WO2023115703A1 - 一种b淋巴细胞发育亚群免疫分型的方法和试剂盒 - Google Patents

一种b淋巴细胞发育亚群免疫分型的方法和试剂盒 Download PDF

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WO2023115703A1
WO2023115703A1 PCT/CN2022/078969 CN2022078969W WO2023115703A1 WO 2023115703 A1 WO2023115703 A1 WO 2023115703A1 CN 2022078969 W CN2022078969 W CN 2022078969W WO 2023115703 A1 WO2023115703 A1 WO 2023115703A1
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antibody
cells
igd
tested
lymphocyte
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French (fr)
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赵耀
牛琳琳
谭冰倩
杨宇婷
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重庆医科大学附属儿童医院
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • 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

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  • the invention relates to the field of biotechnology, in particular to a method and kit for immunotyping B lymphocyte developmental subgroups.
  • B lymphocytes are differentiated and matured from lymphoid precursor cells in the bone marrow of mammals or in the bursa of Fabricius of birds. Mature B cells mainly settled in the lymphoid nodules of the superficial layer of the lymph node cortex and the lymphoid nodules of the red pulp and white pulp of the spleen. In peripheral blood, B cells account for about 20% of the total number of lymphocytes. B cells are cells that produce antibodies in the body and have the function of antigen presentation. B cells and the antibodies they produce are central elements of humoral immunity as part of the adaptive immune system that protects the body against an almost unlimited variety of pathogens. Defects in the development, selection, and function of B cells lead to autoimmunity, malignancy, immunodeficiency, and allergic diseases.
  • B cells can be divided into different developmental subpopulations.
  • Initial development of human B cells occurs in the bone marrow and involves the development of progenitor cells into pre-B cells and pre-B cell populations.
  • transitional B cells develop further in the peripheral blood lymphoid organs and circulate in the blood as naive B cells.
  • Naive B cells encounter antigen and are stimulated in germinal centers and lymphoid organ margins through T-dependent and independent pathways to form memory B cells, plasmablasts, and long-lived plasma cells.
  • Serum immunoglobulin levels are widely regarded as the most reliable surrogate markers of B-cell function in healthy donors and patients with different immune-related diseases.
  • B-cell development is important for understanding the mechanisms underlying B-cell-directed therapy and its impact on B-cell autoantibody production and other functions.
  • B cell-directed therapy In addition to treating hematological malignancies such as non-Hodgkin's lymphoma and chronic lymphocytic leukemia, B cell-directed therapy also plays an important role in the treatment of autoimmune diseases.
  • B lymphocytes in blood samples are closely related to the body's immune system, and the changes in the total number of B cells and the composition of B cells in peripheral blood are more dynamic.
  • B cells have unique differentiation antigens, and flow cytometry is often used to analyze peripheral blood CD19 to detect total B cells.
  • There are a variety of receptors on the surface of B cells such as immunoglobulins on the membrane surface. Most immature naive B cells and mature B cells are detected by indirect fluorescent immunoassay or enzyme immunohistochemistry.
  • the present invention provides a method and kit for immunotyping of B lymphocyte developmental subgroups, which can realize more comprehensive and fine immunophenotyping and quantitative analysis of B lymphocytes, with high efficiency and saving
  • the amount of sample to be tested is short, and it is more suitable for the typing and quantitative analysis of B lymphocyte developmental subgroups.
  • the invention provides a method for immunophenotyping of B lymphocyte developmental subgroups, comprising:
  • Said antibodies include:
  • the determination method of described cell type is:
  • Cell surface markers CD19 + CD10 + CD38 + IgD + represent transitional B cells
  • Cell surface markers CD19 + CD10 - CD38 - IgD - represent memory B cells
  • Cell surface markers CD19 + CD10 - CD38 + IgD - represent plasmablasts
  • Cell surface markers CD19 + CD10 - CD38 - IgD + represent untransformed B cells
  • Cell surface markers CD19 + CD10 - CD38 - CD27 - IgD + represent naive B cells
  • Cell surface markers CD19 + CD10 - CD38 - CD27 + IgD + represent marginal zone B cells.
  • the antibody used in flow cytometry can be an antibody homologous to the sample to be tested or an antibody non-homologous to the sample to be tested, as long as the antibody can bind to the cell surface markers in the sample to be tested
  • the substance can produce antigen-antibody specific binding reaction.
  • the surface markers for identification are expressed on the surface of the cell membrane.
  • fluorescein-labeled monoclonal antibodies as molecular probes, flow cytometry of these cell surface markers can be carried out. Then, the types, subtypes and functional characteristics of cells are analyzed.
  • the present invention provides a method for immunophenotyping of specific T cell developmental subgroups: when the cell surface markers are CD19 + CD10 + CD38 + IgD + , transitional B cells can be immunized Type; when the cell surface markers are CD19 + CD10 - CD38 - IgD - , memory B cells can be immunotyped; when the cell surface markers are CD19 + CD10 - CD38 + IgD - , plasmablasts can be immunotyped ; When the cell surface markers are CD19 + CD10 - CD38 - IgD + , unconverted B cells can be immunophenotyped; when the cell surface markers are CD19 + CD10 - CD38 - CD27 - IgD + , naive B cells can be immunophenotyped B cells in the marginal zone can be immunophenotyped when the cell surface markers are CD19 + CD10 - CD38 - CD27 + IgD + .
  • the present invention designs an antibody combination with optimal cell surface markers, and conducts a more comprehensive immune typing of B lymphocyte developmental subgroups through flow cytometry.
  • the method for immunotyping the development of B lymphocyte subgroups provided by the invention requires less samples to be tested, is simple to operate, has high accuracy, takes a short time, and can be used for immunotyping of B lymphocyte subgroup development.
  • a fluorescence minus one control (FMO control) is also set.
  • the significance of setting the FMO control is to evaluate the interference of other fluorescent dyes on the target channel, which can more accurately Determines the threshold for positive staining to help properly set the positive gate.
  • the control group is a fluorescence minus one control (FMO control).
  • FMO control fluorescence minus one control
  • the FMO control tube is to add all fluorescent antibodies except Percp-cy5.5-CD38.
  • V450-CD27 the FMO control tube is to add all fluorescent antibodies except V450-CD27.
  • the method for immunophenotyping of B lymphocyte developmental subgroups provided by the present invention is specifically:
  • the sample to be tested with erythrocyte lysate, place the resulting mixture in a water bath at 36.5°C to 37.5°C for 6min to 8min, and centrifuge to obtain the first product, which is fluorescently labeled anti-CD19 antibody, anti-CD10 antibody, anti-IgD Antibody, anti-CD27 antibody, anti-CD38 antibody, mixed with the first product, incubated at room temperature (20°C-25°C) for 15min-20min, washed, and tested by flow cytometry to obtain test data, according to the test Data to determine cell type;
  • the determination method of described cell type comprises:
  • Cell surface markers CD19 + CD10 + CD38 + IgD + represent transitional B cells
  • Cell surface markers CD19 + CD10 - CD38 - IgD - represent memory B cells
  • Cell surface markers CD19 + CD10 - CD38 + IgD - represent plasmablasts
  • Cell surface markers CD19 + CD10 - CD38 - IgD + represent untransformed B cells
  • Cell surface markers CD19 + CD10 - CD38 - CD27 - IgD + represent naive B cells
  • Cell surface markers CD19 + CD10 - CD38 - CD27 + IgD + represent marginal zone B cells.
  • the fluorescent labels in each fluorescently labeled antibody are not limited by the present invention, and those skilled in the art can select appropriate fluorescent labels and control methods according to actual conditions.
