Method for detecting T lymphocyte subtype intracellular factors in peripheral blood of rhesus monkey

Abstract

The invention provides a method for detecting T lymphocyte subtype intracellular factors in rhesus peripheral blood, and belongs to the technical field of biological detection. The method for detecting the T lymphocyte subtype intracellular factors in the peripheral blood of the rhesus monkey is constructed by using a fluorescent antibody combination with a specific variety and a specific ratio. 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 immune performance of rhesus monkeys 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 invention relates to the technical field of biological detection, and in particular to a method for detecting intracellular factors of T lymphocyte subtypes in the peripheral blood of rhesus monkeys. Background Art

[0002] T lymphocytes synthesize many lymphokines and many other cytokines, some of which are related to the treatment of tumors, such as interferon, transfer factor, interleukin, etc. Some of these lymphokines can kill tumor cells, and some can stimulate hematopoietic tissue to counteract the side effects of chemotherapy or radiotherapy. The spleen has 25% of the body's circulating T lymphocytes, directly participates in cellular immunity, and has an important regulatory effect on the distribution of T cell subsets in peripheral blood. The spleen's regulatory effect on T lymphocyte immunity is an important part of tumor immunity. Detection of lymphokines in the spleen can be used to evaluate immune function, diagnosis and differential diagnosis, monitor disease progression, evaluate the effectiveness of drug treatment, formulate individualized treatment plans, and evaluate prognosis.

[0003] Rhesus monkeys are one of the most important scientific research experimental animals. They can be 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 intracellular factors of T lymphocyte subtypes in the peripheral blood of rhesus monkeys. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting intracellular factors of T lymphocyte subtypes in the peripheral blood of rhesus monkeys, which has excellent 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 intracellular factors of rhesus monkey T lymphocyte subtypes, comprising the following steps:

[0007] Perform sample processing, including live and dead staining, surface staining, cell membrane permeabilization and intracellular staining;

[0008] The surface staining comprises taking a combination of antibodies 1 with different fluorescent labels and mixing and incubating them with the sample to be detected;

[0009] Through flow cytometry, the detection data results of intracellular factors of rhesus monkey T lymphocyte subtypes were obtained;

[0010] The antibody combination 1 includes: CD45 fluorescent antibody, CD3 fluorescent antibody, CD4 fluorescent antibody and CD8a fluorescent antibody.

[0011] Preferably, the fluorescent marker of the CD45 fluorescent antibody is BV510;

[0012] The fluorescent marker of the CD3 fluorescent antibody is APC;

[0013] The fluorescent label of the CD4 fluorescent antibody is BUV395;

[0014] The fluorescent label of the CD8a fluorescent antibody is AlexaFluor 700.

[0015] Preferably, the volume ratio of the CD45 fluorescent antibody, CD3 fluorescent antibody, CD4 fluorescent antibody and CD8a fluorescent antibody is 0.5-2:8-12:0.5-2:0.5-2.

[0016] Preferably, the dead-live staining uses FixableViability Stain 780.

[0017] Preferably, the cell membrane is disrupted using TF Fix / PermBuffer.

[0018] Preferably, the intracellular staining comprises taking a combination of antibodies 2 with different fluorescent labels and incubating them with the sample to be detected;

[0019] The antibody combination 2 includes: IL-2 fluorescent antibody, TNF-α fluorescent antibody and IFN-γ fluorescent antibody.

[0020] Preferably, the fluorescent label of the IL-2 fluorescent antibody is BV421;

[0021] The fluorescent label of the TNF-α fluorescent antibody is PE;

[0022] The fluorescent label of the IFN-γ fluorescent antibody is FITC.

[0023] Preferably, the volume ratio of the IL-2 fluorescent antibody, TNF-α fluorescent antibody and IFN-γ fluorescent antibody is 0.5-1.5:3-5:0.5-1.5.

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

[0025] The data analysis includes selecting CD45 + CD3 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD3 + CD4 + IFN-γ + 、CD45 + CD3 + CD4 +IL-2 + 、CD45 + CD3 + CD4 + TNF-α + 、CD45 + CD3 + CD8 + IFN-γ + 、CD45 + CD3 + CD8 + IL-2 + 、CD45 + CD3 + CD8 + TNF-α + subgroup expression.

