ELISPOT Protocol

ELISpot Four Parts of Protocol

Note: Use ELISPOT plates and reagents under aseptic conditions (e.g., in laminar flow hood) for Steps 1 – 3. Plates are pre-coated with antibody and ready for the addition of cells. Blocking and wetting are not required.

Cell Activation (under aseptic conditions)

1. Prepare mitogen or antigen, diluted in complete medium (eg, RPMI 1640 with FBS, Pen/Strep, and L-glutamine).

2. Add 100 μl/well to ELISPOT plate.

3. Prepare cell suspensions at different densities, (e.g., 1 × 105 to 2 × 106 cells/ml). Add 100 μl/well of each cell suspension to ELISPOT plate microwells.

4. Replace lid and incubate at 37ºC in a 5% CO2 humidified incubator. The duration of the incubation time will vary (eg, 2h – 24h). Specific activation conditions will vary, depending on cell type, kinetics, and analyte of interest.

Note: Cells may be diluted in a regular tissue culture plate starting at 105/well in triplicate wells with 1:3 or 1:4 serial dilutions down the plate, then transferred to the ELISPOT plate.

Detection Antibody

5. Aspirate cell suspension. Wash wells 2 times with deionized (DI) water. Allow wells to soak for 3 – 5 min at each wash step.

6. Wash wells 3 times with prepared Wash Buffer, 200 μl/well. Discard Wash Buffer.

7. Add prepared Detection Antibody Solution at 100 μl per well.

8. Replace lid and incubate for 2 h at room temperature.


9. Discard Detection Antibody solution. Wash wells 3 times with 200 μl/well prepared Wash Buffer. Allow wells to soak 1 – 2 min at each wash step.

10. Add prepared Streptavidin-HRP Solution at 100 μl/well

11. Replace lid; incubate for 1 h at room temperature.


12. Discard Streptavidin-HRP solution. Wash wells 4 times with 200 μl/well prepared Wash Buffer. Allow wells to soak 1 – 2 min at each wash step.

13. Wash wells 2 times, 200 μl/well, with prepared PBS.

14. Add 100 μl of prepared AEC Substrate Solution to each well. Monitor spot development from 5 – 60 min. Do not let color overdevelop as this will lead to high background.

15. Stop substrate reaction by washing wells with DI water.

16. Air-dry plate at room temperature for 2 h to overnight until it is completely dry. Removal of plastic tray supporting the 96-well plate will facilitate drying. Store plate in a sealed plastic bag, in the dark, until it is analyzed.


17. Enumerate spots manually by inspection under a dissecting microscope (or magnifying glass), or automatically using an ELISPOT plate reader.