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

Abstract

The invention provides a method and a kit for detecting the proportion of T, B and NK cells of cynomolgus monkeys, and belongs to the technical field of biological detection. The method for detecting the proportion of T, B and NK cells of cynomolgus monkeys is constructed by using fluorescent antibody combinations of specific types and proportions. 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 the medicine on the cynomolgus monkey immune system in a non-clinical research stage of a new medicine, is beneficial to deeply understanding the influence of the medicine on the cynomolgus monkey immune response, and has a wide application prospect. Important data is provided for safety and effectiveness evaluation of the medicine.

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 T, B and NK cells in cynomolgus monkeys. 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] Crab-eating macaques are one of the most important scientific research experimental animals. They have a high similarity with humans in their physiological and metabolic mechanisms. 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 of great significance to monitor the ratio of T, B, and NK cells in crab-eating macaques. Summary of the invention

[0004] The object of the present invention is to provide a method and a kit for detecting the ratio of T, B and NK cells in cynomolgus monkeys, 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 and NK cells in cynomolgus monkeys, comprising the following steps:

[0007] Take a combination of antibodies labeled with different fluorescence, mix them with the sample to be tested, incubate them and detect them by flow cytometry to obtain the test data;

[0008] Obtaining the ratio of T, B, and NK cells in cynomolgus monkeys according to the detection data;

[0009] The antibody combination includes:

[0010] CD45 fluorescent antibody to label leukocytes;

[0011] CD3 fluorescent antibody to label total T cells;

[0012] CD4 fluorescent antibody to label helper T cells;

[0013] CD8 fluorescent antibody to label cytotoxic T cells;

[0014] CD19 fluorescent antibody to label B cells;

[0015] CD16 fluorescent antibody to label NK cells;

[0016] NK cells were labeled with CD56 fluorescent antibody.

[0017] Preferably, the fluorescent label of the CD45 fluorescent antibody is FITC;

[0018] The fluorescent label of the CD3 fluorescent antibody is Percp;

[0019] The fluorescent label of the CD4 fluorescent antibody is BUV395;

[0020] The fluorescent marker of the CD8 fluorescent antibody is BV605;

[0021] The fluorescent label of the CD19 fluorescent antibody is PE;

[0022] The fluorescent label of the CD16 fluorescent antibody is APC-Cy7;

[0023] The fluorescent label of the CD56 fluorescent antibody is BV421.

[0024] Preferably, obtaining the detection data includes data acquisition and data analysis;

[0025] The data analysis includes selecting CD45 + CD3 + 、CD45 + CD3 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD19 + 、CD45 + CD3 - CD(16 + 56) + subgroup expression.

[0026] Preferably, the sample to be tested is from the peripheral blood of cynomolgus monkeys.

[0027] Preferably, in the antibody combination, the volume ratio of CD45 fluorescent antibody for labeling leukocytes: CD3 fluorescent antibody for labeling total T cells: CD4 fluorescent antibody for labeling helper T cells: CD8 fluorescent antibody for labeling cytotoxic T cells: CD19 fluorescent antibody for labeling B cells: CD16 fluorescent antibody for labeling NK cells: CD56 fluorescent antibody for labeling NK cells is 4-6:4-6:0.5-1.5:2-3:2-3:2-3:2-3:2-3.

[0028] Preferably, in the antibody combination, the volume ratio of CD45 fluorescent antibody for labeling leukocytes: CD3 fluorescent antibody for labeling total T cells: CD4 fluorescent antibody for labeling helper T cells: CD8 fluorescent antibody for labeling cytotoxic T cells: CD19 fluorescent antibody for labeling B cells: CD16 fluorescent antibody for labeling NK cells: CD56 fluorescent antibody for labeling NK cells is 5:5:1:2.5:2.5:2.5:2.5.

