A flow cytometer detection kit for immune characterization of advanced lung cancer and its detection method

Abstract

A flow cytometry detection kit for immune characterization of advanced lung cancer. Reagent A includes CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody and PD-1 monoclonal antibody labeled with fluorescent microspheres stained with different color dyes. The kit of the present invention uses the peripheral blood of advanced lung cancer patients after receiving immune checkpoint inhibitors as a sample for detection, and uses the kit to detect CD8+PD-1+ / CD8+ T cell %, CD4+PD-1+ / CD4+ T cell %, CD8+HLA-DR+ / CD8+ T cell % in the sample to be tested. Each index can comprehensively characterize the immune level of the patient.

Description

Technical Field

[0001] The invention belongs to the field of flow cytometer detection kits, and in particular relates to a flow cytometer detection kit for peripheral blood immune characterization of advanced lung cancer and a detection method thereof. Background Art

[0002] With the progress of lung cancer treatment research, the clinical success of immune checkpoint inhibitors (antagonists of CTLA-4, PD-1 and PD-L1) has provided new treatment options. A variety of PD-1 / PD-L1 immune checkpoint inhibitors (ICIs) have been approved for the treatment of advanced lung cancer and have achieved remarkable therapeutic effects. In addition, due to continuous tumor antigen stimulation, tumor patients produce exhausted T cells, and the expression of PD-1 in exhausted T cells changes. PD-1 is induced by T cell receptor signals, and PD-1 binds to its ligand, which will inhibit TCR / CD28 signals and T cell activation. Blocking the PD-1 signaling pathway can restore the anti-tumor response of exhausted T cells.

[0003] T lymphocyte subsets are closely related to the body's immune function. When the immune function is disordered, the relative percentage and absolute count of the T lymphocyte subsets will be abnormal. In the case of specific diseases such as lung cancer, T lymphocyte subsets that express PD-1 are extremely important for monitoring the occurrence and development of the disease, patient medication prognosis, and guiding clinical treatment. Using the immunological characteristics of peripheral blood of patients with advanced lung cancer, the patient's T cell immune level can be predicted to assist in determining whether further immunotherapy is suitable, so as to maximize the efficacy of immunotherapy and reduce immune-related adverse reactions.

[0004] Therefore, there is an urgent need for a detection kit and a detection method for characterizing the expression of immune cell subpopulations and immune checkpoint molecules in the peripheral blood of patients with advanced lung cancer. Summary of the invention

[0005] In order to overcome the problems in the prior art, the present invention provides a flow cytometer detection kit for immune characterization of advanced lung cancer and a method of using the kit, which provides an auxiliary basis for judging the effectiveness and safety of immune cell characterization changes in patients receiving immune checkpoint inhibitor treatment.

[0006] The present invention provides the following technical solutions:

[0007] A flow cytometer detection kit for immune characterization of late stage lung cancer, comprising reagent A,

[0008] The reagent A includes CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody and PD-1 monoclonal antibody labeled with fluorescent microspheres dyed with dyes of different colors, and the concentration ratio of each antibody is 1:1:1:1:1, all of which are 0.05-0.1 mg / mL.

[0009] Furthermore, the detection kit also includes reagent B, which is a red blood cell lysate.

[0010] The detection method of the detection kit comprises the following steps:

[0011] Step 1: Take human venous blood, separate and obtain white blood cell solution, and add it to the bottom of the flow tube;

[0012] Step 2, adding the reagent A into the tube, the volume ratio of the reagent A to the white blood cell solution is 1:10, and the reagent A and the white blood cell solution are incubated together;

[0013] Step 3: Add PBS solution for washing, vortex mix, centrifuge at 4°C, discard the supernatant, add PBS solution again to form a cell suspension, and detect using flow cytometer;

[0014] Step 4: Screen the cells with positive CD45 antibody and SSC value in the range of 0-50K to identify them as lymphocytes; screen the cells with positive CD3 antibody from the lymphocytes to identify them as T cells; screen the cells with positive CD4 antibody from the T cells, and screen the cells with positive PD-1 antibody from the cells with positive CD4 antibody to calculate the CD4 + PD-1 + / CD4 + The percentage of T cells; screen CD8 antibody-positive cells from T cells, screen PD-1 antibody-positive cells from CD8 antibody-positive cells, and calculate CD8 + PD-1 + / CD8 + The percentage of T cells.

[0015] Another detection method of the detection kit comprises the following steps:

[0016] Step 1: Use reverse pipetting technique to draw EDTA-K2 anticoagulated whole blood to the bottom of the flow tube to prevent the blood from touching the upper part of the tube wall;

[0017] Step 2: Add reagent A to the tube at a volume ratio of 1:10 between reagent A and whole blood, and mix well on a vortexer; incubate at room temperature away from light; add reagent B to the tube at a volume ratio of 20:1 between reagent B and whole blood; mix well on a vortexer; and react away from light;

[0018] Step 3: After vortex mixing, centrifuge at 4°C and discard the supernatant, add PBS solution for washing, vortex mixing and centrifuge at 4°C, discard the supernatant, add PBS solution again to form a cell suspension, and detect using flow cytometer;

[0019] Step 4: Screen the cells with positive CD45 antibody and SSC value in the range of 0-50K to identify them as lymphocytes; screen the cells with positive CD3 antibody from the lymphocytes to identify them as T cells; screen the cells with positive CD4 antibody from the T cells, and screen the cells with positive PD-1 antibody from the cells with positive CD4 antibody to calculate the CD4 + PD-1 + / CD4 + The percentage of T cells, the cells positive for CD8 antibody were screened from T cells, the cells positive for PD-1 antibody were screened from the cells positive for CD8 antibody, and the CD8 + PD-1 + / CD8 + The percentage of T cells.

[0020] Furthermore, CD8 + PD-1 + / CD8 + The percentage of T cells is <2% and CD4 + PD-1 + / CD4 + The percentage of T cells is <2%, which is a low ratio used to characterize T cell functional exhaustion.

