A kit for detecting PD-1 in tumor patients based on flow cytometry

Abstract

The present invention provides a flow cytometer-based kit for detecting PD-1 in tumor patients. The kit, through antibody reagents containing PD-1-PE and Anti-IgG4-PE, can not only detect the PD-1 expression level in tumor patients, but also detect the PD-1 expression level in tumor patients who have been injected with anti-PD-1 monoclonal antibodies, providing better guidance for clinical medication and therapeutic effects. By adding a blocking agent to the diluent, a one-step incubation method is implemented for detection, shortening the detection time and improving the detection efficiency. At the same time, the Anti-IgG4-PE in the antibody reagent and the blocking agent and antioxidant in the diluent are optimized to improve the specificity and stability of the detection, thereby improving the accuracy of the detection. Using this kit for PD-1 detection has the advantages of simple operation, fast and convenient detection process, and accurate test results.

Description

Technical Field

[0001] The present invention relates to the technical field of flow cytometry, and in particular to a kit for detecting PD-1 in tumor patients based on flow cytometry. Background Art

[0002] Programmed cell death protein 1 (PD-1), a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily, is composed of 288 amino acids and is a key immunosuppressive molecule on the surface of T cells, inhibiting T cell activation. Under normal circumstances, the immune system responds to foreign antigens that accumulate in lymph nodes or the spleen, promoting the proliferation of antigen-specific T cells. The binding of programmed cell death protein 1 (PD-1) to programmed cell death ligand 1 (PD-L1) transmits inhibitory signals, thereby inhibiting T cell activation and inducing apoptosis of tumor-specific T cells. This leads to T cell resistance, contributing to immune escape and immunosuppression in pathological conditions such as tumors and chronic inflammation.

[0003] In clinical applications, PD-1 expression level testing can be used to assess tumor immune function, treatment, and prognosis. PD-1 is often abnormally expressed on the surface of tumor T cells. Different levels of expression correlate with different prognoses, with patients with low expression having a better prognosis than those with high expression. Therefore, measuring PD-1 expression on peripheral blood T cells can be used to assess disease progression. PD-1 expression levels increase or decrease with disease progression or treatment improvement. A gradual decrease in PD-1 during treatment indicates good immune recovery; a gradual increase or persistently high PD-1 level indicates poor anti-tumor immunity and the need for appropriate immunotherapy. Therefore, measuring programmed cell death protein-1 (PD-1) expression can assess the activation of the tumor body and provide a reference for evaluating drug efficacy and clinical treatment plans.

[0004] Some cancer patients receive injections of anti-PD-1 monoclonal antibodies for treatment. Six IgG4-based anti-PD-1 monoclonal antibodies have been approved for marketing by the US FDA: nivolumab, pembrolizumab, cemiplimab, toripalimab, cindilimab, and camrelizumab. Anti-PD-1 monoclonal antibodies bind to the PD-1 site on the cell surface, thereby blocking the PD-1 / PD-L1 signaling pathway, upregulating T cell activation, and activating endogenous anti-tumor immune responses, thereby exerting a therapeutic effect on tumors. However, this binding to the PD-1 site on the cell surface blocks the binding of the PD-1 antibody in PD-1 detection kits, making it impossible to accurately monitor PD-1 expression levels in samples treated with anti-PD-1 monoclonal antibodies.

[0005] The current method for detecting PD-1 expression levels in cancer patients mainly relies on test kits. The test reagents in the test kits are composed of five antibodies conjugated with different fluorescent dyes: CD45, CD3, CD4, CD8, and PD-1. These are used to detect PD-1 expression during patient treatment, thereby guiding clinical treatment. However, this method has drawbacks. When a patient is treated with the above-mentioned PD-1 monoclonal antibody, the PD-1 monoclonal antibody will bind to the PD-1 epitope, thereby blocking the binding of the PD-1 antibody in the test kit to the PD-1 epitope. As a result, the test results may not truly reflect the patient's PD-1 expression level.

[0006] The incubation method used in the test kit for detecting immunological characterization of late-stage lung cancer in CN 114720356 A is a two-step incubation method, wherein an antibody detection reagent is first added for a first incubation, and then a red blood cell lysis solution is added for a second incubation. Multiple incubations result in a long detection time, and the detection efficiency is low, especially when large-scale testing is required.

[0007] Therefore, a kit is urgently needed to solve the above problems. Summary of the Invention

[0008] The present invention provides a kit for detecting PD-1 in tumor patients based on flow cytometry. The kit can detect not only the PD-1 expression level in tumor patients, but also the PD-1 expression level in tumor patients injected with anti-PD-1 monoclonal antibody drugs through antibody reagents containing PD-1-PE and Anti-IgG4-PE, which has better guidance for clinical medication and treatment. By adding a blocking agent to the diluent, a one-step incubation method is implemented for detection, which shortens the detection time and improves the detection efficiency. At the same time, the Anti-IgG4-PE in the antibody reagent and the blocking agent and antioxidant in the diluent are optimized to improve the specificity and stability of the detection, thereby improving the accuracy of the detection. Using this kit for PD-1 detection has the advantages of simple operation, fast and convenient detection process, and accurate detection results.

