Method and kit for detecting proportion of T, B, NK and mononuclear cells of mouse

Abstract

The invention provides a method and a kit for detecting the proportion of T, B, NK and mononuclear cells of mice, and belongs to the technical field of biological detection. The method for detecting the proportion of T, B, NK and mononuclear cells of mice is constructed by using a fluorescent antibody combination with a specific variety and a specific proportion. Experiments prove that the detection method provided by the invention has excellent detection accuracy and relatively high intra-batch precision and inter-batch precision, can be used for evaluating the influence of drugs on a mouse immune system in a non-clinical research stage of new drugs, and provides important data for safety and effectiveness evaluation of the drugs.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method and a kit for detecting the ratio of mouse T, B, NK and monocytes. Background Art

[0002] Lymphocyte subset detection is of great significance in evaluating immune function, diagnosis and differential diagnosis, monitoring disease progression and treatment effects, formulating individualized treatment plans, and evaluating prognosis. In the medical field, lymphocyte subset detection can reflect the various components of the immune system, including T cells, B cells, natural killer cells (NK cells), etc. These cells play different roles in immune response and jointly maintain the body's immune balance. By analyzing the proportion and number of these cells, it is possible to evaluate whether the patient's immune function is normal. Lymphocyte subset detection can help doctors diagnose and differentially diagnose various immune-related diseases. For patients with confirmed immune-related diseases, regular lymphocyte subset detection helps monitor the progression of the disease. Based on the results of lymphocyte subset detection, doctors can formulate individualized immunomodulatory treatment plans for patients. The results of lymphocyte subset detection are closely related to the patient's prognosis and are of great significance for evaluating the patient's prognosis.

[0003] Mice are one of the most important scientific research experimental animals. They are often used as model animals for biological products in the non-clinical research stage of new drugs, which helps to clarify the pharmacological and toxicological characteristics. Therefore, it is important to monitor the proportion of T, B, NK, and monocytes in mice. Summary of the invention

[0004] The purpose of the present invention is to provide a method and a kit for detecting the ratio of mouse T, B, NK and monocytes, with high detection accuracy.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for detecting the ratio of T, B, NK and monocytes in mice, comprising the following steps:

[0007] Take a combination of antibodies labeled with different fluorescence and incubate them with the sample to be tested;

[0008] Process the samples and obtain the test data through flow cytometry;

[0009] According to the detection data, the proportion of mouse T, B, NK and monocytes is obtained;

[0010] The sample processing includes intracellular staining;

[0011] The antibody combination includes:

[0012] CD45 fluorescent antibody to label leukocytes;

[0013] CD3 fluorescent antibody to label total T lymphocytes;

[0014] CD4 fluorescent antibody to label helper T lymphocytes;

[0015] CD25 fluorescent antibody to label regulatory T cells;

[0016] CD8 fluorescent antibody to label cytotoxic T lymphocytes;

[0017] CD45R fluorescent antibody to label total B cells;

[0018] CD14 fluorescent antibody to label monocytes;

[0019] NK cells were labeled with CD49b fluorescent antibody.

[0020] Preferably, the fluorescent marker of the CD45 fluorescent antibody is PE-CY7;

[0021] The fluorescent label of the CD3 fluorescent antibody is FITC;

[0022] The fluorescent label of the CD4 fluorescent antibody is BUV395;

[0023] The fluorescent marker of the CD25 fluorescent antibody is BV786;

[0024] The fluorescent marker of the CD8 fluorescent antibody is PE-CY5;

[0025] The fluorescent label of the CD45R fluorescent antibody is BV711;

[0026] The fluorescent label of the CD14 fluorescent antibody is PE;

[0027] The fluorescent label of the CD49b fluorescent antibody is BV421.

[0028] Preferably, the intracellular staining step further comprises adding FoxP3 fluorescent antibody for marking regulatory T cells.

[0029] Preferably, the fluorescent label of the FoxP3 fluorescent antibody is Alexa Fluor 647.

[0030] Preferably, obtaining the detection data includes data acquisition and data analysis;

[0031] The data analysis includes selecting CD45 + CD3 + 、CD45 + CD3 + CD4 +、CD45 + CD3 + CD8 + 、CD45 + CD3 - CD45R + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + 、CD45 + CD14 + 、CD45 + CD49b + subgroup expression.