[0021] A flow cytometer detection kit for immune characterization of late stage lung cancer, comprising reagent A,

[0022] Reagent A includes CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, PD-1 monoclonal antibody, and HLA-DR monoclonal antibody labeled with fluorescent microspheres dyed with dyes of different colors, and the concentration ratio of each antibody is 1:1:1:1:1:1, all of which are 0.05-1 mg / mL.

[0023] Furthermore, the detection kit also includes reagent B, which is a red blood cell lysate.

[0024] The detection method of the detection kit comprises the following steps:

[0025] Step 1: Use reverse pipetting technique to draw EDTA-K2 anticoagulated whole blood to the bottom of the flow tube to prevent the blood from touching the upper part of the tube wall;

[0026] Step 2: Add reagent A to the tube at a volume ratio of 1:10 between reagent A and whole blood, and mix well on a vortexer; incubate at room temperature away from light; add reagent B to the tube at a volume ratio of 20:1 between reagent B and whole blood; mix well on a vortexer; and react away from light;

[0027] Step 3: After vortex mixing, centrifuge at 4°C and discard the supernatant, add PBS solution for washing, vortex mixing and centrifuge at 4°C, discard the supernatant, add PBS solution again to form a cell suspension, and detect using flow cytometer;

[0028] Step 4: Screen the cells with positive CD45 antibody and SSC value in the range of 0-50K to identify them as lymphocytes; screen the cells with positive CD3 antibody from the lymphocytes to identify them as T cells; screen the cells with positive CD4 antibody from the T cells, and screen the cells with positive PD-1 antibody from the cells with positive CD4 antibody to calculate the CD4 + PD-1 + / CD4 + The percentage of T cells, the cells positive for CD8 antibody were screened from T cells, the cells positive for PD-1 antibody were screened from the cells positive for CD8 antibody, and the CD8 + PD-1 + / CD8 + The percentage of T cells was calculated by screening HLA-DR antibody-positive cells from CD8 antibody-positive cells. + HLA-DR + / CD8 + The percentage of T cells.

[0029] Furthermore, CD8 + PD-1 + / CD8 + The percentage of T cells is <2%, CD4 + PD-1 + / CD4 + The percentage of T cells is <2% and CD8 + HLA-DR + / CD8 + The percentage of T cells was <19.5%, which is used to characterize the low ratio of functional exhaustion and activation of T cells.

[0030] A flow cytometer detection kit for immune characterization of late stage lung cancer, comprising reagent A,

[0031] The reagent A includes CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, PD-1 monoclonal antibody, and CD38 monoclonal antibody labeled with fluorescent microspheres dyed with dyes of different colors. The concentration ratio of each antibody is 1:1:1:1:1:1, all of which are 0.05-1 mg / mL.

[0032] 11. The detection kit according to claim 10, characterized in that the detection kit also includes reagent B, which is a red blood cell lysate.

[0033] The detection method of the detection kit comprises the following steps:

[0034] Step 1: Use reverse pipetting technique to draw EDTA-K2 anticoagulated whole blood to the bottom of the flow tube to prevent the blood from touching the upper part of the tube wall;

[0035] Step 2: Add reagent A to the tube at a volume ratio of 1:10 between reagent A and whole blood, and mix well on a vortexer; incubate at room temperature away from light; add reagent B to the tube at a volume ratio of 20:1 between reagent B and whole blood; mix well on a vortexer; and react away from light;

[0036] Step 3: After vortex mixing, centrifuge at 4°C and discard the supernatant, add PBS solution for washing, vortex mixing and centrifuge at 4°C, discard the supernatant, add PBS solution again to form a cell suspension, and detect using flow cytometer;

[0037] Step 4: Screen the cells with positive CD45 antibody and SSC value in the range of 0-50K to identify them as lymphocytes; screen the cells with positive CD3 antibody from the lymphocytes to identify them as T cells; screen the cells with positive CD4 antibody from the T cells, and screen the cells with positive PD-1 antibody from the cells with positive CD4 antibody to calculate the CD4 + PD-1 + / CD4 + The percentage of T cells, the cells positive for CD8 antibody were screened from T cells, the cells positive for PD-1 antibody were screened from the cells positive for CD8 antibody, and the CD8 + PD-1 + / CD8 + The percentage of T cells was calculated by screening CD38 antibody-positive cells from CD8 antibody-positive cells. + CD38 + / CD8 + The percentage of T cells.

[0038] Furthermore, CD8 + PD-1 + / CD8 + The percentage of T cells is <2%, CD4 +PD-1 + / CD4 + The percentage of T cells is <2% and CD8 + CD38 + / CD8 + The percentage of T cells was <27%, which was used to characterize the exhaustion level of T cells and the low ratio of abnormal activation.

[0039] By adopting the above technical solution, the present invention has the following beneficial effects:

[0040] (1) The flow cytometric analysis kit for peripheral blood immune cell analysis provided by the present invention is used as a sample for testing peripheral blood of patients with advanced lung cancer who have received immune checkpoint inhibitors. The kit is used to detect CD8 + PD-1 + / CD8 + T cell%, CD4 + PD-1 + / CD4 + T cell%, CD8 + HLA-DR + / CD8 + T cell%, each indicator can comprehensively characterize the patient's immune level, thereby assisting in determining the therapeutic effect of the patient's checkpoint inhibitors;

[0041] (2) Using the kit to detect CD8 + PD-1 + / CD8 + T cell%, CD4 + PD-1 + / CD4 + T cell%, CD8 + CD38 + / CD8 + T cell%, each indicator comprehensively represents the patient's immune level, thereby assisting in determining whether the patient has immune-related adverse reactions after checkpoint inhibitor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the flow cytometry result diagram of sample number 24;

[0043] Figure 2 This is the flow cytometry result diagram of sample numbered 13;

[0044] Figure 3 The CD8 + PD-1 + / CD8 + T cell ratio result diagram;

[0045] Figure 4 The CD4 + PD-1 + / CD4 + T cell ratio result diagram;

[0046] Figure 5 This is the flow cytometry result graph of sample number 4;

[0047] Figure 6 This is the flow cytometry result diagram of sample number 13;

[0048] Figure 7 The CD8 + HLA-DR + / CD8 + T cell ratio result diagram;

[0049] Figure 8 The CD4 + HLA-DR + / CD4 + T cell ratio result diagram;

[0050] Fig. 9 This is the flow cytometry result diagram of sample number 13;

[0051] Fig.10 This is the flow cytometry result diagram of sample number 23;

[0052] Fig.11 The CD8 samples of the kit test numbers 9-17 and 18-23 + PD-1 + / CD8 + Cell ratio result diagram;

[0053] Fig.12 The CD4 samples of the kit test numbers 9-17 and 18-23 + PD-1 + / CD4 + Cell ratio results. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the structural diagrams and specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0055] Example 1

[0056] The present invention provides a flow cytometer detection kit for immune characterization of advanced lung cancer, comprising reagent A: reagent A comprises CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody and PD-1 monoclonal antibody labeled with fluorescent microspheres dyed with dyes of different colors. The concentration ratio of each antibody is 1:1:1:1:1, all of which are 0.05-0.1 mg / mL.

