Antibody composition for lymphocyte subpopulation analysis, detection kit, detection method and application

Abstract

The invention relates to the field of medical treatment, and discloses an antibody composition for lymphocyte subpopulation analysis, a detection kit, a detection method and application, and the antibody composition comprises an anti-CD38 antibody, an anti-CD45RA antibody, an anti-CD57 antibody, an anti-CD45 antibody, an anti-CD27 antibody, an anti-CD16 antibody, an anti-CD56 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD19 antibody and an antiTCR-alpha-beta antibody. 12 antibodies are combined, the cell proliferation condition and activation condition of a person to be detected can be detected, the detection items are comprehensive and highly targeted, the prognosis condition of a tumor patient can be well reflected, reference is provided for prevention and relapse, meanwhile, the combination of the antibodies is marked, and the detection accuracy is improved. Under the condition of an existing 10-color flow cytometry instrument, 12 CD can be detected at a time, the requirement for the sample size is obviously reduced, and the detection speed is increased.

Description

Technical Field

[0001] The present invention relates to the field of medical devices, and in particular to an antibody composition, a detection kit, a detection method and applications for lymphocyte subpopulation analysis. Background Art

[0002] Lymphocyte subset analysis is currently the most widely used flow cytometry procedure in clinical practice. It is an important indicator of both cellular and humoral immunity. It provides an overall reflection of the body's current immune function, status, and balance. It can aid in the diagnosis of certain diseases, such as autoimmune diseases, immunodeficiency disorders, malignancies, hematologic disorders, and allergic diseases, and is crucial for analyzing pathogenesis, observing therapeutic efficacy, and assessing prognosis. Lymphocyte subset analysis can assess the relative percentages (%) and absolute cell counts (cells / μl) of CD3+ total T cells, CD3+CD4+ helper T cells (Th), CD3+CD8+ cytotoxic T cells (Tc), CD3-CD19+ B lymphocytes, and CD3(-CD16+CD56)+ NK cells. Currently, there is no universal monitoring reagent for prognostic assessment and prevention of recurrence and metastasis in cancer patients.

[0003] In addition, the current mainstream lymphocyte subset analysis is mainly performed through 6- or 10-color flow cytometers, which can monitor a maximum of 6CD or 10CD fluorescence at a time. Increasing CD requires matching a higher instrument or selecting multiple tubes, which places greater demands on the instrument and sample. However, increasing CD requires matching a higher instrument or using a multi-tube multi-color method. The former has higher requirements for the instrument and requires the purchase of new equipment, resulting in increased testing costs. For some regions, there is currently insufficient funding to support the replacement of equipment, which is one of the reasons for the incompleteness of the analysis method. The latter requires more tests, resulting in longer detection time.

[0004] Therefore, how to provide a product and method that can simultaneously detect multiple cell subpopulations, detect multiple cell subpopulations at a time, and closely monitor the body's health status and self-defense capabilities through the test results has become an urgent problem to be solved. Summary of the Invention

[0005] The present invention aims to overcome the problem of the prior art that tumor patients' prognosis cannot be effectively monitored by the present invention, and provides an antibody composition, detection kit, detection method and application for lymphocyte subset analysis. The antibody composition can effectively monitor the prognosis of tumor patients.

[0006] In order to achieve the above-mentioned objectives, the first aspect of the present invention provides an antibody composition for lymphocyte subpopulation analysis, wherein the antibody composition comprises: the antibody composition comprises: anti-CD38 antibody, anti-CD45RA antibody, anti-CD57 antibody, anti-CD45 antibody, anti-CD27 antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD19 antibody and anti-TCR-αβ antibody.

[0007] By using the above technical solution and combining 12 antibodies, the cell proliferation and activation of the test sample can be tested. The detection items are comprehensive and highly targeted, which can better reflect the prognosis of tumor patients and provide a reference for prevention and recurrence.

[0008] The antibodies of the present invention are all mouse anti-human monoclonal antibodies.

[0009] Preferably, the antibody composition is divided into a first group of antibodies and n second groups of antibodies, where n is a positive integer;

[0010] Wherein, each antibody in the first group of antibodies is independently labeled with a different marker, each second group of antibodies independently includes two antibodies, the two antibodies in the same group of second group of antibodies are labeled with the same marker, and the two antibodies in the same group of second group of antibodies cannot be expressed in the same type of cell population, the markers of each second group of antibodies are different, and the markers of each second group of antibodies are different from the markers of each antibody in the first group of antibodies.

