ELISPOT Protocol

Coat the Plate:
1. Dilute Low-Endotoxin/Azide-Free sterile unlabeled capture antibody (BioLegend’s
LEAF™ format antibodies are specifically designed for this assay) to a
final concentration of 0.5–4 μg/ml in sterile Coating Buffer and transfer 100
μl/well to a high affinity binding PVDF membrane ELISPOT plate (e.g., Millipore;
Cat. No. MAIPS-4510).
2. Store plates overnight in humidified box at 4°C or at 37°C for ≥ 4 hours in
humidified atmosphere.

Block the Plate:
3. Wash plate 3 times with sterile PBS, 200 μl/well.
4. Add 200 μl/well of sterile Blocking Buffer.
5. Seal plate and incubate at room temperature for ≥ 1 hour.
6. Wash plate 3 times with sterile PBS, 200 μl/well.
Set-Up Tissue Culture and Add Antigen or Mitogen:
7. Add appropriate sterile antigen or mitogen solution diluted in appropriate
sterile tissue culture medium (TC) to ELISPOT plate, 100 μl/well.
8. Add cells diluted in sterile TC medium, 100 μl/well. Use 50,000-500,000 cells/
well (the minimum number of cells should be determined in preliminary
experiments).
9. Seal plate and incubate at 37°C 5% CO2 in humidified atmosphere for the
optimum stimulation period. BioLegend recommends a 24 hour incubation
for IFN-γ, IL-2, and TNF-α, and a 48 hour incubation period for IL-4, IL-5, and
IL-10 for most activation conditions.

Add Detection Antibody:
10. Wash plate 3 times with PBS, 200 μl/well.
11. Wash plate 3 times with PBS-Tween, 200 μl/well.
12. Add 100 μl/well of diluted biotinylated detection antibody at 0.25-2 μg/ml in
PBS-Tween-BSA.
13. Seal the plate and incubate at 4°C overnight, or 2 hr at room temperature.

Add Avidin-Horseradish Peroxidase (Av-HRP):
14. Wash plate 4 times with PBS-Tween, 200 μl/well.
15. Add 100 μl per well of the Av-HRP conjugate (Cat. No. 405103) or other
enzyme conjugate diluted to its pre-determined optimal concentration in
PBS-Tween-BSA (usually between 1/500 – 1/2000).

Source: 1. postech.ac.kr

2.http://www.biolegend.com/media_assets/support_protocol/BioLegend_ELISPOT_protocol.pdf