[0057] The amino acid sequences of each antibody binding protein are shown in Table 1.

[0058] Table 1 Amino acid sequence

[0059]

[0060] The antibody model sequences and flow cytometer dye detection signals can be matched as follows: CD45 antibody (2D1 / IgG1, V500-C channel), CD3 antibody (UCHT1 / IgG1, PE-Cy7 channel), CD4 antibody (SK3 / IgG1, AF700 channel), CD8 antibody (53-6.7 / IgG2a, APC-Cy7 channel), PD-1 antibody (J43 / IgG2, BV510 channel).

[0061] The diameter of the fluorescent microspheres is 7-10μm, they can be excited by 488-640nm laser, and the emission wavelength range is 500-719nm.

[0062] Preferably, reagent B is also included. Reagent B is a red blood cell lysis solution, and its components include ammonium chloride (NH4Cl), potassium bicarbonate (KHCO3) and ethylenediaminetetraacetic acid (EDTA), and the ratio of each component is 15.5mM:1nM:0.01mM.

[0063] The present invention provides a detection method of a detection kit, comprising the following steps:

[0064] Step 1: Take human venous blood, separate and obtain white blood cell solution, and add it to the bottom of the flow tube;

[0065] Step 2: Add the reagent A into the tube, the volume ratio of the reagent A to the white blood cell solution is 1:10, and the reagent A and the white blood cell solution are incubated together; the fluorescent labeled antibody in the reagent A will specifically bind to the white blood cell surface antigen.

[0066] Step 3: Add PBS solution for washing, vortex mix, centrifuge at 4°C, discard the supernatant, add PBS solution again to form a cell suspension, and detect it on a flow cytometer; during the flow cytometer detection process, the laser beam causes the labeled antibody bound to the cell surface to excite fluorescence of a specific wavelength. By detecting the fluorescence signal, information about cell size (characterized by forward scatter (FSC) value), internal complexity (characterized by side scatter (SSC) value), and the intensity and abundance of corresponding antigen expression can be determined.

[0067] Step 4: Screen the cells with positive CD45 antibody and SSC value in the range of 0-50K to identify them as lymphocytes; screen the cells with positive CD3 antibody from the lymphocytes to identify them as T cells; screen the cells with positive CD4 antibody from the T cells, and screen the cells with positive PD-1 antibody from the cells with positive CD4 antibody to calculate the CD4 + PD-1 + / CD4 + The percentage of T cells, the cells positive for CD8 antibody were screened from T cells, the cells positive for PD-1 antibody were screened from the cells positive for CD8 antibody, and the CD8 + PD-1 + / CD8 + The percentage of T cells.

[0068] Another detection method of the detection kit, using whole blood as the starting material for the test, comprises the following steps:

[0069] Step 1: Use reverse pipetting technique to draw EDTA-K2 anticoagulated whole blood to the bottom of the flow tube to prevent the blood from touching the upper part of the tube wall;

[0070] Step 2: Add reagent A to the tube at a volume ratio of 1:10 between reagent A and whole blood, and mix well on a vortexer; incubate at room temperature away from light; add reagent B to the tube at a volume ratio of 20:1 between reagent B and whole blood; mix well on a vortexer; and react away from light;

[0071] Step 3: After vortex mixing, centrifuge at 4°C and discard the supernatant, add PBS solution for washing, vortex mixing and centrifuge at 4°C, discard the supernatant, add PBS solution again to form a cell suspension, and detect on flow cytometer;

[0072] Step 4: Screen the cells with positive CD45 antibody and SSC value in the range of 0-50K to identify them as lymphocytes; screen the cells with positive CD3 antibody from the lymphocytes to identify them as T cells; screen the cells with positive CD4 antibody from the T cells, and screen the cells with positive PD-1 antibody from the cells with positive CD4 antibody to calculate the CD4 + PD-1 + / CD4 +The percentage of T cells, the cells positive for CD8 antibody were screened from T cells, the cells positive for PD-1 antibody were screened from the cells positive for CD8 antibody, and the CD8 + PD-1 + / CD8 + The percentage of T cells.

[0073] A total of 23 patients with advanced lung cancer who received immune checkpoint inhibitors were selected. These patients had no infection before and after immunotherapy. At the same time, 11 patients with advanced lung cancer who did not receive immune checkpoint inhibitors were selected as the control group.

[0074] Clinical trial cases are shown in Table 2

[0075] Table 2

[0076]

[0077] The specific progression-free survival (PFS) of these 34 patients is shown in Table 3. Judgment method of immune-related adverse reactions: according to the Common Toxicity Terminology Assessment Criteria (CTCAE4.0) developed by the Cancer Institute of the National Institutes of Health.

[0078] Table 3

[0079]

[0080]

[0081] The above detection kit was used to detect CD8 + PD-1 + / CD8 + The percentage of T cells, CD4 + PD-1 + / CD4 + The percentage of T cells.

