Comparison of three different substrate detection systems for the tumor IFN-γ ELISPOT assay. The AEC, BCIP and TrueBlue™ substrate detection systems were evaluated for spot quality and signal to noise (stimulated to unstimulated) ratio using flu matrix peptide (FMP)-reactive PBMC from normal donors. PBMC (3 × 105 cells/well) alone and stimulated with FMP (5 μg/ml) were run in the ELISPOT assay for 24 hr. Spots were visualized using AEC, BCIP/NBT and TrueBlue™ substrate detection systems according to the manufacturer's instructions. Images from the plate scan generated by the ImmunoSpot analyzing system are shown. Data is represented as average number of spots per well ± SD.