[0011] The existing 10-color flow cytometer can detect 10CDs at a time. The present invention can detect 12CDs at a time through the 10-color flow cytometer, which can meet the needs of some areas with limited detection conditions and shorten the detection time of multiple CDs at a time.

[0012] Preferably, the second group of antibodies is: anti-CD19 antibody and anti-CD8 antibody;

[0013] And / or, the second group of antibodies includes: anti-CD16 antibodies and anti-CD56 antibodies.

[0014] Preferably, the second group of antibodies is: anti-CD19 antibody and anti-CD3 antibody;

[0015] And / or, the second group of antibodies includes: anti-TCR-αβ antibodies and anti-CD8 antibodies.

[0016] Preferably, the second group of antibodies is: anti-CD19 antibody and anti-CD4 antibody;

[0017] And / or, the second group of antibodies includes: anti-CD16 antibodies and anti-CD56 antibodies.

[0018] The above scheme provides multiple options for combined CD detection of the second set of antibodies. The remaining part of the second set of antibodies is the first set of antibodies, which can be selected as needed. When n is 1, only 9 detection channels are required. When n is 2, 12 CDs can be detected using 10 channels.

[0019] Preferably, the label is a fluorescent label.

[0020] Preferably, the fluorescent labels include FITC, PE, BV605 (ECD), PerCP-Cy 5.5, PE-CY7, APC, APC700, APC-Cy7, BV421 and BV510.

[0021] Using the above scheme, appropriate labeling substances can be selected as needed. Generally speaking, CDs with positive or strong positive expression can be matched with weak fluorescent substances to ensure that each CD has an appropriate fluorescence intensity to ensure accurate and reliable detection results.

[0022] Preferably, n is 2, wherein one group of the second group of antibodies comprises: anti-CD19 antibody and anti-CD8 antibody, which are labeled with APC, and another group of the second group of antibodies comprises: anti-CD16 antibody and anti-CD56 antibody, which are labeled with PE;

[0023] The anti-CD38 antibody in the first group of antibodies was labeled with PC5.5, the anti-CD45RA antibody was labeled with BV605 (ECD), the anti-CD57 antibody was labeled with FITC, the anti-CD45 antibody was labeled with APC-Cy7, the anti-CD27 antibody was labeled with APC700, the anti-CD3 antibody was labeled with BV510, the anti-CD4 antibody was labeled with PC7, and the anti-TCR-αβ antibody was labeled with BV421.

[0024] Preferably, n is 2, wherein one group of the second group of antibodies is an anti-CD19 antibody and an anti-CD3 antibody, which are labeled with APC, and another group of the second group of antibodies is an anti-TCR-αβ antibody and an anti-CD8 antibody, which are labeled with PE;

[0025] The anti-CD38 antibody in the first group of antibodies was labeled with PC5.5, the anti-CD45RA antibody was labeled with BV605 (ECD), the anti-CD57 antibody was labeled with FITC, the anti-CD45 antibody was labeled with APC-Cy7, the anti-CD27 antibody was labeled with APC700, the anti-CD4 antibody was labeled with PC7, the anti-CD16 antibody was labeled with BV421, and the anti-CD56 antibody was labeled with BV51.

[0026] Preferably, n is 2, wherein one group of the second group of antibodies is an anti-CD19 antibody and an anti-CD4 antibody, which are labeled with APC, and another group of the second group of antibodies is an anti-CD16 antibody and an anti-CD56 antibody, which are labeled with PE;

[0027] The anti-CD38 antibody in the first group of antibodies was labeled with PC5.5, the anti-CD45RA antibody was labeled with BV605 (ECD), the anti-CD57 antibody was labeled with FITC, the anti-CD45 antibody was labeled with APC-Cy7, the anti-CD27 antibody was labeled with APC700, the anti-CD3 antibody was labeled with BV510, the anti-CD8 antibody was labeled with PC7, and the anti-TCR-αβ antibody was labeled with BV421.

[0028] The above technical solution provides a suitable and specific matching relationship between CD and fluorescent substance for detection, and each marked CD has a relatively balanced fluorescence intensity, which effectively ensures the accuracy of the detection result.

[0029] The second aspect of the present invention provides a detection kit, which includes the antibody composition described in the first aspect of the present invention.

