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ELISPOT Assay to Measure Peptide-specific IFN-γ Production
采用ELISPOT测定法测定肽特异性IFN-γ的生成   

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Abstract

Interferon-gamma (IFN-γ) is crucial for immunity against intracellular pathogens and for tumor control. It is produced predominantly by natural killer (NK) and natural killer T cells (NKT) as well as by antigen-specific Th1 CD4+ and CD8+ effector T cells. When investigating immune responses against pathogens and cancer cells, measuring antigen-specific cytokine-responses by cells of adaptive immunity offers an advantage over total non-specific cytokine responses. Significantly, the measurement of antigen-specific IFN-γ responses against pathogens or cancer cells, when compared to a treatment group, provides a quantitative measure of how well the treatment works. Measuring antigen-specific IFN-γ responses involves culture of the cells being considered (CD4+ or CD8+ T cells) with antigen presenting cells (APC) and a specific peptide from the target pathogen or cancer cell compared to control cultures without a peptide. After a suitable timeframe, the cytokine released is measured by an ELISPOT assay. The difference in the number of cells secreting IFN-γ, with and without peptide, is a measure of antigen-specific IFN-γ responses. This assay can be applied to other cytokines such as IL-10.

Keywords: IFN-gamma(IFN-γ), ELISPOT(ELISPOT), Antigen-specific(抗原特异性), Peptide-specific(肽特异性), T cells(T细胞)

Background

Interferon gamma (IFN-γ) is a dimerized soluble cytokine that is the only member of the type II class of interferons (Gray and Goeddel, 1982). IFN-γ has anti-pathogen, immuno-regulatory, and anti-tumor properties (Schroder et al., 2004) which promotes NK cell activity, increase in antigen presentation, activates inducible nitric oxide synthase, induces the production of IgG2a from activated plasma cells and promotes Th1 differentiation by up-regulating the transcription factor T-bet.

Given the significant role of this cytokine in immune responses, there are several protocols to quantify IFN-γ. Perhaps the simplest measure is an ELISA assay which is used to measure levels of the cytokine in serum samples and tissue culture supernatants by capturing the cytokine with antibodies (Schreiber, 2001). There is also a flow cytometry based-assay where intracellular IFN-γ is detected by flow cytometry following cell-permeabilization (Andersson et al., 1988). The percentage of cells containing the cytokine is usually low, and does not indicate if the protein is functional, if it would be secreted and does not measure if it is in response to a specific target antigen or multiple antigens.

To measure IFN-γ responses to specific antigens, culture assays were developed. Here, CD4+ and CD8+ T cells were stimulated in culture with APC and peptides from the target protein and the supernatants were tested by ELISA for IFN-γ levels (Bradley et al., 1991). Recently, instead of ELISA, several commercial flow cytometry-based bead array assays (e.g., BD Biosciences) are available which offer greater sensitivity to detect low cytokine levels at the nanogram level. However, while the assay can quantify total cytokine secreted, it does not differentiate between a few cells producing a lot of cytokine from a large number of cells secreting little cytokine. The number of cells secreting the cytokine quantifies the number of cells committed to a specific target of immunity. Thus, the enzyme-linked immunospot (ELISPOT) assay is a highly sensitive immunoassay that measures the frequency of cytokine-secreting cells at the single-cell level. An antigen-specific ELISPOT assay allows the quantification of the number of a specific cell type (CD4+ or CD8+ T cells) which secretes IFN-γ in response to a specific antigen (Carvalho et al., 2001; Schmittel et al., 2001)

The IFN-γ–specific antibody on an ELISPOT plate captures the IFN-γ immediately after secretion from the cells with a limit of detection typically around 1 in 100,000 cells. The high sensitivity of the assay makes it particularly useful for studies of the small population of cells found in specific immune responses (Horne-Debets et al., 2013 and 2016; Karunarathne et al., 2016).

Materials and Reagents

  1. Nitrile gloves
  2. Sterile 15 ml and 50 ml polypropylene tubes
  3. Disposable sterile pipettes: 2 ml, 5 ml, 10 ml, 25 ml
  4. Filters for syringes: 0.45 μM and/or 0.22 μM
  5. Multiscreen HTS-IP plates (PVDF membrane) (Merck, catalog number: MSIPS4510 )
  6. Mouse (BioLegend, catalog number: 575402 ) or human (Thermo Fisher Scientific, GibcoTM, catalog number: PHC0026 ) recombinant IL-2
  7. Microbeads (Miltenyi Biotec)
    Note: Catalog numbers depend on the cell type to be tested.
  8. Dynabeads magnetic beads (Thermo Fisher Scientific, USA, https://www.thermofisher.com/kr/en/home/brands/product-brand/dynal.html)
    Note: Catalog numbers depend on the cell type tested.
  9. Ethanol
  10. Peptides or antigen from the protein of choice to stimulate antigen-specific IFN-γ
  11. Rat anti-mouse IFNγ mAb, clone AN18, purified (capture) (50 μg, Thermo Fisher Scientific, eBioscienceTM, catalog number: 14-7313-81 or 500 μg, Thermo Fisher Scientific, eBioscienceTM, catalog number: 14-7313-85 ) mouse anti-human IFNγ mAb, p clone NIB42, purified (capture) (50 μg, BioLegend, catalog number: 502403 or 500 μg, BioLegend, catalog number: 502404 )
    Note: Either titrate or use as recommended by manufacturer. Prepare immediately before coating wells.
  12. Rat anti-mouse IFNγ mAb, clone R4-6A2, biotinylated (detection) (50 μg, Thermo Fisher Scientific, eBioscienceTM, catalog number: 13-7312-81 or 500 μg, Thermo Fisher Scientific, eBioscienceTM, catalog number: 13-7312-85 ) or mouse anti-human IFNγ mAb, clone 4S.B3, biotinylated (detection) (50 μg, BioLegend, catalog number: 502503 or 500 μg, BioLegend, catalog number: 502504 )
    Note: Either titrate or use as recommended by manufacturer.
  13. 0.5% BSA
  14. Streptavidin-horseradish peroxidase (BioLegend, catalog number: 405210 )
  15. 3-amino-9-ethylcarbazole (AEC) substrate/chromogen (BD, catalog number: 551951 )
  16. Milli-Q water
  17. Sodium chloride (NaCl)
  18. Potassium chloride (KCl)
  19. Sodium phosphate dibasic (Na2HPO4)
  20. Potassium phosphate monobasic (KH2PO4)
  21. Hydrochloric acid (HCl)
  22. Sodium bicarbonate (NaHCO3)
  23. Sodium phosphate (Na2CO3)
  24. IMDM-1640
  25. Fetal calf serum (FCS)
    Note: Any brand that is suitable for cell culture and is heat inactivated at 56 °C for 30 min.
  26. Penicillin-streptomycin (Tissue culture grade; Life technologies)
  27. β-mercaptoethanol
  28. Tween 20 (store at room temperature)
  29. IMDM culture medium
  30. L-glutamine (Tissue culture grade; Life technologies)
  31. Phytohemagglutinin-L (PHA, positive control) stock (Roche Diagnostics, catalog number: 11249738001 ). Refer to Recipes section for details
  32. Sterile phosphate buffered saline (1x PBS) (see Recipes)
  33. Coating buffer working solution (see Recipes)
  34. Blocking solution (see Recipes)
  35. Anti-IFN-γ capture working solution (see Recipes)
  36. Biotin-anti-IFN-γ working solution (see Recipes)
  37. Streptavidin-horseradish peroxidase working solution (see Recipes)
  38. Tween 20 working solution (see Recipes)
  39. 10% fetal calf serum in IMDM culture medium (see Recipes)
  40. PHA stock solution (see Recipes)