[0029] The present invention also provides a kit for detecting the ratio of T, B and NK cells in cynomolgus monkeys, wherein the kit comprises the following fluorescently labeled antibodies:

[0030] CD45 fluorescent antibody to label leukocytes;

[0031] CD3 fluorescent antibody to label total T cells;

[0032] CD4 fluorescent antibody to label helper T cells;

[0033] CD8 fluorescent antibody to label cytotoxic T cells;

[0034] CD19 fluorescent antibody to label B cells;

[0035] CD16 fluorescent antibody to label NK cells;

[0036] NK cells were labeled with CD56 fluorescent antibody.

[0037] Preferably, the fluorescent markers include FITC, Percp, BUV395, BV605, PE, APC-Cy7 and BV421.

[0038] Preferably, the fluorescent label of the CD45 fluorescent antibody is FITC;

[0039] The fluorescent label of the CD3 fluorescent antibody is Percp;

[0040] The fluorescent label of the CD4 fluorescent antibody is BUV395;

[0041] The fluorescent marker of the CD8 fluorescent antibody is BV605;

[0042] The fluorescent label of the CD19 fluorescent antibody is PE;

[0043] The fluorescent label of the CD16 fluorescent antibody is APC-Cy7;

[0044] The fluorescent label of the CD56 fluorescent antibody is BV421.

[0045] Preferably, in the kit, the volume ratio of CD45 fluorescent antibody for labeling leukocytes: CD3 fluorescent antibody for labeling total T cells: CD4 fluorescent antibody for labeling helper T cells: CD8 fluorescent antibody for labeling cytotoxic T cells: CD19 fluorescent antibody for labeling B cells: CD16 fluorescent antibody for labeling NK cells: CD56 fluorescent antibody for labeling NK cells is 4-6:4-6:0.5-1.5:2-3:2-3:2-3:2-3:2-3.

[0046] Beneficial effects of the present invention:

[0047] The present invention constructs a method for detecting the ratio of T, B, and NK cells in cynomolgus monkeys 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 inter-batch precision, and can be used to evaluate the effect of drugs on the immune system of cynomolgus monkeys in the non-clinical research stage of new drugs, and helps to deeply understand the effect of drugs on the immune response of cynomolgus monkeys, providing important data for the safety and effectiveness evaluation of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a diagram showing the situation of adjusting the mean fluorescence intensity in Example 1;

[0049] Figure 2 A diagram showing the situation of a single cell population circled in Example 1;

[0050] Figure 3 A diagram of the situation in which the LYMPH group is circled for Example 1;

[0051] Figure 4 This is a diagram of the positive cell population circled in Example 1;

[0052] Figure 5 This is the linear result diagram of Example 6 ①;

[0053] Figure 6 This is the linear result diagram of Example 6 ②

[0054] Figure 7 The linear result diagram of Example 6 is shown in FIG.

[0055] Figure 8 The linear result diagram of Example 6 is shown in FIG.

[0056] Fig. 9 This is the linear result diagram ⑤ of Example 6. DETAILED DESCRIPTION

[0057] 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.

[0058] The sources of the reagents used in the embodiments of the present invention are shown in Table 1 below:

[0059] Table 1 Example reagent information

[0060] Reagent name Manufacturer Part Number FITCMouseAnti-NHPCD45 BD Biosciences 557803 PercpMouseAnti-HumanCD3 BD Biosciences 552851 BUV395MouseAnti-HumanCD4 BD Biosciences 564107 BV605MouseAnti-HumanCD8 BD Biosciences 564116 PEAnti-CD19antibody abcam ab1168 APC-Cy7MouseAnti-HumanCD16 Biolegend 302018 BV421MouseAnti-HumanCD56 Biolegend 318328

[0061] Example 1

[0062] About 2.0 mL of blood was collected from the lower limb vein of the cynomolgus monkey, anticoagulated with sodium heparin, and used as a sample for later use.

[0063] The instrument voltage and threshold are shown in Table 2 below:

[0064] Table 2 Instrument voltage and threshold information

[0065]

[0066]

[0067] The instrument compensation is shown in Table 3 below.