[0032] Preferably, the sample to be tested is from mouse peripheral blood.

[0033] The present invention also provides a kit for detecting the ratio of T, B, NK and monocytes in mice, wherein the kit comprises the following fluorescently labeled antibodies:

[0034] CD45 fluorescent antibody to label leukocytes;

[0035] CD3 fluorescent antibody to label total T lymphocytes;

[0036] CD4 fluorescent antibody to label helper T lymphocytes;

[0037] CD25 fluorescent antibody and FoxP3 fluorescent antibody for labeling regulatory T cells;

[0038] CD8 fluorescent antibody to label cytotoxic T lymphocytes;

[0039] CD45R fluorescent antibody to label total B cells;

[0040] CD14 fluorescent antibody to label monocytes;

[0041] CD49b fluorescent antibody to label NK cells;

[0042] Preferably, the fluorescent markers include PE-CY7, FITC, BUV395, BV786, Alexa Fluor647, PE-CY5, BV711, PE and BV421.

[0043] Preferably, the fluorescent marker of the CD45 fluorescent antibody is PE-CY7;

[0044] The fluorescent label of the CD3 fluorescent antibody is FITC;

[0045] The fluorescent label of the CD4 fluorescent antibody is BUV395;

[0046] The fluorescent marker of the CD25 fluorescent antibody is BV786;

[0047] The fluorescent label of the FoxP3 fluorescent antibody is AlexaFluor 647;

[0048] The fluorescent marker of the CD8 fluorescent antibody is PE-CY5;

[0049] The fluorescent label of the CD45R fluorescent antibody is BV711;

[0050] The fluorescent label of the CD14 fluorescent antibody is PE;

[0051] The fluorescent label of the CD49b fluorescent antibody is BV421.

[0052] Preferably, in the kit, the volume ratio of CD45 fluorescent antibody for labeling leukocytes: CD3 fluorescent antibody for labeling total T lymphocytes: CD4 fluorescent antibody for labeling helper T lymphocytes: CD25 fluorescent antibody for labeling regulatory T cells: FoxP3 fluorescent antibody for labeling regulatory T cells: CD8 fluorescent antibody for labeling cytotoxic T lymphocytes: CD45R fluorescent antibody for labeling total B cells: CD14 fluorescent antibody for labeling monocytes: CD49b fluorescent antibody for labeling NK cells is 0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:1.5-2.5:0.5-1.5.

[0053] Beneficial effects of the present invention:

[0054] The present invention constructs a method for detecting the proportion of mouse T, B, NK, and monocytes by using a combination of fluorescent antibodies of specific types and ratios. Experiments have shown that the detection method provided by the present invention has excellent detection accuracy, high intra-batch precision and high inter-batch precision, and can be used to evaluate the effect of drugs on the mouse immune system in the non-clinical research stage of new drugs, providing important data for the safety and effectiveness evaluation of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a diagram showing the situation of adjusting the mean fluorescence intensity in Example 1;

[0056] Figure 2 A diagram showing the target cell population circled in Example 1;

[0057] Figure 3 A diagram showing the situation of a single cell population circled in Example 1;

[0058] Figure 4This is a diagram of the positive cell population circled in Example 1;

[0059] Figure 5 This is the linear test result diagram of the detection scheme of Example 1;

[0060] Figure 6 This is the linear test result diagram of the detection scheme of Example 1;

[0061] Figure 7 This is the linear test result diagram of the detection scheme of Example 1;

[0062] Figure 8 This is the linear test result diagram of the detection scheme of Example 1 (4);

[0063] Fig. 9 This is the linear test result diagram of the detection scheme of Example 1 ⑤;

[0064] Fig.10 Figure ⑥ is the linear test result of the detection scheme of Example 1;

[0065] Fig.11 Figure ⑦ is the linear test result of the detection scheme in Example 1. DETAILED DESCRIPTION

[0066] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0067] The sources of the reagents used in the embodiments of the present invention are shown in Table 1 below:

[0068] Table 1 Example reagent information

[0069] Reagent name Manufacturer Part Number Dosage (μL / test) PE / Cy7anti-mouseCD45 Biolegend 103114 1.0 FITCanti-mouseCD3 Biolegend 100204 1.0 PE / Cy5anti-mouseCD8a Biolegend 100710 1.0 BUV395RatAnti-MouseCD4 BD Biosciences 565974 1.0 BV711RatAnti-MouseCD45R BD Biosciences 563892 1.0 BV421RatAnti-MouseCD49b BD Biosciences 563063 1.0 PERatAnti-MouseCD14 BD Biosciences 553740 2.0 BV786RatAnti-MouseCD25 BD Biosciences 564368 1.0 AlexaFluor647Ratanti-MouseFoxp3 BD Biosciences 560401 1.0 TFDiluentBuffer BD Pharmingen 51-9008101 1.0 TFFix / PermBuffer(4×) BD Pharmingen 51-9008100 / TFPerm / WashBuffer(5×) BD Pharmingen 51-9008102 /

[0070] Example 1

[0071] About 1.0 mL of whole blood was collected from 4 mice and anticoagulated with heparin sodium or EDTA-K2. The instrument voltage and threshold are shown in Table 2 below:

[0072] Table 2 Instrument voltage and threshold information

[0073] Channel Name Voltage Threshold FSC 357 10000.0 SSC 300 / PE-Cy7 509 / BV711 498 / FITC 495 / BUV395 517 / PE-Cy5 471 / BV786 603 / PE 368 / BV421 455 / APC 606 /

[0074] The instrument compensation is shown in Table 3 below.

[0075] Table 3 Instrument compensation information

[0076]

[0077]

[0078]

[0079] Group area settings

[0080] Select lymphocyte populations and select CD45 + CD3 + 、CD45 + CD3 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD3-CD45R + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + 、CD45 + CD14 + 、CD45 + CD49b + Results for positive areas.

[0081] Testing steps:

[0082] (1) Sample addition: Pipette 50 μL of cell suspension into each flow cytometry tube.

[0083] (2) Add antibodies: pipette 1.0 μL of PE-CY7 RatAnti-Mouse CD45, FITC RatAnti-MouseCD3, BUV395 RatAnti-Mouse CD4, PE-CY5 RatAnti-Mouse CD8, BV711 RatAnti-MouseCD45R, BV786 RatAnti-Mouse CD25, BV421RatAnti-Mouse CD49b, and 2.0 μL of PE RatAnti-Mouse CD14 fluorescent antibody into each flow cytometry tube and incubate at 15-25°C in the dark for 20 min.

[0084] (3) Lysis of red blood cells: Add 1000 μL of 1× NH4Cl red blood cell lysis buffer to the sample tube, mix well, and incubate at 15-25°C in the dark for about 15 minutes.

[0085] (4) Centrifugation: 15-25°C, 400×g, for 5 min.

[0086] (5) Washing: Discard the supernatant and add 1000 μL of 1× PBS buffer for washing.

[0087] (6) Centrifugation: 15-25°C, 400×g, for 5 min.

[0088] (7) Fixation and membrane permeabilization: Discard the supernatant, add 1000 μL Tf 1× Fix / Perm Buffer, and incubate at 2-8°C in the dark for 45 min.

[0089] (8) Washing and centrifugation: Add 1000 μL Tf 1× Perm / Wash Buffer, centrifuge at 400×g, 4°C for 5 min.

[0090] (9) Washing and centrifugation: Discard the supernatant, add 2000 μL Tf 1× Perm / Wash Buffer, and centrifuge at 400 × g, 4°C for 5 min.

[0091] (10) Intracellular staining: discard the supernatant, aspirate 1.0 μL of Alexa Fluor 647 Rat Anti-Mouse FoxP3 fluorescent antibody, and incubate at 2-8°C in the dark for 45 min.

[0092] (11) Washing and centrifugation: Add 2000 μL TF 1× Perm / Wash Buffer and centrifuge at 400 × g at 4°C for 5 min.

[0093] (12) Resuspension: Discard the supernatant, add 500 μL 1× PBS buffer, and test the sample on the instrument.