Equipment

Note: ELISPOTS on the plate can be manually counted under a dissection microscope, or stereomicroscope (For example, Greenough, high-performance zoom stereomicroscope, SMZ 168-series). Alternatively, there are several specialist automated systems for high throughput screening (AELVIS, Autoimmun Diagnostika, Bio-Sys, Cellular Technology and the Zeiss reader) and there pros and cons discussed elsewhere and beyond the scope of this protocol (Janetzki et al., 2015).

  1. Waste container
  2. Gilson pipette and tips: P-2, P-10, P-20, P200, P1000
  3. Gilson multichannel pipette with matched tips
  4. Beckman Allegra 12 refrigerated centrifuge (Beckman Coulter, model: Allegra X-12 )
  5. Class II biohazard hood
  6. Incubator
  7. 1 L bottles

Procedure

There are two ways to measure cell type-specific responses. Firstly, isolate total CD4+ and/or CD8+ T cells and culture with dendritic cells (DCs) and the peptide of choice (Karunarathne et al., 2016). T cells are generally enriched from experimental mice using bead based techniques such as Dynabeads magnetic beads (Untouched Mouse T cells kit; Life Technologies, US) or Miltenyi Biotec (CD90.2 T cell isolation; Germany). DCs are immuno-magnetically isolated from naive mice (for use as antigen-presenting cells; APC) using anti-mouse CD11c Microbeads (Miltenyi Biotec; Germany). DCs should not be isolated from experimental mice where DC function is known to be compromised. Both cell types (T cells and DCs) can also be isolated using flow cytometry-based cell sorting. Approximately 2 x 105 T cells, from individual mice, should be co-cultured with 2 x 104 DC, in 4-8 replicate wells per sample with 20 μg/ml peptides, recombinant protein or no peptide, as previously described (Howland et al., 2013). Alternatively, if peptides are specific for CD4+ but not CD8+ T cells and vice versa, then total spleen cell with no additional DCs or total T cells with DCs may be used. These combined cells are then cultured with the specific peptide on ELISPOT plates coated with anti-IFN-γ antibody. The IFN-γ-specific antibody on an ELISPOT plate captures the IFN-γ immediately after secretion from the cells. For human T cells, total PBMCs containing APC and T cells are cultured with the peptide of choice. For the positive control, just add PHA to total cells or isolated T cells without DCs.

  1. Day 1
    1. Coat Multiscreen HTS-IP plates with primary antibody diluted in coating buffer:
      Before coating the plate with antibody, humidify the membrane in each well with 15 μl of 70% ethanol (in Milli-Q water) for 1 min. Rinse with 150 μl sterile PBS three times before the ethanol evaporates. Then coat plates with 50 μl anti-IFNγ antibody (capture) in sterile coating buffer. Incubate overnight at 4 °C.
      Notes:
      1. Plate may be coated 1 week prior to use but must be stored in humid conditions (e.g., box with tissue drenched in sterile water or PBS).
      2. The number of wells coated with antibody should take into account the all controls listed below and at least 4-8 replicate wells for each sample.
      3. If possible, it is recommended that a titration of cell numbers (Figure 1), in replicates, be tested to ensure an optimal range of cells for counting.
      4. Ensure that sodium azide is never added to any buffer, at any step as this stops horseradish peroxidase activity.


        Figure 1. A representative example of an ELISPOT assay. A titration of cell numbers from negative and positive controls and 2 test samples. Test 1 shows no titration with dilution and thus and cell numbers are similar to negative control. Test 2 has declining number of cells with titration indicating IFN-γ-secreting cells.

    2. The following control wells should be incorporated into the assay:
      1. No antigen stimulation (DC and T cells cultured without peptide).
      2. Positive control with a lymphocyte mitogen (T cells without DC but with mitogen like PHA at 5-10 μg/ml).
      3. No cells; no primary antibody.

  2. Day 2
    1. Block membrane with blocking solution.
      1. Remove primary antibody solution.
      2. Wash off unbound antibody with 150 μl sterile PBS per well, at least twice.
      3. Block membrane with 150 μl per well of cell culture medium (see Recipes) for at least 2 h at 37 °C.
        Note: Keep changing media if it goes deep pink, until orange.
    2. Prepare mouse splenocytes (MacPherson et al., 2001; Wykes et al., 2007) or isolate T cells/DCs, suspended in cell culture medium (see Recipes). Plan for each sample to have 4-8 replicate wells.
      1. For assays using total splenocytes or PBMCs, cells are resuspended in cell culture medium, at final concentrations range of 2 x 106 to 6 x 106 cells/ml for testing at possible testing range of 1 x 105 to 3 x 105 splenocytes or PBMCs per well.
      2. Isolated T cells are usually resuspended at 4 x 106 cells/ml for testing 2 x 105 T cells cultured with 2 x 104 DC per well.
        Note: The desired cell concentration is dependent on the intensity of the immune response. If the expected response is not known, a serial dilution of cell concentrations is recommended.
      3. Antigens are diluted (20 μg/ml) in culture medium eventually containing recombinant IL2 (60 U/ml).
    3. Cell stimulation
      1. Discard blocking medium.
      2. Add 50 μl of the cell suspension and 50 μl of diluted peptide (antigen) per well (final volume: 100 μl/well, corresponding to 10 μg/ml for the antigen and 30 U/ml for recombinant IL2).
        Note: To minimize overseeding of the wells, it is recommended to not add more than 3 x 105 cells/well.
      3. Incubate for 18 to 24 h at 37 °C, 5% CO2, 95% humidity.
        Note: Do not move the plates while the cells are culturing. This will lead to ‘snail trail’ spots that will not be well defined. Don’t stack the plates if you have more than one to prevent edge effects.

