Method for detecting the expression level of immune cell factors in the body after vaccination

Abstract

The application discloses a detection method for expression level of immune cytokines in a body after vaccination, which comprises the following steps: after resting of recovered peripheral blood mononuclear cell cryopreservation samples to be detected, specific peptide pools are added for culture; a protein transport blocking agent is added during the culture process; after the culture is completed, the samples are transferred into a flow tube; after the cells are fixed and the cells are subjected to membrane permeation treatment, a monoclonal antibody reagent combination is added for incubation in the dark; and the expression level of immune cytokines is detected by flow cytometry. The detection method can improve detection accuracy and sensitivity, greatly shorten an experimental period, improve experimental efficiency, avoid false positive results, overcome antigen endocytosis caused by a protein transport blocking agent, and accurately evaluate the immune state of the body after vaccination.

Description

Technical Field

[0001] This invention belongs to the field of immunology technology, and more specifically, this invention relates to a method for detecting the expression level of immune cytokines in the body after vaccination. Background Technology

[0002] Cellular immune responses depend on the interaction of T lymphocytes and antigen-presenting cells. T lymphocytes are divided into two types: CD8+ T lymphocytes and CD4+ T lymphocytes, each mediating different types of immune responses. CD8+ T lymphocytes are cytotoxic T cells; they can directly kill infected cells by releasing granzymes, perforin, etc., or by releasing various cytokines, playing the most direct role in the immune response. CD4+ T lymphocytes are helper T cells; they can stimulate the activation of CD8+ T lymphocytes and macrophages, or promote the activation and differentiation of B cells, by releasing different cytokines.

[0003] Cytokines play a crucial role in physiological and pathological processes such as immune response, immune regulation, and tumor metastasis. Detecting cytokines secreted by immune cells can reflect the functional state of immune cells and is also an indicator for clinical exploration of disease pathogenesis, prognosis assessment, and efficacy evaluation. Therefore, in vaccine evaluation, the secretion of intracellular cytokines is often used to reflect the state of the body's cellular immune function.

[0004] Cellular immunity assessment mainly involves detecting the frequency of T cells secreting IFN-γ using enzyme-linked immunosorbent assay (ELISA) and ELISA spot assay, and detecting intracellular cytokine levels using flow cytometry. ELISA detects free cytokines or antibodies in body fluids. However, due to the varying half-lives of free circulating antibodies or cytokines, they are continuously metabolized or bound to target organs in body fluids. Therefore, ELISA cannot accurately reflect the actual levels of antibodies and cytokines in vivo. Furthermore, it requires large sample volumes, can only measure one cytokine at a time, and involves lengthy procedures and testing times. Additionally, ELISA amplification technology can easily distort background signals, leading to numerous interfering factors and false positive results. ELISA spot assays have a long experimental cycle, can only detect one antigen or antibody target at a time, require overnight stimulation and culture, which can easily cause cell contamination, affecting result interpretation, and cannot detect the phenotype of secreted cytokines. Flow cytometry can differentiate cell subtypes that secrete cytokines based on cellular immunophenotypes and perform multi-parameter correlation analysis. However, during the experiment, issues may arise such as poor staining results from cytokine-labeled antibodies (traditional flow cytometry stains for cell viability and death are cell membrane-permeable nucleic acid dyes, such as PI, 7-AAD, and DAPI. These nucleic acid dyes can penetrate the cell membrane of dead cells, fluoresce after binding to nucleic acids, and when cells need to be fixed and ruptured, the nucleic acid dye will stain all cells, making it difficult to distinguish between viability and death signals) and antigen endocytosis.

[0005] Therefore, there is a clinical need for a cell-mediated immune assessment method that is highly accurate, specific, sensitive, simple, rapid, stable, and reproducible. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a method for detecting the expression level of immune cytokines in the body after vaccination. The detection method of the present invention can assess cellular immunity with higher accuracy and stronger sensitivity.

[0007] The technical solutions for achieving the above-mentioned objectives include the following.

[0008] In a first aspect, the present invention provides a method for detecting the expression level of immune cytokines in the body after vaccination, comprising the following steps:

[0009] (1) When the frozen peripheral blood mononuclear cell samples to be tested are resting for 0-1 h after revival, add the specific peptide pool and culture for 5-7 h.

[0010] (2) Add protein transport inhibitor 4 hours before the end of culture; after the culture is completed, thoroughly pipette the cells and transfer them to flow cytometry tubes; centrifuge and discard the supernatant, then add phosphate buffer, centrifuge and discard the supernatant again;

[0011] (3) Add cell surface marker antibodies CD3, CD8 and CD45, as well as Fixable ViabilityStain700 to the flow cytometer and mix well; incubate at 3-5°C in the dark for 25-35 min; add phosphate buffer containing newborn calf serum, gently pipette to mix, centrifuge and discard the supernatant, wash again and discard the supernatant.

[0012] (4) After fixing the cells with cell permeabilization reagent and performing permeabilization treatment, the cells were divided into detection tubes and isotype control tubes. Monoclonal antibody reagent combination 1 and monoclonal antibody reagent combination 2 were added to the detection tubes and isotype control tubes, respectively, and incubated at 3-5°C in the dark for 25-35 min. The monoclonal antibody reagent combination 1 includes: FITC IFN-γ, PE IL-5, and PerCP-Cy. TM 5.5 TNF, APC IL-4, BV421 IL-2, BV605 CD4; the monoclonal antibody combination reagent 2 includes: FITC IgG1, PE IgG1, PerCP-Cy TM 5.5 IgG1, APC IgG1, BV421 IgG2a, BV605 CD4;

[0013] (5) The expression levels of immune cytokines were detected by flow cytometry.

