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.

7. Analysis

Spots are counted in an automated ELISpot reader or under a dissection microscope, and the frequency of secreting cells is calculated.