  3. Day 3
    1. Secondary antibody
      1. Remove culture medium with cells.
      2. Wash plate 3 times with PBS and 3 times with PBS/0.01% Tween 20.
        Note: Ensure you include Tween 20 in the wash buffer. Some cells will have started attaching after culture overnight. Tween 20 will help wash these off the membrane. Do not use a plate washer at this stage if available.
      3. Dilute biotinylated anti-IFNγ antibody (detection) in PBS/0.5% BSA. Pass the detection antibody mixture through a 0.45 μm or 0.22 μm filter to remove aggregates. Some of the mixture will be lost by filtration and original volume must be scaled up accordingly. Add 50 μl of detection antibody mixture to each well.
        Note: Failure to filter secondary antibody may results in non-specific spot formation due to protein aggregates.
      4. Incubate for 2 h at 37 °C, 5% CO2, 95% humidity.
      5. Then incubate overnight at 4 °C, in a sealed box with tissues moistened with sterile water or PBS which provide humidity.

  4. Day 4
    1. Wash plate 6 times with PBS/0.01% Tween 20.
    2. Enzyme conjugate and substrate development
      1. Prepare streptavidin-horseradish peroxidase dilution in PBS.
      2. Add 50 μl per well of streptavidin-horseradish peroxidase. Incubate for 45 min at room temperature.
        Note: Exceeding 1 h incubation with enzyme conjugate could result in increased background color.
      3. Remove streptavidin solution, wash 3 times with PBS/0.01% Tween 20, followed by 3 washes with PBS.
        Notes:
        1. The final washes with only PBS are important as Tween 20 will interfere with the spot development.
        2. The plastic base should be taken off the bottom of the plate to enable thorough washing of the membrane before adding substrate/chromogen. For example, after incubation with the streptavidin horseradish peroxidase conjugate, remove the base and wash both sides of the membrane under running distilled water. This helps to prevent high background as some reagents can leak through the membrane into the bottom tray of the plate.
      4. Add 75 μl/well AEC substrate (after passing through 0.45 μM or 0.22 μM filter). Incubate for 5 min in the dark (under aluminum foil or in a drawer) and then check for spots. If spots are not clearly visible, incubate again and check regularly. Stop spot development when spots are clearly visible but before background membrane color becomes too dark and the contrast between spot and membrane color is lost.
        Note: Optimization of the time of substrate development is critical. Time of development may vary. Over-development will result in increased background.
      5. Stop spot development using running tap water and wash extensively. While washing, remove the bottom of the plate and continue rinsing.
      6. Press the plate thoroughly to an absorbent tissue paper.
      7. Let plate dry overnight in the dark. Spot intensity may decrease with exposure to light.
        Note: Spots may become sharper if membranes are stored overnight. If storing, wrap membranes in foil.

Data analysis

  1. Spots are counted under a dissection microscope or in an automated ELISPOT reader, and the frequency of secreting cells is calculated:
    One cell = One spot
    Number of spots = number of cells secreting cytokines
    Intensity and size of spot = relative cytokine-secreting ability of cells
  2. Background subtraction
    For ELISPOT data analysis, the background value is subtracted from measured results. Each specimen group should have a single background spot mean/median value, calculated from the negative control (T cells and DCs without antigen). Then for each antigen group in the sample, the mean/median for the group is calculated, the background mean/median is then subtracted and the count is normalized by the number of cells per well.
  3. Assay
    1. Positive should be > 50 spots/106 cells.
    2. Negative control should ideally be 0 spots/106 cells. If > 0 and < 50 spots/106 cells, subtract the negative control reading from the test count to correct for background staining. If > 50 spots/106 cells, repeat the assay.
    3. Contamination:
      1. If one of the replicate wells of one assay condition clearly appears contaminated, ignore that particular well and use the result from the other wells.
      2. If multiple wells clearly appear to be contaminated, repeat the assay.
  4. Results
    For each test antigen, results are reported as spot forming cells (SFC)/million for human PBMC or SFC/spleen for mouse spleen cells.

Notes

  1. As horseradish peroxidase activity is completely stopped by sodium azide, please ensure it is never added to any buffer or reagents associated with this assay.
  2. There are several brands of antibodies available that are suitable for this assay and can even be purchased as antibody pairs. However, each new antibody vial should be tested in a checkerboard titration to determine the optimal dilution (Asai, 2000 #14220).