[0068] Table 3 Instrument compensation information

[0069]

[0070]

[0071]

[0072] Sample processing steps

[0073] Antibody preparation: prepare mixed antibodies according to the ratio of 5.0 μL of FITC Mouse Anti-NHP CD45, Percp MouseAnti-Human CD3, 1.0 μL of BUV395 MouseAnti-Human CD4, 2.5 μL of BV605 MouseAnti-Human CD8, PEAnti-CD19 antibody, APC-Cy7 Mouse Anti-Human CD16, BV421 MouseAnti-Human CD56 fluorescent antibodies per tube;

[0074] Sample preparation: 50 μL of whole blood sample was respectively drawn into the flow tube;

[0075] Add antibodies: add 21.0 μL of mixed antibodies to each counterstaining tube, mix well, and incubate at 15-25°C in the dark for 20 min;

[0076] Add lysis buffer: Add 1000 μL of 1×NH4Cl red blood cell lysis buffer, mix well and lyse for 15 min at 15-25°C in the dark;

[0077] Centrifugation after lysis: 15-25°C, 400g, centrifugation for 5 minutes;

[0078] After washing, centrifuge: discard the supernatant, add 1000 μL of 1× PBS buffer, mix well, and centrifuge at 15-20°C, 400 g for 5 min;

[0079] Sample detection: discard the supernatant, add 500μL 1×PBS buffer, and test the sample on the machine.

[0080] Test control tube

[0081] (1) Test the blank control tube: adjust the voltage of each channel and set the threshold of the FSC channel.

[0082] (2) Detect the compensation control tubes and adjust the compensation between the FITC-CD45, Percp-CD3, BUV395-CD4, BV605-CD8, PE-CD19, APC-Cy7-CD16, and BV421-CD56 channels in turn. The difference in the mean fluorescence intensity (Mean) value between the channels is less than 20. Taking the compensation adjustment between the BUV395 and FITC 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.

[0083] (3) Circle each positive cell population in the counterstained tube: Select a single cell population on the FSC-H / FSC-A dual parameter scatter plot, such as Figure 2 . Circle the leukocyte and lymphocyte populations on the SSC-A / FITC-A dual parameter scatter plot, such as Figure 3 On the SSC-A / Percp-A graph, lymphocytes are shown and the positive cell population is circled, defined as CD45 + CD3 + 、CD45 + CD3 - ; On BV605-A / Percp-A, lymphocytes are shown, and the positive cell population is circled, defined as CD45 + CD3 + CD8 + ;

[0084] In the BUV395-A / Percp-A graph, lymphocytes are shown. The positive cell population is circled and defined as CD45 + CD3 + CD4 + ; On SSC-A / PE-A, lymphocytes are shown, and the positive cell population is circled, defined as CD45 + CD19 + ; In APC-Cy7-CD16 / BV421-CD56, lymphocyte population is displayed, and the positive cell population is circled, which is defined as CD45 + CD3 - CD(16+56) + ,like Figure 4 shown.

[0085] Data analysis and results

[0086] The detection data were analyzed using BD FACSDiva Software v8.0.3. + CD3 + 、CD45 + CD3 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD19 + The indicator uses % Parent value as the sample result, CD45 + CD3 - CD(16+56) + The indicator %Grand Parent value is given as a sample result.

[0087] In this example, the experimental animal number is 1F001, and the test results show that the relative number of total T cells in this sample (as a percentage of the total number of lymphocytes) is 54.84%;

[0088] The relative number of helper T cells (percentage of total lymphocytes) was 27.74%;

[0089] The relative number of cytotoxic T cells (percentage of total lymphocytes) was 24.98%;

[0090] The relative number of B cells (percentage of total lymphocytes) was 23.48%;

[0091] The relative number of NK cells (percentage of total lymphocytes) was 17.32%.