  • the fluorescent labels of the five antibodies include APC, BV510, FITC, V450 or Percp-cy5.5, including but not limited thereto.
  • the method for immunotyping B lymphocyte developmental subgroups provided by the present invention further includes the step of detecting the number of B lymphocytes in the sample.
  • the present invention provides a method for immunophenotyping B lymphocyte developmental subgroups, the step of detecting the number of B lymphocyte developmental groups in the sample to be tested includes:
  • the cell number of the B lymphocyte development subgroup in the sample to be tested can be obtained.
  • the total number of lymphocytes is multiplied by the percentage of B lymphocytes therein to obtain the number of B lymphocytes.
  • the absolute number of cells of each B cell subgroup can be obtained by multiplying the absolute number of B lymphocytes by the immunotype and relative statistics of each B cell developmental subgroup in the sample to be tested.
  • the step of detecting the percentage of B lymphocytes in the sample includes:
  • the antibody used in detecting the percentage of B lymphocytes in the sample to be tested is an anti-CD19 antibody.
  • the fluorescent label of the anti-CD19 antibody is APC.
  • the step of detecting the percentage of B lymphocytes in the sample is a routine lymphocyte immunophenotyping and quantitative analysis method, which is not limited by the present invention, and those skilled in the art can according to The actual situation chooses the method of determining the percentage of B lymphocytes in the sample to be tested.
  • the method of detecting the total number of lymphocytes in the sample to be tested is counting by using a blood cell counter or a cell counter.
  • the method for detecting the total number of lymphocytes in the sample to be tested is a conventional method, which is not limited by the present invention, and those skilled in the art can choose to detect the sample to be tested according to the actual situation. The total number of lymphocytes in the method.
  • the present invention also provides a method for immunotyping of B lymphocyte developmental subgroups, which includes the step of immunotyping of B lymphocyte developmental subgroups provided by the present invention;
  • the methods for immunophenotyping of the B lymphocyte developmental subsets include:
  • Said antibodies include:
  • Anti-CD19 antibody anti-CD10 antibody, anti-IgD antibody, anti-CD27 antibody, anti-CD38 antibody;
  • the method of described cell typing comprises:
  • Cell surface markers CD19 + CD10 + CD38 + IgD + represent transitional B cells
  • Cell surface markers CD19 + CD10 - CD38 - IgD - represent memory B cells
  • Cell surface markers CD19 + CD10 - CD38 + IgD - represent plasmablasts
  • Cell surface markers CD19 + CD10 - CD38 - IgD + represent untransformed B cells
  • Cell surface markers CD19 + CD10 - CD38 - CD27 - IgD + represent naive B cells
  • Cell surface markers CD19 + CD10 - CD38 - CD27 + IgD + represent marginal zone B cells
  • the present invention also provides a kit for immunophenotyping of B lymphocyte developmental subgroups, including the following different fluorescently labeled antibodies:
  • the difference here refers to the different fluorescent labels used by the above five antibodies.
  • the fluorescently labeled antibody in the kit for immunophenotyping of B lymphocyte developmental subgroups provided by the present invention, can be placed separately with the fluorescent marker and the antibody, and when used, the two are coupled to obtain fluorescence Labeled antibody; it can also be directly fluorescently labeled antibody, which can be used directly when used.
  • the kit for immunophenotyping of B lymphocyte developmental subgroups provided by the present invention includes fluorescent markers and antibodies:
  • This antibody includes:
  • the fluorescent markers include APC, BV510, FITC, V450 or Percp-cy5.5, including but not limited to the above fluorescent markers.
  • the fluorescent label of the anti-CD19 antibody is APC.
  • the fluorescent label of the anti-CD10 antibody is BV510.
  • the fluorescent label of the anti-IgD antibody is FITC.
  • the fluorescent label of the anti-CD27 antibody is V450.
  • the fluorescent label of the anti-CD38 antibody is Percp-cy5.5.
  • the control group is a fluorescence minus one control (FMO control).
  • FMO control fluorescence minus one control
  • the FMO control tube is to add all fluorescent antibodies except Percp-cy5.5-CD38.
  • V450-CD27 the FMO control tube is to add all fluorescent antibodies except V450-CD27.
  • the fluorescent markers in each fluorescently labeled antibody are not limited by the present invention, and those skilled in the art can select appropriate fluorescent markers and corresponding controls according to actual conditions.
  • the accuracy of the target cells can be judged by the target cell grouping and the fluorescence intensity of the target cells; the target cell grouping is clear, and the target cell fluorescence intensity is accurate and high, indicating that the accuracy is high .
  • the invention provides a method and kit for immunophenotyping of B lymphocyte developmental subgroups.
  • the method for immunophenotyping of B lymphocyte developmental subgroups provided by the present invention comprises: taking different fluorescently labeled antibodies, mixing them with the samples to be tested, and after incubation, detecting by flow cytometry to obtain detection data, and analyzing the obtained detection data ;
  • the antibody includes: anti-CD19 antibody, anti-CD10 antibody, anti-IgD antibody, anti-CD27 antibody, anti-CD38 antibody.
  • the method of this analysis includes: cell surface markers CD19 + CD10 + CD38 + IgD + represent transitional B cells; cell surface markers CD19 + CD10 - CD38 - IgD - represent memory B cells; cell surface markers CD19 + CD10 - CD38 + IgD - Represents plasmablasts; cell surface markers CD19 + CD10 - CD38 - IgD + represent unconverted B cells; cell surface markers CD19 + CD10 - CD38 - CD27 - IgD + represent naive B cells; cell surface markers CD19 + CD10 - CD38 - CD27 + IgD + stands for marginal zone B cells.
  • the present invention designs and obtains the optimal antibody combination of cell surface markers, and realizes a more comprehensive immune typing and quantitative analysis of B lymphocyte developmental subgroups by flow cytometry.
  • the method provided by the present invention requires a small amount of samples to be tested, simple operation, obvious clustering, and short time-consuming.
  • the method provided by the present invention has good repeatability, obvious grouping and high accuracy, and can be widely used in immunophenotyping and quantitative analysis of B lymphocyte developmental subgroups.
  • Fig. 1 is the result of B lymphocyte classification in the implementation case 1;
  • Fig. 2 is the typing result of B lymphocyte developmental subpopulation in embodiment case 1;
  • Fig. 3 is the result of B lymphocyte classification in the implementation case 2;
  • Fig. 4 is the typing result of B lymphocyte developmental subpopulation in embodiment case 2;
  • Fig. 5 is the result of B lymphocyte classification in the implementation case 3.
  • Fig. 6 is the typing result of the B lymphocyte developmental subpopulation in the implementation case 3;
  • Fig. 7 is the result of B lymphocyte classification in the implementation case 4.
  • Fig. 8 is the typing result of B lymphocyte developmental subpopulation in embodiment case 4.
  • Fig. 9 is the result of B lymphocyte classification in implementation case 5.
  • Fig. 10 is the typing result of B lymphocyte developmental subgroups in Example 5.
  • the invention provides a method and kit for immunophenotyping of B lymphocyte developmental subgroups.
  • Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. What needs to be pointed out in particular is that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.
  • the reagents and raw materials used in the method and kit for immunotyping B lymphocyte developmental subgroups provided by the present invention can be purchased from the market.
  • the fluorescent labels FITC, Percp-cy5.5, BV510, V450, and APC used in the present invention are all common fluorescent labels and can be purchased from the market, and the antibodies of each fluorescent label can also be purchased from the market.
  • Sample to be tested anticoagulated peripheral blood sample, derived from healthy volunteers, which is the peripheral blood of normal people.
