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

Abstract

The invention provides a method and a kit for detecting the proportion of T, B and NK cells of a dog, and belongs to the technical field of biological detection. The method for detecting the proportion of the T, B and NK cells of the dog 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 the canine 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 canine T, B and NK cells. 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] Dogs 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 of great significance to monitor the ratio of T, B, and NK cells in dogs. Summary of the invention

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

[0007] Take a combination of antibodies 1 labeled with different fluorescence and mix them with the sample to be tested for incubation;

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

[0009] Obtaining the ratio of canine T, B, and NK cells according to the detection data;

[0010] The sample processing includes taking a combination of antibodies 2 with different fluorescent labels and mixing and incubating them with the sample to be detected;

[0011] The antibody combination 1 comprises:

[0012] CD3 fluorescent antibody to label total T lymphocytes;

[0013] CD4 fluorescent antibody to label helper T lymphocytes;

[0014] CD8 fluorescent antibody to label cytotoxic T lymphocytes;

[0015] NK cells were labeled with CD5 fluorescent antibody.

[0016] Preferably, the fluorescent label of the CD3 fluorescent antibody is FITC;

[0017] The fluorescent label of the CD4 fluorescent antibody is PE-CY7;

[0018] The fluorescent marker of the CD8 fluorescent antibody is EF450;

[0019] The fluorescent marker of the CD5 fluorescent antibody is APC.

[0020] Preferably, the antibody combination 2 includes a CD45 fluorescent antibody for marking leukocytes and a CD21 fluorescent antibody for marking B lymphocytes.

[0021] Preferably, the fluorescent label of the CD45 fluorescent antibody is Alexa Fluor 700;

[0022] The fluorescent label of the CD21 fluorescent antibody is PE.

[0023] Preferably, obtaining the detection data includes data acquisition and data analysis;

[0024] The data analysis includes selecting CD45 + CD3 + 、CD45 + CD21 + 、CD45 + CD3 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD3 - CD5 + subgroup expression.

[0025] Preferably, the sample to be tested is from canine peripheral blood.

[0026] The present invention also provides a kit for detecting the ratio of canine T, B, and NK cells, wherein the kit comprises the following fluorescently labeled antibodies:

[0027] CD3 fluorescent antibody to label total T lymphocytes;

[0028] CD4 fluorescent antibody to label helper T lymphocytes;

[0029] CD8 fluorescent antibody to label cytotoxic T lymphocytes;

[0030] CD5 fluorescent antibody to label NK cells;

[0031] CD45 fluorescent antibody to label leukocytes;

[0032] CD21 fluorescent antibody is used to label B lymphocytes.

[0033] Preferably, the fluorescent markers include Alexa Fluor 700, FITC, PE-CY7, EF450, APC and PE.

[0034] Preferably, the fluorescent label of the CD3 fluorescent antibody is FITC;

[0035] The fluorescent label of the CD4 fluorescent antibody is PE-CY7;

[0036] The fluorescent marker of the CD8 fluorescent antibody is EF450;

[0037] The fluorescent marker of the CD5 fluorescent antibody is APC;

[0038] The fluorescent label of the CD45 fluorescent antibody is Alexa Fluor 700;

[0039] The fluorescent label of the CD21 fluorescent antibody is PE.

[0040] Preferably, in the kit, the volume ratio of the CD3 fluorescent antibody for labeling total T lymphocytes: the CD4 fluorescent antibody for labeling helper T lymphocytes: the CD8 fluorescent antibody for labeling cytotoxic T lymphocytes: the CD5 fluorescent antibody for labeling NK cells: the CD45 fluorescent antibody for labeling leukocytes: and the CD21 fluorescent antibody for labeling B lymphocytes is 2.4-2.6:2.4-2.6:2.4-2.6:4-6:1.8-2.2:2.4-2.6.

[0041] Beneficial effects of the present invention:

[0042] The present invention constructs a method for detecting the proportion of canine T, B, and NK cells 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 effects of drugs on the canine 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

[0043] Figure 1 This is a diagram showing the situation of adjusting the mean fluorescence intensity in Example 1;

[0044] Figure 2 A diagram of the situation in which the lymphocyte population is circled for Example 1;

[0045] Figure 3 A diagram showing the situation of a single cell population circled in Example 1;

[0046] Figure 4 This is a diagram of the positive cell population circled in Example 1.

[0047] Figure 5 This is the linear result diagram of Example 1①;

[0048] Figure 6 This is the linear result diagram of Example 1②;

[0049] Figure 7 It is the linear result diagram of Example 1 ③;

[0050] Figure 8 This is the linear result diagram of Example 1④;

[0051] Fig. 9 This is the linear result diagram ⑤ of Example 1. DETAILED DESCRIPTION

[0052] 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.

