| 1. | Coat membrane with antibodies. Add immune cells and incubate |
| 2. | Responding cells produce cytokines. The cytokine of interest is then bound by the antibody |
| 3. | Wash to remove cells. Add biotinylated antibodies which bind to the cytokine-antibody complex |
| 4. | Add avidin-enzyme conjugate |
| 5. | Add enzyme substrate and each responding cell will result in one spot |