Recipes

  1. Sterile phosphate buffered saline (1x PBS)
    Start with 800 ml of distilled water, to which add 8 g of NaCl; 0.2 g of KCl; 1.44 g of Na2HPO4 and 0.24 g of KH2PO4
    Adjust the pH to 7.4 with HCl
    Add distilled water to a total volume of 1 L
    Stock is stored in 1 L bottles
    Store at room temperature
  2. Coating buffer working solution (0.5 M carbonate-bicarbonate buffer pH 9.6)
    3.7 g NaHCO3 and 0.6 g Na2CO3 (anhydrous) made up to 100 ml with distilled water
    Make sure that the carbonate bicarbonate is dissolved by gently mixing it until there is no powder residue left and filter sterilized with 0.22 μM filter
    Check pH which should be ~9.6 and occasionally needs to be adjusted
    Store at room temperature in the dark
  3. Blocking solution
    IMDM culture medium
    10% fetal calf serum
    1% penicillin-streptomycin
    0.1% β-mercaptoethanol
  4. Anti-IFN-γ capture working solution
    This is the capture antibody. Prepare immediately before coating wells. Need 50 μl per well, at a concentration according to manufacturer's instruction or by titration for each antibody stock
  5. Biotin-anti-IFN-γ working solution
    This is the detection antibody. Prepare as in PBS. Need 50 μl per well, at a concentration according to manufacturer's instruction or by titration for each antibody stock
  6. Streptavidin-horseradish peroxidase working solution
    Prepare a dilution of the stock in PBS. Need 50 μl per well, at a concentration according to manufacturer's instruction or by titration for each antibody stock
  7. Tween 20 working solution
    Add 0.5 ml Tween 20 stock to 1,000 ml 1x PBS
    Store at room temperature
  8. 10% fetal calf serum in IMDM culture medium (store at 4 °C in the dark)
    1. Fetal calf serum stock
      Thaw and dispense into 50 ml aliquots. Store at -20 °C
    2. IMDM culture medium (store at 4 °C in the dark)
    3. L-glutamine stock
      Supplied as 50 ml at 200 mM concentration. Store at -20 °C
      Thaw and dispense into 1 or 5 ml aliquots depending on volume of culture medium to be prepared later. Store at -20 °C in the dark until needed
    4. Penicillin-streptomycin (5,000 U/ml)
      Supplied as 100 ml at 5,000 U/ml concentration. Store at -20 °C
      Thaw and dispense into 1 or 5 ml aliquots depending on volume of culture medium to be prepared later. Store at -20 °C in the dark until needed
    5. 10% fetal calf serum in IMDM culture medium (complete medium)
      For 100 ml: add 10 ml FCS (a), 1 ml L-glutamine (c), 1 ml penicillin-streptomycin (d), 100 μl β-mercaptoethanol, and to 88 ml IMDM (b). Store at 4 °C in dark
  9. PHA stock solution
    Add to 10 ml PBS to the PHA powder for a concentration of 500 μg/ml
    Aliquot and freeze in volumes of 10 μl. Store at -80 °C
    Notes:
    1. Do not re-freeze thawed PHA.
    2. PHA (Lectin, positive control) stock is supplied as 5 mg of purified, salt-free lyophilized powder.
    3. In the assay, use at a final concentration of 5-10 μg/ml. Add 1-2 μl of stock solution to each well containing cells in a 100 μl volume

Acknowledgments

MNW was supported by ARC Future Fellowship and NHMRC. L.R. was supported by Singapore’s A*STAR and NRF Singapore (NRF2007NRF-RF001-226).The authors wish to thank Mr Josh Horne-Debets for the ELISPOT image.
The ELISPOT method was originally developed to detect antigen-specific antibodies from B cells (Czerkinsky et al., 1983) and has over the years been modified by several groups to detect cytokines produced by antigen-specific cells.

References

  1. Andersson, U., Hallden, G., Persson, U., Hed, J., Moller, G. and DeLey, M. (1988). Enumeration of IFN-γ-producing cells by flow cytometry. Comparison with fluorescence microscopy. J Immunol Methods 112(1): 139-142.
  2. Bradley, L. M., Duncan, D. D., Tonkonogy, S. and Swain, S. L. (1991). Characterization of antigen-specific CD4+ effector T cells in vivo: immunization results in a transient population of MEL-14-, CD45RB- helper cells that secretes interleukin 2 (IL-2), IL-3, IL-4, and interferon gamma. J Exp Med 174(3): 547-559.
  3. Carvalho, L. H., Hafalla, J. C. and Zavala, F. (2001). ELISPOT assay to measure antigen-specific murine CD8+ T cell responses. J Immunol Methods 252(1-2): 207-218.
  4. Czerkinsky, C. C., Nilsson, L. A., Nygren, H., Ouchterlony, O. and Tarkowski, A. (1983). A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65(1-2): 109-121.
  5. Gray, P. W. and Goeddel, D. V. (1982). Structure of the human immune interferon gene. Nature 298(5877): 859-863.
  6. Horne-Debets, J. M., Faleiro, R., Karunarathne, D. S., Liu, X. Q., Lineburg, K. E., Poh, C. M., Grotenbreg, G. M., Hill, G. R., MacDonald, K. P., Good, M. F., Renia, L., Ahmed, R., Sharpe, A. H. and Wykes, M. N. (2013). PD-1 dependent exhaustion of CD8+ T cells drives chronic malaria. Cell Rep 5(5): 1204-1213.
  7. Horne-Debets, J. M., Karunarathne, D. S., Faleiro, R. J., Poh, C. M., Renia, L. and Wykes, M. N. (2016). Mice lacking Programmed cell death-1 show a role for CD8+ T cells in long-term immunity against blood-stage malaria. Sci Rep 6: 26210.
  8. Howland, S. W., Poh, C. M., Gun, S. Y., Claser, C., Malleret, B., Shastri, N., Ginhoux, F., Grotenbreg, G. M. and Renia, L. (2013). Brain microvessel cross-presentation is a hallmark of experimental cerebral malaria. EMBO Mol Med 5(7): 984-999.
  9. Janetzki, S., Price, L., Schroeder, H., Britten, C. M., Welters, M. J. and Hoos, A. (2015). Guidelines for the automated evaluation of Elispot assays. Nat Protoc 10(7): 1098-1115.
  10. Karunarathne, D. S., Horne-Debets, J. M., Huang, J. X., Faleiro, R., Leow, C. Y., Amante, F., Watkins, T. S., Miles, J. J., Dwyer, P. J., Stacey, K. J., Yarski, M., Poh, C. M., Lee, J. S., Cooper, M. A., Renia, L., Richard, D., McCarthy, J. S., Sharpe, A. H. and Wykes, M. N. (2016). Programmed death-1 ligand 2-mediated regulation of the PD-L1 to PD-1 axis is essential for establishing CD4+ T cell immunity. Immunity 45(2): 333-345.
  11. MacPherson, G. G., Wykes, M., Huang, F. P. and Jenkins, C. D. (2001). Isolation of dendritic cells from rat intestinal lymph and spleen. In: Robinson, S. P. and Stagg, A. J. (Eds.). Dendritic Cells Protocols. Humana Press.
  12. Schmittel, A., Keilholz, U., Bauer, S., Kuhne, U., Stevanovic, S., Thiel, E. and Scheibenbogen, C. (2001). Application of the IFN-γ ELISPOT assay to quantify T cell responses against proteins. J Immunol Methods 247(1-2): 17-24.
  13. Schreiber, R. D. (2001). Measurement of mouse and human interferon gamma. Curr Protoc Immunol Chapter 6: Unit 6 8.
  14. Schroder, K., Hertzog, P. J., Ravasi, T. and Hume, D. A. (2004). Interferon-γ: an overview of signals, mechanisms and functions. J Leukoc Biol 75(2): 163-189.
  15. Wykes, M. N., Liu, X. Q., Jiang, S., Hirunpetcharat, C. and Good, M. F. (2007). Systemic tumor necrosis factor generated during lethal Plasmodium infections impairs dendritic cell function. J Immunol 179(6): 3982-3987.
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简介