[0092] Example 2

[0093] The same detection method as in Example 1 was used, except that the experimental animal was replaced by 1M001.

[0094] The test results showed that the relative number of total T cells in this sample (as a percentage of the total number of lymphocytes) was 57.85%;

[0095] The relative number of helper T cells (percentage of total lymphocytes) was 36.95%;

[0096] The relative number of cytotoxic T cells (percentage of total lymphocytes) was 18.82%;

[0097] The relative number of B cells (percentage of total lymphocytes) is 22.75%;

[0098] The relative number of NK cells (percentage of total lymphocytes) was 14.53%.

[0099] Example 3

[0100] The same detection method as in Example 1 was used, except that the experimental animal was replaced by 2F001.

[0101] The test results showed that the relative number of total T cells in this sample (as a percentage of the total number of lymphocytes) was 55.82%;

[0102] The relative number of helper T cells (percentage of total lymphocytes) was 30.79%;

[0103] The relative number of cytotoxic T cells (percentage of total lymphocytes) was 22.13%;

[0104] The relative number of B cells (percentage of total lymphocytes) was 25.79%;

[0105] The relative number of NK cells (percentage of total lymphocytes) was 14.92%.

[0106] Example 4

[0107] The same detection method as in Example 1 was used, except that the experimental animal was replaced by 2M001.

[0108] The test results showed that the relative number of total T cells in this sample (as a percentage of the total number of lymphocytes) was 56.82%;

[0109] The relative number of helper T cells (percentage of total lymphocytes) was 30.91%;

[0110] The relative number of cytotoxic T cells (percentage of total lymphocytes) was 25.84%;

[0111] The relative number of B cells (percentage of total lymphocytes) was 24.06%;

[0112] The relative number of NK cells (percentage of total lymphocytes) was 17.07%.

[0113] Experimental example

[0114] The detection method in Example 1 was verified, and the precision of the analytical method was investigated to evaluate the repeatability of the method;

[0115] The linearity of the analytical method was investigated to evaluate the relative accuracy of the method.

[0116] Intra-batch precision:

[0117] 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 0.23-3.83%, which was less than 25% and met the acceptance criteria. The results are shown in Table 4.

[0118] Batch-to-batch precision:

[0119] 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 0.03-2.44%, which was less than 25%, meeting the acceptance criteria. The results are shown in Table 5.

[0120] Linear evaluation method:

[0121] 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.

[0122] Table 4 Intra-batch precision of flow cytometry for detecting the proportion of T, B, and NK cells in the peripheral blood of cynomolgus monkeys

[0123]

[0124]

[0125]

[0126] Table 5 Inter-batch precision of flow cytometry for detecting the proportion of T, B and NK cells in peripheral blood of cynomolgus monkeys

[0127]

[0128]

[0129]

[0130]

[0131] Table 6 Linear results of flow cytometry detection of the proportion of T, B, and NK cells in the peripheral blood of cynomolgus monkeys

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] The results are summarized as follows:

[0139] The intra-batch precision CV ranged from 0.23 to 3.83%. The inter-batch precision CV ranged from 0.03 to 2.44%. The linear slope ranged from 1.0022 to 1.0467, R 2 The range was 0.9887-0.9978, and the recovery range was 80.48-115.52, both of which met the acceptance criteria, indicating that it had good linearity. The results showed that this bioanalytical method can be used to detect the proportion of T, B, and NK cells in the peripheral blood of cynomolgus monkeys with good repeatability and high detection accuracy.

[0140] 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 and NK cells in cynomolgus monkeys, characterized in that: The following steps are involved: Take a combination of antibodies labeled with different fluorescence, mix them with the sample to be tested, incubate them and detect them by flow cytometry to obtain the test data; Obtaining the ratio of T, B, and NK cells in cynomolgus monkeys according to the detection data; The antibody combination includes: CD45 fluorescent antibody to label leukocytes; CD3 fluorescent antibody to label total T cells; CD4 fluorescent antibody to label helper T cells; CD8 fluorescent antibody to label cytotoxic T cells; CD19 fluorescent antibody to label B cells; CD16 fluorescent antibody to label NK cells; NK cells were labeled with CD56 fluorescent antibody.