  • Anti-CD19 antibodies, anti-CD10 (BV510) antibodies, and anti-CD38 (Percp-Cy5.5) antibodies labeled with fluorescent APC were purchased from BioLegend, and anti-IgD (FITC) antibodies and anti-CD27 (V450) antibodies were purchased from BDbiosciences.
  • Red blood cell lysate (cat RT122-02) was purchased from Tiangen Biochemical Technology Co., Ltd.
  • the remaining 100 ⁇ L was used to detect the developmental subgroups of B lymphocytes, and perform immunotype and quantitative analysis of the subgroups of B lymphocyte development.
  • B-1 and B-2 are the control group and B-2 is the test group, and put them into the two flow tubes B-1 and B-2.
  • the relative number (percentage) and the absolute number of lymphocytes of each B lymphocyte development subgroup are obtained.
  • Table 2 The cell surface markers used for immunophenotyping of each B cell subset, and the relative and absolute numbers of each B cell subset
  • the method used in this case can accurately divide B lymphocytes into transitional B cells, memory B cells, plasmablasts, untransformed B cells, naive B cells, marginal zone B cells, and Quantitative analysis was carried out to obtain the relative and absolute numbers of each B lymphocyte development subpopulation.
  • the method provided by the present invention can realize the immunophenotyping of the developmental subgroups of B lymphocytes in the samples to be tested by using a small amount of samples to be tested, and count the content of each cell subgroup, and the method can Accurate and clear immunophenotyping of each B lymphocyte developmental subgroup.
  • Table 2 the absolute number of each cell subgroup is basically consistent with the reference range of normal people. It shows that the experimental results of the present invention are stable and accurate. Except that the values of plasmablasts and untransformed B cells are not within the reference range, it may be related to the source of normal people in the literature, and normal people are from foreign countries.
  • the method provided by the present invention uses fewer samples to be tested to achieve lymphocyte immunophenotyping of the sample to be tested, and counts the content of each cell subgroup; and the method provided by the present invention can accurately Individual cell subsets were immunotyped.
  • Samples to be tested anticoagulated peripheral blood samples, derived from venous blood samples from children who came to our hospital (with signed informed consent), and peripheral blood from children with suspected neutrophil-mediated autoinflammation.
  • Anti-CD19 antibodies, anti-CD10 (BV510) antibodies, and anti-CD38 (Percp-Cy5.5) antibodies labeled with fluorescent APC were purchased from BioLegend, and anti-IgD (FITC) antibodies and anti-CD27 (V450) antibodies were purchased from BDbiosciences.
  • Red blood cell lysate (cat RT122-02) was purchased from Tiangen Biochemical Technology Co., Ltd.
  • the remaining 100 ⁇ L was used to detect the developmental subgroups of B lymphocytes, and perform immunotype and quantitative analysis of the subgroups of B lymphocyte development.
  • APC-labeled anti-CD19 antibody BV510-labeled anti-CD10 antibody
  • Percp-Cy5.5-labeled anti-CD38 antibody FITC-labeled anti-IgD antibody
  • V450-labeled anti-CD27 antibody Antibody after fully vortexing, incubate at room temperature in the dark for 20min.
  • Table 3 The cell surface markers used for immunophenotyping of each B cell subset, and the relative and absolute numbers of each B cell subset
  • the method used in this case can accurately divide B lymphocytes into transitional B cells, memory B cells, plasmablasts, untransformed B cells, naive B cells, marginal zone B cells, and Quantitative analysis was carried out to obtain the relative and absolute numbers of each B lymphocyte development subpopulation.
  • the results showed that the relative numbers and absolute values of memory B cells and plasmablasts were lower than normal values, while the absolute values of naive B cells and untransformed B cells were higher than normal values, suggesting humoral immune dysfunction in children.
  • the method provided by the present invention can realize the immunophenotyping of the developmental subgroups of B lymphocytes in the samples to be tested by using a small amount of samples to be tested, and count the content of each cell subgroup, and the method can Accurate and clear immunophenotyping of each B lymphocyte developmental subgroup.
  • Samples to be tested anticoagulated peripheral blood samples, derived from venous blood samples from children who came to our hospital (with signed informed consent), and peripheral blood from children with suspected liver failure.
  • Anti-CD19 antibodies, anti-CD10 (BV510) antibodies, and anti-CD38 (Percp-Cy5.5) antibodies labeled with fluorescent APC were purchased from BioLegend, and anti-IgD (FITC) antibodies and anti-CD27 (V450) antibodies were purchased from BDbiosciences.
  • Red blood cell lysate (cat RT122-02) was purchased from Tiangen Biochemical Technology Co., Ltd.
  • the remaining 100 ⁇ L was used to detect the developmental subgroups of B lymphocytes, and perform immunotype and quantitative analysis of the subgroups of B lymphocyte development.
  • APC-labeled anti-CD19 antibody BV510-labeled anti-CD10 antibody
  • Percp-Cy5.5-labeled anti-CD38 antibody FITC-labeled anti-IgD antibody
  • V450-labeled anti-CD27 antibody Antibody after fully vortexing, incubate at room temperature in the dark for 20min.
  • Table 4 The cell surface markers used for immunophenotyping of each B cell subset, and the relative and absolute numbers of each B cell subset
  • the method used in this case can accurately divide B lymphocytes into transitional B cells, memory B cells, plasmablasts, untransformed B cells, naive B cells, marginal zone B cells, and Quantitative analysis was carried out to obtain the relative and absolute numbers of each B lymphocyte development subpopulation. It can be seen from the results that the relative and absolute numbers of total B cells and transitional B cells increased, the absolute values of untransformed B cells were higher than normal, and the relative and absolute numbers of plasmablasts decreased, suggesting that the humoral immune function of the children obstacle.
  • the method provided by the present invention can realize the immunophenotyping of the developmental subgroups of B lymphocytes in the samples to be tested by using a small amount of samples to be tested, and count the content of each cell subgroup, and the method can Accurate and clear immunophenotyping of each B lymphocyte developmental subgroup.
  • Samples to be tested anticoagulated peripheral blood samples, derived from venous blood samples of children who came to our hospital (with signed informed consent), peripheral blood of children with biliary atresia to be diagnosed.
  • Anti-CD19 antibodies, anti-CD10 (BV510) antibodies, and anti-CD38 (Percp-Cy5.5) antibodies labeled with fluorescent APC were purchased from BioLegend, and anti-IgD (FITC) antibodies and anti-CD27 (V450) antibodies were purchased from BDbiosciences.
  • Red blood cell lysate (cat RT122-02) was purchased from Tiangen Biochemical Technology Co., Ltd.
  • the remaining 100 ⁇ L was used to detect the developmental subgroups of B lymphocytes, and perform immunotype and quantitative analysis of the subgroups of B lymphocyte development.
  • APC-labeled anti-CD19 antibody BV510-labeled anti-CD10 antibody
  • Percp-Cy5.5-labeled anti-CD38 antibody FITC-labeled anti-IgD antibody
  • V450-labeled anti-CD27 antibody Antibody after fully vortexing, incubate at room temperature in the dark for 20min.
  • Table 5 The cell surface markers used for immunophenotyping of each B cell subset, and the relative and absolute numbers of each B cell subset
  • the method used in this case can accurately divide B lymphocytes into transitional B cells, memory B cells, plasmablasts, untransformed B cells, naive B cells, marginal zone B cells, and Quantitative analysis was carried out to obtain the relative and absolute numbers of each B lymphocyte development subpopulation. From the results, it can be seen that the relative number and absolute number of transitional B cells decreased, the absolute value of untransformed B cells was higher than the normal value, and the relative number of naive B cells and memory B cells decreased, but the absolute value was normal, suggesting that the children Humoral immune dysfunction.