[0030] Preferably, the antibody composition is a powder or liquid formulation, and when it is a liquid formulation, it also includes a buffer. The detection antibody in the kit can be in powder form or liquid form, and there is no particular requirement for its presence. When it is a liquid formulation, the buffer can be PBS buffer.

[0031] The third aspect of the present invention provides a method for lymphocyte subpopulation analysis, wherein the antibody composition of the first aspect of the present invention or the detection kit of the second aspect of the present invention is used to treat cells to be tested, and then data analysis is performed, wherein the data analysis includes dividing the cell population into a granulocyte region, a monocyte region, a lymphocyte region, a CD45 weakly positive region, and a CD45 negative region according to the expression of CD45, and then performing gating analysis on the cells in the cell region obtained in each partition, and performing cell population analysis according to the expression of the antibody pair, wherein the expression of the antibody pair includes:

[0032] CD45 bri+ SSC low CD3+、CD45 bri+ CD3+CD4+CD8-、CD45 bri+ CD3+CD8+CD4-、CD45 bri+ CD3+CD4-CD8-、CD45 bri+ CD3+CD4+CD8+、CD45 bri+ SSC low CD3+TCRaβ+、CD45 bri+ SSClow CD3+TCRaβ-, CD45 bri+ CD3+CD4-CD8-TCRaβ+, CD4+CD3+ / CD8+CD3+, CD3+CD4+CD45RA+CD27+, CD3+CD4+CD45RA-CD27+, CD3+CD4+CD45RA-CD27-, CD3+CD4+CD45RA+CD27-, CD45+CD3+CD4+CD38+, CD45+CD3+CD4+CD57+, CD3+CD8+CD45RA+CD27+, CD3+CD8+CD45RA-CD27+, CD3+CD8+CD45RA-CD27-, CD3+CD8+CD45RA+CD27-, CD45+CD3+CD8+CD38+, CD45+CD3+CD8+CD57+, CD45+CD3-CD16+ / CD56+, CD3-CD16+ / CD56+CD57+, CD45+CD3-CD19+, CD45+CD19+CD27+CD38 dim / - .

[0033] Among them, bri+ indicates that the corresponding cell population is divided into three zones, including a strong positive zone, an intermediate zone, and a negative zone, and bri+ indicates a strong positive;

[0034] Low means that the corresponding cell population is divided into three zones, including the strong positive zone, the intermediate zone, and the negative zone, and low means the intermediate zone;

[0035] + and − indicate that the corresponding cell populations were divided into two zones, the positive zone (+) and the negative zone (−);

[0036] High means high expression;

[0037] dim / - indicates low expression or no expression;

[0038] / represents or, such as CD45+CD3-CD16+ / CD56 represents CD45 positive, CD3 negative, CD16 positive or CD56 positive.

[0039] Preferably, the process of treating the cells to be tested includes: performing a first dark-proof incubation on the antibody composition or the detection kit and the human blood sample to be tested, then adding hemolysin for a second dark-proof incubation, obtaining the cells to be tested by centrifugation, and then adding a buffer solution to perform flow cytometry on the cells to be tested.

[0040] Preferably, the first dark-proof incubation is performed at room temperature for 15-25 minutes.

[0041] Preferably, the second dark-proof incubation is performed at room temperature for 15-25 minutes.

[0042] The fourth aspect of the present invention provides an application of the antibody composition provided by the first aspect of the present invention, or the detection kit provided by the second aspect of the present invention, or the detection method provided by the third aspect of the present invention in lymphocyte subset analysis.

[0043] The antibody composition provided by the present invention covers markers of multiple immune cells of the granulocyte, monocyte and lymphocyte lineages, establishes a reasonable antibody expression pattern, and can explain many phenotypic and functional changes of NK cells associated with healthy aging, comprehensively judge the body's immune function, and evaluate the immunosuppressive state and degree of aging.

[0044] Through a specific fluorescent CD combination and with the help of flow cytometry technology, 12CDs can be detected at a time under the conditions of the existing 10-color flow cytometer, and the sample size requirement is significantly reduced. The test results obtained can be used to closely monitor the body's health status and autoimmune defense capabilities, assess the body's health risks, understand the immunity level and changes of people at risk of disease, and take measures to intervene in time based on the individual's own situation. The test results obtained can also play an auxiliary role in reflecting the disease condition, exploring the pathogenesis of the disease, and forming a closed-loop management of disease prevention, control, and intervention.

