A1 (zh) 一种用于流式细胞仪分析的淋巴细胞样品的制备方法
WO2021012925A1 (zh) 一种人外周血淋巴细胞的检测方法
CN107402296A (zh) 一种循环肿瘤细胞的免疫荧光染色及判读方法
WO2023159343A1 (zh) 一种用于检测cd141 +树突状细胞亚群表型和功能的方法及其应用试剂盒
CN115166252A (zh) 淋巴细胞亚群分群与定量检测试剂盒及其检测方法与应用
CN112098646B (zh) 一种定量检测淋巴细胞亚群的试剂盒及其检测方法
Templeman et al. Analytical performance of the FDA-cleared Parsortix® PC1 system
Mura et al. Optimized flow cytometric protocol for the detection of functional subsets of low frequency antigen-specific CD4+ and CD8+ T cells
Houyhongthong et al. Automated nucleated red blood cell count using the Mindray BC‐6800 hematology analyzer
Tsai et al. High throughput pSTAT signaling profiling by fluorescent cell barcoding and computational analysis
Qiu et al. Changes and clinical significance of detailed peripheral lymphocyte subsets in evaluating the immunity for cancer patients
McLaughlin et al. Nine‐color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part II: Panel performance across different instrument platforms
Venable et al. An analysis of endothelial microparticles as a function of cell surface antibodies and centrifugation techniques
Fang et al. Circulating immune cell phenotypes are associated with age, sex, CMV, and smoking status in the Framingham Heart Study offspring participants
WO2022246764A1 (zh) 一种分析cd303+树突状细胞亚群表型和功能的方法及试剂盒
US20220317120A1 (en) Use of proteins pd-1 and cd38 as markers of an active auto-immune pathology
Popko et al. Disturbances in NK cells in various types of hemophagocytic lymphohistiocytosis in a population of polish children
Liu et al. Approaches for Performance Verification Toward Standardization of Peripheral Blood Regulatory T-Cell Detection by Flow Cytometry

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 2022436042

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2022436042

Country of ref document: AU

Date of ref document: 20220222

Kind code of ref document: A

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22927640

Country of ref document: EP

Kind code of ref document: A1