  • the method provided by the present invention can realize the immunophenotyping of the developmental subgroups of B lymphocytes in the samples to be tested by using a small amount of samples to be tested, and count the content of each cell subgroup, and the method can Accurate and clear immunophenotyping of each B lymphocyte developmental subgroup.
  • Sample to be tested an anticoagulated peripheral blood sample, derived from a healthy volunteer (female, 26 years old), which is the peripheral blood of a normal person.
  • Anti-CD19 antibodies, anti-CD10 (BV510) antibodies, and anti-CD38 (Percp-Cy5.5) antibodies labeled with fluorescent APC were purchased from BioLegend, and anti-IgD (FITC) antibodies and anti-CD27 (V450) antibodies were purchased from BDbiosciences.
  • Lymphocyte separation medium was purchased from Dakowei Biotechnology Co., Ltd.
  • peripheral blood mononuclear cells PBMC
  • PBMC peripheral blood mononuclear cells
  • lymphocyte separation medium was added to the diluted peripheral blood, and centrifuged at 800g for 20min. After centrifugation, the buffy coat in the middle was sucked, which was PBMC.
  • APC-labeled anti-CD19 antibody Take 1 ⁇ 10 6 flow tubes and add antibodies: APC-labeled anti-CD19 antibody, BV510-labeled anti-CD10 antibody, Percp-Cy5.5-labeled anti-CD38 antibody, FITC-labeled anti-IgD antibody, V450-labeled anti-CD27 After fully vortexing, incubate at room temperature in the dark for 20 min.
  • Table 6 The cell surface markers used for immunophenotyping of each B cell subset, the relative number and absolute number of each B cell subset
  • the method used in this case can accurately divide B lymphocytes into transitional B cells, memory B cells, plasmablasts, untransformed B cells, naive B cells, marginal zone B cells, and Quantitative analysis was carried out to obtain the relative and absolute numbers of each B lymphocyte development subpopulation. It can be seen from Table 2 that the absolute number of each cell subgroup is basically consistent with the reference range of normal people. It shows that the experimental results of the present invention are stable and accurate.
  • the method provided by the present invention can realize the immunophenotyping of the developmental subgroups of B lymphocytes in the samples to be tested by using a small amount of samples to be tested, and count the content of each cell subgroup, and the method can Accurate and clear immunophenotyping of each B lymphocyte developmental subgroup.

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Abstract

一种B淋巴细胞发育亚群免疫分型的方法和试剂盒。B淋巴细胞发育亚群免疫分型的方法包括：取不同的荧光标记的抗体，与待检测样品混合，孵育后，经流式细胞术检测，得检测数据；抗体包括：抗CD19的抗体、抗CD10的抗体、抗IgD的抗体、抗CD27的抗体、抗CD38的抗体。实现了对B淋巴细胞发育亚群进行更加全面的免疫分型，所需待测样品少，操作简单、所需时间短、准确性高。

Description

一种B淋巴细胞发育亚群免疫分型的方法和试剂盒
本申请要求于2021年12月22日提交中国专利局、申请号为202111584004.5、发明名称为“一种B淋巴细胞发育亚群免疫分型的方法和试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，尤其涉及一种B淋巴细胞发育亚群免疫分型的方法和试剂盒。
背景技术
B淋巴细胞，简称B细胞，是由哺乳动物骨髓或鸟类法氏囊中的淋巴样前体细胞分化成熟而来。成熟B细胞主要定居于淋巴结皮质浅层的淋巴小结和脾脏红髓及白髓的淋巴小结内。在外周血中，B细胞约占淋巴细胞总数的20％左右。B细胞是体内产生抗体的细胞，并具有抗原提呈功能。B细胞及其产生的抗体是体液免疫的核心要素，作为适应性免疫系统的一部分，可以保护人体抵抗几乎无限多种病原体。B细胞的发育、选择和功能缺陷导致自身免疫、恶性肿瘤、免疫缺陷和过敏性疾病。
根据B细胞不同的细胞表面标志，可以将B细胞分为不同的发育亚群。人B细胞的最初发育发生在骨髓中，包括将祖细胞发育为前B细胞和前B细胞群体。离开骨髓后，过渡性B细胞在外周血淋巴器官中进一步发育，并作为初始B细胞在血液中循环。初始B细胞遇到抗原，在生发中心和淋巴器官边缘区通过T依赖和独立的途径受到刺激，形成记忆B细胞、浆母细胞和长寿浆细胞。