[0053] The sources of the reagents used in the embodiments of the present invention are shown in Table 1 below:

[0054] Table 1 Example reagent information

[0055] Reagent name Manufacturer Part Number Dosage (μL / Test) Ratanti-CD45(YKIX716.13)[AlexaFluor700] Novus NB100-65906AF700 2.0 MOUSEANTOGCD3:FITC BIO-RAD MCA1774F 2.5 ANTI-DOGCD4YKIX302.9PE-CY7 Invitrogen 25-5040-42 2.5 ANTI-DOGCD8AYCATE55.9EF450 Invitrogen 48-5080-42 2.5 RATAntiDOGCD5:APC BIO-RAD MCA1037APC 5.0 <![CDATA[MOUSEANTICANINECD21:RPE * ]]> BIO-RAD MCA1781PE 2.5

[0056] Example 1

[0057] About 3.0 mL of blood was collected from the dog's forelimb vein and anticoagulated with heparin sodium. The instrument voltage and threshold are shown in Table 2 below:

[0058] Table 2 Instrument voltage and threshold information

[0059] Channel Name Voltage Threshold FSC 255 20000 SSC 282 / FITC 530 / PE-Cy7 600 / BV421 450 / APC 520 / AlexaFlour700 602 / PE 490 /

[0060] The instrument compensation is shown in Table 3 below.

[0061] Table 3 Instrument compensation information

[0062]

[0063]

[0064] Group area settings

[0065] Select lymphocyte populations and select CD45 + CD3 + 、CD45 + CD21 + 、CD45 + CD3 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD3 - CD5 + Results for positive areas.

[0066] Testing steps:

[0067] (1) Antibody preparation: Mixed antibody 1 was prepared according to the ratio of 2.0 μL of Rat anti-CD45 (YKIX716.13) [Alexa Fluor 700], 2.5 μL of MOUSE ANTIDOG CD3: FITC, ANTI-DOG CD4 YKIX302.9PE-CY7, ANTI-DOG CD8A YCATE55.9EF450, and 5.0 μL of RAT Anti DOG CD5: APC fluorescent antibody; mixed antibody 2 was prepared according to the ratio of 2.0 μL of Rat anti-CD45 (YKIX716.13) [Alexa Fluor 700], 2.5 μL of MOUSE ANTI CANINE CD21: RPE fluorescent antibody.

[0068] (2) Adding antibodies: Add 14.5 μL of mixed antibody 1 or 4.5 μL of mixed antibody 2 to each flow tube, and discard the remaining antibodies.

[0069] (3) Sample preparation: 50 μL of whole blood sample was added to the flow cytometry tube, mixed, and incubated at 15-25°C in the dark for 20 min.

[0070] (4) Lysis: Add 1000 μL of 1× NH4CL red blood cell lysis buffer to the flow tube, mix well, and incubate at 15-25°C in the dark for 15 min.

[0071] (5) Centrifugation: 15-25°C, 400 g, centrifuge for 5 min, and discard the supernatant.

[0072] (6) Washing: Add 1000 μL of 1× PBS buffer, mix well, centrifuge at 15-25°C, 400 g for 5 min, and discard the supernatant.

[0073] (7) Resuspend: Add 500 μL 1× PBS buffer and test the sample on the instrument.

[0074] Test control tube

[0075] (1) Test the blank control tube: adjust the voltage of each channel.

[0076] (2) Test compensation control tube: adjust the compensation between Alexa Fluor 700-CD45, FITC-CD3, PE-CY7-CD4, BV421-CD8, APC-CD5 channels, and the compensation between Alexa Fluor700-CD45 and PE-CD21 channels, and set the threshold of the FSC channel. The difference between the mean fluorescence intensity (Mean) values ​​of each channel is less than 20. Taking the compensation adjustment between BUV395 and BV4605 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.

[0077] (3) Detection of counterstained tube 1: Select a single cell population on the FSC-H / FSC-A dual parameter scatter plot, such as Figure 2 . Circle the lymphocyte population on the SSC-A / Alexa Fluor 700-A dual parameter scatter plot, such as Figure 3 Circle the target cell population: On the FITC-A / SSC-A graph, a single cell population is displayed, circle the positive cell population, defined as CD45 + CD3 + On FITC-A / PE-CY7-A, single cell populations were visualized and double positive cell populations were circled, defined as CD45 + CD3 + CD4 + On FITC-A / BV421-A, a single cell population is visualized and the double positive cell population is circled, defined as CD45 + CD3 + CD8 + On FITC-A / APC-A, a single cell population was visualized and the single positive cell population was circled, defined as CD45 + CD3 - CD5 + .like Figure 4 .

[0078] Detect counterstained tube 2: Select a single cell population on the FSC-H / FSC-A dual parameter scatter plot, such as Figure 2 . Circle the lymphocyte population on the SSC-A / Alexa Fluor 700-A dual parameter scatter plot, such as Figure 3Circle the target cell population: On the FITC-A / SSC-A graph, a single cell population is displayed, circle the positive cell population, defined as CD45 + CD21 + .like Figure 4 .

[0079] Based on the results, the expression of total T lymphocytes, helper T lymphocytes, cytotoxic T cells, total B cells and NK cells in the dog's peripheral blood was obtained.

