Fig. 1
Principle of ELISPOT. For cytokine ELISPOT assay, the membrane of an ELISPOT plate is coated first with capture antibody as shown in (a). (b) Cytokines that are produced by cells of interest (e.g., T cells) with or without specific stimulation (antigen or mitogen) during culture will bind specifically to the plate-bound capture antibody. (c) After the cells are removed, biotin-conjugated detection antibody will be added and binds to the captured cytokines. (d) Upon subsequent addition of streptavidin-enzyme conjugate and corresponding substrate, precipitates will form around the site of the antibody-cytokine-antibody complex and appear as visible spots, as shown in (e) in the case of BCIP/NBT substrate. (e) Example of a well from a developed and analyzed mouse-IFN-γ ELISPOT plate using day 9 draining lymph node cells restimulated with specific antigen
2 Materials
2.1 Capture and Detection Antibodies (See Note 1)
1.
Mouse cytokine ELISPOT: use suggested clones for each monoclonal antibody (mAb) pair and the optimal working concentration or titration range listed in Table 1.
Table 1
Recommended antibody pairs for mouse cytokine ELISPOT assay
Cytokine | Capture mAb | Detection mAb (biotin-conjugated) |
|---|---|---|
IFN-γ | AN-18 (1–2 μg/ml) | R4-6A2 (0.25–0.5 μg/ml) |
IL-2 | JES6-1A12 (4 μg/ml) | JES6-5H4 (0.5–1 μg/ml) |
IL-4 | 11B11 (4 μg/ml) | BVD6-24G2 (2 μg/ml) |
IL-5 | TRFK (5 μg/ml) | TRFK4 (0.5 μg/ml) |
IL-13 | eBio 13A (2–4 μg/ml) | eBio 1316H (0.25–1 μg/ml) |
IL-17 | 17 F3 (2 μg/ml) | TC11-8H4 (0.125 μg/ml) |
GM-CSF | MPI-22E9 (2 μg/ml) | MP1-31G6 (0.25 μg/ml) |
2.
Human cytokine ELISPOT: use suggested clones for each mAb pair and the optimal working concentration or titration range listed in Table 2.
Table 2
Recommended antibody pairs for human cytokine ELISPOT assay
Cytokine | Capture mAb | Detection mAb (biotin-conjugated) |
|---|---|---|
IFN-γ | 2G1 (4 μg/ml) | B133.5 (0.75 μg/ml) |
IL-17 | eBio64CAP17 (2 μg/ml) | eBio64DEC17 (0.75 μg/ml) |
IL-2 | MT2A91/2C95 (5–10 μg/ml) | MT8G10 (0.25 μg/ml) |
TNF-a | TNF3/4 (10–15 μg/ml) | TNF5 (0.5 μg/ml) |
2.2 Medium and Buffers
1.
1× PBS, sterile and non-sterile.
2.
Blocking buffer: sterile PBS with 1 % BSA (Sigma Aldrich).
3.
Serum-free medium:
For mouse cytokine ELISPOT: HL-1 medium (Lonza, BioWhittaker).
For human cytokine ELISPOT using PBMCs or T cell line: CTL-Test Medium (Cellular Technology Limited, CTL).
Medium must be supplemented with 1 % l-glutamine (2 mM) and preferably prepared fresh, since the half-life of l-Glu is only 7–10 days at 4 °C.
4.
PBST: 1× PBS containing 0.05 % Tween-20.
2.3 ELISPOT Plates, Enzyme, and Substrate
1.
Millipore MultiScreen® sterile IP plates with high protein binding membrane (Millipore, Cat no. MSIPS4W10) are recommended for both human and mouse cytokine ELISPOT assays.
2.
Streptavidin alkaline phosphatase conjugate (e.g., Life technologies/Invitrogen, Cat no. S-921, as used in author’s laboratory) or streptavidin-horseradish peroxidase (HRP) if 3,3′,5,5′-Tetramethylbenzidine (TMB) or 3-Amino-9-Ethylcarbazole (AEC) substrate will be used (e.g., BD Biosciences).
3.
Substrate: 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) Phosphatase pre-mixed solution (e.g., Kirkegaard & Perry Laboratories, KPL, Cat no. 50-81-08, as used in author’s laboratory), TMB or AEC substrate (if streptavidin-HRP is used) (see Note 2 ).
2.4 Plate Washer and ELISPOT Analyzer (Recommended)
1.
Plate washer (e.g., BioTek Elx405 plate washer, as used in author’s laboratory).
2.
ELISPOT plate analyzer and corresponding software (e.g., CTL ImmunoSpot analyzer, as used in author’s laboratory).
3 Methods
3.1 Coating of Plates with Capture Antibody
1.
Dilute capture mAb in sterile PBS at suggested or titrated optimal working concentration.
2.
Coat the ELISPOT plate with diluted capture mAb at 100 μl/well; seal and store plate with lid in humid box at 4 °C, overnight.
3.2 Antigen Recall and Cell Culture (See Note 5)
1.
Wash plate four times with sterile PBS at 200 μl/well.
2.
Block plate with blocking buffer at 200 μl/well at room temperature, for 1 h.
3.
Wash plate again three times with sterile PBS at 200 μl/well.
4.
Add antigens (1–20 μg/ml or desired concentration for particular experiment) or positive control (e.g., PHA at 5 μg/ml; anti-CD3 mAb at 1 μg/ml) diluted in serum-free medium at 100 μl/well.
5.
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Resuspend cells harvested from particular tissue/organ (e.g., spleen, lymph nodes, or see Notes 6 and 7 ) at proper concentration with serum-free medium and add onto the plate at 100 μl/well using a pipettor with 200 μl large-orifice pipet tips (if two types of cells will be plated, e.g., when APCs are required, then add 50 μl/well with double the cell concentration for each type) (see Notes 8 and 9 ).
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