血清免疫球蛋白水平被广泛认为是健康供体和不同免疫相关疾病患者中最可靠的B细胞功能替代标志物。而B细胞发育对理解B细胞定向治疗的潜在机制及其对B细胞自身抗体产生和其他功能的影响非常重要。除了治疗血液系统恶性肿瘤如非霍奇金淋巴瘤和慢性淋巴细胞白血病外， B细胞定向疗法在自身免疫性疾病的治疗中也发挥着重要作用。最近在类风湿关节炎和系统性红斑狼疮患者中进行的临床研究表明，利用利妥昔单抗治疗记忆细胞的损耗是有疗效的，但是记忆B细胞并不是消耗抗体的唯一目标，因此，这些干预措施可能会调节其他具有治疗相关性的B细胞群(Joshua A.Boyce et.al..B cell–directed therapies for autoimmune disease and correlates of disease response and relapse.J ALLERGY CLIN IMMUNOL,2008,121:13-21.)。因此，对B细胞亚群的研究更为迫切。
血液样本中的B淋巴细胞与机体的免疫系统密切相关，利用外周血检测B细胞总数和B细胞组成的变化更为动态。B细胞具有独特的分化抗原，常用流式细胞术分析外周血CD19检测总B细胞，但是，没有对B细胞的发育情况进行进一步检测。B细胞表面有多种受体，如膜表面免疫球蛋白，大多采用间接荧光免疫法或酶免疫组织化学法检测未成熟的幼稚B细胞和成熟B细胞。不管是利用流式细胞术检测总B细胞，还是利用间接荧光免疫法或酶免疫组织化学法，这些方法对B细胞的检测面窄，操作复杂，样品需要量大，耗时也比较长。如果要对B细胞发育进行更加精细的免疫分型，需要采用更为简单、省时和样品量需求小的方法和定量分析。
发明内容
有鉴于此，本发明提供了一种B淋巴细胞发育亚群免疫分型的方法和试剂盒，该方法可实现B淋巴细胞更全面、精细地免疫分型和定量分析，效率高，且节约了待测样品的用量，用时短，更适合B淋巴细胞发育亚群的分型和定量分析。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种B淋巴细胞发育亚群免疫分型的方法，包括：
取不同荧光标记的抗体，与待检测样品混合，孵育后，经流式细胞术检测，得检测数据，根据检测数据判定细胞型别；
所述抗体包括：
抗CD19的抗体、抗CD10的抗体、抗IgD的抗体、抗CD27的抗体 和抗CD38的抗体；
所述细胞型别的判定方法为：
细胞表面标志CD19 +CD10 +CD38 +IgD +代表过渡性B细胞；
细胞表面标志CD19 +CD10 -CD38 -IgD -代表记忆B细胞；
细胞表面标志CD19 +CD10 -CD38 +IgD -代表浆母细胞；
细胞表面标志CD19 +CD10 -CD38 -IgD +代表未转换B细胞；
细胞表面标志CD19 +CD10 -CD38 -CD27 -IgD +代表初始B细胞；
细胞表面标志CD19 +CD10 -CD38 -CD27 +IgD +代表边缘区B细胞。
在本发明中，流式细胞术中所用到的抗体可以为与待测样品同源的抗体或也可以为与待测样品非同源的抗体，只要该抗体能够与待测样品中细胞表面标志物产生抗原-抗体特异性结合反应即可。
在本发明中，“+”代表阳性，即表示该抗原在细胞表面有表达；
“++”代表强阳性，即表示该抗原在细胞表面高表达；
“-”代表阴性，即表示该抗原在细胞表面不表达。
淋巴细胞在各自正常分化成熟的不同阶段及活化过程中，其细胞膜表面均表达可供鉴别的表面标志，利用荧光素标记的单克隆抗体作为分子探针，可对这些细胞表面标志进行流式细胞术检测，进而对细胞的种类、亚类以及功能特性进行分析。本发明经过大量创造性的研究和试验验证，提供了一种针对的T细胞发育亚群免疫分型的方法：细胞表面标志为CD19 +CD10 +CD38 +IgD +时，可对过渡性B细胞进行免疫分型；细胞表面标志为CD19 +CD10 -CD38 -IgD -时，可对记忆B细胞进行免疫分型；细胞表面标志为CD19 +CD10 -CD38 +IgD -时，可对浆母细胞进行免疫分型；细胞表面标志为CD19 +CD10 -CD38 -IgD +时，可对未转换B细胞进行免疫分型；细胞表面标志为CD19 +CD10 -CD38 -CD27 -IgD +时，可对初始B细胞进行免疫分型；细胞表面标志为CD19 +CD10 -CD38 -CD27 +IgD +时，可对边缘区B细胞进行免疫分型。本发明根据对各个细胞所对应的细胞表面标志，设计获得了最优的细胞表面标志的抗体组合，通过流式细胞术，对B淋巴细胞发育亚群进行了更为全面的免疫分型。本发明提供的B淋巴细胞 亚群发育免疫分型的方法所需待测样品少、操作简单、准确性高，用时短、能够用于B淋巴细胞发育亚群的免疫分型。
优选地，本发明提供B淋巴细胞发育亚群的免疫分型方法中，还设置荧光减一对照(FMO对照)，设置FMO对照的意义在于评估其他荧光染料对目的通道的干扰，能较为准确地确定阳性染色的阈值，帮助正确设置阳性门。
在本发明的实施例中，本发明提供的B淋巴细胞发育亚群的免疫分型的方法中，对照组采用的是荧光减一对照(FMO对照)。当检测Percp-cy5.5-CD38时，FMO对照管就是加入除去Percp-cy5.5-CD38以外的所有荧光抗体。当检测V450-CD27时，FMO对照管就是加入除去V450-CD27以外的所有荧光抗体。
本发明的实施例中，本发明提供的B淋巴细胞发育亚群免疫分型的方法，具体为：
取待测样品与红细胞裂解液混合，并将所得混合液至于36.5℃～37.5℃的水浴6min～8min，离心，得第一产品，取荧光标记的抗CD19的抗体、抗CD10的抗体、抗IgD的抗体、抗CD27的抗体、抗CD38的抗体，与第一产品混合，室温(即20℃～25℃)条件下，孵育15min～20min，洗涤，流式上机检测，得到检测数据，根据检测数据判定细胞型别；
所述细胞型别的判定方法包括：
细胞表面标志CD19 +CD10 +CD38 +IgD +代表过渡性B细胞；
细胞表面标志CD19 +CD10 -CD38 -IgD -代表记忆B细胞；
细胞表面标志CD19 +CD10 -CD38 +IgD -代表浆母细胞；
细胞表面标志CD19 +CD10 -CD38 -IgD +代表未转换B细胞；
细胞表面标志CD19 +CD10 -CD38 -CD27 -IgD +代表初始B细胞；
细胞表面标志CD19 +CD10 -CD38 -CD27 +IgD +代表边缘区B细胞。
在本发明中，本发明提供的方法中，各个荧光标记抗体中的荧光标记不受本发明的限制，本领域的技术人员可以根据实际情况选择合适的荧光标记和对照方法。在本发明的实施例中，所述五种抗体所述荧光标记包括 APC、BV510、FITC、V450或Percp-cy5.5，包括但不限于此。
本发明提供的B淋巴细胞发育亚群的免疫分型方法中，还包括检测样品中B淋巴细胞数目的步骤。
在本发明的实案例中，本发明提供B淋巴细胞发育亚群免疫分型的方法中，检测待测样品中B淋巴细胞发育群数目的步骤包括：
检测待测样品中淋巴细胞的总数；
检测待测样品中B淋巴细胞发育亚群占所述淋巴细胞的百分比；
通过计算，即得待测样品中B淋巴细胞发育亚群的细胞数目。
本发明提供的方法中，检测样品中B淋巴细胞的数目中，淋巴细胞总数乘以其中B淋巴细胞的百分比，即得B淋巴细胞的数目。在本发明中，经过对待测样品中各B细胞发育亚群的免疫分型和相对数统计，再乘以B淋巴细胞的绝对数即可获得每个B细胞亚群的细胞的绝对数。
在本发明的实施例中，本发明提供的方法中，检测样品中B淋巴细胞的百分比的步骤，包括：
取荧光标记的抗体，与所述待测样品混合，孵育后，经流式细胞术检测，得检测数据，分析所述检测数据，计算待测样品中B淋巴细胞的百分比；
其中，检测待测样品中B淋巴细胞的百分比中所用的抗体为抗CD19的抗体。
在本发明的一些实施例中，本发明提供的制备方法中，检测样品中B淋巴细胞的百分比的步骤中，抗CD19的抗体的荧光标记为APC。
在本发明中，本发明提供的方法中，检测样品中B淋巴细胞的百分比的步骤为常规的淋巴细胞免疫分型和定量分析方法，该方法不受本发明的限制，本领域技术人员可以根据实际情况选择测定待测样品中B淋巴细胞的百分比的方法。
在本发明的实施例中，本发明提供的方法中，检测待测样品中淋巴细胞的总数的方法为采用血细胞计数仪或细胞计数仪进行计数。在本发明中，本发明提供的方法中，检测待测样品中淋巴细胞的总数的方法为常规的方法，该方法不受本发明的限制，本领域技术人员可以根据实际情况选 择检测待测样品中淋巴细胞的总数的方法。
本发明还提供了一种B淋巴细胞发育亚群免疫分型的方法，其包括本发明提供的B淋巴细胞发育亚群免疫分型的步骤；