[0080] The test results showed that the relative number of total T lymphocytes in this sample (as a percentage of the total number of lymphocytes) was 58.41%;

[0081] The relative number of helper T lymphocytes (percentage of total lymphocytes) was 29.84%;

[0082] The relative number of cytotoxic T lymphocytes (percentage of total lymphocytes) was 20.44%;

[0083] The relative number of total B cells (percentage of total lymphocytes) was 26.21%;

[0084] The relative number of NK cells (percentage of total lymphocytes) was 20.85%.

[0085] Experimental example

[0086] The detection method in Example 1 was verified to examine the detection accuracy of the analytical method.

[0087] Intra-batch precision:

[0088] 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 0.14-6.80%, which was less than 25% and met the acceptance criteria. The results are shown in Table 4.

[0089] Batch-to-batch precision:

[0090] 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 0-4.37%, which was less than 25% and met the acceptance criteria. The results are shown in Table 5.

[0091] Linear:

[0092] 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.

[0093] Table 4 Intra-batch precision verification results

[0094]

[0095]

[0096]

[0097]

[0098] Table 5 Results of inter-batch precision verification

[0099]

[0100]

[0101]

[0102]

[0103] Table 6 Linearity of the ratio of T and NK cells in dog peripheral blood detected by flow cytometry

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] The results are summarized as follows:

[0111] The intra-batch precision CV ranged from 0.14 to 6.80%. The inter-batch precision CV ranged from 0 to 4.37%, and the linear slope ranged from 0.9793 to 1.0192. 2 The range of is 0.9968~0.9993, and the recovery range is 82.57~109.86; the results show that this bioanalytical method can be used to detect the proportion of T, B, and NK cells in canine peripheral blood with high detection accuracy.

[0112] 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 canine T, B and NK cells, characterized in that: The following steps are involved: Take a combination of antibodies 1 labeled with different fluorescence and mix them with the sample to be tested for incubation; Process the samples and obtain the test data through flow cytometry; Obtaining the ratio of canine T, B, and NK cells according to the detection data; The sample processing includes taking a combination of antibodies 2 with different fluorescent labels and mixing and incubating them with the sample to be detected; The antibody combination 1 comprises: CD3 fluorescent antibody to label total T lymphocytes; CD4 fluorescent antibody to label helper T lymphocytes; CD8 fluorescent antibody to label cytotoxic T lymphocytes; NK cells were labeled with CD5 fluorescent antibody.

2. The detection method according to claim 1, characterized in that: The fluorescent label of the CD3 fluorescent antibody is FITC; The fluorescent label of the CD4 fluorescent antibody is PE-CY7; The fluorescent marker of the CD8 fluorescent antibody is EF450; The fluorescent marker of the CD5 fluorescent antibody is APC.

3. The detection method according to claim 1 or 2, characterized in that: The antibody combination 2 includes a CD45 fluorescent antibody for marking leukocytes and a CD21 fluorescent antibody for marking B lymphocytes.

4. The detection method according to claim 3, characterized in that: The fluorescent label of the CD45 fluorescent antibody is Alexa Fluor 700; The fluorescent label of the CD21 fluorescent antibody is PE.

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 + CD21 + 、CD45 + CD3 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD3 - CD5 + subgroup expression.

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

7. A kit for detecting the ratio of canine T, B, and NK cells, characterized in that: The kit includes the following fluorescently labeled antibodies: CD3 fluorescent antibody to label total T lymphocytes; CD4 fluorescent antibody to label helper T lymphocytes; CD8 fluorescent antibody to label cytotoxic T lymphocytes; CD5 fluorescent antibody to label NK cells; CD45 fluorescent antibody to label leukocytes; CD21 fluorescent antibody is used to label B lymphocytes.

8. The kit according to claim 7, characterized in that The fluorescent labels include Alexa Fluor 700, FITC, PE-CY7, EF450, APC and PE.

9. The kit according to claim 7, characterized in that The fluorescent label of the CD3 fluorescent antibody is FITC; The fluorescent label of the CD4 fluorescent antibody is PE-CY7; The fluorescent marker of the CD8 fluorescent antibody is EF450; The fluorescent marker of the CD5 fluorescent antibody is APC; The fluorescent label of the CD45 fluorescent antibody is Alexa Fluor 700; The fluorescent label of the CD21 fluorescent antibody is PE.

10. The kit according to claim 7, characterized in that In the kit, the volume ratio of the CD3 fluorescent antibody for marking total T lymphocytes: the CD4 fluorescent antibody for marking helper T lymphocytes: the CD8 fluorescent antibody for marking cytotoxic T lymphocytes: the CD5 fluorescent antibody for marking NK cells: the CD45 fluorescent antibody for marking leukocytes: and the CD21 fluorescent antibody for marking B lymphocytes is 2.4-2.6:2.4-2.6:2.4-2.6:4-6:1.8-2.2:2.4-2.6.

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