[0082] Figure 1 This is the flow cytometry result of patient No. 24 who had advanced lung cancer and did not receive immune checkpoint inhibitors; Figure 2 This is the flow cytometry result of the patient No. 13 who had good efficacy in immune checkpoint inhibitor treatment and no immune-related adverse reactions. Gating strategy: (a) Use SSC and CD45 channels to circle lymphocytes (LYM); (b) Circle CD3 antibody-positive cells in LYM as T cells (T cells); (c) Circle CD4-positive cells in T cells as CD4 + T cells (CD4 + T cells), CD8 antibody positive cells as CD8 + T cells (CD8+ T cells), (d) in CD4 + Among the T cells, cells positive for PD-1 antibody were selected as PD-1 + CD4 + T cells, the value in the upper right corner is the percentage of CD4 + The percentage of T cells. (g) In CD8 + Among the T cells, cells positive for PD-1 antibody were selected as PD-1 + CD8 + T cells, the value in the upper right corner is the percentage of CD8 + The percentage of T cells.

[0083] CD8 + PD-1 + / CD8 + The results of T cell ratio are as follows Figure 3 As shown in the figure, patients with advanced lung cancer (No. 9-17) who received immune checkpoint inhibitors and had good efficacy and no immune-related adverse reactions had peripheral blood CD8 + PD-1 + / CD8 + The proportion of T cells was significantly decreased in advanced lung cancer patients who did not receive immune checkpoint inhibitor treatment (0.1 (0.1-0.6) vs 9.3 (6.1-12.1), p=0.002). Statistical method: Independent sample t-test was used for comparison.

[0084] Patients with advanced lung cancer who respond well to immune checkpoint inhibitors have CD8 + +PD-1 + / CD8 + The proportion of T cells was significantly lower than that in patients with advanced lung cancer who did not receive immunotherapy.

[0085] CD4 + PD-1 + / CD4 + The results of T cell ratio are as follows Figure 4 As shown in the figure, patients with advanced lung cancer (No. 9-17) who received immune checkpoint inhibitors and had good efficacy and no immune-related adverse reactions had peripheral blood CD4 + PD-1 + / CD4 + The proportion of T cells was significantly decreased in advanced lung cancer patients who did not receive immune checkpoint inhibitor treatment (0.1 (0.1-0.5) vs 15.7 (13.8-17.7), p < 0.05). Statistical method: Independent sample t-test was used for comparison.

[0086] Patients who responded well to immune checkpoint inhibitors had a lower CD4 + PD-1 + / CD4 + The proportion of T cells decreased significantly compared with advanced lung cancer patients who did not receive immune checkpoint inhibitor treatment.

[0087] Therefore, using the kit of this embodiment, CD8 + PD-1 + / CD8 + The percentage of T cells is <2% and CD4 + PD-1 + / CD4 + The percentage of T cells is <2%, which is used to characterize the functional exhaustion of T cells, or to assist in characterizing the treatment with immune checkpoint inhibitors with good efficacy.

[0088] Example 2

[0089] The present invention provides another flow cytometer detection kit for late stage lung cancer immune characterization, including reagent A, which includes CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, PD-1 monoclonal antibody, and HLA-DR monoclonal antibody labeled with fluorescent microspheres dyed with dyes of different colors, and the amino acid sequence of each antibody binding protein is shown in Table 1. The sequence of each antibody model and the dye detection signal are matched as follows: CD45 antibody (2D1 / IgG1, V500-C channel), CD3 antibody (UCHT1 / IgG1, PE-Cy7 channel), CD4 antibody (SK3 / IgG1, AF700 channel), CD8 antibody (53-6.7 / IgG2a, APC-Cy7 channel), PD-1 antibody (J43 / IgG2, BV510 channel), HLA-DR antibody (L243 / IgG1a, PerCp5.5 channel). The concentration ratio of each antibody was 1:1:1:1:1:1, all ranging from 0.05 to 1 mg / mL.

[0090] The detection kit also includes reagent B, which is a red blood cell lysis solution.

[0091] The detection method of the detection kit comprises the following steps:

[0092] Step 1: Use reverse pipetting technique to draw EDTA-K2 anticoagulated whole blood to the bottom of the flow tube to prevent the blood from touching the upper part of the tube wall;

[0093] Step 2: Add reagent A to the tube at a volume ratio of 1:10 between reagent A and whole blood, and mix well on a vortexer; incubate at room temperature away from light; add reagent B to the tube at a volume ratio of 20:1 between reagent B and whole blood; mix well on a vortexer; and react away from light;

[0094] Step 3: After vortex mixing, centrifuge at 4°C and discard the supernatant, add PBS solution for washing, vortex mixing and centrifuge at 4°C, discard the supernatant, add PBS solution again to form a cell suspension, and detect using flow cytometer;

[0095] Step 4: Screen the cells with positive CD45 antibody and SSC value in the range of 0-50K to identify them as lymphocytes; screen the cells with positive CD3 antibody from the lymphocytes to identify them as T cells; screen the cells with positive CD4 antibody from the T cells, and screen the cells with positive PD-1 antibody from the cells with positive CD4 antibody to calculate the CD4 + PD-1 + / CD4 + The percentage of T cells, the cells positive for CD8 antibody were screened from T cells, the cells positive for PD-1 antibody were screened from the cells positive for CD8 antibody, and the CD8 + PD-1 + / CD8 + The percentage of T cells was calculated by screening HLA-DR antibody-positive cells from CD8 antibody-positive cells. + HLA-DR + / CD8 + The percentage of T cells.

[0096] The above detection kit was used to detect CD8 + HLA-DR + / CD8 + The percentage of T cells.

[0097] The peripheral blood immune cell subtype results of patients with advanced lung cancer who had good efficacy in immune checkpoint inhibitor treatment (No. 9-23) and patients with poor efficacy in advanced lung cancer (No. 1-8) were compared, as shown in Table 4.

