Easy comprehension of a brief description of ELISpot principle
Initially, the cells are cultured directly on the bottom of 96-well ELISpot plates or ones with PVDF or NC membranes which are pre-coated with specific primary antibodies to detect secreted cytokines. (The primary antibody applied in ELISpot assay should be high standard compared to the ones applied in general ELISA assay, e.g. without endotoxin, high affinity) Then it goes to adding of culture medium (without serum) and cells, and antigen stimuli (if any).
Responding to stimulation of specific antigen or non-specific mitogen, within hours T cells secret different cytokines. Rapidly cytokines are captured by antibodies on the membranes. After cells are washed off, captured cytokines are bond by biotin-conjugated secondary antibodies. Enzyme-avidin combines with biotin and reveals colors with certain substrate.
So far there will be visual spots on membranes. Every spot represents single cell that secrets certain cytokine. These cells are called sports forming cells, SFCs. Positive ratio can be calculated by a divide of total cell number by sports number.