ELISpot tutorial: step-by-step full assay protocol

Gather all necessary materials including MSIP plates, reagent troughs, ethanol, water, and multichannel pipettes. Activate the PVDF membrane in the ELISpot plate by adding 20 μl of 35% ethanol per well for maximum 1 minute, observing the color change from bright white to grey. Wash the plate 5 times with sterile water using a dispenser pipette.

Prepare diluted capture antibody from Mabtech in a reagent trough, calculating 100 μl per well plus excess. Add 100 μl of diluted capture antibody to each well using a multichannel pipette, then wrap the plate in aluminum foil and refrigerate overnight.

Remove aluminum foil and discard excess capture antibody into waste. Wash the plate 5 times with 200 μl PBS per well using a dispenser pipette. Add 200 μl of cell culture medium to each well and incubate at room temperature for at least 30 minutes to block non-specific binding.

Add 25 μl of cell culture medium to each well, followed by 25 μl of prepared stimuli (peptide pools diluted in culture medium), then 50 μl of cell suspension at approximately 250,000 cells per well. Wrap the plate in aluminum foil and incubate in a 37°C humidified CO₂ incubator for the appropriate duration based on cell type and antigen.

Discard cells and wash the plate 5 times with 200 μl PBS per well. Filter biotinylated detection antibody diluted to 1 μg/ml in PBS with 0.5% FCS through a 0.2 μm filter, then add 100 μl per well and incubate at room temperature for 2 hours. Wash 5 times with PBS, then add 100 μl of streptavidin-ALP diluted 1:1000 per well and incubate for 1 hour at room temperature.

Wash the plate 5 times with 200 μl PBS per well. Filter BCIP/NBT-plus substrate through a 0.45 μm filter into a reagent trough, then add 100 μl per well. Monitor color development under a microscope for 5-30 minutes until clear spots appear, then stop the reaction by thoroughly rinsing with tap water and allow the plate to dry completely.

Once the plate is completely dry, insert it into an automated ELISpot reader such as IRIS or ASTOR to count the spot-forming cells. The reader will quantify the immune response based on the number and intensity of developed spots.