[0045] The detection method provided by the present invention is simple to operate and has high detection efficiency, and there is no problem of mutual inhibition of expression between the antibodies in the composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 is a scatter diagram of the cell immune phenotype in Example 1, wherein Figure 1 (a) is a schematic diagram of FSC-A / FSC-H expression scatter plots and Figure 1 (b) is a scatter diagram of CD45 / SSC expression;

[0047] Figure 2 The expression of fluorescent antibody of cellular immune phenotype CD3 antibody in Example 1;

[0048] Figure 3 The expression of fluorescent antibodies for the cellular immune phenotype (CD19+CD8) / CD4 antibody pair in Example 1;

[0049] Figure 4 The fluorescent antibody expression of the cellular immune phenotype TCR-αβ antibody in Example 1;

[0050] Figure 5 The fluorescent antibody expression of the cell immunophenotype DNT cell immunophenotype TCR-αβ / CD3 antibody pair in Example 1;

[0051] Figure 6 The expression of fluorescent antibodies of the CD45RA / CD27 antibody pair for the cell immunophenotype T4 cell immunophenotype in Example 1;

[0052] Figure 7 The expression of fluorescent antibodies for the cellular immune phenotype CD38 / CD4 antibody pair in Example 1;

[0053] Figure 8 The expression of fluorescent antibodies for the cellular immunophenotype CD57 / CD4 antibody pair in Example 1;

[0054] Figure 9 The expression of fluorescent antibodies of the cell immunophenotype CD45RA / CD27 antibody pair in Example 1;

[0055] Figure 10 The expression of fluorescent antibodies for the cell immune phenotype CD38 / (CD19+CD8) antibody pair in Example 1;

[0056] Figure 11 The expression of fluorescent antibodies for the cell immune phenotype CD57 / (CD19+CD8) antibody pair in Example 1;

[0057] Figure 12 The expression of fluorescent antibodies for the cell immune phenotype (CD19+CD8) / (CD16+CD56) antibody pair in Example 1;

[0058] Figure 13 The expression of fluorescent antibodies for the CD57 / (CD16+CD56) antibody pair in the NK cell immunophenotype of the example;

[0059] Figure 14 The expression of fluorescent antibodies for the cellular immunophenotype CD27 / CD38 antibody pair in Example B;

[0060] Figure 15 These are the test data in Example 1 and the clinical normal reference interval. DETAILED DESCRIPTION

[0061] The following describes the specific embodiments of the present invention in detail with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0062] The sources of the instruments and raw materials used in the following examples are as follows:

[0063] Antibodies against CD38, CD45RA, CD57, CD45, CD27, CD16, CD56, CD3, CD4, CD8, and CD19 were purchased from Henan Kepre Biotechnology Co., Ltd. and are all mouse anti-human monoclonal antibodies;

[0064] Anti-TCR-αβ antibody was purchased from BioLegend and is a mouse anti-human monoclonal antibody;

[0065] MateCyte flow cytometer (10 channels) was purchased from Beijing Layer Biotechnology Co., Ltd.;

[0066] Cell lysis buffer (hemolysin) was purchased from BD;

[0067] PBS buffer was prepared by ourselves.

[0068] Preparation Example 1

[0069] Each antibody was combined to form an antibody composition. The components of the antibody composition are shown in Table 1.

[0070] Table 1

[0071]

[0072] Example 1

[0073] The antibody composition prepared in Preparation Example 1 was placed in a flow cytometry tube, and 100 μL of the anticoagulated human blood sample to be tested (EDTAK2K anticoagulated whole blood from a patient with tumor prognosis) was added to the bottom of the tube. The mixture was incubated in the dark at room temperature for 20 minutes, and 2 mL of hemolysin was added and mixed. After incubation in the dark at room temperature for 20 minutes, the mixture was centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. 2 mL of PBS buffer was added and mixed, and the mixture was centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the operation was repeated until the liquid had no obvious color. 0.5 mL of PBS buffer was added again and the mixture was tested on the machine.