干扰素-γ(IFN-γ)对细胞内病原体免疫和肿瘤控制至关重要。它主要由天然杀伤剂(NK)和天然杀伤T细胞(NKT)以及抗原特异性Th1 CD4 + / sup和CD8 + /效应T细胞产生。当研究针对病原体和癌细胞的免疫应答时,通过适应性免疫测量细胞的抗原特异性细胞因子反应提供了优于总非特异性细胞因子反应的优点。值得注意的是,与治疗组相比,抗病原体或癌细胞的抗原特异性IFN-γ反应的测量提供了治疗效果的定量测量。测量抗原特异性IFN-γ应答涉及用抗原呈递细胞(APC)和特异性肽从正在考虑的细胞(CD4 + sup +或CD8 + sup + T细胞)与没有肽的对照培养物相比,靶病原体或癌细胞。在合适的时间范围之后,通过ELISPOT测定来测量释放的细胞因子。有或无肽分泌IFN-γ的细胞数量的差异是抗原特异性IFN-γ应答的量度。该测定可以应用于其它细胞因子如IL-10。

背景 干扰素γ(IFN-γ)是二型可溶性细胞因子,其是II类干扰素的唯一成员(Gray和Goeddel,1982)。 IFN-γ具有抗病原体,免疫调节和抗肿瘤特性(Schroder等人,2004),其促进NK细胞活性,增加抗原呈递,激活诱导型一氧化氮合酶,诱导从激活的浆细胞产生IgG2a,并通过上调转录因子T-bet来促进T 1分化。
&NBSP;鉴于这种细胞因子在免疫应答中的重要作用,有几个方法来量化IFN-γ。也许最简单的措施是ELISA测定法,用于通过用抗体捕获细胞因子来测量血清样品和组织培养上清液中细胞因子的水平(Schreiber,2001)。还有一种基于流式细胞术的测定法,其中细胞内IFN-γ在细胞透化后通过流式细胞术检测(Andersson等人,1988)。含有细胞因子的细胞的百分比通常较低,并且不表示蛋白质是否是功能性的,如果其被分泌并且不测量其是否响应于特异性靶抗原或多种抗原。
&NBSP;为了测量对特定抗原的IFN-γ反应,开发了培养测定。在这里,来自靶蛋白的APC和肽的培养物刺激CD4 + sup + T细胞和CD8 + T细胞,并通过ELISA测定IFN-γ水平的上清液(Bradley < em>等人,1991)。最近,代替ELISA,可以使用几种基于商业流式细胞仪的珠阵列测定(例如BD Biosciences),其提供更高的灵敏度以在纳克级水平检测低细胞因子水平。然而,虽然该测定可以量化分泌的总细胞因子,但是它不会从分泌少量细胞因子的大量细胞中产生大量细胞因子的少数细胞分化。分泌细胞因子的细胞数量化了致力于特异性免疫目标的细胞数量。因此,酶联免疫斑点(ELISPOT)测定是高度灵敏的免疫测定,其测量单细胞水平的细胞因子分泌细胞的频率。抗原特异性ELISPOT测定允许定量分泌IFN-γ以响应于特异性的特异性细胞类型(CD4 + 或CD8 + T细胞)的数目抗原(Carvalho et al。,2001; Schmittel等人,2001)
&NBSP; ELISPOT板上的IFN-γ特异性抗体在从细胞分泌后立即捕获IFN-γ,检测限通常在100,000个细胞中约为1。该测定的高灵敏度使其对于在特异性免疫应答中发现的细胞群的研究特别有用(Horne-Debets等人,2013和2016; Karunarathne等人, em>。,2016)。

关键字:IFN-γ, ELISPOT, 抗原特异性, 肽特异性, T细胞

材料和试剂

  1. 丁腈手套
  2. 无菌15毫升和50ml聚丙烯管
  3. 一次性无菌移液管:2ml,5ml,10ml,25ml
  4. 注射器过滤器:0.45μM和/或0.22μM
  5. 多屏HTS -IP板(PVDF膜)(Merck,目录号:MSIPS4510)
  6. (BioLegend,目录号:575402)或人(Thermo Fisher Scientific,Gibco TM,目录号:PHC0026)重组IL-2
  7. 微珠(Miltenyi Biotec)
    注意:目录号码取决于要测试的单元格类型。
  8. Dynabeads磁珠(Thermo Fisher Scientific,USA, https://www.thermofisher.com/kr/en/home/brands/product-brand/dynal.html
    注意:目录号取决于测试的单元格类型。
  9. 乙醇
  10. 肽或抗原从选择的蛋白质刺激抗原特异性IFN-γ
  11. 大鼠抗小鼠IFNγmAb,克隆AN18,纯化(捕获)(50μg,Thermo Fisher Scientific,eBioscience ,目录号:14-7313-81或500μg,Thermo Fisher Scientific,eBioscience目录号:14-7313-85)小鼠抗人IFNγmAb,p克隆NIB42,纯化(捕获)(50μg,BioLegend,目录号:502403或500μg,BioLegend,目录号:502404)
    注意:按制造商的推荐滴定或使用。在涂布井前立即准备。
  12. 大鼠抗小鼠IFNγmAb,克隆R4-6A2,生物素化(检测)(50μg,Thermo Fisher Scientific,eBioscience ,目录号:13-7312-81或500μg,Thermo Fisher Scientific, eBioscience TM,目录号:13-7312-85)或小鼠抗人IFNγmAb,克隆4S.B3,生物素化(检测)(50μg,BioLegend,目录号:502503或500μg, BioLegend,目录号:502504)
    注意:按照制造商的推荐,滴定或使用。
  13. 0.5%BSA
  14. 链霉亲和素 - 辣根过氧化物酶(BioLegend,目录号:405210)
  15. 3-氨基-9-乙基咔唑(AEC)底物/色原体(BD,目录号:551951)
  16. Milli-Q水
  17. 氯化钠(NaCl)
  18. 氯化钾(KCl)
  19. 磷酸氢二钠(Na 2 HPO 4)
  20. 磷酸二氢钾(KH 2 PO 4)
  21. 盐酸(HCl)
  22. 碳酸氢钠(NaHCO 3)
  23. 磷酸钠(Na 2 CO 3 3)
  24. IMDM-1640
  25. 胎牛血清(FCS)
    注意:任何适合细胞培养并在56°C热灭活30分钟的品牌。
  26. 青霉素 - 链霉素(组织培养级;生命技术)
  27. β-巯基乙醇
  28. 吐温20(室温下储存)
  29. IMDM培养基
  30. L-谷氨酰胺(组织培养等级;生命技术)
  31. 植物凝集素-L(PHA,阳性对照)(Roche Diagnostics,目录号:11249738001)。有关详细信息,请参阅食谱部分
  32. 无菌磷酸盐缓冲盐水(1x PBS)(见食谱)
  33. 涂层缓冲液工作液(见配方)
  34. 阻塞解决方案(见配方)
  35. 抗IFN-γ捕获工作液(参见食谱)
  36. 生物素 - 抗IFN-γ工作液(参见食谱)
  37. 链霉亲和素 - 辣根过氧化物酶工作液(见配方)
  38. 吐温20工作液(见配方)
  39. 10%胎牛血清在IMDM培养基(见食谱)
  40. PHA储备溶液(参见食谱)