[0009] The kit for detecting the PD-1 expression level in tumor patients is composed of five antibodies coupled with different fluorescent dyes: CD45, CD3, CD4, CD8, and PD-1. It is used to detect the expression of PD-1 during the patient's treatment, thereby guiding clinical treatment. However, this method has disadvantages. When a patient is injected with an anti-PD-1 monoclonal antibody for treatment, the anti-PD-1 monoclonal antibody will bind to the PD-1 epitope, thereby blocking the binding of the PD-1 antibody in the kit to the PD-1 epitope, and thus causing the test results to fail to truly reflect the patient's PD-1 expression level. Since the structure of the anti-PD-1 monoclonal antibody drug is IgG4, the kit of the present invention adds an Anti-IgG4 antibody to the five detection antibodies. The Anti-IgG4 monoclonal antibody binds to the anti-PD-1 monoclonal antibody that has already bound to the PD-1 epitope. In this way, the Anti-IgG4 monoclonal antibody acts as a secondary antibody and indirectly binds to the PD-1 epitope. Specifically, the detection principle of the kit of the present invention is to add antibody reagents to the sample and detect it by flow cytometry. The six detection antibodies in the antibody reagents are coupled with different fluorescent dyes, CD45-PE-Cy7 distinguishes T lymphocytes, CD3-FITC, CD4-APC, and CD8-Percp-cy5.5 distinguish CD3 + T lymphocyte population, CD4 + T lymphocyte population, CD8 +T lymphocyte population. If the test sample comes from a tumor patient who has not been injected with an anti-PD-1 monoclonal antibody, PD-1-PE binds to the PD-1 epitope of a different T lymphocyte population, and Anti-IgG4-PE has no effect. If the test sample comes from a tumor patient who has been injected with an anti-PD-1 monoclonal antibody, PD-1-PE binds to the PD-1 epitope of a different T lymphocyte population, and Anti-IgG4-PE binds to the anti-PD-1 monoclonal antibody that has already bound to the PD-1 epitope. This allows the Anti-IgG4 monoclonal antibody to indirectly bind to the PD-1 epitope as a secondary antibody. Therefore, the kit of the present invention can not only detect the PD-1 expression level of tumor patients, but also detect the PD-1 expression level of tumor patients who have been injected with an anti-PD-1 monoclonal antibody.

[0010] A kit for detecting PD-1 expression levels in tumor patients based on flow cytometry, comprising antibody reagents, wherein the antibody reagents include PD-1 monoclonal antibody and anti-IgG4 monoclonal antibody.

[0011] Furthermore, the antibody reagents also include CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody and CD8 monoclonal antibody.

[0012] CD (Cluster of Differentiation) is a differentiation cluster or differentiation group, also called leukocyte differentiation antigen, which refers to the cell surface markers that appear or disappear during the different stages of normal differentiation and maturation and activation of white blood cells of different lineages. They are a class of proteins or glycoproteins on the cell membrane. In physiology, CD molecules have many uses and are usually used as important receptors or ligands of cells. They can not only be used as surface markers for cell identification and separation, but also widely participate in cell growth, maturation, differentiation, development, migration, and activation. CD molecules are usually used as cell markers for immune antigen recognition. This technology can be used to observe molecules on the cell surface and identify the cell. Different blood cells have different CD molecules distributed on their surfaces. With the help of these CD molecules, different blood cells can be identified and separated. T cells express CD45, CD3, and CD28 on their surface, so they can be recorded as CD45. + 、CD3 + 、CD28 + ; Helper T cells express CD45, CD3, and CD4 on their surface, so they can be referred to as CD45 + 、CD3 + 、CD4 + ; Cytotoxic T cells express CD45, CD3, CD8, and CD28 on their surface, so they can be recorded as CD45 + 、CD3 + 、CD8 + 、CD28 + Thus, this combination of CDs can be used to distinguish T cells from other cells.

[0013] Furthermore, the kit further comprises a diluent, the diluent comprises a blocking agent, and the blocking agent comprises one or more of mouse IgG, mouse Poly IgG, and fetal bovine serum.

[0014] The sample generally used to detect PD-1 is whole blood, and the test kit usually uses a two-step incubation method. In the two-step incubation method, the antibody reagent (without blocking agent) is added for the first incubation to allow the antibody reagent (without blocking agent) to bind to the antigen epitope on the T cells in the sample. The hemolysin is added for the second incubation to lyse the red blood cells. Since the antibody has already bound to the antigen epitope on the T cell, the protein released by the lysis of the red blood cells has no effect on the detection. If the two-step incubation method is combined into a one-step incubation method, the non-specific binding effect caused by the protein released by the lysis of the red blood cells must be avoided. Because the antibodies in the antibody reagent bind to the T cell surface antigen and the red blood cells are lysed to release proteins, the kit of the present invention avoids the non-specific binding effect caused by the protein released by the lysis of the red blood cells by adding a blocking agent to the antibody reagent, thereby realizing the one-step incubation method for detection. Since the basic structure of the anti-PD-1 monoclonal antibody drug is IgG4 and the antibody in the detection reagent is Anti-IgG4-PE, the components of the blocking agent must not only avoid the nonspecific binding caused by the addition of Anti-IgG4-PE as a secondary antibody, but also avoid the nonspecific binding effect caused by proteins released by red blood cell lysis. After a large number of experimental verification and screening, the selected blocking agent must include one or more of mouse IgG, mouse Poly IgG, and fetal bovine serum to ensure that the test results are within the allowable deviation range.