[0014] The present invention has the following beneficial effects:

[0015] The detection method of this invention, after culturing in a specific peptide pool for a certain period of time, can stimulate and amplify the expression of intracellular cellular secretory factors, inducing a more significant cellular immune response, thereby improving detection accuracy and sensitivity, and significantly shortening the experimental cycle and increasing experimental efficiency. By setting up isotype control tubes, false positive interference caused by non-specific antibody binding to cells can be eliminated. At the same time, by adding the active dye Fixed Viability Stain 700 (FVS700) to label dead cells, spontaneous and non-specific fluorescence interference is removed, experimental background signal is reduced, and false positive results are avoided. By first fixing and permeabilizing the cells before adding anti-human CD4 antibody, antigen endocytosis caused by protein transport blockers can be overcome, and CD4 signal can be detected better. Under the combined effect of all the above key steps, the immune status of the body after vaccination can be accurately assessed. Attached Figure Description

[0016] Figure 1 This is a graph showing the percentage of CD3+CD4+IFN-r expression intensity after the sample wells in Experiment Example 3 of this invention were stimulated with co-stimulatory antibodies.

[0017] Figure 2This is a graph showing the percentage of CD3+CD4+IFN-r expression intensity after the sample wells in Experiment Example 3 of this invention were cultured without the addition of co-stimulatory antibodies.

[0018] Figure 3 This is a graph showing the percentage of expression intensity of CD3+CD4+IFN-r in the control wells of Experiment Example 3 of this invention after culture without the addition of co-stimulatory antibodies.

[0019] Figure 4 The graph shows the percentage of CD3+CD4+IFN-r expression intensity after the negative control wells in Experiment Example 3 of this invention were stimulated with co-stimulatory antibodies.

[0020] Figure 5 This is a graph showing the percentage of CD3+CD4+IFN-r expression intensity in the negative control wells after culture without co-stimulatory antibody in Experiment Example 3 of this invention.

[0021] Figure 6 This is a flow cytometry diagram showing the intracellular cytokine CD3+CD4+IL-4 after adding the active dye Fixable Viability Stain 700 (FVS700) in Experimental Example 5 of this invention.

[0022] Figure 7 This is a flow cytometry diagram of the intracellular cytokine CD3+CD4+IL-4 after no addition of the active dye Fixable Viability Stain 700 (FVS700) in Experiment Example 5 of this invention.

[0023] Figure 8 This is a flow cytometry diagram showing the intracellular cytokine CD3+CD4+IFN-r in the detection tube and the isotype control tube in Experiment Example 6 of the present invention.

[0024] Figure 9 The image shows the effect of adding anti-human CD4 antibody before immobilization and permeabilization in Experiment Example 6 of this invention, as shown in the cell flow cytometry diagram.

[0025] Figure 10 The flow cytometry results are shown in Experiment 6 of this invention, after immobilization and permeabilization, with the addition of anti-human CD4 antibody. Detailed Implementation

[0026] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0027] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0028] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0029] In this invention, the inventors discovered that resting revived cryopreserved cells for 0 hours (when cell activity and state are optimal) and then culturing them in a specific peptide pool for a certain period (6 hours yields the best results) can stimulate and amplify the expression of intracellular secretory factors. Adding co-stimulatory antibodies CD49d and CD28 to the specific peptide pool effectively activates antigen-specific cytokine responses through co-stimulation, further stimulating and amplifying the ability of cells to secrete factors and inducing a more significant cellular immune response (under co-stimulatory culture with co-stimulatory antibodies, the signal of intracellular cytokine IFN-γ is more concentrated and obvious). The expression levels of intracellular secretory factors are then detected by flow cytometry, simultaneously measuring multiple cytokines in a single sample and studying the changes in the levels of specific cytokines in CD4+ T cells and CD8+ T cells. Because the specific peptide pool and co-stimulatory antibodies increase the signal detection intensity, the accuracy and sensitivity of detection can be improved, and the experimental cycle can be significantly shortened, increasing experimental efficiency.

[0030] In the detection method of this invention, by setting up an isotype control tube, false positive interference caused by the non-specific binding of antibodies to cells can be eliminated (positive rate = intracellular cytokine positive rate - isotype control positive rate); at the same time, by adding the active dye Fixed Viability Stain 700 (FVS700), dead cells are labeled, spontaneous and non-specific fluorescence interference is removed, experimental background signal is reduced, and false positive results are avoided (without adding the active dye to remove dead cell interference, a large group of dead cells can be seen in the positive area of ​​intracellular cytokine IL-4, after adding the active dye to remove dead cells, only clean intracellular cytokine IL-4 signal remains in the positive area).