[0026] Preferably, the sample is from peripheral blood of rhesus monkeys.

[0027] Beneficial effects of the present invention:

[0028] The present invention constructs a method for detecting intracellular factors of T lymphocyte subtypes in the peripheral blood of rhesus 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; the intra-batch precision of the positive drug stimulation experimental group also meets the requirements; it can be used to evaluate the effect of drugs on the immune performance of the peripheral blood of rhesus monkeys in the non-clinical research stage of new drugs, and provide important data for the safety and effectiveness evaluation of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A logical relationship diagram used in the embodiment;

[0030] Figure 2 This is a diagram of the stimulation blocking culture negative test result;

[0031] Figure 3 This is a diagram of the positive test results of stimulation blocking culture. DETAILED DESCRIPTION

[0032] Summary of the present invention:

[0033] Peripheral blood of rhesus monkeys was collected, mononuclear cells were extracted, and the cell concentration was adjusted. Specific stimulatory peptides and Phorbol 12-myristate 13-acetate (PMA) / ionomycin were used to stimulate the cells to secrete cytokines. The protein transport inhibitor Brefeldin A (BFA) was used to block the intracellular cytokines from being transported to the extracellular space mediated by the Golgi apparatus. After a certain period of stimulation and blocking, T lymphocytes were co-labeled with CD45, CD3, CD4, and CD8 fluorescent antibodies. After incubation, the cells were fixed and holes were punched on the cell membrane. IL-2, IFN-γ, and TNF-α fluorescent antibodies were added to label the intracellular cytokines. The antibodies entered the cells through the artificial holes on the cell membrane and combined with the cytokines for detection on the machine. After the cells to be tested enter the flow cytometer, they form a single cell bundle wrapped in sheath fluid. The cells are irradiated by the excitation light emitted by the exciter in turn, emitting scattered light and fluorescence of different intensities. These emitted lights are collected by the optical microscope system through the filter and reach the photodetector (photomultiplier tube). The fluorescence signal of the photodetector is converted into an electrical signal, which is converted into a digital signal by a digital converter. The lymphocyte population is selected, and the lymphocytes containing cytokines such as IL-2, IFN-γ, and TNF-α are collected through fluorescent antibody labeling, and CD45 is recorded. + CD3 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD3 + CD4 + IFN-γ + 、CD45 + CD3 + CD4 + IL-2 + 、CD45 + CD3 + CD4 + TNF-α + 、CD45 + CD3 + CD8 + IFN-γ + 、CD45 + CD3 + CD8 + IL-2 + 、CD45 + CD3 + CD8 + TNF-α + , calculated CD45 + CD3 + CD4+ and CD45 + CD3 + CD8 + The expression of IFN-γ, IL-2, and TNF-α on cells.

[0034] The sources of the reagents used in the embodiments of the present invention are shown in Table 1 below:

[0035] Table 1 Example reagent information

[0036]

[0037]

[0038] 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.

[0039] Example 1

[0040] Rhesus monkey individual blank whole blood sample collection: about 8.0 mL of blood was collected from the jugular vein, anticoagulated with sodium heparin, and the whole blood was mixed by inversion before use;

[0041] PBMC extraction:

[0042] (1) Mix the sodium heparin anticoagulated whole blood by inversion.

[0043] (2) Take 2 mL of anticoagulated whole blood and add it to 2 mL of normal saline (tube A) (make 4 sets according to this ratio) and mix well.

[0044] (3) Slowly add the cells in tube A along the wall of the tube into the tube B containing 3 mL of LSM so that the liquid is spread on the LSM. Centrifuge at 400 × g, 15-25°C for 30 min.

[0045] (4) After centrifugation, a middle white cell layer is visible. Discard the upper liquid layer and transfer the white cell layer to a centrifuge tube (tube C) containing 3 mL of 1× PBS. Mix well and centrifuge at 250×g, 15-25°C for 10 min.

[0046] (5) After centrifugation, discard the supernatant and add 1000 μL of RPMI1640 complete medium to the remaining cells at the bottom of the tube. Mix gently to prepare a cell suspension.