[0015] In some methods, mouse IgG and mouse Poly IgG not only avoid the nonspecific binding effects caused by proteins released by red blood cell lysis, but can also effectively avoid the nonspecific binding caused by the addition of Anti-IgG4-PE secondary antibody, thereby improving specific binding; and fetal bovine serum provides a stable environment for proteins, allowing antigens and antibodies to bind more fully, thereby improving the accuracy of detection.

[0016] Furthermore, the diluent further comprises an antioxidant, and the antioxidant comprises one or more of zinc chloride, magnesium chloride, and sodium orthovanadate.

[0017] In some methods, through a large number of experiments, it was found that one or more of the antioxidants selected from a large number of antioxidants, including zinc chloride, magnesium chloride, and sodium orthovanadate, are most effective in improving the detection stability of this test kit. Antioxidants are a class of substances that can help capture and neutralize free radicals. Their main function is to scavenge free radicals, reduce oxidative stress, and protect cells. During the detection process of this test kit, antioxidants mainly protect the proteins on the surface of T cells and the detection proteins in the antibody reagent. If the protein loses its activity due to oxidation, whether it is the antibody protein in the detection reagent or the antigen protein in the sample being tested, it will lead to inaccurate test results. Therefore, the addition of antioxidants can improve the stability of the protein and thus improve the accuracy of the test.

[0018] Furthermore, the antibody reagent also includes fluorescent dyes PE-Cy7, FITC, APC, Percp-cy5.5 and PE, and the CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, PD-1 monoclonal antibody, and Anti-IgG4 monoclonal antibody are respectively coupled with PE-Cy7, FITC, APC, Percp-cy5.5, PE, and PE to form CD45-PE-Cy7, CD3-FITC, CD4-APC, CD8-Percp-cy5.5, PD-1-PE, and Anti-IgG4-PE.

[0019] In some methods, PE-Cy7, FITC, APC, Percp-cy5.5, and PE are different fluorescent dyes used to distinguish different fluorescent signals; CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, PD-1 monoclonal antibody, and Anti-IgG4 monoclonal antibody are coupled with PE-Cy7, FITC, APC, Percp-cy5.5, PE, and PE, respectively, and PD-1 monoclonal antibody and Anti-IgG4 monoclonal antibody are coupled with the same fluorescent dye PE, because both monoclonal antibodies are used to detect the expression level of PD-1, PD-1-PE is a direct detection, and Anti-IgG4-PE is an indirect detection.

[0020] Furthermore, the ratio of CD45-PE-Cy7:CD3-FITC:CD4-APC:CD8-Percp-cy5.5:PD-1-PE:Anti-IgG4-PE:diluent in the antibody reagent is (3-12):(3-12):(3-12):(3-12):(1-6):(3-12):(30-68).

[0021] The ratio of the six detection proteins and diluent in the antibody reagent has no significant difference in the detection of the kit within the above range.

[0022] The typical process for detecting PD-1 includes a two-step incubation method, where an antibody detection reagent is first added for the first incubation, and then hemolysin is added for the second incubation. Multiple incubations result in a long detection time, and the detection efficiency is low, especially when large-scale testing is required.

[0023] Furthermore, a method for detecting the PD-1 content in a blood sample is provided, using the above-mentioned kit for detection, the detection method comprising the following steps:

[0024] (1) Centrifuge and clean the sample;

[0025] (2) Add antibody reagents containing PD-1-PE and Anti-IgG4-PE and mix with hemolysin, incubate, and centrifuge for washing;

[0026] (3) Detection using flow cytometry.

[0027] In some embodiments, the hemolysin is added in step (2) of the method to dissolve the red blood cells in the sample and eliminate the interference of the red blood cells on the detection. At the same time, the red blood cell dissolution releases proteins, which increase nonspecific binding and affect the accuracy of the detection. Therefore, a blocking agent is added to the diluent to eliminate the nonspecific effect on the detection.

[0028] Furthermore, a blocking agent composition is used to prepare a reagent for improving the accuracy of detecting PD-1 in tumor patients injected with anti-PD-1 monoclonal antibody drugs, thereby achieving one-step incubation, wherein the composition includes mouse Poly IgG and fetal bovine serum.

[0029] In some embodiments, through experimental screening, mouse Poly IgG and fetal bovine serum are the best composition formula for improving the detection specificity of the kit and thus improving the detection accuracy.

[0030] Furthermore, an antibody is used to prepare a reagent for improving the specificity of detecting PD-1 in tumor patients injected with anti-PD-1 monoclonal antibody drugs, and the antibody is Anti-IgG4.

[0031] In some methods, Anti-IgG4 antibodies of different sequences are screened experimentally. Anti-IgG4 antibodies of different sequences have different binding abilities to anti-PD-1 monoclonal antibody drugs. An Anti-IgG4 antibody with the strongest binding ability to anti-PD-1 monoclonal antibody drugs is screened out, thereby improving the specific reaction and thus improving the accuracy of detection.