[0094] Test control tube

[0095] (1) Test the blank control tube: adjust the voltage of each channel and set the threshold of the FSC channel.

[0096] (2) Detection compensation control tube: adjust the compensation between the channels of PE-CY7, BV510, FITC, BUV395, PE-CY5, BV711, BV786, PE, BV421, and Alexa Fluor 647. The difference in the mean fluorescence intensity (Mean) value between the channels is less than 20. Taking the compensation adjustment between the PE-CY7 and BUV395 channels as an example, the difference between the mean values ​​of the Q3 quadrant and the Q4 quadrant of BUV395 is less than 20. Figure 1 shown.

[0097] (3) Detection of counterstained tubes: Circle the target cell population on the SSC-A / CD45-PE-CY7 dual parameter scatter plot, such as Figure 2 . Select a single cell population on the FSC-H / FSC-A dual parameter scatter plot, such as Figure 3 On CD3-FITC / CD45R-BV711, a single cell population was visualized and the positive cell population was circled, which was defined as CD45 + CD3 + 、CD45 +CD3 - CD45R + On the CD4-BUV395 / CD3-FITC graph, single cell populations are displayed, and CD45 + CD3 + CD4 + , on the CD8-PE-CY5 / CD3-FITC graph, display the single cell population and circle CD45 + CD3 + CD8 + In the FoxP3-APC / CD25-BV786 diagram, CD45 + CD3 + CD4 + , circle CD45 + CD3 + CD4 + CD25 + FoxP3 + On the SSA / CD49b-BV421 graph, single cell populations are displayed, and CD45 + CD49b + On the SSA / CD14-PE graph, single cell population 2 is displayed, and CD45 + CD14 + ,like Figure 4 .

[0098] Based on the results, the expression of total T lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, regulatory T cells, total B cells, monocytes, and NK cells in the peripheral blood of mice was obtained.

[0099] Experimental example

[0100] The detection method in Example 1 was verified to examine the detection precision and linearity of the analytical method.

[0101] Intra-batch precision:

[0102] Four samples were analyzed in the same analysis batch, and each sample was repeated three times. The CV of each measured value was calculated, and the CV range was 0.05-11.34%, which was less than 20% and met the acceptance criteria. The results are shown in Table 4.

[0103] Batch-to-batch precision:

[0104] Three samples were analyzed in two independent analysis batches on one day, with three replicates for each sample. The CV of the average of the measured values ​​of each sample in the two batches was calculated, and the CV range was 0.01-9.15%, which was less than 15%, meeting the acceptance criteria. The results are shown in Table 5.

[0105] Perform a linear test on the above scheme:

[0106] A high-concentration sample was stained with CD45 alone and completely stained, and then the CD45 alone and completely stained samples were serially diluted in a proportional gradient (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0), and each diluted sample was measured twice. The mean value of the high-value sample was used as the benchmark result, and the theoretical results of each series of diluted samples were calculated according to the dilution ratio relationship as the expected value. The average value of the test results was taken as the measured value. The horizontal axis X represents the expected value of each sample, and the vertical axis Y represents the measured value of each sample. Linear regression statistics were performed to obtain Y=bX+a, the slope was obtained, and the determination coefficient R was calculated. 2 The measured value was compared with the expected value to calculate the recovery rate. The slope, determination coefficient recovery rate (%) all met the standards. The results are shown in Table 6.

[0107] Table 4 Intra-batch precision verification results

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] Table 5 Results of inter-batch precision verification

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] Table 6 Linearity verification results

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] The results are summarized as follows:

[0131] The intra-assay CV ranged from 0.05 to 11.34%. The inter-assay CV ranged from 0.01 to 9.15%, and the linear slope ranged from 0.9579 to 1.0052. 2 The range of is 0.9755~0.9998, and the range of recovery is 83.74~115.20; the results show that this bioanalytical method can be used to detect the proportion of T, B, NK and monocytes in the peripheral blood of mice with high detection accuracy.