[0098] Table 4

[0099] Patients with poor response to immunotherapy Patients who respond well to immunotherapy P-value CD8+% 26.8(20.4-33.2) 29.0(21.2-31.3) 0.900 CD8+HLA-DR+% 36.6±16.7 14.2±5.8 0.002* CD8+CD38+% 20.4±12.9 22.9±14.0 0.675 CD8+PD-1+% 0.6(0.23-0.88) 0.4(0.1-0.9) 0.925 CD4+% 27.4(22.0-38.3) 33.5(27-40) 0.506 CD4+HLA-DR+% 27.7±18.6 11.5±7.7 0.008* CD4+CD38+% 8.8(5.2-16.72) 7.5(4.9-11.2) 0.636 CD4+PD-1+% 0.35(0.23-0.90) 0.1(0.1-1.2) 0.975

[0100] Among them, the streaming result of sample number 4 is Figure 5 The streaming result of sample No. 13 is Figure 6 Gating strategy: (a) Use SSC and CD45 channels to gate lymphocytes (LYM); (b) Select CD3 antibody-positive cells in LYM as T cells (T cells); (c) Select CD4-positive cells in T cells as CD4 + T cells (CD4 +T cells), CD8 antibody-positive cells as CD8 + T cells (CD8 + T cells); (d, e) in CD4 + Among T cells, cells positive for PD-1 / HLA-DR antibodies were selected as PD-1 + CD4 + T cells / HLA-DR + CD4 + T cells, the value in the upper right corner is the percentage of CD4 + The percentage of T cells; (gh) in CD8 + Among the T cells, cells positive for PD-1 / HLA-DR antibodies were selected as PD-1 + CD + T cells / HLA-DR + CD8 + T cells, the value in the upper right corner is the percentage of CD8 + The percentage of T cells.

[0101] according to Figure 7 The results showed that patients with advanced lung cancer (No. 9-23) who received immune checkpoint inhibitors had a good response and peripheral blood CD8 + HLA-DR + / CD8 + Peripheral blood CD8 T cell ratio in patients with advanced lung cancer with poor efficacy + HLA-DR + / CD8 + The proportion of T cells was significantly lower (14.2±5.8% vs 36.6±16.7%, p<0.05).Statistical methods: Independent sample t test was used for comparison.

[0102] according to Figure 8 The results showed that patients with advanced lung cancer (No. 9-23) who received immune checkpoint inhibitors and had good responses had a lower peripheral blood CD4 + HLA-DR + / CD4 + Peripheral blood CD4 T cell ratio in patients with advanced lung cancer with poor efficacy + HLA-DR + / CD4 + The proportion of T cells was significantly lower (11.5±7.7% vs 27.7±18.6%, p<0.05). Statistical methods: Independent sample t test was used for comparison.

[0103] The logistic regression analysis method was used to conduct a multivariate analysis of the patient group with better immunotherapy efficacy and the patient group with advanced lung cancer with poor immunotherapy efficacy (as shown in Table 5). It was found that the proportion of peripheral blood CD8+HLA-DR+ cells <19.5% after immunotherapy was an independent predictor of better efficacy of continued treatment with immune checkpoint inhibitors.

[0104] Table 5

[0105] OR 95% CI P-value CD8+HLA-DR+%<19.5% 0.004 0.001-0.479 0.024* age 1.135 0.850-1.517 0.391 gender 2.814 0.046-172.93 0.623 Whether there is mutation 0.054 0.001-5.560 0.217 Whether it is non-small cell lung cancer 0.004 0015-86.159 0.960

[0106] Therefore, using the detection kit of this embodiment, CD8 + PD-1 + / CD8 + The percentage of T cells is <2%, CD4 + PD-1 + / CD4 + The percentage of T cells is <2% and CD8 + HLA-DR + / CD8 + The percentage of T cells is <19.5%, which can be used to characterize a low ratio of T cell functional exhaustion to activation. The relevant patients continue to receive immune checkpoint inhibitor treatment with better efficacy.

[0107] Example 3

[0108] A flow cytometer detection kit for immune characterization of advanced lung cancer, including reagent A. Reagent A includes CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, PD-1 monoclonal antibody, and CD38 monoclonal antibody labeled with fluorescent microspheres stained with dyes of different colors. The amino acid sequence of each antibody binding protein is shown in Table 1. The sequence of each antibody model and the dye detection signal are matched as follows: CD45 antibody (2D1 / IgG1, V500-C channel), CD3 antibody (UCHT1 / IgG1, PE-Cy7 channel), CD4 antibody (SK3 / IgG1, AF700 channel), CD8 antibody (53-6.7 / IgG2a, APC-Cy7 channel), PD-1 antibody (J43 / IgG2, BV510 channel), CD38 antibody (HB7 / IgG1, V450 channel). The concentration ratio of each antibody is 1:1:1:1:1:1, all of which are 0.05-1 mg / mL. The diameter of the fluorescent microspheres is 7-10 μm, can be excited by 488-640 nm laser, and the emission wavelength range is 500-719 nm.

[0109] The detection kit also includes reagent B, which is a red blood cell lysis solution. The components include ammonium chloride (NH4Cl), potassium bicarbonate (KHCO3) and ethylenediaminetetraacetic acid (EDTA), and the ratio of each component is 15.5mM:1nM:0.01mM.

[0110] The detection method of the detection kit comprises the following steps:

[0111] Step 1: Use reverse pipetting technique to draw EDTA-K2 anticoagulated whole blood to the bottom of the flow tube to prevent the blood from touching the upper part of the tube wall;

[0112] Step 2: Add reagent A to the tube at a volume ratio of 1:10 between reagent A and whole blood, and mix well on a vortexer; incubate at room temperature away from light; add reagent B to the tube at a volume ratio of 20:1 between reagent B and whole blood; mix well on a vortexer; and react away from light;

[0113] Step 3: After vortex mixing, centrifuge at 4°C and discard the supernatant, add PBS solution for washing, vortex mixing and centrifuge at 4°C, discard the supernatant, add PBS solution again to form a cell suspension, and detect using flow cytometer;

[0114] Step 4: Screen the cells with positive CD45 antibody and SSC value in the range of 0-50K to identify them as lymphocytes; screen the cells with positive CD3 antibody from the lymphocytes to identify them as T cells; screen the cells with positive CD4 antibody from the T cells, and screen the cells with positive PD-1 antibody from the cells with positive CD4 antibody to calculate the CD4 + PD-1 + / CD4 + The percentage of T cells, the cells positive for CD8 antibody were screened from T cells, the cells positive for PD-1 antibody were screened from the cells positive for CD8 antibody, and the CD8 + PD-1 + / CD8 + The percentage of T cells was calculated by screening CD38 antibody-positive cells from CD8 antibody-positive cells. + CD38 + / CD8 + The percentage of T cells.