设备

注意:板上的ELISPOTS可以在夹层显微镜或立体显微镜下手动计数(例如,Greenough,高性能变焦立体显微镜,SMZ 168系列)。或者,有几个专门的高通量筛选自动化系统(AELVIS,Autoimmun Diagnostika,Bio-Sys,Cellular Technology和Zeiss reader),其他地方讨论的优缺点超出了本协议的范围(Janetzki et al。,2015 )。

  1. 垃圾箱
  2. Gilson移液器和提示:P-2,P-10,P-20,P200,P1000
  3. Gilson多通道移液器与匹配的提示
  4. Beckman Allegra 12冷冻离心机(Beckman Coulter,型号:Allegra X-12)
  5. II类生物危害罩
  6. 孵化器
  7. 1升瓶子

程序

有两种测量细胞类型特异性反应的方法。首先,分离总CD4 + / sup和/或CD8 + sup细胞并用树突状细胞(DC)和所选择的肽(Karunarathne等人。,2016)。 T细胞通常使用珠基技术如Dynabeads磁珠(Untouched Mouse T cells kit; Life Technologies,US)或Miltenyi Biotec(CD90.2T细胞分离;德国)从实验小鼠中富集。 DC使用抗小鼠CD11c Microbeads(Miltenyi Biotec; Germany)与幼稚小鼠(用作抗原呈递细胞; APC)免疫磁性分离。 DC不应与已知DC功能受损的实验小鼠分离。也可以使用基于流式细胞术的细胞分选来分离细胞类型(T细胞和DC)。来自各个小鼠的约2×10 5个T细胞应与2×10 4个DC共培养,每个样品的4-8个重复孔中,20μg/ ml肽,重组蛋白或无肽,如前所述(Howland等人,2013)。或者,如果肽对CD4 + T细胞而言不是特异性的,反之亦然,则可以使用没有额外的DC或具有DC的总T细胞的总脾细胞。然后将这些组合的细胞与特异性肽在用抗-IFN-γ抗体包被的ELISPOT平板上培养。 ELISPOT板上的IFN-γ特异性抗体从细胞分泌后立即捕获IFN-γ。对于人T细胞,含有APC和T细胞的总PBMC与选择的肽一起培养。对于阳性对照,只需将PHA添加到总细胞或没有DC的分离的T细胞。

  1. 第1天
    1. 涂覆缓冲液稀释一次抗体的多层HTS薄膜-IP板:
      在用抗体涂覆板之前,用15μl70%乙醇(Milli-Q水)将每个孔中的膜加湿1分钟。在乙醇蒸发之前用150μl无菌PBS冲洗3次。然后用无菌涂层缓冲液中的50μl抗-IFNγ抗体(捕获)涂覆平板。在4℃下孵育过夜。
      注意:
      1. 板可以在使用前一周涂覆,但必须储存在潮湿条件下(例如,用无菌水或PBS中浸湿的组织的盒子)。
      2. 涂有抗体的孔数应考虑下列所有对照和每个样品至少4-8个重复孔。
      3. 如果可能,建议对重复的细胞编号(图1)进行滴定,以确保计数细胞的最佳范围。

      4. 确保叠氮化钠不会添加到任何缓冲液中,任何步骤都会停止辣根过氧化物酶活性。

        图1. ELISPOT测定的代表性实例。从阴性和阳性对照和2个测试样品中滴定细胞数。测试1显示没有稀释滴定,因此细胞数量与阴性对照相似。测试2具有下降的细胞数,滴定指示IFN-γ分泌细胞。

    2. 以下对照孔应纳入试验:
      1. 无抗原刺激(DC和T细胞培养无肽)
      2. 淋巴细胞有丝分裂原阳性对照(T细胞无DC,但有丝分裂原如PHA为5-10μg/ ml)。
      3. 没有细胞;无一抗
  2. 第2天
    1. 阻塞膜与阻塞溶液
      1. 取出一抗溶液
      2. 用150μl无菌PBS /孔冲洗未结合的抗体,至少两次。
      3. 将细胞培养基(参见食谱)每孔150μl的阻断膜在37℃下至少2小时。
        注意:如果介质变粉红色,直到橙色,请继续更换。
    2. 准备小鼠脾细胞(MacPherson等人,2001; Wykes等人,2007),或分离悬浮在细胞培养基中的T细胞/ DC(参见食谱)。计划每个样本有4-8个重复井。
      1. 对于使用总脾细胞或PBMC的测定,将细胞重悬于细胞培养基中,终浓度范围为2×10 6至6×10 6细胞/ ml,用于在1×10 5至3×10 5个脾细胞或每个孔的PBMC的可能测试范围。
      2. 分离的T细胞通常以4×10 6个细胞/ ml重悬浮,用于测试用2×10 4个DC / DC培养的2×10 5个细胞每口
        注意:所需的细胞浓度取决于免疫应答的强度。如果预期的反应未知,建议连续稀释细胞浓度。
      3. 在最终含有重组IL2(60U / ml)的培养基中将抗原稀释(20μg/ ml)
    3. 细胞刺激
      1. 放弃阻挡介质。
      2. 加入50μl细胞悬浮液和50μl每孔稀释的肽(抗原)(最终体积:100μl/孔,对应于抗原为10μg/ ml,重组IL2为30U / ml)。
        注意:为了最大限度地减少井的播种,建议不要添加超过3×10 5 细胞/孔。
      3. 在37℃,5%CO 2,95%湿度下孵育18至24小时。
        注意:细胞培养时不要移动板。这将导致“蜗牛径”点不明确。如果您有不止一个以防止边缘效应,请不要堆叠板。