[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting the ratio of T, B, NK and monocytes in mice, characterized in that: The following steps are involved: Take a combination of antibodies labeled with different fluorescence and incubate them with the sample to be tested; Process the samples and obtain the test data through flow cytometry; According to the detection data, the proportion of mouse T, B, NK and monocytes is obtained; The sample processing includes intracellular staining; The antibody combination includes: CD45 fluorescent antibody to label leukocytes; CD3 fluorescent antibody to label total T lymphocytes; CD4 fluorescent antibody to label helper T lymphocytes; CD25 fluorescent antibody to label regulatory T cells; CD8 fluorescent antibody to label cytotoxic T lymphocytes; CD45R fluorescent antibody to label total B cells; CD14 fluorescent antibody to label monocytes; NK cells were labeled with CD49b fluorescent antibody.

2. The detection method according to claim 1, characterized in that: The fluorescent label of the CD45 fluorescent antibody is PE-CY7; The fluorescent label of the CD3 fluorescent antibody is FITC; The fluorescent marker of the CD4 fluorescent antibody is BUV395; The fluorescent marker of the CD25 fluorescent antibody is BV786; The fluorescent marker of the CD8 fluorescent antibody is PE-CY5; The fluorescent label of the CD45R fluorescent antibody is BV711; The fluorescent label of the CD14 fluorescent antibody is PE; The fluorescent label of the CD49b fluorescent antibody is BV421.

3. The detection method according to claim 1, characterized in that: The intracellular staining step also includes adding FoxP3 fluorescent antibody to mark regulatory T cells.

4. The detection method according to claim 3, characterized in that: The fluorescent label of the FoxP3 fluorescent antibody is AlexaFluor 647.

5. The detection method according to claim 3, characterized in that: The obtaining of detection data includes data acquisition and data analysis; The data analysis includes selecting CD45 + CD3 + 、CD45 + CD3 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD3 - CD45R + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + 、CD45 + CD14 + 、CD45 + CD49b + subgroup expression.

6. The detection method according to any one of claims 1 to 5, characterized in that: The sample to be tested comes from mouse peripheral blood.

7. A kit for detecting the ratio of T, B, NK and monocytes in mice, characterized in that: The kit includes the following fluorescently labeled antibodies: CD45 fluorescent antibody to label leukocytes; CD3 fluorescent antibody to label total T lymphocytes; CD4 fluorescent antibody to label helper T lymphocytes; CD25 fluorescent antibody and FoxP3 fluorescent antibody for labeling regulatory T cells; CD8 fluorescent antibody to label cytotoxic T lymphocytes; CD45R fluorescent antibody to label total B cells; CD14 fluorescent antibody to label monocytes; NK cells were labeled with CD49b fluorescent antibody.

8. The kit according to claim 7, characterized in that The fluorescent labels include PE-CY7, FITC, BUV395, BV786, Alexa Fluor 647, PE-CY5, BV711, PE and BV421.

9. The kit according to claim 7, characterized in that The fluorescent label of the CD45 fluorescent antibody is PE-CY7; The fluorescent label of the CD3 fluorescent antibody is FITC; The fluorescent label of the CD4 fluorescent antibody is BUV395; The fluorescent marker of the CD25 fluorescent antibody is BV786; The fluorescent label of the FoxP3 fluorescent antibody is AlexaFluor 647; The fluorescent marker of the CD8 fluorescent antibody is PE-CY5; The fluorescent label of the CD45R fluorescent antibody is BV711; The fluorescent label of the CD14 fluorescent antibody is PE; The fluorescent label of the CD49b fluorescent antibody is BV421.

10. The kit according to claim 7, characterized in that In the kit, the volume ratio of CD45 fluorescent antibody for labeling leukocytes: CD3 fluorescent antibody for labeling total T lymphocytes: CD4 fluorescent antibody for labeling helper T lymphocytes: CD25 fluorescent antibody for labeling regulatory T cells: FoxP3 fluorescent antibody for labeling regulatory T cells: CD8 fluorescent antibody for labeling cytotoxic T lymphocytes: CD45R fluorescent antibody for labeling total B cells: CD14 fluorescent antibody for labeling monocytes: CD49b fluorescent antibody for labeling NK cells is 0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:1.5-2.5:0.5-1.5.

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