[0031] Secreted cytokines are continuously secreted into the extracellular space in response to stimulation. Conventional flow cytometry detects cytokines within cells; if all cytokines are secreted, they become undetectable. Therefore, to ensure detection within cells, it's necessary to prevent their secretion. This is achieved by adding protein transport inhibitors that block Golgi-mediated transport, causing the stimulated cytokines to accumulate in the endoplasmic reticulum within the cytoplasm. This facilitates accurate detection of the cell's ability to produce cytokines. However, phorbol ester stimulants or protein transport inhibitors can lead to antigen endocytosis. This invention first fixes and permeates cells (the fixative protects the integrity of cell morphology and the antigenicity of intracellular substances, and also prevents the permeabilizing agent from penetrating the cell; the permeabilizing agent increases cell membrane permeability, forming a transmission channel on the cell membrane, allowing fluorescein-labeled antibodies to penetrate through the cell membrane and cytoplasm into the Golgi complex on the endoplasmic reticulum and bind to cytokines or other cytoplasmic molecules), and then adds anti-human CD4 antibody. This results in better detection of CD4 signals because when anti-human CD4 antibody is added before fixation and permeabilization, it binds to CD4 on the cell membrane, resulting in unclear cell clustering, which is considered to be antigen endocytosis caused by the influence of phorbol ester stimulants and protein transport inhibitors; after fixation and permeabilization, the addition of anti-human CD4 antibody allows it to bind to cytoplasmic CD4, thus enabling better detection of CD4 signals.

[0032] In some embodiments of the present invention, a method for detecting the expression level of immune cytokines in the body after vaccination is disclosed, comprising the following steps:

[0033] (1) When the frozen peripheral blood mononuclear cell sample to be tested is resting for 0-1h, add the specific peptide pool and culture for 5-7h; the nucleotide sequence of the specific peptide pool is shown in SEQ ID NO:1-SEQ ID NO:67 or SEQ ID NO:68-SEQ ID NO:110.

[0034] (2) Add protein transport inhibitors during the last 4 hours of culture; after culture, thoroughly pipette the cells, transfer them to flow cytometry tubes, centrifuge and discard the supernatant, then add phosphate buffer, centrifuge and discard the supernatant.

[0035] (3) Add immune cell surface marker antibodies CD3, CD8 and CD45, and FixableViability Stain 700 to the flow cytometer and mix well; incubate at 3-5°C in the dark for 25-35 min; add phosphate buffer containing newborn calf serum, gently pipette to mix, centrifuge and discard the supernatant, wash again and discard the supernatant.

[0036] (4) Fix cells with cell permeabilization reagent and perform permeabilization treatment. Centrifuge and discard the supernatant. Add washing buffer and mix well by pipetting. Divide the mixture into detection tubes and isotype control tubes. Add monoclonal antibody reagent combination 1 and monoclonal antibody reagent combination 2 to the detection tubes and isotype control tubes, respectively. Incubate at 3-5°C in the dark for 25-35 min. Monoclonal antibody reagent combination 1 includes: FITC IFN-γ, PE IL-5, and PerCP-Cy. TM 5.5 TNF, APC IL-4, BV421 IL-2, BV605 CD4; the monoclonal antibody combination reagent 2 includes: FITC IgG1, PE IgG1, PerCP-Cy TM 5.5 IgG1, APC IgG1, BV421IgG2a, BV605 CD4;

[0037] (5) The expression levels of immune cytokines were detected by flow cytometry.

[0038] In some embodiments, the working concentration of the specific peptide pool in step (1) is 1 ug / ml to 4 ug / ml.

[0039] In some embodiments, step (1) also includes co-culturing with co-stimulatory antibodies, wherein the co-stimulatory antibodies are CD49d and CD28.

[0040] In some embodiments, the working concentrations of CD49d and CD28 are both 0.8–1.2 ug / ml.

[0041] In some embodiments, the resting time of the frozen peripheral blood mononuclear cell sample to be tested in step (1) is 0 h.

[0042] In some embodiments, the amounts of the immune cell surface marker antibodies CD3, CD8, and CD45 added in step (3) are 10 μL, 5 μL, and 5 μL, respectively; and / or, the amounts of FITC IFN-γ, PE IL-5, and PerCP-Cy added in step (5) are... TM 5.5 The amounts of TNF, APC IL-4, BV421 IL-2, and BV605 CD4 added were 20 μL, 5 μL, 5 μL, 5 μL, and 5 μL, respectively; the FITC IgG1, PE IgG1, and PerCP-Cy were also added. TM 5.5 The amounts of IgG1, APC IgG1, BV421 IgG2a, and BV605 CD4 added were 20 μL, 5 μL, 5 μL, 5 μL, and 5 μL, respectively.

[0043] In some embodiments, the culture time in step (1) is 5.5 to 6.5 hours, preferably 6 hours.

[0044] In some embodiments, step (1) involves incubation in a 36–38°C, 4–6% carbon dioxide incubator.

[0045] In some embodiments, the protein transport inhibitor in step (2) is brefiltrant A or monensin, and 1.5 to 2.5 μL of protein transport inhibitor is added to every 3 mL of cell culture system.

[0046] In some embodiments, the centrifugation speed in steps (2) and (3) is 1600-1800 rpm, and the centrifugation time is 4-6 min.

[0047] The reagents used in the following examples are shown in Tables 1 and 2.

[0048] Table 1

[0049]

[0050] Table 2

[0051]

[0052]

[0053] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0054] Example 1: Method for detecting the expression level of immune cytokines in the body after vaccination

[0055] This embodiment provides a method for detecting the expression level of immune cytokines in the body after vaccination, including the following steps:

[0056] 1. Add 10 ml of culture medium (90% RPMI 1640 medium + 20 mM HEPES + 10% fetal bovine serum + 1% Gibco antibiotics) to a 15 ml centrifuge tube and preheat it to 37°C in a water bath. Remove the cryovial of the peripheral blood mononuclear sample to be tested from the -80°C freezer (liquid nitrogen) and quickly transfer it to a 37°C water bath to thaw (until a small ice cube remains). Then quickly add the peripheral blood mononuclear sample to be tested to the preheated 15 ml centrifuge tube containing the culture medium and mix well. Centrifuge at 250 g at room temperature for 10 min. Discard the supernatant and gently tap the cell clumps. Add 1 ml of the above culture medium and gently pipette to prepare a cell suspension. Add the suspension to a culture dish (note aseptic operation) to obtain the thawed peripheral blood mononuclear sample to be tested.

