1. Antibody coating
Cytokine-specific monoclonal capture antibodies are immobilized on an ethanol-treated PVDF membrane plate. In our plate coating guide you can find detailed information on how to perform this step.
2. Cell incubation
Cells are added to the wells in the presence or absence of activating stimuli, and then incubated to allow for cytokine secretion. See our cell handling guide.
3. Cytokine capture
Secreted cytokines bind to the capture antibodies on the membrane
immediately surrounding the activated cells.
4. Detection antibodies
Following removal of the cells and washing of the plate wells, biotinylated
cytokine-specific detection antibodies are added to the wells.
5. Streptavidin-enzyme conjugate
To enable the formation of spots on the membrane, a streptavidin-enzyme conjugate is added to the wells. See our guide for more information about enzymes and substrates.
6. Addition of substrate
Colorimetric substrate is added to the wells and will form an insoluble precipitate when catalyzed by the enzyme; a visible representation of cytokine release by a single activated cell.
Spots are counted in an automated ELISpot reader or under a dissection microscope, and the frequency of secreting cells is calculated.