  3. 第3天
    1. 二抗
      1. 用细胞去除培养基
      2. 用PBS洗涤板3次,PBS / 0.01%吐温20洗3次 注意:确保在洗涤缓冲液中加入吐温20。一些细胞在培养过夜后已经开始附着。吐温20将有助于将其从膜上清洗掉。如果可用,请勿在此阶段使用洗板机。
      3. 在PBS / 0.5%BSA中稀释生物素化的抗-γγ抗体(检测)。将样品通过0.45μM或0.22μM过滤器去除聚集体。一些样品将通过过滤而丢失,原始体积必须相应地缩小。每孔加入50μl检测抗体 注意:由于蛋白质聚集,未能过滤二抗可能导致非特异性点形成。
      4. 在37℃,5%CO 2,95%湿度下孵育2小时。
      5. 然后在4℃下孵育过夜,在密封的盒子里,用无菌水或PBS湿润的组织湿润。

  4. 第4天
    1. 用PBS / 0.01%吐温20洗涤板6次。
    2. 酶结合物和底物发育
      1. 在PBS中制备链霉抗生物素蛋白 - 辣根过氧化物酶稀释液
      2. 每孔加入50μl链霉抗生物素蛋白 - 辣根过氧化物酶。在室温下孵育45分钟。
        注意:用酶偶联物孵育超过1小时可能会导致背景颜色增加。
      3. 去除链霉亲和素溶液,用PBS / 0.01%吐温20洗涤3次,然后用PBS洗涤3次。
        注意:

        1. 只有PBS的最后洗涤是重要的,因为吐温20会干扰现场发展。
        2. 塑料底座应从板的底部取下,以便在加入基质/色原体之前彻底清洗膜。例如,在与链霉抗生物素蛋白辣根过氧化物酶缀合物孵育后,除去碱并在运行的蒸馏水下洗涤膜的两侧。这有助于防止高背景,因为一些试剂可能通过膜泄漏到板的底部托盘中。
      4. 加入75μl/孔AEC底物(经过0.45μM或0.22μM过滤)。在黑暗中孵育5分钟(铝箔或抽屉内),然后检查斑点。如果斑点不清楚,请再次孵育并定期检查。当斑点清晰可见但背景膜颜色变得太暗并且斑点和膜颜色之间的对比度丢失时,停止斑点发育。
        注意:优化底物开发的时间至关重要。发展时间可能会有所不同。过度开发将导致背景增加。
      5. 停止现场开发使用自来水和洗涤广泛。洗涤时,取出底板,继续冲洗。
      6. 将板彻底压到吸收性纸巾上。
      7. 让板在黑暗中一夜之间干燥。曝光时光斑强度可能会下降。
        注意:如果膜过夜储存,斑点可能会变得更加尖锐。如果储存,将膜包裹在箔中。

数据分析

  1. 斑点计数在解剖显微镜或自动ELISPOT阅读器中,并计算分泌细胞的频率:
    一个单元格=一个点
    斑数=分泌细胞因子的细胞数量
    斑点的强度和大小=细胞的相对细胞因子分泌能力
  2. 背景减法
    对于ELISPOT数据分析,从测量结果中减去背景值。每个样本组应具有单个背景斑点平均值/中值,由阴性对照(T细胞和没有抗原的DC)计算。然后对于样品中的每个抗原组,计算组的平均值/中位数,然后减去背景平均值/中值,并通过每孔的细胞数归一化计数。
  3. 测定
    1. 积极应该是&gt; 50个点/ 10个/ 6个细胞。
    2. 阴性对照应理想地为0个/ 10个细胞。如果&gt; 0和&lt; 50个/ 10个细胞,从测试计数中减去阴性对照,校正背景染色。如果&gt; 50个/ 10个细胞,重复测定。
    3. 污染:
      1. 如果一个测定条件的一个重复孔清楚地显示为污染,则忽略该特定孔,并使用其他孔的结果。
      2. 如果多个井清楚地被污染,重复测定。
  4. 结果
    对于每种测试抗原,结果报告为用于人PBMC或SFC /脾的小鼠脾细胞的斑点形成细胞(SFC)/百万。

笔记

  1. 由于辣根过氧化物酶活性被叠氮化钠完全停止,请确保其不会添加到与此测定相关的任何缓冲液或试剂中。
  2. 有几个品牌的抗体可用,适用于此测定,甚至可以作为抗体对购买。然而,每个新的抗体小瓶应在棋盘滴定中进行测试以确定最佳稀释度(Asai,2000#14220)。