[0057] 2. Add 2 μL of a specific peptide pool (0.5 mg of synthetic peptide library 1 dissolved in 500 μL of dimethyl sulfoxide and added to 2 μL of the peripheral blood mononuclear cryopreserved sample to be tested, i.e., the working concentration of the specific peptide pool is 2 μg / mL; the amino acid sequence of synthetic peptide library 1 is shown in SEQ ID NO: 1-67, and the specific sequence is shown in Table 3), 1 μg / mL of anti-human CD49d, and 1 μg / mL of anti-human CD28 to the sample well (1 mL, 10^6 cells) of the peripheral blood mononuclear cryopreserved sample to be tested; place in an incubator at 37°C and 5% CO2 and incubate for 6 hours.

[0058] Table 3

[0059]

[0060]

[0061]

[0062] 3. After 2 hours of stimulation culture, remove the culture dish from the CO2 incubator, add a protein transport inhibitor (brevidin A or monensin, 4uL per 6mL of cell culture system (~10^6 cells / mL), and continue to culture in the CO2 incubator; after stimulation culture, remove the culture dish from the CO2 incubator, thoroughly pipette the cells, and transfer the cells from the culture dish to a flow cytometer; centrifuge at 1700 rpm for 5 min, and discard the supernatant;

[0063] 4. Add 1 ml of phosphate buffer (dissolve one packet of phosphate powder in 500 mL of deionized water at room temperature and adjust the pH to 7.2 ± 0.2), gently mix by pipetting, centrifuge at 1700 rpm for 5 min, and discard the supernatant.

[0064] 5. Add immune cell surface marker antibodies, APC-ALexaFluor 750CD3 (10ul), PE-Cy TM 7 CD8 (5ul), BV510 CD45 (5ul), Fixable Viability Stain 700 (4ul), mix well; incubate at 4°C in the dark for 30 minutes;

[0065] 6. Add 2 ml of phosphate buffer containing 1% newborn calf serum (inactivate the newborn calf serum in a 56°C water bath for 30 minutes, and add a certain amount of phosphate buffer to make the final concentration 1%), gently pipette to mix, centrifuge at 1700 rpm for 5 min, discard the supernatant, and repeat the washing once more, discarding the supernatant again.

[0066] 7. Add 500 μL of Fixation / Permeabilization solution to fix the cells, gently shake to mix, and incubate at 4°C in the dark for 20–30 minutes (permeabilization treatment); centrifuge at 1700 rpm for 5 minutes and discard the supernatant. Add 1 ml of BD Perm / Washbuffer washing buffer (BD Perm / Washbuffer stock solution: distilled water = 1:9), mix well by pipetting, and divide into two equal tubes, labeled as test tubes and isotype control tubes respectively. Centrifuge at 1700 rpm for 5 minutes and discard the supernatant.

[0067] 8. Add monoclonal antibody reagent combination 1 and monoclonal antibody reagent combination 2 to the test tube and isotype control tube respectively, and incubate at 4°C in the dark for 30 min.

[0068] Monoclonal antibody combination reagent 1: FITC IFN-γ (20ul), PE IL-5 (5ul), PerCP-Cy TM 5.5 TNF(5ul), APC IL-4(5ul), BV421 IL-2(5ul), BV605 CD4(5ul)

[0069] Monoclonal antibody combination reagent 2: FITC IgG1 (20ul), PE IgG1 (5ul), PerCP-Cy TM 5.5 IgG1(5ul), APC IgG1(5ul), BV421 IgG2a(5ul), BV605 CD4(5ul)

[0070] 9. Add 1 ml of BD Perm / Washbuffer to wash the cells, centrifuge at 1700 rpm for 5 min, discard the supernatant, and resuspend the cells in 400 μl of BD Perm / Washbuffer.

[0071] 10. The expression of each cytokine (IFN-γ, IL-5, TNF-α, CD8, IL-4, FVS700, CD3, IL-2, CD45, cCD4) was detected by flow cytometry, and the intracellular cytokine positivity rate was calculated.

[0072] Positive rate = Positive rate of intracellular cytokines detected - Positive rate of intracellular cytokines detected in isotype control tracheocytes

[0073] Example 2: Method for detecting the expression level of immune cells in the body after vaccination

[0074] This embodiment provides a method for detecting the expression level of immune cells in the body after vaccination. Except for step (1), in which 2 μg of specific peptide pool is added (0.5 mg of synthetic peptide library 2 is dissolved in 500 μl of dimethyl sulfoxide and added to the sample to be tested; the amino acid sequence of synthetic peptide library 2 is shown in SEQ ID NO: 68-108, and the specific sequence is shown in Table 4), all other steps are the same as in Example 1.