[0074] The test results were processed in the analysis software. Cell debris was first removed within the cell gate, and then adherent cells were removed. The CD45-SSC gate was set, and the cell population was divided into granulocyte area (Gra), monocyte area (MON), lymphocyte area (Lym), CD45 weak positive area and CD45 negative area according to the expression of CD45. The target cells in each area were then gated and analyzed. Cell population analysis was performed according to the expression of the following antibodies:

[0075] CD45 bri+ SSC low CD3+、CD45 bri+ CD3+CD4+CD8-、CD45 bri+ CD3+CD8+CD4-、CD45 bri+CD3+CD4-CD8-、CD45 bri+ CD3+CD4+CD8+、CD45 bri+ SSC low CD3+TCRaβ+、CD45 bri+ SSC low CD3+TCRaβ-, CD45 bri+ CD3+CD4-CD8-TCRaβ+, CD4+CD3+ / CD8+CD3+, CD3+CD4+CD45RA+CD27+, CD3+CD4+CD45RA-CD27+, CD3+CD4+CD45RA-CD27-, CD3+CD4+CD45RA+CD27-, CD45+CD3+CD4+CD38+, CD45+CD3+CD4+CD57+, CD3+CD8+CD45RA+CD27+, CD3+CD8+CD45RA-CD27+, CD3+CD8+CD45RA-CD27-, CD3+CD8+CD45RA+CD27-, CD45+CD3+CD8+CD38+, CD45+CD3+CD8+CD57+, CD45+CD3-CD16+ / CD56+, CD3-CD16+ / CD56+CD57+, CD45+CD3-CD19+, CD45+CD19+CD27+CD38 dim / - .

[0076] Among them, bri+ indicates that the corresponding cell population is divided into three zones, including a strong positive zone, an intermediate zone, and a negative zone, and bri+ indicates a strong positive;

[0077] Low means that the corresponding cell population is divided into three zones, including the strong positive zone, the intermediate zone, and the negative zone, and low means the intermediate zone;

[0078] + and − indicate that the corresponding cell populations were divided into two zones, the positive zone (+) and the negative zone (−);

[0079] High means high expression;

[0080] dim / - indicates low expression or no expression;

[0081] / represents or, such as CD45+CD3-CD16+ / CD56 represents CD45 positive, CD3 negative, CD16 positive or CD56 positive.

[0082] CD45 bri+ SSC low CD3+ analyzed the total T lymphocyte population;

[0083] CD45 bri+ CD3+CD4+CD8- analyzes helper / inducer T lymphocytes (T4); CD45 bri+ CD3+CD8+CD4- analyzes suppressor / cytotoxic T lymphocytes (T8); CD45 bri+ CD3+CD4-CD8- analyzed double negative T lymphocytes (DNT); CD45 bri+ CD3+CD4+CD8+ analyzed double-positive T lymphocytes (DPT); CD45 bri+ SSC low CD3+TCRaβ+ analyzed TCRaβ+ T lymphocytes;

[0084] CD45 bri+ SSC low CD3+TCRaβ- analyzed were TCRaβ- T cells;

[0085] CD45 bri+ The CD3+CD4-CD8-TCRaβ+ cells analyzed were TCRaβ+DNT cells;

[0086] CD4+CD3+ / CD8+CD3+ analyzed the CD4:CD8 ratio;

[0087] CD3+CD4+CD45RA+CD27+ analyzed CD4+ naive T cells; CD3+CD4+CD45RA-CD27- analyzed CD4+ effector memory T cells;

[0088] CD3+CD4+CD45RA+CD27- analyzed CD4+ terminal effector T cells;

[0089] CD45+CD3+CD4+CD38+ analyzed activated T4 lymphocytes; CD45+CD3+CD4+CD57+ analyzed senescent T4 lymphocytes (CD57+); CD3+CD8+CD45RA+CD27+ analyzed CD8+ naive T cells; CD3+CD8+CD45RA-CD27+ analyzed CD8+ central memory T cells;

[0090] CD3+CD8+CD45RA-CD27- analyzed CD8+ effector memory T cells;

[0091] CD3+CD8+CD45RA+CD27- analyzed CD8+ terminal effector T cells;

[0092] CD45+CD3+CD8+CD38+ analyzed activated T8 lymphocytes (CD38+); CD45+CD3+CD8+CD57+ analyzed senescent T8 lymphocytes (CD57+); CD45+CD3-CD16+ / CD56+ analyzed NK lymphocytes;

[0093] CD3-CD16+ / CD56+CD57+ analyzed CD57+ NK cells;

[0094] CD45+CD3-CD19+ analyzed B lymphocytes;

[0095] CD45+CD19+CD27+CD38 dim / - Memory B lymphocytes were analyzed;

[0096] The specific analysis is as follows:

[0097] First, set the FSC / SSC gate to remove cell debris, and then set the FSC-A / FSC-H gate to remove adherent cells. Figure 1 (a) As shown. The gate is set with CD45-SSC. The CD45+ cell area is the white blood cells, and the CD45 positive area is circled. bri+ SSC low Lymphocytes (Lym), monocytes (E), granulocytes (D), such as Figure 1 (b) shown.