[0047] (6) Mix the four sets of PBMCs and mix them evenly. Take out 50 μL of the cell suspension, dilute it 10 times, and use a fully automatic five-differentiation cell analyzer to count the cells and detect the white blood cell concentration.

[0048] (7) According to the counting results, the cell suspension was diluted with RPMI1640 complete medium to make the concentration of the diluted cell suspension of each animal 4-4.5×10 6 / mL, to be cultured.

[0049] Cell stimulation and culture

[0050] (1) A negative control group, a positive control group, and a sample peptide stimulation sample group were set up: the negative control group only added Protein Transport Inhibitor (Containing BrefeldinA), the positive control group added PMA, Ionomycin, and Protein Transport Inhibitor (Containing BrefeldinA), and the sample peptide stimulation sample group added specific stimulatory peptide and Protein Transport Inhibitor (Containing BrefeldinA).

[0051] (2) Take the diluted cell suspension of each sample (concentration 4×10 6 / mL) 460 μL was added to the corresponding sample peptide group well plate;

[0052] Take the diluted cell suspension of each sample (concentration 4×10 6 / mL) 500 μL was added to the negative control group well plate;

[0053] Take a diluted cell suspension (concentration 4×10 6 / mL) was added into the positive control group well plate.

[0054] (3) Add 40 μL of specific stimulatory peptide (final concentration 8.0 μg / mL / well) to the peptide group sample wells, and add 5 μL of PMA dilution (final concentration 10 ng / mL / well) and 5 μL of Lonomycin dilution (final concentration 1.0 μg / mL / well) to the positive control wells, and mix gently.

[0055] (4) Place the plate after adding the sample at 37°C and 5% CO 2 , and stimulated culture in a saturated humidity carbon dioxide incubator for 2 hours.

[0056] (5) After 2 hours of stimulation and culture, remove each well plate, add 1 μL of Protein Transport Inhibitor (containing Brefeldin A) to each well, mix gently, and incubate at 37°C and 5% CO 2 , and continue stimulation culture in a saturated humidity carbon dioxide incubator for 12 to 16 hours.

[0057] Cell collection

[0058] After the culture is completed, take out the culture well plate, gently blow and mix the cells in the well plate, collect the cells in the well plate into the flow tube, add 1000μL 1×PBS, 400×g, 15-25℃, centrifuge for 5min, discard the supernatant, and leave the cells at the bottom of the tube, add 1000μL 1×PBS again, wash, 400×g, 15-25℃, centrifuge for 5min, discard the supernatant, and leave about 100μL of cells at the bottom of the tube, and mix to form a cell suspension.

[0059] Sample processing and testing

[0060] Dead and alive staining

[0061] (1) Pipette 100 μL of cell suspension into each flow cytometry tube.

[0062] (2) Place 1000 μL of the diluted Fixable Viability Stain 780 in each flow cytometry tube, mix well, and incubate at room temperature in the dark for 10 min.

[0063] (3) Add 1000 μL 1× PBS to each tube, wash and centrifuge at 20°C, 400×g, 5 min, and discard the supernatant.

[0064] Surface staining

[0065] (1) Staining: Pipette 1.0 μL BV510 Mouse Anti-NHP CD45, 10.0 μL APC Mouse Anti-Human CD3, 1.0 μL BUV395 Mouse Anti-Human CD4, and 1.0 μL Alexa Fluor 700 anti-human CD8a fluorescent antibody into each flow cytometry tube, mix well, and incubate at 15-25°C in the dark for 20 min.

[0066] (2) Centrifugation after washing: Add 1000 μL 1× PBS for washing. Centrifuge under the following conditions: temperature 20°C, speed 400×g, time 5 min, and discard the supernatant.

[0067] intracellular staining

[0068] (1) Reagent preparation: Use Diluent Buffer to prepare Fix / Perm Buffer (4×) into Fix / Perm Buffer (1×). Use pure water to prepare Perm / Wash Buffer (5×) into Perm / Wash Buffer (1×). Store at 2-8°C in dark for later use.

[0069] (2) Fixation and permeabilization: Add 1000 μL of TF Fix / Perm Buffer (1×) to each flow cytometry tube, vortex to mix, and incubate at 2-8°C in the dark for 45 min.