该B淋巴细胞发育亚群免疫分型的方法包括：
取不同的荧光标记的抗体，与待测样品混合，孵育后，经流式细胞术检测，得检测数据，根据检测数据判定细胞分型；
所述抗体包括：
抗CD19的抗体、抗CD10的抗体、抗IgD的抗体、抗CD27的抗体、抗CD38的抗体；
所述细胞分型的方法包括：
细胞表面标志CD19 +CD10 +CD38 +IgD +代表过渡性B细胞；
细胞表面标志CD19 +CD10 -CD38 -IgD -代表记忆B细胞；
细胞表面标志CD19 +CD10 -CD38 +IgD -代表浆母细胞；
细胞表面标志CD19 +CD10 -CD38 -IgD +代表未转换B细胞；
细胞表面标志CD19 +CD10 -CD38 -CD27 -IgD +代表初始B细胞；
细胞表面标志CD19 +CD10 -CD38 -CD27 +IgD +代表边缘区B细胞；
本发明还提供了一种用于B淋巴细胞发育亚群的免疫分型的试剂盒，包括如下不同荧光标记的抗体：
抗CD19的抗体、抗CD10的抗体、抗IgD的抗体、抗CD27的抗体、抗CD38的抗体。
这里的不同是指以上五种抗体采用的荧光标记不同。
在本发明中，本发明提供的用于B淋巴细胞发育亚群免疫分型的试剂盒中，荧光标记的抗体，可以是荧光标记物与抗体单独放置，在使用时将两者偶联获得荧光标记的抗体；也可以是直接荧光标记的抗体，在使用时，直接使用即可。
在本发明的一些实施例中，本发明提供的用于B淋巴细胞发育亚群免疫分型的试剂盒包括荧光标记物和抗体：
该抗体包括：
抗CD19的抗体、抗CD10的抗体、抗IgD的抗体、抗CD27的抗体、抗CD38的抗体。
所述荧光标记包括APC、BV510、FITC、V450或Percp-cy5.5，包括但不限于以上荧光标记。
在本发明一些实施例中，本发明提供的用于B淋巴细胞发育亚群免疫分型的试剂盒中，抗CD19的抗体的荧光标记为APC。
在本发明一些实施例中，本发明提供的用于B淋巴细胞发育亚群免疫分型的试剂盒中，抗CD10的抗体的荧光标记为BV510。
在本发明一些实施例中，本发明提供的用于B淋巴细胞发育亚群免疫分型的试剂盒中，抗IgD的抗体的荧光标记为FITC。
在本发明一些实施例中，本发明提供的用于B淋巴细胞发育亚群免疫分型的试剂盒中，抗CD27的抗体的荧光标记为V450。
在本发明一些实施例中，本发明提供的用于B淋巴细胞发育亚群免疫分型的试剂盒中，抗CD38的抗体的荧光标记为Percp-cy5.5。
在本发明的实施例中，本发明提供的B淋巴细胞发育亚群的免疫分型的方法中，对照组采用的是荧光减一对照(FMO对照)。当检测Percp-cy5.5-CD38时，FMO对照管就是加入除去Percp-cy5.5-CD38以外的所有荧光抗体。当检测V450-CD27时，FMO对照管就是加入除去V450-CD27以外的所有荧光抗体。
在本发明中，本发明提供的试剂盒中，各个荧光标记的抗体中的荧光标记物不受本发明的限制，本领域技术人员可以根据实际情况选择合适的荧光标记物，以及对应的对照。
在本发明中，流式细胞术检测过程中，通过目标细胞分群和目标细胞的荧光强度可以对目标细胞进行准确性的判断；目标细胞分群清楚、目标细胞荧光强度准确高，则表示准确性高。
本发明提供了一种B淋巴细胞发育亚群免疫分型的方法和试剂盒。本发明提供的B淋巴细胞发育亚群免疫分型的方法，包括：取不同的荧光标记的抗体，与待测样品混合，孵育后，经流式细胞术检测，得到检测数据，分析所得检测数据；该抗体包括：抗CD19的抗体、抗CD10的抗 体、抗IgD的抗体、抗CD27的抗体、抗CD38的抗体。该分析的方法包括：细胞表面标志CD19 +CD10 +CD38 +IgD +代表过渡性B细胞；细胞表面标志CD19 +CD10 -CD38 -IgD -代表记忆B细胞；细胞表面标志CD19 +CD10 -CD38 +IgD -代表浆母细胞；细胞表面标志CD19 +CD10 -CD38 -IgD +代表未转换B细胞；细胞表面标志CD19 +CD10 -CD38 -CD27 -IgD +代表初始B细胞；细胞表面标志CD19 +CD10 -CD38 -CD27 +IgD +代表边缘区B细胞。实验结果证实，本发明设计获得了最优的细胞表面标志的抗体组合，通过流式细胞术，实现了对B淋巴细胞发育亚群进行了更加全面的免疫分型和定量分析。在本发明的一些实施例中，本发明提供的方法，所需待测样本量少、操作简单、分群明显、用时短。在本发明的一些实施例中，本发明提供的方法重复性好、分群明显、准确性高、可广泛用于B淋巴细胞发育亚群免疫分型和定量分析。
附图说明
图1为实施案例1中B淋巴细胞分类的结果；
图2为实施案例1中B淋巴细胞发育亚群的分型结果；
图3为实施案例2中B淋巴细胞分类的结果；
图4为实施案例2中B淋巴细胞发育亚群的分型结果；
图5为实施案例3中B淋巴细胞分类的结果；
图6为实施案例3中B淋巴细胞发育亚群的分型结果；
图7为实施案例4中B淋巴细胞分类的结果；
图8为实施案例4中B淋巴细胞发育亚群的分型结果；
图9为实施案例5中B淋巴细胞分类的结果；
图10为实施案例5中B淋巴细胞发育亚群的分型结果。
具体实施方式
本发明提供了一种B淋巴细胞发育亚群免疫分型的方法和试剂盒。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它 们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明中，中英文对照如下：
Figure PCTCN2022078969-appb-000001
本发明提供的一种B淋巴细胞发育亚群免疫分型的方法和试剂盒中所用到的试剂和原料均可由市场购得。
本发明中用到的荧光标记FITC、Percp-cy5.5、BV510、V450、APC均为常见的荧光标记可以由市场购得，各个荧光标记的抗体也可以由市场购得。
下面结合实施例，进一步阐述本发明：
实施案例1 B淋巴细胞发育亚群免疫分型和定量分析
实验材料：
待测样品：抗凝外周血样品，来源于健康志愿者，为正常人的外周血。
荧光标记APC的抗CD19抗体、抗CD10(BV510)的抗体、抗CD38(Percp-Cy5.5)的抗体购买于BioLegend，抗IgD(FITC)的抗体、抗CD27(V450)的抗体购买于BDbiosciences。
红细胞裂红液(cat RT122-02)购买于天根生化科技有限公司。
实验方法：
取300μL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为2.58×10 9个/L。
剩下100μL用于检测B淋巴细胞发育亚群，进行B淋巴细胞发育亚群免疫分型和定量分析。
B淋巴细胞发育亚群免疫分型
1、取两根流式管，分别标记为B-1、B-2，其中B-1为对照组，B-2为待检测组，向B-1、B-2两根流式管中各加入50μL待测样品，然后分别向流式管中加入1mL的红细胞裂红液，充分涡旋后，37℃水浴6min～8min，离心500g，5min，倒去上清液。
2、按照表1向B-1、B-2流式管中加入以下抗体，充分涡旋后，室温避光孵育20min；
表1各个流式管中加入的抗体的类别和加入的体积
Figure PCTCN2022078969-appb-000002
3、各加入1mL PBS，500g，离心5min，洗涤一次后，加入200μLPBS垂悬，流式上机并分析结果。
结果分析：
通过以上操作，得到各B淋巴细胞发育亚群相对数(百分数)和淋巴细胞绝对数。各B淋巴细胞发育亚群相对数(百分数)×淋巴细胞绝对数，即得到B淋巴细胞各亚群绝对数。
B淋巴细胞发育亚群免疫分型和定量分析结果(采用FlowJo分析软件分析)
B淋巴细胞免疫分型结果见图1-A，图2-A、2-B、2-C，从图中可知，B细胞以及各类B细胞发育亚群的相对数(百分数)，根据淋巴细胞的绝对数、和各个B细胞亚群的相对数(百分数)，计算获得各个B细胞亚群的绝对数，具体实验结果见表2。
表2各个B细胞亚群免疫分型所用的细胞表面标志、各个B细胞亚群的相对数和绝对数
Figure PCTCN2022078969-appb-000003
Figure PCTCN2022078969-appb-000004