[0032] Furthermore, an antioxidant composition is used to prepare a reagent for improving the stability of PD-1 in tumor patients injected with anti-PD-1 monoclonal antibody drugs, wherein the composition comprises zinc chloride, magnesium chloride and sodium orthovanadate.

[0033] In some embodiments, through experimental screening, an antioxidant composition comprising zinc chloride, magnesium chloride, and sodium orthovanadate is most effective in improving the detection stability of the kit and thus improving the detection accuracy.

[0034] The present invention has the following beneficial effects:

[0035] (1) The present invention provides a kit for detecting PD-1 in tumor patients based on flow cytometry. The kit can detect not only the PD-1 expression level in tumor patients but also the PD-1 expression level in tumor patients who have been injected with anti-PD-1 monoclonal antibody drugs by using antibody reagents containing PD-1-PE and Anti-IgG4-PE, thereby providing better guidance for clinical medication and treatment.

[0036] (2) By adding a blocking agent to the diluent, a one-step incubation method is achieved for detection, shortening the detection time and improving the detection efficiency;

[0037] (3) Optimize the Anti-IgG4 antibody in the antibody reagent and the blocking agent and antioxidant in the diluent to improve the specificity and stability of the test, thereby improving the accuracy of the test;

[0038] (4) The use of this kit for PD-1 detection has the advantages of simple operation, fast and convenient detection process, and accurate detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a scatter plot of the isotype control test for sample 1 in Example 2;

[0040] Figure 2 This is a scatter plot of reagent No. ① detection of sample 1 in Example 2;

[0041] Figure 3 This is a scatter plot of the reagent No. ② test for sample 1 in Example 2;

[0042] Figure 4 This is a scatter plot of the isotype control test for Sample 2 in Example 2;

[0043] Figure 5 This is a scatter plot of the test of reagent No. ① for sample 2 in Example 2;

[0044] Figure 6 This is a scatter plot of the test results of reagent No. ② for sample 2 in Example 2;

[0045] Figure 7 This is a scatter plot of the isotype control test of sample 3 in Example 2;

[0046] Figure 8 This is a scatter plot of the test of reagent No. ① for sample 3 in Example 2;

[0047] Figure 9 This is a scatter plot of the reagent No. ② test for sample 3 in Example 2;

[0048] Figure 10 This is a scatter plot of the isotype control test of sample 4 in Example 2;

[0049] Figure 11 This is a scatter plot of reagent No. ① detection of sample 4 in Example 2;

[0050] Figure 12 This is a scatter plot of the reagent No. ② test for sample 4 in Example 2;

[0051] Figure 13 This is a scatter plot of the isotype control test of sample 5 in Example 2;

[0052] Figure 14 This is a scatter plot of the test of reagent No. ① for sample 5 in Example 2;

[0053] Figure 15 This is a scatter plot of the reagent No. ② test for sample 5 in Example 2;

[0054] Figure 16 This is a scatter plot of the isotype control test of sample 6 in Example 2;

[0055] Figure 17 This is a scatter plot of the test of reagent No. ① for sample 6 in Example 2;

[0056] Figure 18 This is a scatter plot of the reagent No. ② test for sample 6 in Example 2;

[0057] Figure 19 This is a scatter plot of the low-value quality control product isotype control test in Example 2;

[0058] Figure 20 This is a scatter plot of the low-value quality control reagent No. 1 test in Example 2;

[0059] Figure 21 This is a scatter plot of the low-value quality control reagent No. ② test in Example 2;

[0060] Figure 22 This is a scatter plot of the high-value quality control product isotype control test in Example 2;

[0061] Figure 23 This is a scatter plot of the high-value quality control product No. 1 reagent test in Example 2;

[0062] Figure 24 This is a scatter plot of the detection of the high-value quality control product No. ② reagent in Example 2. DETAILED DESCRIPTION

[0063] The present invention will be further described in detail below with reference to the examples. It should be noted that the examples described below are intended to facilitate understanding of the present invention and do not have any limiting effect on the present invention.

[0064] Example 1: Kit and detection method provided by the present invention

[0065] 1. Antibody reagent preparation

[0066] Antibody fluorescent dye conjugates CD45-PE-Cy7 (purchased from Biolegend), CD3-FITC (purchased from Biolegend), CD4-APC (purchased from Biolegend), CD8-Percp-cy5.5 (purchased from Biolegend), and PD-1-PE (purchased from Biolegend) were diluted to 0.0125 mg / mL.

[0067] Anti-IgG4 (purchased from Hangzhou Saiji Biotechnology Co., Ltd., catalog number HZ010503202) was coupled with PE to form Anti-IgG4-PE. The coupling steps are as follows:

[0068] (1) Dilute the antibody to 1-5 mg / mL with pH 9.8 sodium bicarbonate buffer, or dialyze the antibody sufficiently with buffer to prevent lysine dissociation (removal of its positive charge), but ensure that most of the protein remains intact;

[0069] (2) Add the diluted or dialyzed antibody from step (1) to PE and place it in a dialysis bag. Place the bag in a beaker and dialyze it with 100 mL of freshly prepared pH 9.8 sodium bicarbonate buffer containing 0.1 mg / mL. Cover the beaker with aluminum platinum to protect it from light and stir at 4°C overnight.