[0098] In CD45 bri+ SSC low Based on the lymphocytes (Lym), CD3+T lymphocytes (CD3+T) and CD3- lymphocytes (CD3-Lym) (CD3- lymphocytes include B cells and NK cells) are gated by CD3 / FSC. Figure 2 shown.

[0099] On the basis of CD3-lymphocytes (Lym), the gate was set to (CD19+CD8) / (CD16+CD56) to divide the cells into four areas. The upper left corner area was CD45 bri+ CD3+CD4+CD8- is the T4 lymphocyte group, and the area in the lower right corner is CD45 bri+ CD3+CD8+CD4- is the T8 lymphocyte group, and the lower left corner area is CD45 bri+ CD3+CD4-CD8- is the double negative T lymphocyte (DNT) population, and the upper right corner area is CD45 bri+ CD3+CD4+CD8+ is a double positive T lymphocyte (DPT) population, such as Figure 3 shown.

[0100] On the basis of CD3+ lymphocytes (Lym), the gate was set by TCRaβ / SSC, and the upper right corner area was CD45 bri+ SSC low CD3+TCRaβ+ is the TCRaβ+ T lymphocyte population, and the upper left corner area is CD45 bri+ SSC low CD3+TCRaβ- is the TCRaβ-T lymphocyte population, such as Figure 4 shown.

[0101] On the basis of double negative T lymphocytes (DNT), gate was set based on TCRaβ / CD3, and CD45 bri+ The CD3+CD4-CD8-TCRaβ+ region is TCRaβ+DNT cells, such as Figure 5 shown.

[0102] On the basis of T4 lymphocyte population, a cross gate was set with CD45RA / CD27 to divide the cells into four areas: the upper left area was CD3+CD4+CD45RA-CD27+, which was the CD4+ central memory T cell population; the lower right area was CD3+CD4+CD45RA+CD27-, which was the CD4+ terminal effector T cell population; the lower left area was CD3+CD4+CD45RA-CD27-, which was the CD4+ effector memory T cell population; and the upper right area was CD3+CD4+CD45RA+CD27+, which was the initial T cell population. Figure 6 shown.

[0103] On the basis of T4 lymphocyte population, set the gate based on CD38 / CD4 and circle the CD45+CD3+CD4+CD38+ area in the upper right corner as activated T4 lymphocytes. Figure 7 shown.

[0104] On the basis of T4 lymphocyte population, a cross gate was set based on CD57 / CD4 to divide the cells into four areas. The CD45+CD3+CD4+CD57+ area in the upper right corner was circled as senescent T4 lymphocytes. Figure 8 shown.

[0105] On the basis of T8 lymphocyte population, a cross gate was set with CD45RA / CD27 to divide the cells into four areas: the upper left area was CD3+CD8+CD45RA-CD27+, which was the CD8+ central memory T cell population; the lower right area was CD3+CD8+CD45RA+CD27-, which was the CD8+ terminal effector T cell population; the lower left area was CD3+CD8+CD45RA-CD27-, which was the CD8+ effector memory T cell population; the upper right area was CD3+CD8+CD45RA+CD27+, which was the CD8+ initial T cell population. Figure 9 shown.

[0106] On the basis of T8 lymphocyte population, set the gate with CD38 / (CD19+CD8) and circle the CD45+CD3+CD8+CD38+ area in the upper right corner as activated T8 lymphocytes. Figure 10 shown.

[0107] On the basis of T8 lymphocyte population, a cross gate was set to divide the cells into four areas based on CD57 / (CD19+CD8), and the CD45+CD3+CD8+CD57+ area in the upper right corner was circled as senescent T8 lymphocytes. Figure 11 shown.

[0108] On the basis of CD3- lymphocytes (Lym), the gate was set based on (CD19+CD8) / (CD16+CD56). The lower right area was CD45+CD3-CD19+ B lymphocytes, and the upper left area was CD45+CD3-CD16+ / CD56+ NK cells. Figure 12 shown.