[0075] Table 4

[0076] SC1208 Chemical Peptide Synthesis:S-1 CTFEYVSQPFLMDLE(SEQ ID NO:68) SC1208 Chemical Peptide Synthesis:S-2 EFVFKNIDGYFKIYS(SEQ ID NO:69) SC1208 Chemical Peptide Synthesis:S-3 KHTPIIVREPEDLPQGFS(SEQ ID NO:70) SC1208 Chemical Peptide Synthesis:S-4 IVREPEDLPQGFSALEP(SEQ ID NO:71) SC1208 Chemical Peptide Synthesis:S-5 YAWNRKRISNCVADY(SEQ ID NO:72) SC1208 Chemical Peptide Synthesis:S-6 GVSPTKLNDLCFTNV(SEQ ID NO:73) SC1208 Chemical Peptide Synthesis:S-7 SGNYNYLYRLFRKSN(SEQ ID NO:74) SC1208 Chemical Peptide Synthesis:S-8 YLYRLFRKSNLKPFE(SEQ ID NO:75) SC1208 Chemical Peptide Synthesis:S-9 VVLSFELLHAPATVC(SEQ ID NO:76) SC1208 Chemical Peptide Synthesis:S-10 GPKKSTNLVKNKCVN(SEQ ID NO:77) SC1208 Chemical Peptide Synthesis:S-11 SVTTEILPVSMTKTS(SEQ ID NO:78) SC1208 Chemical Peptide Synthesis:S-12 STECSNLLLQYGSFC(SEQ ID NO:79) SC1208 Chemical Peptide Synthesis:S-13 NLLLQYGSFCTQLKR(SEQ ID NO:80) SC1208 Chemical Peptide Synthesis:S-14 NFSQILPDPSKPSKR(SEQ ID NO:81) SC1208 Chemical Peptide Synthesis:S-15 TDEMIAQYTSALLAG(SEQ ID NO:82) SC1208 Chemical Peptide Synthesis:S-16 GINASVVNIQKEIDR(SEQ ID NO:83) SC1208 Chemical Peptide Synthesis:S-17 LIDLQELGKYEQYI(SEQ ID NO:84) SC1208 Chemical Peptide Synthesis:S-18 YEQYIKWPWYIWLGF(SEQ ID NO:85) SC1208 Chemical Peptide Synthesis:NP-1 MSDNGPQNQRNALRITF(SEQ ID NO:86) SC1208 Chemical Peptide Synthesis:NP-2 NQRNALRITFGGPSDSTG(SEQ ID NO:87) SC1208 Chemical Peptide Synthesis:NP-3 DQIGYYRRATRRIR(SEQ ID NO:88) SC1208 Chemical Peptide Synthesis:NP-4 MKDLSPRWYFYYL(SEQ ID NO:89) SC1208 Chemical Peptide Synthesis:NP-5 LSPRWYFYYLGTGPEAGL(SEQ ID NO:90) SC1208 Chemical Peptide Synthesis:NP-6 AFFGMSRIGMEVTPSGTW(SEQ ID NO:91) SC1208 Chemical Peptide Synthesis:NP-7 GMEVTPSGTWLTYTGAIK(SEQ ID NO:92) SC1208 Chemical Peptide Synthesis:NP-8 TWLTYTGAIKLDDKDPNF(SEQ ID NO:93) SC1208 Chemical Peptide Synthesis:NP-9 PNFKDQVILLNKHIDAYK(SEQ ID NO:94) SC1208 Chemical Peptide Synthesis:NP-10 LLNKHIDAYKTFPPTEPK(SEQ ID NO:95) SC1208 Chemical Peptide Synthesis:M-1 LLESELVIGAVILRGHLR(SEQ ID NO:96) SC1208 Chemical Peptide Synthesis:M-2 GAVILRGHLRIAGHHLGR(SEQ ID NO:97) SC1208 Chemical Peptide Synthesis:M-3 LRIAGHHLGRCDIKDLPK(SEQ ID NO:98) SC1208 Chemical Peptide Synthesis:M-4 PKEITVATSRTLSYYKL(SEQ ID NO:99) SC1208 Chemical Peptide Synthesis:M-5 TSRTLSYYKLGASQRVA(SEQ ID NO:100) SC1208 Chemical Peptide Synthesis:M-6 IGNYKLNTDHSSSSDNIA(SEQ ID NO:101) SC1208 Chemical Peptide Synthesis:ORF3a-1 YFLCWHTNCYDYCIPY(SEQ ID NO:102) SC1208 Chemical Peptide Synthesis:ORF3a-2 KDCVVLHSYFTSDYYQLY(SEQ ID NO:103) SC1208 Chemical Peptide Synthesis:ORF3a-3 YFTSDYYQLYSTQLSTDTGV(SEQ ID NO:104) SC1208 Chemical Peptide Synthesis:ORF3a-4 GVEHVTFFIYNKIVDEPEEH(SEQ ID NO:105) SC1208 Chemical Peptide Synthesis:ORF7a-1 LITLATCELYHYQECVR(SEQ ID NO:106) SC1208 Chemical Peptide Synthesis:ORF7a-2 FHPLADNKFALTCFSTQF(SEQ ID NO:107) SC1208 Chemical Peptide Synthesis:ORF7a-3 DGVKHVYQLRARSVSPKL(SEQ ID NO:108)

[0077] Experimental Example 1: Effect of Resting Culture Time on Cell Viability of Peripheral Blood Mononuclear Cells after Resuscitation

[0078] Peripheral blood mononuclear frozen specimens from two healthy volunteers were thawed and added to cell culture dishes, which were then placed in a CO2 cell culture incubator. After thawing, the cells were incubated at rest for 0H, 6H, 12H, and 24H, respectively, following the same procedures as steps 2-10 in Example 1. Dead cells were labeled with the cell viability assay dye Fixed Viability Stain 700, and the cell viability in each specimen was detected by flow cytometry to calculate the percentage of live cells. The results are shown in Table 5.