[0115] The above detection kit was used to detect the CD8 + CD38 + / CD8 + The percentage of T cells.

[0116] The streaming result of sample No. 13 is Fig. 9 The streaming result of sample No. 23 is Fig.10. Gating strategy: (a) Use SSC and CD45 channels to circle lymphocytes (LYM); (b) In LYM, cells positive for CD3 antibodies are circled as T cells (T cells); (c) In T cells, cells positive for CD4 are circled as CD4+ T cells (CD4+ T cells), and cells positive for CD8 antibodies are circled as CD8+ T cells (CD8+ T cells); (df) In CD4+ T cells, cells positive for PD-1 / HLA-DR / CD38 antibodies are circled as PD-1+CD4+ T cells / HLA-DR+CD4+ T cells / CD38+CD4+ T cells, and the value in the upper right corner is the percentage of this group of cells in CD4+ T cells; (gi) In CD8+ T cells, cells positive for PD-1 / HLA-DR / CD38 antibodies are circled as PD-1+CD+ T cells / HLA-DR+CD8+ T cells / CD38+CD8+ T cells, the value in the upper right corner is the percentage of this group of cells in CD8+ T cells.

[0117] In patients with advanced lung cancer who developed immune-related adverse reactions (ir-AEs) after treatment with immune checkpoint inhibitors, peripheral blood CD8 + PD-1 + / CD8 + The percentage of T cells in patients with advanced lung cancer who did not receive immune checkpoint inhibitors was significantly decreased [0.7 (0.2-1.4) vs 9.3 (6.1-12.1), p < 0.05]. Statistical method: independent sample t-test was used for comparison. The results are shown in Fig.11 .

[0118] In patients with advanced lung cancer who experience immune-related adverse reactions, CD4 + PD-1 + / CD4 + The percentage of T cells in advanced lung cancer patients who did not receive immune checkpoint inhibitors was significantly decreased [0.6 (0.1-1.3) vs 15.7 (13.8-17.7), p < 0.05]. Statistical method: independent sample t test was used for comparison. Fig.12 shown.

[0119] The results of peripheral blood immune cell subtypes in patients with advanced lung cancer who had immune-related adverse reactions after receiving immune checkpoint inhibitors were compared with those in patients with advanced lung cancer who did not have immune-related adverse reactions after immunotherapy, as shown in Table 6. It was found that the proportion of peripheral blood CD8+CD38+ cells in patients with advanced lung cancer who had immune-related adverse reactions after receiving immune checkpoint inhibitors was significantly higher than that in patients with advanced lung cancer who did not have ir-AEs (37.9 (33.5-40.7) vs 13.8 (7.3-19.2), p = 0.001).

[0120] Table 6

[0121]

[0122]

[0123] Logistic regression analysis was used to conduct multivariate analysis on the group of patients with no immune-related adverse reactions to immunotherapy and the group of patients with advanced lung cancer who did not experience ir-AEs, as shown in Table 7. It was found that the proportion of CD8+CD38+ cells in peripheral blood >27% after immunotherapy was an independent predictor of immune-related adverse reactions.

[0124] Table 7

[0125] OR 95% CI P-value CD8+CD38+%>27% 0.025 0.001-0.496 0.016

[0126] Therefore, using the detection kit of this embodiment, CD8 + PD-1 + / CD8 + The percentage of T cells is <2%, CD4 + PD-1 + / CD4 + The percentage of T cells is <2% and CD8 + CD38 + / CD8 + The percentage of T cells is <27%, which is used to characterize a low ratio of T cell exhaustion level to abnormal activation. The patients are less likely to experience immune-related adverse reactions when continuing to receive immune checkpoint inhibitor treatment.