“*”正常值范围参考文献：Elena Blanco et al..Age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood.2018,J ALLERGY CLIN IMMUNOL.,141(6):2208-2219。
“-”代表未从文献查到边缘区B细胞占B细胞的绝对数。
根据图1和图2可知，本案例所用的方法能够准确的将B淋巴细胞分为过渡性B细胞、记忆B细胞、浆母细胞、未转换B细胞、初始B细胞、边缘区B细胞，并进行定量分析，获得了各个B淋巴细胞发育亚群的相对数和绝对数。
综上所述，本发明提供的方法采用少量的待测样品就可以实现对待检测样品的B淋巴细胞的发育亚群的免疫分型，并统计了每个细胞亚群的含量，且本方法可以准确、清晰的将各个B淋巴细胞发育亚群进行免疫分型。从表2中可知，各个细胞亚群的绝对数与正常人的参考范围基本一致。说明本发明实验结果稳定、准确。除了浆母细胞和未转换B细胞的值未在参考范围内，可能跟文献中的正常人来源有关，正常人来源于国外。
综上所述，本发明提供的方法采用较少的待测样品就实现了对待测样品的淋巴细胞免疫分型，并统计出了每个细胞亚群的含量；且本发明提供的方法能够准确将各个细胞亚群进行免疫分型。
实施案例2 B淋巴细胞发育亚群免疫分型和定量分析
实验材料：
待测样品：抗凝外周血样品，来源于来我院就诊患儿静脉血样品(已签知情同意书)，为拟诊中性粒细胞介导的自身炎症患儿的外周血。
荧光标记APC的抗CD19抗体、抗CD10(BV510)的抗体、抗CD38(Percp-Cy5.5)的抗体购买于BioLegend，抗IgD(FITC)的抗体、抗CD27(V450)的抗体购买于BDbiosciences。
红细胞裂红液(cat RT122-02)购买于天根生化科技有限公司。
实验方法：
取300μL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为2.92×10 9个/L。
剩下100μL用于检测B淋巴细胞发育亚群，进行B淋巴细胞发育亚群免疫分型和定量分析。
B淋巴细胞发育亚群免疫分型
与实施案例1中记载的B淋巴细胞分类方法相同，流式管中加入50μL待测样品，然后向流式管中加入1mL的红细胞裂红液，充分涡旋后，37℃水浴6min～8min，离心500g，5min，倒去上清液。
然后向流式管中加入抗体：APC标记的抗CD19抗体、BV510标记的抗CD10的抗体、Percp-Cy5.5标记的抗CD38的抗体，FITC标记的抗IgD的抗体、V450标记的抗CD27的抗体，充分涡旋后，室温避光孵育20min。
B淋巴细胞免疫分型和定量分析结果(采用FlowJo分析软件分析)
B淋巴细胞免疫分型结果见图3-A，3-B，图4-A、4-B、4-C，从图中可知，B细胞和各个B细胞亚群的相对数(百分数)，计算获得各个B细胞亚群的绝对数，具体实验结果见表3。
表3各个B细胞亚群免疫分型所用的细胞表面标志、各个B细胞亚群的相对数和绝对数
Figure PCTCN2022078969-appb-000005
Figure PCTCN2022078969-appb-000006
“*”正常值范围参考文献：Elena Blanco et al..Age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood.2018,JALLERGY CLIN IMMUNOL.,141(6):2208-2219；Yuan Ding et al..Reference values for peripheral blood lymphocyte subsets of healthy children in China.2018,J ALLERGY CLIN IMMUNOL.,1-4。“-”代表未从文献查到边缘区B细胞占B淋巴细胞的相对数和绝对数。
根据图3和图4可知，本案例所用的方法能够准确的将B淋巴细胞分为过渡性B细胞、记忆B细胞、浆母细胞、未转换B细胞、初始B细胞、边缘区B细胞，并进行定量分析，获得了各个B淋巴细胞发育亚群的相对数和绝对数。从结果可知，记忆B细胞和浆母细胞的相对数和绝 对值低于正常值，而初始B细胞和未转换B细胞的绝对值高于正常值，提示患儿体液免疫功能障碍。
综上所述，本发明提供的方法采用少量的待测样品就可以实现对待检测样品的B淋巴细胞的发育亚群的免疫分型，并统计了每个细胞亚群的含量，且本方法可以准确、清晰的将各个B淋巴细胞发育亚群进行免疫分型。
实施案例3 B淋巴细胞发育亚群免疫分型和定量分析
实验材料：
待测样品：抗凝外周血样品，来源于来我院就诊患儿静脉血样品(已签知情同意书)，为拟诊肝功能衰竭患儿的外周血。
荧光标记APC的抗CD19抗体、抗CD10(BV510)的抗体、抗CD38(Percp-Cy5.5)的抗体购买于BioLegend，抗IgD(FITC)的抗体、抗CD27(V450)的抗体购买于BDbiosciences。
红细胞裂红液(cat RT122-02)购买于天根生化科技有限公司。
实验方法：
取300μL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为5.14×10 9个/L。
剩下100μL用于检测B淋巴细胞发育亚群，进行B淋巴细胞发育亚群免疫分型和定量分析。
B淋巴细胞发育亚群免疫分型
与实施案例1中记载的B淋巴细胞分类方法相同，流式管中加入50μL待测样品，然后向流式管中加入1mL的红细胞裂红液，充分涡旋后，37℃水浴6min～8min，离心500g，5min，倒去上清液。
然后向流式管中加入抗体：APC标记的抗CD19抗体、BV510标记的抗CD10的抗体、Percp-Cy5.5标记的抗CD38的抗体，FITC标记的抗IgD的抗体、V450标记的抗CD27的抗体，充分涡旋后，室温避光孵育20min。
B淋巴细胞免疫分型和定量分析结果(采用FlowJo分析软件分析)
B淋巴细胞免疫分型结果见图5-A，5-B，图6-A、6-B、6-C，从图中可知，B细胞和各个B细胞亚群的相对数(百分数)，计算获得各个B细胞亚群的绝对数，具体实验结果见表4。
表4各个B细胞亚群免疫分型所用的细胞表面标志、各个B细胞亚群的相对数和绝对数
Figure PCTCN2022078969-appb-000007
注：*正常值范围参考文献：Elena Blanco et al..Age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood.2018,J ALLERGY CLIN IMMUNOL.,141(6):2208-2219。Yuan Ding et al..Reference values for peripheral blood lymphocyte subsets of healthy children  in China.2018,J ALLERGY CLIN IMMUNOL.,1-4。“-”代表未从文献查到边缘区B细胞占B淋巴细胞的相对数和绝对数。
根据图5和图6可知，本案例所用的方法能够准确的将B淋巴细胞分为过渡性B细胞、记忆B细胞、浆母细胞、未转换B细胞、初始B细胞、边缘区B细胞，并进行定量分析，获得了各个B淋巴细胞发育亚群的相对数和绝对数。从结果可知，总B细胞和过渡性B细胞的相对数和绝对数增加，未转换B细胞的绝对值高于正常值，而浆母细胞的相对数和绝对数降低，提示患儿体液免疫功能障碍。
综上所述，本发明提供的方法采用少量的待测样品就可以实现对待检测样品的B淋巴细胞的发育亚群的免疫分型，并统计了每个细胞亚群的含量，且本方法可以准确、清晰的将各个B淋巴细胞发育亚群进行免疫分型。
实施案例4 B淋巴细胞发育亚群免疫分型和定量分析
实验材料：
待测样品：抗凝外周血样品，来源于来我院就诊患儿静脉血样品(已签知情同意书)，为拟诊胆道闭锁患儿的外周血。
荧光标记APC的抗CD19抗体、抗CD10(BV510)的抗体、抗CD38(Percp-Cy5.5)的抗体购买于BioLegend，抗IgD(FITC)的抗体、抗CD27(V450)的抗体购买于BDbiosciences。