[0079] Table 5

[0080]

[0081] Table 5 shows that the optimal cell viability is achieved when the resting time is 0H, i.e., immediately after the specimen is revived and the experiment is performed.

[0082] Experimental Example 2: Effect of the type and concentration of specific peptide pools on the percentage of CD4+IL-4+ expression stimulated production

[0083] After resuscitation of healthy volunteer samples, specific peptide pools 1 through 6 were added to the sample wells; PMA stimulant (2 μL per 1 mL of cell culture system (~10^6 cells / mL)) was added to the positive control wells. Leukocyte Activation Cocktail with BD GolgiPlug TM Dimethyl sulfoxide (2 μL, Sigma) was added to the negative control wells.

[0084] Among them, specific peptide pools 1 to 3 are synthetic peptide libraries 1 with concentrations of 1 μg / ml, 2 μg / ml, and 4 μg / ml, respectively. 0.5 mg of synthetic peptide library 1 was dissolved in 500 μl of dimethyl sulfoxide, and 1 μl, 2 μl, and 4 μl were added to the sample wells, respectively (the sequence of synthetic peptide library 1 is shown in Table 3, C0827HG150-68 / PE5174). Specific peptide pools 4 to 6 are synthetic peptide libraries 2 with concentrations of 1 μg / ml, 2 μg / ml, and 4 μg / ml, respectively. 0.5 mg of synthetic peptide library 2 was dissolved in 500 μl of dimethyl sulfoxide, and 1 μl, 2 μl, and 4 μl were added to the sample wells, respectively (the sequence of synthetic peptide library 2 is shown in Table 4, C9281HG200-42 / PE2975).

[0085] All other steps are the same as steps 2-10 in Example 1. The percentage of expression intensity of cytokines CD4+IL-4+ after stimulation with different specific peptide pools is shown in Table 7.

[0086] Table 6

[0087]

[0088] Table 6 shows that there was no significant difference in the expression percentage of CD4+IL-4+ generated by stimulation with different concentrations of the specific peptide pool, with CV values ​​all less than 5%; and there was also no significant difference in the expression percentage of CD4+IL-4+ generated by stimulation with different specific peptide pools, with CV values ​​less than 5%. This indicates that the specific peptide pool stimulation of the present invention is suitable for enhancing and amplifying the ability of cells to secrete factors.

[0089] The percentage of expression intensity of cytokines CD4+IFN-r+ in different wells is shown in Table 7.

[0090] Table 7

[0091] Specific peptide pool 1 0.03％ Specific peptide pool 2 0.04％ Specific peptide pool 3 0.03％ Specific peptide pool 4 0.04％ Specific peptide pool 5 0.04％ Specific peptide pool 6 0.05％ Positive control 17.53％ negative control 0.00％

[0092] Normally, unstimulated leukocytes do not express cytokines or express them at levels so low as to be undetectable. When detecting T cell cytokine expression after PMA stimulation, a positive control is a very useful experimental staining control, as it can confirm the specificity of the staining process and the cytokine antibody used (see positive control results in Table 7). Simultaneously, a no-stimulator control (i.e., a negative control) is added to understand the background signal.

[0093] Experimental Example 3: Effect of co-stimulatory antibodies on the expression intensity of cytokines CD3+CD4+IFN-r

[0094] After the healthy volunteer samples were resuscitated, 1 μg / ml of Ultra-LEAF specific peptide pool 1 co-stimulatory antibody was added to the sample wells, negative control wells, and unrelated peptide control wells. TMPurified anti-human CD49d and 1ug / ml Ultra-LEAF TM Purified anti-human CD28 was used, with a control group using no co-stimulatory antibody. All other steps were the same as steps 2-10 of Example 1. Experimental results are as follows: Figures 1 to 6 As shown.

[0095] After 0 hours of stimulation with 1 μg / ml CD49d and CD28 co-stimulatory antibody, the expression intensity percentage of CD3+CD4+IFN-r in the sample wells was 0.15%. Figure 1 Without the addition of CD49d and CD28 co-stimulatory antibodies, the expression intensity percentage of CD3+CD4+IFN-r was 0.01%. Figure 2 Without the addition of CD49d and CD28 co-stimulatory antibodies, the expression intensity percentage of CD3+CD4+IFN-r was 0.02%. Figure 3 The percentage of expression intensity of CD3+CD4+IFN-r in the negative unrelated peptide control well was 0.03%. Figure 4 Without the addition of CD49d and CD28 co-stimulatory antibodies, the expression intensity percentage of CD3+CD4+IFN-r was 0.00%. Figure 5 ).

[0096] The results showed that under the co-stimulation of the co-stimulatory antibody, the signal of the intracellular cytokine IFN-r was more concentrated and obvious, and the co-stimulatory antibody could stimulate and amplify the cell's ability to secrete factors.

[0097] Experiment 4: Effect of Co-stimulatory Antibody Stimulation Time on Cytokine Expression

[0098] After resuscitation of healthy volunteer samples, specific peptide pool 1 and co-stimulatory antibody 1ug / ml Ultra-LEAF were added to the sample wells. TM Purified anti-human CD49d and 1ug / ml Ultra-LEAF TM Purified anti-human CD28 was placed in a 37°C, 5% CO2 incubator and cultured for 6H, 12H and 18H respectively. All other steps were the same as steps 2 to 10 of Example 1. The percentage of expression intensity of each cytokine is shown in Table 8.