[0109] On the basis of CD16+ / CD56+ NK cell population, a gate was set based on CD57 / (CD16+CD56), and the CD3-CD16+ / CD56+CD57+ region was identified as CD57+ NK cells. Figure 13 shown.

[0110] On the basis of CD19+B lymphocytes, gate was set based on CD27 / CD38 to circle CD45+CD19+CD27+CD38 dim / - The region is the memory B lymphocyte population, such as Figure 14 shown.

[0111] The results of this cell analysis are summarized as follows Figure 15 shown.

[0112] From the analysis of statistics Figure 15 The various lymphocyte subsets can be clearly distinguished and their proportions can be calculated, which facilitates the analysis and interpretation of many phenotypic and functional changes of lymphocytes associated with healthy aging, and the comprehensive judgment of the body's immune function and the assessment of immunosuppression status and degree of aging.

[0113] The antibody combination of the present invention covers expression markers of three types of cells: T cells, B cells, and monocytes. In addition, normal, aging, and activated antibody expression patterns have been established. This allows for monitoring tumor patients, assessing their immune function, determining their prognosis, and predicting tumor recurrence and metastasis. It can also be used for screening for autoimmune lymphoproliferative syndrome and for immune monitoring to facilitate precise post-transplant management. Extensive experimental data demonstrates that the antibodies in each combination of the present invention do not inhibit each other's expression.

[0114] The antibody combination and detection method provided by this invention can detect 12 CD cells at a time using existing 10-color flow cytometers. This method requires minimal equipment and shortens detection time. The test results obtained help understand the current state and level of immunity, assess health risks, understand immune levels and changes, formulate personalized health interventions, screen for potential diseases, and accurately assess clinical efficacy and unexpected risks. By referring to changes in relevant indicators, the scope and methods of disease risk assessment can be improved, and the body's immunity can be accurately assessed. This can fully leverage the role of TBNK lymphocyte testing in preventing disease and better support health management.

[0115] The antibody composition, kit and detection method provided by the present invention can be used for monitoring the prognosis of tumor patients and can also be used for health monitoring of healthy people.

[0116] Other situations of the second antibody of the present invention (such as when the second antibody is a combination of other CDs) are similar to those in Example 1, and can well detect and assist in assessing the health risks of the body and understanding the body's immune level and changes, and will not be repeated here.

[0117] The preferred embodiments of the present invention are described in detail above with reference to the accompanying drawings. However, the present invention is not limited thereto. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention. However, these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and fall within the scope of protection of the present invention.

Claims

1. An antibody composition for lymphocyte subset analysis, characterized in that: The antibody composition includes: anti-CD38 antibody, anti-CD45RA antibody, anti-CD57 antibody, anti-CD45 antibody, anti-CD27 antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD19 antibody and anti-TCR-αβ antibody.

2. The antibody composition according to claim 1, wherein Dividing the antibody composition into a first group of antibodies and n second groups of antibodies, where n is a positive integer; Wherein, each antibody in the first group of antibodies is independently labeled with a different marker, each second group of antibodies independently includes two antibodies, the two antibodies in the same group of second group of antibodies are labeled with the same marker, and the two antibodies in the same group of second group of antibodies cannot be expressed in the same type of cell population, the markers of each second group of antibodies are different, and the markers of each second group of antibodies are different from the markers of each antibody in the first group of antibodies.

3. The antibody composition according to claim 2, wherein The second group of antibodies includes: anti-CD19 antibody and anti-CD8 antibody; And / or, the second group of antibodies includes: anti-CD16 antibodies and anti-CD56 antibodies.

4. The antibody composition according to claim 2, wherein The second group of antibodies includes: anti-CD19 antibody and anti-CD3 antibody; And / or, the second group of antibodies includes: anti-TCR-αβ antibodies and anti-CD8 antibodies.

5. The antibody composition according to claim 2, wherein The second group of antibodies includes: anti-CD19 antibody and anti-CD4 antibody; And / or, the second group of antibodies includes: anti-CD16 antibodies and anti-CD56 antibodies.

6. The antibody composition according to any one of claims 2 to 5, wherein The label is a fluorescent label; Preferably, the fluorescent labels include FITC, PE, BV605 (ECD), PerCP-Cy 5.5, PE-CY7, APC, APC700, APC-Cy7, BV421 and BV510.

