IL-2 ELISPOT

Materials Required
·  Peptides of desired specificity (stock at 10mM in DMSO - may be aliquotted and stored at -20ºC)
·  Phytohemagglutinin (PHA; optional) for use as a control for cytokine production (Sigma #L8902)
·  Purified anti-IL-2 capture antibody (e.g. clone IL2-I, Mabtech #3440-3)
·  Biotin-conjugated anti-IL-2 detection antibody (e.g. clone IL2-II, Mabtech #3440-6)
·  Streptavidin-alkaline phosphatase 1mg/ml (e.g. Vector Labs SA-S100)
·  BCIP/NBT alkaline phosphatase kit (e.g. Vector Labs SK-5400)
·  100mM Tris -Hcl, pH9.5 for color development
·  35% ethanol (v/v in distilled water)
·  Sterile phosphate buffered saline (PBS)
·  Sterile distilled water
·  Complete R10 medium (RPMI-1640 supplemented with 10mM HEPES, 50mM 2-mercapto-ethanol, 100U/ml Penicillin,0.1mg/ml streptomycin, 2mM L-Glutamine and 10% fetal calf serum)
·  96 well PVDF membrane ELISPOT plates (Millipore #MSIPS4510)

Recommended Experimental Controls
·  Negative Control – no antigen stimulation / stimulation with known negative peptide
·  Positive Control – Stimulation with PHA

Protocol applicable for IL-2 ELISPOT assay
Aseptic Procedures (Use sterile buffers and aseptic conditions; use laminar flow hood for procedures)
1. Pre-wet plate wells with 15ml 35% ethanol for 1 minute. Aspirate, then wash twice with 200ml dH2O before the ethanol evaporates.
Once the membrane is pre-wet with alcohol, do not allow membrane to dry for the duration of the assay.
2. Dilute anti-IL-2 capture antibody to 15mg/ml in sterile PBS. Coat plate with 50ml/well. Incubate at 4°C overnight.
3. Decant or aspirate coating antibody from plate.
4. Wash plates 5 times with 300 ml/well sterile PBS. Decant.
5. Block plate with 200 ml/well of complete R10 medium at room temperature for at least 1 hour. Decant or aspirate plate.
6. Aliquot up to 300,000 cells/well in 100 ml complete R10. Aliquot peptide ([5mM] final) or controls diluted in complete R10 medium to appropriate wells.
The desired cell concentration depends on the intensity of the immune response. If the expected response is unknown, then we recommend a serial dilution of cell concentrations.
7. Incubate for 23-36 hours at 37°C, 5% CO2 and 95% humidity.

Incubation times should be developed and evaluated by the user. However, antigen-specific stimulation of cells results in detectable spots within 23 hours.

Non-Aseptic Procedures
8. Decant cells and medium from plates. Wash plate 5 times with 300ml PBS.
9. Dilute biotin-conjugated anti-Il-2 detection antibody in PBS and filter. Add 50ml/well to plate and incubate at room temperature for 3 hours.
Failure to filter the detection antibody may result in non-specific spot formation due to protein aggregates.
10. Decant antibody solution. Wash 5 times with 300ml PBS. Allow wells to soak for 1 minute for each wash.
11. Dilute alkaline-phosphatase reagent in PBS (final concentration 1mg/ml). Add 50 ml to each well and incubate at room temperature for 1 hour.
Exceeding 1 hour incubation with enzyme conjugate will result in increased background.
12. Discard solution. Wash plate 5 times with 300ml PBS.
13. Add NBT/BCIP reagent as per the manufacturers instructions and leave the substrate to develop until spots are clearly visible (approximately 30 minutes)
Optimization of substrate development time is critical, since over-development will result in increased background.
14. Stop the substrate development by washing wells 3 times with dH20.
15. Remove the under-drain from the plate and allow to air-dry.
16. Count spots by eye, or using an automated ELISPOT plate reader.