[0127] The above-mentioned embodiments only express the implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Sequence Listing <110> Li Taisheng <120> A flow cytometer detection kit for immune characterization of advanced lung cancer and its detection method <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 552 <212> PRT <213> CD45 antibody (artificial sequence) <400> 1 Gln Ser Pro Thr Pro Ser Pro Thr Gly Leu Thr Thr Ala Lys Met Pro 1 5 10 15 Ser Val Pro Leu Ser Ser Asp Pro Leu Pro Thr His Thr Thr Ala Phe 20 25 30 Ser Pro Ala Ser Thr Phe Glu Arg Glu Asn Asp Phe Ser Glu Thr Thr 35 40 45 Thr Ser Leu Ser Pro Asp Asn Thr Ser Thr Gln Val Ser Pro Asp Ser 50 55 60 Leu Asp Asn Ala Ser Ala Phe Asn Thr Thr Gly Val Ser Ser Val Gln 65 70 75 80 Thr Pro His Leu Pro Thr His Ala Asp Ser Gln Thr Pro Ser Ala Gly 85 90 95 Thr Asp Thr Gln Thr Phe Ser Gly Ser Ala Ala Asn Ala Lys Leu Asn 100 105 110 Pro Thr Pro Gly Ser Asn Ala Ile Ser Asp Val Pro Gly Glu Arg Ser 115 120 125 Thr Ala Ser Thr Phe Pro Thr Asp Pro Val Ser Pro Leu Thr Thr Thr 130 135 140 Leu Ser Leu Ala His His Ser Ser Ala Ala Leu Pro Ala Arg Thr Ser 145 150 155 160 Asn Thr Thr Ile Thr Ala Asn Thr Ser Asp Ala Tyr Leu Asn Ala Ser 165 170 175 Glu Thr Thr Thr Leu Ser Pro Ser Gly Ser Ala Val Ile Ser Thr Thr 180 185 190 Thr Ile Ala Thr Thr Pro Ser Lys Pro Thr Cys Asp Glu Lys Tyr Ala 195 200 205 Asn Ile Thr Val Asp Tyr Leu Tyr Asn Lys Glu Thr Lys Leu Phe Thr 210 215 220 Ala Lys Leu Asn Val Asn Glu Asn Val Glu Cys Gly Asn Asn Thr Cys 225 230 235 240 Thr Asn Asn Glu Val His Asn Leu Thr Glu Cys Lys Asn Ala Ser Val 245 250 255 Ser Ile Ser His Asn Ser Cys Thr Ala Pro Asp Lys Thr Leu Ile Leu 260 265 270 Asp Val Pro Pro Gly Val Glu Lys Phe Gln Leu His Asp Cys Thr Gln 275 280 285 Val Glu Lys Ala Asp Thr Thr Ile Cys Leu Lys Trp Lys Asn Ile Glu 290 295 300 Thr Phe Thr Cys Asp Thr Gln Asn Ile Thr Tyr Arg Phe Gln Cys Gly 305 310 315 320 Asn Met Ile Phe Asp Asn Lys Glu Ile Lys Leu Glu Asn Leu Glu Pro 325 330 335 Glu His Glu Tyr Lys Cys Asp Ser Glu Ile Leu Tyr Asn Asn His Lys 340 345 350 Phe Thr Asn Ala Ser Lys Ile Ile Lys Thr Asp Phe Gly Ser Pro Gly 355 360 365 Glu Pro Gln Ile Ile Phe Cys Arg Ser Glu Ala Ala His Gln Gly Val 370 375 380 Ile Thr Trp Asn Pro Pro Gln Arg Ser Phe His Asn Phe Thr Leu Cys 385 390 395 400 Tyr Ile Lys Glu Thr Glu Lys Asp Cys Leu Asn Leu Asp Lys Asn Leu 405 410 415 Ile Lys Tyr Asp Leu Gln Asn Leu Lys Pro Tyr Thr Lys Tyr Val Leu 420 425 430 Ser Leu His Ala Tyr Ile Ile Ala Lys Val Gln Arg Asn Gly Ser Ala 435 440 445 Ala Met Cys His Phe Thr Thr Lys Ser Ala Pro Pro Ser Gln Val Trp 450 455 460 Asn Met Thr Val Ser Met Thr Ser Asp Asn Ser Met His Val Lys Cys 465 470 475 480 Arg Pro Pro Arg Asp Arg Asn Gly Pro His Glu Arg Tyr His Leu Glu 485 490 495 Val Glu Ala Gly Asn Thr Leu Val Arg Asn Glu Ser His Lys Asn Cys 500 505 510 Asp Phe Arg Val Lys Asp Leu Gln Tyr Ser Thr Asp Tyr Thr Phe Lys 515 520 525 Ala Tyr Phe His Asn Gly Asp Tyr Pro Gly Glu Pro Phe Ile Leu His 530 535 540 His Ser Thr Ser Tyr Asn Ser Lys 545 550 <210> 2 <211> 104 <212> PRT <213> CD3 antibody (Artificial Sequence) <400> 2 Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys Val 1 5 10 15 Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro Gly 20 25 30 Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp Glu 35 40 45 Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys Glu 50 55 60 Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly 65 70 75 80 Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg Val 85 90 95 Cys Glu Asn Cys Met Glu Met Asp 100 <210> 3 <211> 371 <212> PRT <213> CD4 antibody (Artificial Sequence) <400> 3 Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr Val Glu Leu Thr Cys 1 5 10 15 Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His Trp Lys Asn Ser Asn 20 25 30 Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro 35 40 45 Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp Asp Gln 50 55 60 Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys Ile Glu Asp Ser Asp 65 70 75 80 Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu Glu Val Gln Leu Leu 85 90 95 Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His Leu Leu Gln Gly Gln 100 105 110 Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly Ser Ser Pro Ser Val 115 120 125 Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln Gly Gly Lys Thr Leu 130 135 140 Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly Thr Trp Thr Cys Thr 145 150 155 160 Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys Ile Asp Ile Val Val 165 170 175 Leu Ala Phe Gln Lys Ala Ser Ser Ile Val Tyr Lys Lys Glu Gly Glu 180 185 190 Gln Val Glu Phe Ser Phe Pro Leu Ala Phe Thr Val Glu Lys Leu Thr 195 200 205 Gly Ser Gly Glu Leu Trp Trp Gln Ala Glu Arg Ala Ser Ser Ser Lys 210 215 220 Ser Trp Ile Thr Phe Asp Leu Lys Asn Lys Glu Val Ser Val Lys Arg 225 230 235 240 Val Thr Gln Asp Pro Lys Leu Gln Met Gly Lys Lys Leu Pro Leu His 245 250 255 Leu Thr Leu Pro Gln Ala Leu Pro Gln Tyr Ala Gly Ser Gly Asn Leu 260 265 270 Thr Leu Ala Leu Glu Ala Lys Thr Gly Lys Leu His Gln Glu Val Asn 275 280 285 Leu Val Val Met Arg Ala Thr Gln Leu Gln Lys Asn Leu Thr Cys Glu 290 295 300 Val Trp Gly Pro Thr Ser Pro Lys Leu Met Leu Ser Leu Lys Leu Glu 305 310 315 320 Asn Lys Glu Ala Lys Val Ser Lys Arg Glu Lys Ala Val Trp Val Leu 325 330 335 Asn Pro Glu Ala Gly Met Trp Gln Cys Leu Leu Ser Asp Ser Gly Gln 340 345 350 Val Leu Leu Glu Ser Asn Ile Lys Val Leu Pro Thr Trp Ser Thr Pro 355 360 365 Val Gln Pro 370 <210> 4 <211> 149 <212> PRT <213> CD8 antibody (artificial sequence) <400> 4 Leu Gln Gln Thr Pro Ala Tyr Ile Lys Val Gln Thr Asn Lys Met Val 1 5 10 15 Met Leu Ser Cys Glu Ala Lys Ile Ser Leu Ser Asn Met Arg Ile Tyr 20 25 30 Trp Leu Arg Gln Arg Gln Ala Pro Ser Ser Asp Ser His His Glu Phe 35 40 45 Leu Ala Leu Trp Asp Ser Ala Lys Gly Thr Ile His Gly Glu Glu Val 50 55 60 Glu Gln Glu Lys Ile Ala Val Phe Arg Asp Ala Ser Arg Phe Ile Leu 65 70 75 80 Asn Leu Thr Ser Val Lys Pro Glu Asp Ser Gly Ile Tyr Phe Cys Met 85 90 95 Ile Val Gly Ser Pro Glu Leu Thr Phe Gly Lys Gly Thr Gln Leu Ser 100 105 110 Val Val Asp Phe Leu Pro Thr Thr Ala Gln Pro Thr Lys Lys Ser Thr 115 120 125 Leu Lys Lys Arg Val Cys Arg Leu Pro Arg Pro Glu Thr Gln Lys Gly 130 135 140 Pro Leu Cys Ser Pro 145 <210> 5 <211> 147 <212> PRT <213> PD-1 antibody (Artificial Sequence) <400> 5 Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro 1 5 10 15 Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser 20 25 30 Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser 35 40 45 Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser 50 55 60 Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly 65 70 75 80 Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly 85 90 95 Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys 100 105 110 Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val 115 120 125 Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln 130 135 140 Thr Leu Val 145 <210> 6 <211> 191 <212> PRT <213> HLA-DR antibody (Artificial Sequence) <400> 6 Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro 1 5 10 15 Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe 20 25 30 His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe 35 40 45 Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala 50 55 60 Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr 65 70 75 80 Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro 85 90 95 Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe 100 105 110 Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val 115 120 125 Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu 130 135 140 Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val 145 150 155 160 Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys 165 170 175 His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu 180 185 190 <210> 7 <211> 258 <212> PRT <213> CD38 antibody (Artificial Sequence) <400> 7 Val Pro Arg Trp Arg Gln Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg 1 5 10 15 Phe Pro Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Ile His 20 25 30 Pro Glu Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys 35 40 45 Gly Ala Phe Ile Ser Lys His Pro Cys Asn Ile Thr Glu Glu Asp Tyr 50 55 60 Gln Pro Leu Met Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Ile 65 70 75 80 Leu Leu Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val 85 90 95 Gln Arg Asp Met Phe Thr Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala 100 105 110 Asp Asp Leu Thr Trp Cys Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr 115 120 125 Gln Ser Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser 130 135 140 Val Phe Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp 145 150 155 160 Val Val His Val Met Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys 165 170 175 Asn Ser Thr Phe Gly Ser Val Glu Val His Asn Leu Gln Pro Glu Lys 180 185 190 Val Gln Thr Leu Glu Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser 195 200 205 Arg Asp Leu Cys Gln Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile 210 215 220 Ser Lys Arg Asn Ile Gln Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp 225 230 235 240 Lys Phe Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser 245 250 255 Glu Ile