[0099] Table 8

[0100] CD4+IFN-γ+ 0.46％ 0.01％ 0％ CD4+IL5+ 0.04％ 0.01％ 0.15％ CD4+TNF-α+ 0.05％ 0.03％ 0.06％ CD4+IL-4+ 0.93％ 0.9％ 0.62％ CD3+CD4+IL-2+ 0.23％ 0.08％ 0.28％ CD8+IFN-γ+ 0.39％ 0.02％ 0％ CD8+TNF-α+ 0.01％ 0％ 0.01％ CD8+IL-2+ 0.05％ 0％ 0％

[0101] As shown in Table 8, the ability of cells to amplify secreted factors is best when 1 μg / ml of CD49d and CD28 co-stimulatory antibody is added and cultured for 6 hours.

[0102] Experimental Example 5: The effect of the reactive dye FixedViability Stain 700 on the detection results

[0103] In step (3) of Example 1, the active dye Fixable Viability Stain 700 was added, and the flow cytometry effect of intracellular cytokines CD3+CD4+IL-4 was observed (all other steps were the same as in Example 1). Figure 6 As shown, after removing dead cells by using the active dye FixableViability Stain 700, only clean intracellular cytokine IL-4 signaling remained in the positive area.

[0104] In step (3) of Example 1, without the addition of the active dye Fixable Viability Stain 700, the flow cytometry results of intracellular cytokines CD3+CD4+IL-4 (other steps were the same as in Example 1) are as follows: Figure 7 As shown, a large number of dead cells were observed in the positive area, indicating the presence of the intracellular cytokine IL-4.

[0105] Therefore, adding the active dye Fixed Viability Stain 700 can remove dead cells, reduce experimental background signal, and avoid false positive results.

[0106] Experiment 6: Effects of Cell Fixation and Membrane Permeation on Detection Results

[0107] 1. Add protein transport blocker

[0108] After resuscitation of healthy volunteer samples, specific peptide pool 1 and co-stimulatory antibody 1ug / ml Ultra-LEAF were added to the sample wells. TM Purified anti-human CD49d and 1ug / ml Ultra-LEAF TM Purified anti-human CD28 was placed in an incubator at 37°C with 5% carbon dioxide and cultured for 6 hours.

[0109] During the last 4 hours of stimulation culture (i.e., after 2 hours of culture), the culture dish was removed from the CO2 incubator and a protein transport inhibitor (brevidin A, 4uL per 6mL of cell culture system (~10^6 cells / mL)) was added, and the dish was placed back into the CO2 incubator for further culture.

[0110] 2. Cell collection and washing

[0111] After stimulation culture, remove the culture dish from the CO2 incubator, thoroughly pipette the cells, and transfer the cells from the culture dish to a flow cytometry tube; centrifuge at 1700 rpm for 5 min, and discard the supernatant; add 1 ml of phosphate buffer (dissolve one packet of phosphate powder in 500 mL of deionized water at room temperature and adjust the pH to 7.2 ± 0.2), centrifuge at 1700 rpm for 5 min, gently pipette to mix, and discard the supernatant.

[0112] 3. Staining on the cell membrane

[0113] Add APC-ALexaFluor 750CD3 (10ul) and PE-Cy TM 7 CD8 (5ul), BV510 CD45 (5ul), and Fixed Viability Stain 700 (4ul) were mixed. The mixture was incubated at 4°C in the dark for 30 minutes. 2 ml of phosphate buffer containing 1% newborn calf serum was added (newborn calf serum was inactivated in a 56°C water bath for 30 minutes, and a certain amount of phosphate buffer was added to make the final concentration 1%). The mixture was gently pipetted and aspirated to mix. The mixture was centrifuged at 1700 rpm for 5 minutes, and the supernatant was discarded. The washing was repeated once, and the supernatant was discarded again.

[0114] 4. Fixation and membrane permeation treatment

[0115] Add 500 μL of Fixation / Permeabilization solution, gently shake to fix the cells, and incubate at 4°C in the dark for 20–30 minutes (permeabilization treatment). Centrifuge at 1700 rpm for 5 minutes and discard the supernatant.

[0116] Prepare BD Perm / Wash buffer (BD Perm / Wash buffer stock solution: distilled water = 1:9), add 1 ml of BD Perm / Wash buffer and mix well by pipetting and aspirating, then divide into two equal tubes, labeling them as test tubes and isotype control tubes respectively (setting up test tubes and isotype control tubes eliminates false positive interference caused by non-specific binding of antibodies to cells), centrifuge at 1700 rpm for 5 min, and discard the supernatant;

[0117] 5. Cell cytoplasmic staining

[0118] a. Add the following monoclonal antibody to the test tube and incubate at 4°C in the dark for 30 minutes.

[0119] FITC IFN-γ(20ul), PE IL-5(5ul), PerCP-Cy TM5.5 TNF(5ul), APC IL-4(5ul), BV421 IL-2(5ul), BV605 CD4(5ul)

[0120] b. Add the following monoclonal antibody (using immunoglobulin of the same source, label and subtype as the fluorescently labeled antibody to eliminate background staining caused by nonspecific binding of the antibody to the cell surface) to the isotype control tube and incubate at 4°C in the dark for 30 minutes.

