Quantifying Antigen-Specific CTL Responses

The antigen specific CTL response was determined by interferon-γ (IFN-γ) ELISpot assay according to the manufacturer’s protocol (Mabtech, Stockholm, Sweden). Briefly, 96-well plates with nitrocellulose membranes (Millipore, Bedford, MA) were pre-coated with primary anti-IFN-γ antibody (1-D1K) at 4 °C overnight. The plates were blocked with AIM-V medium containing 5% human serum (Invitrogen). Target cells (2 × 10⁴/well) and CTL clones (2 × 10³/well) were co-cultured in 200 µL of culture medium for 24 h in triplicate. These wells were treated with biotinylated secondary anti-IFN-γ antibody (7-B6-1), followed by incubation with HRP-reagent and stained with TMB (Mabtech). The spots were then quantified with automated ELISpot reader, ImmunoSPOT S4 (Cellular Technology Ltd, Cleveland, OH). Positive CTL responding specifically to the vaccinated peptide were evaluated and classified according to a previously described algorithm [29 (link)].