[0070] (3) Centrifugation after washing: Add 1000 μL of TF Perm / Wash Buffer (1×) for washing. Centrifuge at 4°C, 400×g, for 5 min. Discard the supernatant.

[0071] (4) Centrifugation after washing: add 2000 μL of TF Perm / Wash Buffer (1×) for washing. Centrifugation conditions: temperature 4°C, speed 400×0, time 5 min, and discard the supernatant.

[0072] (5) Intracellular staining: Add 100 μL TF Perm / Wash Buffer (1×) into each flow cytometry tube, and pipette 2.5 μL BV421 Rat Anti-Human IL-2, 10.0 μL PE Mouse Anti-Human TNF-α, and 2.5 μL FITC Mouse Anti-Human IFN-γ fluorescent antibodies into each flow cytometry tube, respectively. Vortex to mix, and incubate at 2-8°C in the dark for 45 min.

[0073] (6) Centrifugation after washing: Add 2000 μL of TF Perm / Wash Buffer (1×) for washing. Centrifuge at 4°C, 400×g, for 5 min. Discard the supernatant.

[0074] (7) Sample testing: Take out the centrifuged sample, discard the supernatant, add 500 μL 1× PBS buffer to resuspend, shake to mix, and store in the dark for testing.

[0075] Data processing and analysis

[0076] Use the CytoFLEX LX flow cytometer to detect samples, and use the instrument's built-in CytExpert software to analyze the test data. Use histograms, scatter plots, and contour plots to determine the target cell population, adjust fluorescence compensation, gate position and other analysis parameters as needed, confirm the distribution of each cell population, and obtain the required data. Obtain the % Parent value as the sample result

[0077] In this embodiment, a set of logical relationships is as follows: Figure 1 shown.

[0078] The instrument parameters are shown in Tables 2 and 3 below:

[0079] Table 2 Instrument voltage, threshold

[0080] Channel Name Voltage Threshold FSC 266 50000 SSC 174 / B525-FITC 127 / Y585-PE 404 R660-APC 306 R712-APCA700 616 / R673-APCA750 360 / V450-PB 32 V610 100 / NUV450 200

[0081] Table 3 Instrument compensation

[0082]

[0083] Different stimulant concentrations and different stimulation times were set, and the method of the present invention was used for detection. The result data are shown in Table 4 below:

[0084] Table 4 Test results of different stimulant concentrations and different stimulation times

[0085]

[0086] Results: PBMCs were stimulated with specific stimulants. Compared with the negative control, after 6 hours of stimulation at different concentrations, the results showed no obvious stimulation effect; after 12 hours of stimulation at different concentrations of 2μg, 4μg, and 8μg, each index had obvious intracellular cytokine release, and the results increased, and the 8μg concentration was the highest, which was consistent with the expected situation. This shows that the detection method provided by the present invention can obtain accurate results.

[0087] Experimental example

[0088] Methodological verification of the technical solution provided in the embodiment:

[0089] With-run Precision (WP)

[0090] Four samples were tested in the same analytical batch and the test was repeated three times. The %CV of each test value of each sample was calculated, and the %CV was between 0.00 and 12.00% (satisfying CV ≤ 25%). The intra-batch precision met the requirements. The specific results are shown in Table 5.

[0091] When stimulated by peptide, negative group and peptide group were performed separately, 4 samples were tested in the same analysis batch, and the test was repeated 3 times. The %CV of each test value of each sample was calculated, and the %CV of the negative group was between 0.07 and 34.64% (satisfying CV≤25%, low proportion cells met CV≤35%), and the %CV of the peptide group was between 0.00 and 25.00% (satisfying CV≤25%, low proportion cells met CV≤35%). The intra-batch precision met the requirements. The specific results are shown in Tables 6 and 7.

[0092] Between-run Precision (BP)

[0093] The same three samples were analyzed in two independent analysis batches within one day, and each sample in each batch was tested three times. The %CV of each test value of the same sample in the two analysis batches was calculated, and the %CV was between 0.04 and 9.65% (satisfying CV ≤ 25%), and the inter-batch precision met the requirements. The specific results are shown in Table 8.