[0070] (3) Replace the 0.1 mg / mL pH 9.8 sodium bicarbonate buffer with PBS buffer and dialyze at 4°C to terminate the reaction. During this process, replace the PBS buffer at least three times until the absorption at 480 nm is zero.

[0071] Anti-IgG4-PE was purified using an affinity chromatography column, and the purified Anti-IgG4-PE antibody was diluted to 0.0125 mg / mL.

[0072] The preparation of the diluent comprises the following steps:

[0073] (1) Prepare 1× PBS solution in a beaker;

[0074] (2) Add 2-5% of blocking agent to the 1× PBS solution in step (1). The blocking agent includes mouse PolyIgG (purchased from Beyotime) and fetal bovine serum (purchased from Gibco). The ratio of mouse PolyIgG to fetal bovine serum is 5:3. Stir thoroughly to mix.

[0075] (3) Add antioxidants zinc chloride (purchased from Sinopharm Chemical Reagent Co., Ltd.), magnesium chloride (purchased from Sinopharm Chemical Reagent Co., Ltd.), and sodium orthovanadate (purchased from Sinopharm Chemical Reagent Co., Ltd.) to make the final concentrations of zinc chloride 0.1 mm / L, magnesium chloride 0.5 mm / L, and sodium orthovanadate 0.01 mm / L, and stir thoroughly to mix.

[0076] Six antibodies coupled with fluorescent dyes were diluted and mixed with diluent, and the ratio of CD45-PE-Cy7:CD3-FITC:CD4-APC:CD8-Percp-cy5.5:PD-1-PE:Anti-IgG4-PE: diluent was (3-12):(3-12):(3-12):(3-12):(1-6):(3-12):(30-68).

[0077] 2. Prepare quality control products

[0078] The steps for preparing quality control products are as follows:

[0079] (1) Fresh blood samples without any disease symptoms and normal blood routine were selected as low-value PD-1 quality control products. + Fresh blood samples from patients with T lymphocyte cytopenia are used as high-value PD-1 quality control products;

[0080] (2) Screen out fresh blood samples with low and high PD-1 values ​​that meet the quality control requirements. Take 1 mL of each blood sample and mix them separately according to the high value group and the low value group.

[0081] The target value ranges of low-value quality control products and high-value quality control products are shown in Table 1 below.

[0082] Table 1. Target value range of quality control products

[0083]

[0084] 4. Testing process

[0085] The detection steps are as follows:

[0086] (1) Take 100 μL of blood sample, add 1 mL of PBS, mix well, centrifuge at 400 g for 5 min, and discard the supernatant;

[0087] (2) Repeat step (1) and prepare the blood sample;

[0088] (3) Take 50 μL of the blood sample that has been washed in step (2), add 20 μL of antibody reagent and 1 mL of hemolysin, incubate for 15 minutes, centrifuge at 400 g for 5 minutes, and discard the supernatant;

[0089] (4) Add 1 mL of PBS, centrifuge at 400 g for 5 min, discard the supernatant, add 500 μL of PBS, and set aside;

[0090] (5) On-machine detection: First, use fluorescent calibration microspheres to calibrate the various parameters of the flow cytometer (purchased from Beckman). After passing the test, the sample is tested. Circle the T lymphocyte population (CD45) on the scatter plot of CD45 PE-Cy7 (X axis) vs SSC (Y axis). + ); then circle CD3 on the CD3 FITC (X-axis) vs SSC (Y-axis) scatter plot. + T lymphocyte population; circle CD4 on the scatter plot of CD4 APC (X-axis) vs. CD8 Percp-cy5.5 (Y-axis) + T lymphocyte populations and CD8 + T lymphocyte population; CD3 FITC (X axis) vs PE (Y axis), CD4 APC (X axis) vs PE (Y axis), CD8 Percp-cy5.5 (X axis) vs PE (Y axis) scatter plots are associated with CD3 + 、CD4 + 、CD8 + T lymphocyte population, draw PE gate at the position of negative blood sample, obtain CD3 + 、CD4 + 、CD8 + The PD-1-PE positive percentage within the T lymphocyte population gate is recorded.

[0091] 4. Linear

[0092] A high-concentration cell sample (requires quadruplicate initial cell concentration ≥10 million cells / mL) (cell concentration range and cell quantity determined) was diluted 2-fold to five different cell concentrations (each concentration replicated four times). Linearity was assessed by analyzing the percentage of positive cells relative to the concentration. The median of the replicate values ​​for each dilution should be within 3% of the median value for all dilutions tested, or within 10% of the median value for all dilutions tested. When the percentage of positive cells was >30%, the relative deviation was calculated; when the percentage of positive cells was <30%, the absolute deviation was calculated. The test results are shown in Table 2 below.

[0093] Table 2. Linearity test results

[0094]

[0095] According to the results in Table 2, CD3 + PD-1, CD4 + PD-1 and CD8 + The median of the repeated test values ​​of PD-1 at each dilution should be within 3% of the absolute value or 10% of the relative value of the median of the test values ​​of all dilutions, indicating that the kit of the present invention has good linear performance, has accurate measurement capabilities within the expected concentration range, and its performance meets the quality standards.