7. The antibody composition according to claim 6, wherein n is 2, wherein the second group of antibodies in one group includes anti-CD19 antibody and anti-CD8 antibody, which are labeled with APC, and the second group of antibodies in another group includes anti-CD16 antibody and anti-CD56 antibody, which are labeled with PE; The anti-CD38 antibody in the first group of antibodies is labeled with PC5.5, the anti-CD45RA antibody is labeled with BV605 (ECD), the anti-CD57 antibody is labeled with FITC, the anti-CD45 antibody is labeled with APC-Cy7, the anti-CD27 antibody is labeled with APC700, the anti-CD3 antibody is labeled with BV510, the anti-CD4 antibody is labeled with PC7, and the anti-TCR-αβ antibody is labeled with BV421; or, n is 2, wherein one group of the second group of antibodies comprises anti-CD19 antibody and anti-CD3 antibody, which are labeled with APC, and the other group of the second group of antibodies comprises anti-TCR-αβ antibody and anti-CD8 antibody, which are labeled with PE; In the first group of antibodies, the anti-CD38 antibody is labeled with PC5.5, the anti-CD45RA antibody is labeled with BV605 (ECD), the anti-CD57 antibody is labeled with FITC, the anti-CD45 antibody is labeled with APC-Cy7, the anti-CD27 antibody is labeled with APC700, the anti-CD4 antibody is labeled with PC7, the anti-CD16 antibody is labeled with BV421, and the anti-CD56 antibody is labeled with BV51; Or, n is 2, wherein one group of the second group of antibodies is an anti-CD19 antibody and an anti-CD4 antibody, which are labeled with APC, and the other group of the second group of antibodies is an anti-CD16 antibody and an anti-CD56 antibody, which are labeled with PE; The anti-CD38 antibody in the first group of antibodies was labeled with PC5.5, the anti-CD45RA antibody was labeled with BV605 (ECD), the anti-CD57 antibody was labeled with FITC, the anti-CD45 antibody was labeled with APC-Cy7, the anti-CD27 antibody was labeled with APC700, the anti-CD3 antibody was labeled with BV510, the anti-CD8 antibody was labeled with PC7, and the anti-TCR-αβ antibody was labeled with BV421.

8. A detection kit, characterized in that: An antibody composition comprising any one of claims 1 to 7; Preferably, the antibody composition is in the form of a powder or liquid preparation, and when it is in the form of a liquid preparation, it further comprises a buffer.

9. A method for detecting lymphocyte subpopulation analysis, characterized in that: The antibody composition according to any one of claims 1 to 7 or the detection kit according to claim 8 is used to treat the cells to be tested, and then data analysis is performed, wherein the data analysis includes dividing the cell population into a granulocyte region, a monocyte region, a lymphocyte region, a CD45 weakly positive region, and a CD45 negative region according to the expression of CD45, and then performing gating analysis on the cells in the cell regions obtained in each partition, and performing cell population analysis according to the expression of the antibody pair, wherein the expression of the antibody pair includes: CD45 bri+ SSC low CD3+、CD45 bri+ CD3+CD4+CD8-、CD45 bri+ CD3+CD8+CD4-、CD45 bri+ CD3+CD4-CD8-、CD45 bri+ CD3+CD4+CD8+、CD45 bri+ SSC low CD3+TCRaβ+、CD45 bri+ SSC low CD3+TCRaβ-、CD45 bri+ CD3+CD4-CD8-TCRaβ+、CD4+CD3+ / CD8+CD3+、CD3+CD4+CD45RA+CD27+、CD3+CD4+CD45RA-CD27+、CD3+CD4+CD45RA-CD27-、CD3+CD4+CD45RA+CD27-、CD45+CD3+CD4+CD38+、CD45+CD3+CD4+CD57+、CD3+CD8+CD45RA+CD27+、CD3+CD8+CD45RA-CD27+、CD3+CD8+CD45RA-CD27-、CD3+CD8+CD45RA+CD27-、CD45+CD3+CD8+CD38+、CD45+CD3+CD8+CD57+、CD45+CD3-CD16+ / CD56+、CD3-CD16+ / CD56+CD57+、CD45+CD3-CD19+、CD45+CD19+CD27+CD38 dim / - 。 10. Use of the antibody composition according to any one of claims 1 to 7, or the detection kit according to claim 8, or the detection method according to claim 9 in lymphocyte subset analysis.

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