Comparison between qPCR and ELISA based assays in the functional detection of antigen specific T cells. Between 1.5 and 3000 gp100154–162 reactive CD8+ T cell clones (C6E4) were spiked into 150,000 nonreactive autologous PBMC in individual microwells (n = 8) of a 96 well plate and immediately tested for T cell recognition of T2 cells pulsed with gp100154–162 peptide (1 μM) and HIVpol peptide (1 μM). (A.) qPCR Assay. Cellular IFN-γ mRNA production was measured by qPCR at 3 hours and reported as a stimulation index (SI). SI = IFN-γ mRNA (gp100154–162)/IFN-γ mRNA (HIVpol). Reactive wells (SI > 2) could be identified at every dilution down to 1.5 cells spiked into 150,000 PBMC. (B.) ELISA Assay. Supernatant IFN-γ protein production was measured at 24 hours by standard ELISA. SI = IFN-γ protein (gp100154–162)/IFN-γ protein (HIVpol). Reactive wells (SI > 2) could be identified at dilutions down to 300 cells spiked into 150,000 PBMC. (O) represents the SI for each microwell. Shaded area represents range of non-specific reactivity (SI ≤ 2).