[0096] 5. Accuracy

[0097] Positive and negative cells were mixed in a certain ratio. The test results should be within the expected value ± 10%. The test results are shown in Table 3 below.

[0098] Table 3. Accuracy test results

[0099]

[0100] According to the results analysis in Table 3, the relative deviations between the detection results of all tested cells and the expected values ​​were within the range of ±10%, indicating that the kit of the present invention has high accuracy.

[0101] 6. Repeatability

[0102] Low-value positive cells and high-value positive cells were taken and tested 10 times using the kit. SD and CV were calculated. The test results are shown in Table 4 below.

[0103] Table 4. Repeatability test results

[0104]

[0105] According to the results analysis in Table 4, when the positive percentage is greater than or equal to 30%, the CV value is no more than 10%; when the positive percentage is less than 30%, the CV value is no more than 10%, indicating that the kit of the present invention has high precision and the test results are reliable and consistent.

[0106] Example 2: Comparison of test results with antibody reagents without Anti-IgG4-PE

[0107] The difference between this embodiment and embodiment 1 is that reagent No. ① is the same as embodiment 1, with Anti-IgG4-PE, and another reagent No. ② is set without Anti-IgG4-PE. Three samples that were clearly treated with monoclonal antibody drugs and three samples that were not treated were respectively taken, and the detection was carried out according to the detection process described in embodiment 1. Each sample was detected three times, and the average value was taken. The detection result of the antibody reagent with Anti-IgG1-PE added but without PD-1-PE and Anti-IgG4-PE was used as the isotype control. The isotype control is used to eliminate the background staining caused by the non-specific binding of the antibody to the Fc receptor on the cell surface and electrostatic binding. At the same time, the low-value quality control product and the high-value quality control product are detected to calibrate the detection accuracy. The detection results are shown in Tables 5 and 6 below. The scatter plots of the isotype control detection of samples 1 to 6, the detection of reagent No. ①, and the detection of reagent No. ② are shown below. Figures 1-18 As shown, the scatter plots of low-value quality control products and high-value quality control products are as follows: Figures 19-24 shown.

[0108] Table 5. Test results of whether the antibody reagent contains Anti-IgG4-PE

[0109]

[0110] Table 6. Results of the Anti-IgG4-PE Quality Control Test to Check if the Antibody Reagent Contains Anti-IgG4-PE

[0111]

[0112] According to the analysis of the results in Tables 5 and 6, the comparison of the test results of reagents ① and ② for samples treated with monoclonal antibodies and samples not treated with monoclonal antibodies found that in the samples treated with monoclonal antibodies, the test results of reagent ② were much lower than those of reagent ①, indicating that after the injection of monoclonal antibodies, the PD-1 epitope on the cell surface was bound by the monoclonal antibodies. If the antibody reagent did not contain Anti-IgG4-PE as a secondary antibody to bind to the monoclonal antibodies, the expression level of PD-1 in the samples treated with monoclonal antibodies would not be detected. The comparison of the test results of reagents ① and ② for samples not treated with monoclonal antibodies, low-value quality control products, and high-value quality control products showed no significant difference in the test results. At the same time, the test results of the quality control products were within the target value range, indicating that this kit can not only detect the PD-1 expression level in tumor patients, but also detect the PD-1 expression level in tumor patients injected with anti-PD-1 monoclonal antibodies.

[0113] Example 3: Screening of blocking agents

[0114] The difference between this example and Example 1 is that different blocking agent combinations are used to prepare the diluent. The different blocking agent combinations are shown in Table 7 below. The quality control products were tested according to the detection process described in Example 1. Each quality control product was tested three times and the average value was taken. The test results of the antibody reagent without adding PD-1-PE and Anti-IgG4-PE but adding Anti-IgG1-PE were used as isotype controls. The isotype control is used to eliminate background staining caused by nonspecific binding of the antibody to Fc receptors on the cell surface and electrostatic binding. The test results are shown in Table 8 below.

[0115] Table 7. Types of sealants

[0116]

[0117] Table 8. Effects of different blocking agent combinations on detection

[0118]

[0119] According to the data analysis in Table 8, comparing the quality control results with Blockers 1, 2, 3, and 4, the results of Blockers 1 to 3 exceeded the target range of the quality control or only partially fell within the target range. Only Blocker 4 had all the test results within the target range of the quality control. This indicates that Blocker 4 has the best blocking effect and the strongest ability to eliminate nonspecific binding, which can improve detection accuracy. Therefore, a combination of mouse poly IgG and fetal bovine serum was selected as the blocking agent. Mouse poly IgG not only avoids nonspecific binding caused by proteins released by erythrocyte lysis, but also avoids nonspecific binding caused by the addition of the Anti-IgG4-PE secondary antibody, thereby improving specific binding and thus improving detection accuracy. Fetal bovine serum provides a stable environment for proteins, thereby improving detection stability.