红细胞裂红液(cat RT122-02)购买于天根生化科技有限公司。
实验方法：
取300μL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为7.31×10 9个/L。
剩下100μL用于检测B淋巴细胞发育亚群，进行B淋巴细胞发育亚群免疫分型和定量分析。
B淋巴细胞发育亚群免疫分型
与实施案例1中记载的B淋巴细胞分类方法相同，流式管中加入50μL待测样品，然后向流式管中加入1mL的红细胞裂红液，充分涡旋后，37℃水浴6min～8min，离心500g，5min，倒去上清液。
然后向流式管中加入抗体：APC标记的抗CD19抗体、BV510标记的抗CD10的抗体、Percp-Cy5.5标记的抗CD38的抗体，FITC标记的抗IgD的抗体、V450标记的抗CD27的抗体，充分涡旋后，室温避光孵育20min。
B淋巴细胞免疫分型和定量分析结果(采用FlowJo分析软件分析)
B淋巴细胞免疫分型结果见图7-A，7-B，图8-A、8-B、8-C，从图中可知，B细胞和各个B细胞亚群的相对数(百分数)，计算获得各个B细胞亚群的绝对数，具体实验结果见表5。
表5各个B细胞亚群免疫分型所用的细胞表面标志、各个B细胞亚群的相对数和绝对数
Figure PCTCN2022078969-appb-000008
*正常值范围参考文献：Elena Blanco et al..Age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood.2018,J  ALLERGY CLIN IMMUNOL.,141(6):2208-2219。Yuan Ding et al..Reference values for peripheral blood lymphocyte subsets of healthy children in China.2018,J ALLERGY CLIN IMMUNOL.,1-4。
“-”代表未从文献查到边缘区B细胞占B淋巴细胞的相对数和绝对数。
根据图7和图8可知，本案例所用的方法能够准确的将B淋巴细胞分为过渡性B细胞、记忆B细胞、浆母细胞、未转换B细胞、初始B细胞、边缘区B细胞，并进行定量分析，获得了各个B淋巴细胞发育亚群的相对数和绝对数。从结果可知，过渡性B细胞的相对数和绝对数都降低，未转换B细胞的绝对值高于正常值，而初始B细胞和记忆B细胞的相对数降低，但绝对值正常，提示患儿体液免疫功能障碍。
综上所述，本发明提供的方法采用少量的待测样品就可以实现对待检测样品的B淋巴细胞的发育亚群的免疫分型，并统计了每个细胞亚群的含量，且本方法可以准确、清晰的将各个B淋巴细胞发育亚群进行免疫分型。
实施案例5 B淋巴细胞发育亚群免疫分型和定量分析
实验材料：
待测样品：抗凝外周血样品，来源于健康志愿者(女，26岁)，为正常人的外周血。
荧光标记APC的抗CD19抗体、抗CD10(BV510)的抗体、抗CD38(Percp-Cy5.5)的抗体购买于BioLegend，抗IgD(FITC)的抗体、抗CD27(V450)的抗体购买于BDbiosciences。
淋巴细胞分离液购买于达科为生物技术有限公司。
实验方法：
取3mL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为1.13×10 9个/L。
剩下的外周血用于提取外周血单核细胞(PBMC)，PBMC用于检测B淋巴细胞发育亚群，进行B淋巴细胞发育亚群免疫分型和定量分析。
B淋巴细胞发育亚群免疫分型
外周血用PBS稀释后，在稀释的外周血中加入淋巴细胞分离液，离心800g，20min，离心完后，吸取中间的白膜层，即为PBMC。
取1í10 6流式管中加入抗体：APC标记的抗CD19抗体、BV510标记的抗CD10的抗体、Percp-Cy5.5标记的抗CD38的抗体，FITC标记的抗IgD的抗体、V450标记的抗CD27的抗体，充分涡旋后，室温避光孵育20min。
B淋巴细胞免疫分型和定量分析结果(采用FlowJo分析软件分析)
B淋巴细胞免疫分型结果见图9-A，9-B，图10-A、10-B、10-C，从图中可知，B细胞和各个B细胞亚群的相对数(百分数)，计算获得各个B细胞亚群的绝对数，具体实验结果见表6。
表6各个B细胞亚群免疫分型所用的细胞表面标志、各个B细胞亚群的相对数和绝对数
Figure PCTCN2022078969-appb-000009
*正常值范围参考文献：Elena Blanco et al..Age-associated distribution of normal B-cell andplasma cell subsets in peripheral blood.2018,J ALLERGY CLIN IMMUNOL.,141(6):2208-2219。Yuan Ding et al..Reference values for peripheral blood lymphocyte subsets of healthy children  in China.2018,J ALLERGY CLIN IMMUNOL.,1-4。“-”代表未从文献查到边缘区B细胞占B细胞的绝对数。
根据图9和图10可知，本案例所用的方法能够准确的将B淋巴细胞分为过渡性B细胞、记忆B细胞、浆母细胞、未转换B细胞、初始B细胞、边缘区B细胞，并进行定量分析，获得了各个B淋巴细胞发育亚群的相对数和绝对数。从表2中可知，各个细胞亚群的绝对数与正常人的参考范围基本一致。说明本发明实验结果稳定、准确。
综上所述，本发明提供的方法采用少量的待测样品就可以实现对待检测样品的B淋巴细胞的发育亚群的免疫分型，并统计了每个细胞亚群的含量，且本方法可以准确、清晰的将各个B淋巴细胞发育亚群进行免疫分型。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (6)

  1. 一种B淋巴细胞发育亚群免疫分型的方法，其特征在于，包括：
    取不同荧光标记的抗体，与待检测样品混合，孵育后，经流式细胞术检测，得检测数据，根据检测数据判定细胞型别；
    所述抗体包括：
    抗CD19的抗体、抗CD10的抗体、抗IgD的抗体、抗CD27的抗体和抗CD38的抗体；
    所述细胞型别的判定方法为：
    细胞表面标志CD19 +CD10 +CD38 +IgD +代表过渡性B细胞；
    细胞表面标志CD19 +CD10 -CD38 -IgD -代表记忆B细胞；
    细胞表面标志CD19 +CD10 -CD38 +IgD -代表浆母细胞；
    细胞表面标志CD19 +CD10 -CD38 -IgD +代表未转换B细胞；
    细胞表面标志CD19 +CD10 -CD38 -CD27 -IgD +代表初始B细胞；
    细胞表面标志CD19 +CD10 -CD38 -CD27 +IgD +代表边缘区B细胞。
  2. 根据权利要求1所述的方法，其特征在于，所述荧光标记包括APC、BV510、FITC、V450或Percp-cy5.5，包括但不限于以上荧光标记。
  3. 根据权利要求1所述的方法，其特征在于，还包括统计所述待测样品中B淋巴细胞数目的步骤，包括：
    1)、检测所述待测样品中淋巴细胞的总数；
    2)、检测所述待测样品中B淋巴细胞占所述淋巴细胞的百分比；
    3)、通过计算，获得所述待测样品中B淋巴细胞的数目。
  4. 根据权利要求3所述的方法，其特征在于，所述步骤2)包括：
    取荧光标记的抗体，与所述待测样品混合，孵育后，经流式细胞术检测，得检测数据，分析检测数据；
    所述抗体为抗CD19的抗体。
  5. 一种用于B淋巴细胞发育亚群免疫分型的试剂盒，其特征在于，包括荧光标记的如下抗体：
    抗CD19的抗体、抗CD10的抗体、抗IgD的抗体、抗CD27的抗体 和抗CD38的抗体。
  6. 根据权利要求5所述的试剂盒，其特征在于，所述荧光标记包括APC、BV510、FITC、V450或Percp-cy5.5。
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