[0121] FITC IgG1 (20ul), PE IgG1 (5ul), PerCP-Cy TM 5.5 IgG1(5ul), APC IgG1(5ul), BV421IgG2a(5ul), BV605 CD4(5ul)

[0122] 6. On-machine testing

[0123] After fixation and permeabilization, the test cells were washed with 1 ml of BD Perm / Wash buffer, centrifuged at 1700 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 400 μL of BD Perm / Wash buffer. The expression of each cytokine was detected by flow cytometry. The flow cytometry results of intracellular cytokines CD3+CD4+IFN-γ in the test tube and the isotype control tube are shown below. Figure 8 As shown. The positive rate was calculated by subtracting the positive rate of isotype controls from the positive rate of intracellular cytokines, resulting in an IFN positive rate of 15.60% - 0.02% = 15.58%.

[0124] The flow cytometry effects of adding anti-human CD4 antibody before and after immobilization permeabilization are as follows: Figure 9 and Figure 10 As shown. Figures 9-10 The results showed that when anti-human CD4 antibody was added before fixation and permeabilization, it would bind to CD4 on the cell membrane, and cell clustering was not obvious. This was considered to be an antigen endocytosis phenomenon caused by the influence of phorbol ester stimulant and protein transport blocker. After fixation and permeabilization, when anti-human CD4 antibody was added, it would bind to cytoplasmic CD4, and CD4 signal could be detected better.

[0125] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for detecting the expression level of immune cytokines in the body after vaccination, characterized in that, Includes the following steps: (1) When the frozen peripheral blood mononuclear cell sample to be tested is at rest for 0 h, add the specific peptide pool, co-stimulatory antibodies CD49d and CD28, and culture for 5-7 h; the nucleotide sequence of the specific peptide pool is shown in SEQ ID NO:1~SEQ ID NO:67 or SEQ ID NO:68~SEQ ID NO:

110. (2) Add protein transport inhibitor 4 hours before the end of culture; after the culture is completed, thoroughly pipette the cells and transfer them to flow cytometry tubes; centrifuge and discard the supernatant, then add phosphate buffer, centrifuge and discard the supernatant again; (3) Add immune cell surface marker antibodies CD3, CD8 and CD45, and Fixed ViabilityStain 700 to the flow cytometer and mix well; incubate at 3~5 ℃ in the dark for 25~35 min; add phosphate buffer containing newborn calf serum, gently pipette to mix, centrifuge and discard the supernatant, wash again, centrifuge and discard the supernatant. (4) After fixing the cells with cell permeabilization reagent and performing permeabilization treatment, the cells were divided into detection tubes and isotype control tubes. Monoclonal antibody reagent combination 1 and monoclonal antibody reagent combination 2 were added to the detection tubes and isotype control tubes, respectively, and incubated at 3-5°C in the dark for 25-35 min. Monoclonal antibody reagent combination 1 includes: FITC IFN-γ, PE IL-5, and PerCP-Cy. TM 5.5 TNF, APC IL-4, BV421 IL-2, BV605 CD4; the monoclonal antibody combination reagent 2 includes: FITC IgG1, PE IgG1, PerCP-Cy TM 5.5 IgG1, APC IgG1, BV421 IgG2a, BV605 CD4; (5) The expression levels of immune cytokines in frozen peripheral blood mononuclear cell samples were detected by flow cytometry.

2. The method for detecting the expression level of immune cytokines in the body after vaccination according to claim 1, characterized in that, The working concentration of the specific peptide pool in step (1) is 1ug / ml to 4ug / ml.

3. The method for detecting the expression level of immune cytokines in the body after vaccination according to claim 1, characterized in that, The working concentrations of CD49d and CD28 are both 0.8~1.2 ug / ml.

4. The method for detecting the expression level of immune cytokines in the body after vaccination according to any one of claims 1 to 3, characterized in that, In step (3), the amounts of the immune cell surface marker antibodies CD3, CD8, and CD45 added are 10 μL, 5 μL, and 5 μL, respectively; and / or, in step (5), the amounts of FITC IFN-γ, PE IL-5, and PerCP-Cy are added. TM 5.5 The amounts of TNF, APC IL-4, BV421 IL-2, and BV605 CD4 added were 20 μL, 5 μL, 5 μL, 5 μL, and 5 μL, respectively; the amounts of FITC IgG1, PE IgG1, and PerCP-Cy were also added. TM 5.5 The amounts of IgG1, APC IgG1, BV421 IgG2a, and BV605 CD4 added were 20 μL, 5 μL, 5 μL, 5 μL, and 5 μL, respectively.

5. The method for detecting the expression level of immune cytokines in the body after vaccination according to any one of claims 1 to 3, characterized in that, The culture time mentioned in step (1) is 5.5~6.5 h.

6. The method for detecting the expression level of immune cytokines in the body after vaccination according to claim 5, characterized in that, The incubation time in step (1) is 6 hours.

7. The method for detecting the expression level of immune cytokines in the body after vaccination according to any one of claims 1 to 3, characterized in that, In step (1), the food is placed in an incubator at 36~38℃ and 4~6% carbon dioxide for incubation.

8. The method for detecting the expression level of immune cytokines in the body after vaccination according to any one of claims 1 to 3, characterized in that, The protein transport inhibitor mentioned in step (2) is brefiltrant A or monensin, and 1.5~2.5 uL of protein transport inhibitor is added to every 3 mL of cell culture system; and / or the centrifugation speed mentioned in steps (2) and (3) is 1600~1800 rpm and the centrifugation time is 4~6 min.

9. The method for detecting the expression level of immune cytokines in the body after vaccination according to any one of claims 1 to 3, characterized in that, The permeabilization process described in step (3) includes the following steps: incubation at 3~5 ℃ in the dark for 20~30 minutes.

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