[0120] Example 4: Adding a blocking agent to the diluent to achieve one-step incubation detection

[0121] In the two-step incubation method, the first incubation allows the antibody reagent (without blocking agent) to bind to the antigen epitope on the T cells in the sample, and the second incubation causes the red blood cells to lyse. Since the antibody has already bound to the antigen epitope on the T cell, the protein released by the red blood cell lysis has no effect on the detection. If the two-step incubation method is combined into a one-step incubation method, the non-specific binding effect brought by the protein released by the red blood cell lysis must be avoided. Therefore, a blocking agent is added to the antibody reagent to avoid the non-specific binding effect brought by the protein released by the red blood cell lysis, thereby realizing the one-step incubation method for detection.

[0122] The difference between the two-step incubation method and the one-step incubation method in Example 1 is that the antibody reagent and hemolysin are incubated separately. The antibody reagent (without blocking agent) is first added to the sample for the first incubation, and then the hemolysin is added after centrifugation for the second incubation. The specific detection steps are as follows:

[0123] (1) Take 100 μL of blood sample, add 1 mL of PBS, mix well, centrifuge at 400 g for 5 min, and discard the supernatant;

[0124] (2) Repeat step (1) and prepare the blood sample;

[0125] (3) Take 50 μL of the blood sample that has been cleaned in step (2), add 20 μL of antibody reagent (without blocking agent), mix, incubate for 15 minutes, centrifuge at 400g for 5 minutes, and discard the supernatant;

[0126] (4) Add 1 mL of hemolysin to hemolyze, incubate for 15 minutes, centrifuge at 400 g for 5 minutes, and discard the supernatant;

[0127] (4) Add 1 mL of PBS, centrifuge at 400 g for 5 min, discard the supernatant, add 500 μL of PBS, and set aside;

[0128] (5) Detection using flow cytometry.

[0129] The quality control products were tested using the one-step incubation method and the two-step incubation method. Each quality control product was tested three times and the average value was taken. The test results of the antibody reagent with no PD-1-PE or Anti-IgG4-PE but with Anti-IgG1-PE added were used as isotype controls. The isotype control was used to eliminate background staining caused by nonspecific binding of the antibody to Fc receptors on the cell surface and electrostatic binding. The test results are shown in Table 9 below.

[0130] Table 9. Effect of blocking agent on incubation

[0131]

[0132] According to the data analysis in Table 9, the results of the one-step incubation method (the antibody reagent does not contain a blocking agent) and the one-step incubation method (the antibody reagent contains a blocking agent) for detecting the quality control product are compared. The test results of the one-step incubation method (the antibody reagent does not contain a blocking agent) exceed the target value range of the quality control product, while the test results of the one-step incubation method (the antibody reagent contains a blocking agent) and the two-step incubation method are within the target value range of the quality control product, indicating that the addition of a blocking agent to the antibody reagent can indeed avoid the nonspecific binding of proteins released by red blood cell lysis; further comparing the results of the one-step incubation method (the antibody reagent contains a blocking agent) and the two-step incubation method for detecting the quality control product, there is no significant difference between the two, indicating that adding a blocking agent to the antibody reagent for one-step incubation method detection can completely replace the two-step incubation method detection, shortening the detection time and improving the detection efficiency.

[0133] In this example, the four blocking agents provided in Table 7 of Example 3 were subjected to the above-mentioned one-step incubation method (the antibody reagent does not contain a blocking agent), one-step incubation method (the antibody reagent contains a blocking agent), and two-step incubation method experiments. The results showed that only the one-step incubation method (the antibody reagent contains a blocking agent) in which a combination of blocking agent 4 mouse poly IgG: fetal bovine serum = 5:3 was added to the diluent resulted in a quality control product test result within the target value range. The test results of the other three blocking agent combinations were not all within the target value range and had deviations. Therefore, only by adding a combination of blocking agent 4 mouse poly IgG: fetal bovine serum = 5:3 to the diluent could the two-step incubation method be combined into a one-step incubation method.

[0134] Example 5: Optimization of Anti-IgG4-PE Antibody

[0135] In this example, four types of Anti-IgG4-PE antibodies are provided, namely Anti-IgG4-PE antibody 1 (Anti-IgG4 was purchased from Biolegend, product number 411202, Anti-IgG4 was coupled to PE but not purified), Anti-IgG4-PE antibody 2 (same as Example 1 but not purified), purified Anti-IgG4-PE antibody 1, and purified Anti-IgG4-PE antibody 2 (same as Example 1). After the antibody is coupled with the fluorescent dye, the antibody solution contains not only Anti-IgG4-PE but also some impurities such as Anti-IgG4 and PE. Impurities may cause false positive test results, and the impurities can be removed by affinity chromatography.

[0136] The above four antibodies were respectively combined with antibody reagents containing CD45-PE-Cy7, CD3-FITC, CD4-APC, CD8-Percp-cy5.5, and PD-1-PE, numbered as antibody reagent No. ①, antibody reagent No. ②, antibody reagent No. ③, and antibody reagent No. ④, as shown in Table 10 below. The quality control products were tested according to the detection process described in Example 1. Each sample was tested three times and the average value was taken. The test results of the antibody reagent without adding PD-1-PE and Anti-IgG4-PE but adding Anti-IgG1-PE were used as isotype controls. The isotype controls were used to eliminate background staining caused by nonspecific binding of antibodies to Fc receptors on the cell surface and electrostatic binding. The test results are shown in Table 11 below.