[0094] Table 5 Intra-batch precision of the detection scheme for intracellular factors of T lymphocyte subtypes in peripheral blood of rhesus monkeys

[0095]

[0096]

[0097]

[0098]

[0099]

[0100] Table 6 Intra-batch precision of negative samples for detection of intracellular factors of T lymphocyte subtypes in peripheral blood of rhesus monkeys

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] Table 7 Intra-batch precision of positive samples for detection of intracellular factors of T lymphocyte subtypes in peripheral blood of rhesus monkeys

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] Table 8 Inter-batch precision of intracellular factors of T lymphocyte subtypes in peripheral blood of rhesus monkeys

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] The verification results show that the intra-batch precision CV range of the scheme provided by the present invention is 0.24-14.21%; the inter-batch precision CV range is 0.03-16.03%; when stimulated by polypeptide, the negative group and polypeptide group are respectively carried out, and 4 samples are respectively detected in the same analysis batch, and the detection is repeated 3 times. The %CV of each test value of each sample is calculated, and it is obtained that the %CV of the negative group is between 0.07 and 34.64% (satisfying CV≤25%, and low-proportion cells meet CV≤35%), and the %CV of the polypeptide group is between 0.00 and 25.00% (satisfying CV≤25%, and low-proportion cells meet CV≤35%). The intra-batch precision meets the requirements, indicating that the detection results of this method are accurate and can be used for the detection of intracellular factors of T lymphocyte subtypes in the peripheral blood of rhesus monkeys.

[0125] 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 intracellular factors of rhesus monkey T lymphocyte subtypes, characterized in that: The following steps are involved: Perform sample processing, including live and dead staining, surface staining, cell membrane permeabilization and intracellular staining; The surface staining comprises taking a combination of antibodies 1 with different fluorescent labels and mixing and incubating them with the sample to be detected; Through flow cytometry, the detection data results of intracellular factors of rhesus monkey T lymphocyte subtypes were obtained; The antibody combination 1 includes: CD45 fluorescent antibody, CD3 fluorescent antibody, CD4 fluorescent antibody and CD8a fluorescent antibody.

2. The detection method according to claim 1, characterized in that: The fluorescent marker of the CD45 fluorescent antibody is BV510; The fluorescent marker of the CD3 fluorescent antibody is APC; The fluorescent label of the CD4 fluorescent antibody is BUV395; The fluorescent label of the CD8a fluorescent antibody is AlexaFluor 700.

3. The detection method according to claim 2, characterized in that: The volume ratio of the CD45 fluorescent antibody, CD3 fluorescent antibody, CD4 fluorescent antibody and CD8a fluorescent antibody is 0.5-2:8-12:0.5-2:0.5-2.

4. The detection method according to claim 1, characterized in that: The described dead-live staining adopts FixableViability Stain 780.

5. The detection method according to claim 1, characterized in that: The cell membrane was permeabilized using TF Fix / PermBuffer.

6. The detection method according to claim 7, characterized in that: The intracellular staining comprises taking a combination of antibodies 2 with different fluorescent labels and mixing and incubating them with the sample to be detected; The antibody combination 2 includes: IL-2 fluorescent antibody, TNF-α fluorescent antibody and IFN-γ fluorescent antibody.

7. The detection method according to claim 6, characterized in that: The fluorescent label of the IL-2 fluorescent antibody is BV421; The fluorescent label of the TNF-α fluorescent antibody is PE; The fluorescent label of the IFN-γ fluorescent antibody is FITC.

8. The detection method according to claim 6, characterized in that: The volume ratio of the IL-2 fluorescent antibody, TNF-α fluorescent antibody and IFN-γ fluorescent antibody is 0.5-1.5:3-5:0.5-1.

5.

9. 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 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD3 + CD4 + IFN-γ + 、CD45 + CD3 + CD4 + IL-2 + 、CD45 + CD3 + CD4 + TNF-α + 、CD45 + CD3 + CD8 + IFN-γ + 、CD45 + CD3 + CD8 + IL-2 + 、CD45 + CD3 + CD8 + TNF-α + subgroup expression.

10. The detection method according to any one of claims 1 to 9, characterized in that: The samples were obtained from peripheral blood of rhesus monkeys.

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