2. The detection method according to claim 1, characterized in that: The fluorescent label of the CD45 fluorescent antibody is FITC; The fluorescent label of the CD3 fluorescent antibody is Percp; The fluorescent label of the CD4 fluorescent antibody is BUV395; The fluorescent marker of the CD8 fluorescent antibody is BV605; The fluorescent label of the CD19 fluorescent antibody is PE; The fluorescent label of the CD16 fluorescent antibody is APC-Cy7; The fluorescent label of the CD56 fluorescent antibody is BV421.

3. The detection method according to claim 1, 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 + CD19 + 、CD45 + CD3 - CD(16 + 56) + subgroup expression.

4. The detection method according to any one of claims 1 to 3, characterized in that: The sample to be tested is from the peripheral blood of cynomolgus monkeys.

5. The detection method according to claim 1, characterized in that: In the antibody combination, the volume ratio of CD45 fluorescent antibody for marking leukocytes: CD3 fluorescent antibody for marking total T cells: CD4 fluorescent antibody for marking helper T cells: CD8 fluorescent antibody for marking cytotoxic T cells: CD19 fluorescent antibody for marking B cells: CD16 fluorescent antibody for marking NK cells: CD56 fluorescent antibody for marking NK cells is 4-6:4-6:0.5-1.5:2-3:2-3:2-3:2-3:2-3.

6. The detection method according to claim 5, characterized in that: In the antibody combination, the volume ratio of CD45 fluorescent antibody for labeling leukocytes: CD3 fluorescent antibody for labeling total T cells: CD4 fluorescent antibody for labeling helper T cells: CD8 fluorescent antibody for labeling cytotoxic T cells: CD19 fluorescent antibody for labeling B cells: CD16 fluorescent antibody for labeling NK cells: CD56 fluorescent antibody for labeling NK cells is 5:5:1:2.5:2.5:2.5:2.

5.

7. A kit for detecting the ratio of T, B and NK cells in cynomolgus monkeys, characterized in that: The kit includes the following fluorescently labeled antibodies: CD45 fluorescent antibody to label leukocytes; CD3 fluorescent antibody to label total T cells; CD4 fluorescent antibody to label helper T cells; CD8 fluorescent antibody to label cytotoxic T cells; CD19 fluorescent antibody to label B cells; CD16 fluorescent antibody to label NK cells; NK cells were labeled with CD56 fluorescent antibody.

8. The kit according to claim 4, characterized in that The fluorescent labels include FITC, Percp, BUV395, BV605, PE, APC-Cy7 and BV421.

9. The kit according to claim 4, characterized in that The fluorescent label of the CD45 fluorescent antibody is FITC; The fluorescent label of the CD3 fluorescent antibody is Percp; The fluorescent label of the CD4 fluorescent antibody is BUV395; The fluorescent marker of the CD8 fluorescent antibody is BV605; The fluorescent label of the CD19 fluorescent antibody is PE; The fluorescent label of the CD16 fluorescent antibody is APC-Cy7; The fluorescent label of the CD56 fluorescent antibody is BV421.

10. The kit according to claim 4, characterized in that In the kit, the volume ratio of CD45 fluorescent antibody for labeling leukocytes: CD3 fluorescent antibody for labeling total T cells: CD4 fluorescent antibody for labeling helper T cells: CD8 fluorescent antibody for labeling cytotoxic T cells: CD19 fluorescent antibody for labeling B cells: CD16 fluorescent antibody for labeling NK cells: CD56 fluorescent antibody for labeling NK cells is 4-6:4-6:0.5-1.5:2-3:2-3:2-3:2-3:2-3.

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