[0137] Table 10. Different Anti-IgG4-PE Antibody Reagent Combinations

[0138]

[0139] Table 11. Test results of different Anti-IgG4-PE antibody reagents

[0140]

[0141] According to the data analysis in Table 11, comparing the test results of Antibody Reagent 1 and Antibody Reagent 2 quality control, and the test results of Antibody Reagent 3 and Antibody Reagent 4 quality control, the test results of Antibody Reagent 1 and Antibody Reagent 2 quality control were below the target range, while the test results of Antibody Reagent 3 and Antibody Reagent 4 quality control were within the target range, with no significant difference. This indicates that both purified Anti-IgG4-PE Antibody 1 and Anti-IgG4-PE Antibody 2 can be used as test antibodies. Unpurified antibodies contain impurities and may result in false positive results. Purified antibodies, on the other hand, have stronger binding to anti-PD-1 monoclonal antibodies and increase the effective antibody content. Therefore, the purified Anti-IgG4-PE Antibody can more accurately detect a patient's actual PD-1 expression.

[0142] Furthermore, the low-value quality control product and the high-value quality control product were tested 10 times each using antibody reagent No. ③ and antibody reagent No. ④, respectively, and the CV values ​​were calculated. The results showed that the CV values ​​of the low-value quality control product and the high-value quality control product tested by antibody reagent No. ④ were both smaller than those of antibody reagent No. ③, indicating that the detection precision and stability of antibody reagent No. ④ were higher.

[0143] Based on the above analysis, the purified Anti-IgG4-PE antibody 2 is preferred.

[0144] Example 6: Effect of Antioxidants on Detection

[0145] Antioxidants can protect the antigen molecules on the surface of T cells and the protein molecules in antibody reagents. Whether the antibody protein in the antibody reagent or the antigen protein in the test sample is oxidized and loses its activity, it will lead to inaccurate test results. Therefore, adding antioxidants can improve the stability of the protein and thus improve the accuracy of the test.

[0146] In this example, different antioxidant combinations are set, as shown in Table 12 below. The quality control products are tested according to the detection process described in Example 1. Each quality control product is tested three times, and the average value is taken. The detection results of the antibody reagent without adding PD-1-PE and Anti-IgG4-PE but adding Anti-IgG1-PE are used as isotype controls. The isotype control is used to eliminate background staining caused by nonspecific binding of the antibody to Fc receptors on the cell surface and electrostatic binding. The test results are shown in Table 13 below.

[0147] Table 12. Combinations of different antioxidants

[0148]

[0149] Table 13. Test results of different antioxidant combinations

[0150]

[0151] According to the data analysis in Table 13, the test quality control results of antioxidant combinations 1 to 3 and combinations 4 to 6 were compared. The test results of the two combinations of combinations 4 to 6 were significantly higher than those of combinations 1 to 3. Although the test results of combinations 1 to 6 were all outside the target value range, the test results of combinations 4 to 6 were closer to the target value range. Comparing the test quality control results of combinations 4 to 6 and combination 7, only the test results of the quality control of combination 7 were within the target value range, indicating that combination 7 can significantly improve the detection stability and thereby improve the binding ability of the antibody reagent to the target protein. The three antioxidants in combination 7 work together to protect the detected protein on the surface of T cells and the detection protein in the antibody reagent to improve the detection accuracy. Therefore, combination 7 was selected as the antioxidant.

[0152] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by the claims.

Claims

1. A kit for detecting PD-1 in tumor patients based on flow cytometry, characterized in that: It includes an antibody reagent and a diluent; the antibody reagent includes PD-1 monoclonal antibody and Anti-IgG4 monoclonal antibody; the diluent includes a blocking agent, and the blocking agent includes one or more of mouse IgG and fetal bovine serum; the diluent also includes zinc chloride, magnesium chloride, and sodium orthovanadate; the concentration of the zinc chloride is 0.1 mm / L, the concentration of the magnesium chloride is 0.5 mm / L, and the concentration of the sodium orthovanadate is 0.01 mm / L.

2. The kit according to claim 1, wherein The antibody reagents also include CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody and CD8 monoclonal antibody.

3. The kit according to claim 2, wherein The antibody reagent also includes fluorescent dyes PE-Cy7, FITC, APC, Percp-cy5.5, and PE. The CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, PD-1 monoclonal antibody, and Anti-IgG4 monoclonal antibody are coupled with PE-Cy7, FITC, APC, Percp-cy5.5, PE, and PE to form CD45-PE-Cy7, CD3-FITC, CD4-APC, CD8-Percp-cy5.5, PD-1-PE, and Anti-IgG4-PE, respectively.

4. The kit according to claim 3, wherein The ratio of CD45-PE-Cy7:CD3-FITC:CD4-APC:CD8-Percp-cy5.5:PD-1-PE:Anti-IgG4-PE:diluent in the antibody reagent is (3-12):(3-12):(3-12):(3-12):(1-6):(3-12):(30-68).

5. A method for detecting the PD-1 content in a blood sample, characterized in that: The method comprises the following steps: (1) Centrifuge and clean the sample; (2) Add antibody reagent containing PD-1-PE and Anti-IgG4-PE and mix with hemolysin, incubate, and centrifuge for washing; (3) Detection using flow cytometry.

Images