食谱

  1. 无菌磷酸缓冲盐水(1x PBS)
    开始用800毫升蒸馏水,加入8克NaCl; 0.2克KCl; 1.44g的Na 2 HPO 4和0.24g的KH 2 PO 4 用HCl将pH调节至7.4 加入蒸馏水至1L的总体积 库存储存为1 L瓶
    在室温下存放
  2. 涂层缓冲液工作溶液(0.5M碳酸氢盐 - 碳酸氢盐缓冲液pH9.6) 3.7g NaHCO 3和0.6g Na 2 CO 3(无水),用蒸馏水补足至100ml 100ml / 通过轻轻混合碳酸盐碳酸氢盐溶解,直到没有留下粉末残留物,并用0.22μM过滤器进行过滤器
    检查pH应为〜9.6,偶尔需要调整
    在室温下存放在黑暗中
  3. 阻塞解决方案
    IMDM培养基
    10%胎牛血清
    1%青霉素 - 链霉素
    0.1%β-巯基乙醇
  4. 抗IFN-γ捕获工作解决方案
    这是捕获抗体。在涂布井前立即准备。每个孔需要50μl,按制造商的浓度或每种抗体库存的滴定量
  5. 生物素 - 抗IFN-γ工作液
    这是检测抗体。准备像PBS一样。每个孔需要50μl,按制造商的浓度或每种抗体库存的滴定量
  6. 链霉亲和素 - 辣根过氧化物酶工作溶液
    准备PBS中的稀释液。每个孔需要50μl,按制造商的浓度或每种抗体库存的滴定量
  7. 吐温20工作方案
    加入0.5ml吐温20储备液至1,000ml 1x PBS 在室温下存放
  8. 10%胎牛血清在IMDM培养基中(在4℃暗处存储)
    1. 胎牛血清库存
      解冻并分配到50ml等分试样中。储存于-20°C
    2. IMDM培养基(在黑暗中存储在4℃)
    3. L-谷氨酰胺库存
      以200mM浓度提供为50ml。储存于-20°C
      根据随后要制备的培养基的体积,解冻并分配成1或5ml等分试样。储存于-20°C,黑暗中直至需要
    4. 青霉素 - 链霉素(5,000 U / ml)
      以5,000U / ml浓度提供为100ml。储存于-20°C
      根据随后要制备的培养基的体积,解冻并分配成1或5ml等分试样。储存于-20°C,黑暗中直至需要
    5. 10%胎牛血清在IMDM培养基(完全培养基)中 对于100ml:加入10ml FCS(a),1ml L-谷氨酰胺(c),1ml青霉素 - 链霉素(d),100μlβ-巯基乙醇和88ml IMDM(b)。在黑暗中储存4°C
  9. PHA库存解决方案
    加入10ml PBS至浓度为500μg/ ml的PHA粉末 等分并冷冻10μl体积。储存于-80°C
    注意:
    1. 不要重新冻结解冻的PHA。
    2. 以5mg纯化的无盐冻干粉末提供PHA(凝集素,阳性对照)原料。
    3. 在测定中,使用终浓度为5-10μg/ ml。向含有100μl体积的细胞的每个孔中加入1-2μl储备溶液

致谢

MNW得到ARC Future Fellowship和NHMRC的支持。 L.R.得到新加坡A * STAR和NRF新加坡(NRF2007NRF-RF001-226)的支持。作者希望感谢Josh Horne-Debets先生的ELISPOT形象。
ELISPOT方法最初是用于检测来自B细胞的抗原特异性抗体(Czerkinsky等人,1983),并且多年来被几组修饰以检测由抗原特异性细胞产生的细胞因子。

参考

  1. Andersson,U.,Hallden,G.,Persson,U.,Hed,J.,Moller,G。和DeLey,M。(1988)。流式细胞计数法检测IFN-γ产生细胞。与荧光显微镜比较。 J Immunol Methods 112(1):139-142。
  2. Bradley,LM,Duncan,DD,Tonkonogy,S.and Swain,SL(1991)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/ 1678774“target =”_ blank“>体内抗原特异性CD4 效应T细胞的表征:免疫导致MEL-14-,CD45RB辅助剂的瞬时群体分泌白介素2(IL-2),IL-3,IL-4和干扰素γ的细胞.J Exp Med 174(3):547-559。
  3. Carvalho,LH,Hafalla,JC和Zavala,F。(2001)。 ELISPOT测定以测量抗原特异性小鼠CD8 + T细胞应答。 J Immunol Methods 252(1-2):207-218。
  4. Czerkinsky,CC,Nilsson,LA,Nygren,H.,Ouchterlony,O.和Tarkowski,A.(1983)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm。 nih.gov/pubmed/6361139“target =”_ blank“>用于计数特异性抗体分泌细胞的固相酶联免疫斑点(ELISPOT)测定。免疫方法 65 (1-2):109-121
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  6. Horne-Debets,JM,Faleiro,R.,Karunarathne,DS,Liu,XQ,Lineburg,KE,Poh,CM,Grotenbreg,GM,Hill,GR,MacDonald,KP,Good,MF,Renia,L.,Ahmed, R.,Sharpe,AH和Wykes,MN(2013)。&nbsp; PD-1依赖性耗尽CD8 + T细胞驱动慢性疟疾。细胞Rep 5(5):1204-1213。
  7. Horne-Debets,JM,Karunarathne,DS,Faleiro,RJ,Poh,CM,Renia,L。和Wykes,MN(2016)。&nbsp; 缺乏程序性细胞死亡的小鼠-1显示CD8 + T细胞在长期免疫血液疟疾中的作用 Sci Rep 6:26210.
  8. Howard,SW,Poh,CM,Gun,SY,Claser,C.,Malleret,B.,Shastri,N.,Ginhoux,F.,Grotenbreg,GM and Renia,L。(2013)。&nbsp; 脑微血管交叉呈现是实验性脑疟疾的标志。 EMBO Mol Med 5(7):984-999。
  9. Janetzki,S.,Price,L.,Schroeder,H.,Britten,CM,Welters,MJ和Hoos,A。(2015)。&nbsp; Elispot测定的自动评估指南。 Nat Protoc <10>(10):1098-1115。
  10. Karunarathne,DS,Horne-Debets,JM,Huang,JX,Faleiro,R.,Leow,CY,Amante,F.,Watkins,TS,Miles,JJ,Dwyer,PJ,Stacey,KJ,Yarski,M.,Poh ,CM,Lee,JS,Cooper,MA,Renia,L.,Richard,D.,McCarthy,JS,Sharpe,AH和Wykes,MN(2016)。&nbsp; 编程性死亡-1配体2介导的PD-L1对PD-1轴的调节对于建立CD4 + T细胞免疫。 免疫 45(2):333-345。
  11. MacPherson,GG,Wykes,M.,Huang,FP和Jenkins,CD(2001)。从大鼠肠淋巴和脾脏中分离树突细胞。在Robinson,SP和Stagg,AJ(Eds。)。树突细胞方案。人文出版社。
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  • English
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wykes, M. N. and Renia, L. (2017). ELISPOT Assay to Measure Peptide-specific IFN-γ Production. Bio-protocol 7(11): e2302. DOI: 10.21769/BioProtoc.2302.
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