Claims

1. A detection method of a flow cytometer detection kit for immune characterization of advanced lung cancer, the method is a method for non-diagnostic purposes, characterized in that: The following steps are involved: Step 1: Use reverse pipetting technique to draw EDTA-K2 anticoagulated whole blood to the bottom of the flow tube to prevent the blood from touching the upper part of the tube wall; Step 2, add reagent A into the tube, the volume ratio of reagent A to whole blood is 1:10, and mix on a vortexer; incubate at room temperature away from light; add reagent B into the tube, the volume ratio of reagent B to whole blood is 20:1; mix on a vortexer; react in a dark place; reagent A includes CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, PD-1 monoclonal antibody, HLA-DR monoclonal antibody labeled with fluorescent microspheres dyed with dyes of different colors, and the concentration ratio of each antibody is 1:1:1:1:1:1:1, all of which are 0.05-1 mg / mL; reagent B is red blood cell lysate; Step 3: After vortex mixing, centrifuge at 4°C and discard the supernatant, add PBS solution for washing, vortex mixing and centrifuge at 4°C, discard the supernatant, add PBS solution again to form a cell suspension, and detect using flow cytometer; Step 4: Screen cells that are CD45 antibody positive and have an SSC value in the range of 0-50K to determine that they are lymphocytes; CD3 antibody-positive cells were screened from lymphocytes and identified as T cells; CD4 antibody-positive cells were screened from T cells, and PD-1 antibody-positive cells were screened from CD4 antibody-positive cells to calculate CD4 + PD-1 + / CD4 + The percentage of T cells, the cells positive for CD8 antibody were screened from T cells, the cells positive for PD-1 antibody were screened from the cells positive for CD8 antibody, and the CD8 + PD-1 + / CD8 + The percentage of T cells was calculated by screening HLA-DR antibody-positive cells from CD8 antibody-positive cells. + HLA-DR + / CD8 + The percentage of T cells.

2. A detection method of a flow cytometer detection kit for immune characterization of advanced lung cancer, the method is a method for non-diagnostic purposes, characterized in that: The following steps are involved: Step 1: Use reverse pipetting technique to draw EDTA-K2 anticoagulated whole blood to the bottom of the flow tube to prevent the blood from touching the upper part of the tube wall; Step 2, add reagent A into the tube, the volume ratio of reagent A to whole blood is 1:10, and mix with a vortexer; incubate at room temperature away from light; add reagent B into the tube, the volume ratio of reagent B to whole blood is 20:1; mix with a vortexer; react in a dark place; the reagent A includes CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, PD-1 monoclonal antibody, CD38 monoclonal antibody labeled with fluorescent microspheres dyed with dyes of different colors, and the concentration ratio of each antibody is 1:1:1:1:1:1, all of which are 0.05-1 mg / mL; reagent B is red blood cell lysate; Step 3: After vortex mixing, centrifuge at 4°C and discard the supernatant, add PBS solution for washing, vortex mixing and centrifuge at 4°C, discard the supernatant, add PBS solution again to form a cell suspension, and detect using flow cytometer; Step 4: Screen cells that are CD45 antibody positive and have an SSC value in the range of 0-50K to determine that they are lymphocytes; CD3 antibody-positive cells were screened from lymphocytes and identified as T cells; CD4 antibody-positive cells were screened from T cells, and PD-1 antibody-positive cells were screened from CD4 antibody-positive cells to calculate CD4 + PD-1 + / CD4 + The percentage of T cells, the cells positive for CD8 antibody were screened from T cells, the cells positive for PD-1 antibody were screened from the cells positive for CD8 antibody, and the CD8 + PD-1 + / CD8 + The percentage of T cells was calculated by screening CD38 antibody-positive cells from CD8 antibody-positive cells. + CD38 + / CD8 + The percentage of T cells.

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