Elispot assay

IFN-γ ELISPOT assays were performed using cytokine capture and detection reagents according to the manufacturer’s instructions (ELISpotPRO for human IFN-γ, Mabtech, Stockholm, Sweden). Briefly, 96-well nitrocellulose plates pre-coated with anti- IFN-γ mAb were seeded with PBMCs at a density of 2 × 10⁵ cells in 200 μl medium per well, followed by addition of 4 μl (1 μg/μl) peptides or 40 μl mixed peptide (pools), the final concentration of each peptide is 4 μg/well, CD3 antibody was used as positive control in a dilution of 1:1000. After incubation for 48 h at 37 °C, the cells were discarded, and captured IFN-γ was detected with a biotinylated anti- IFN-γ Ab, followed by addition of an alkaline phosphatase substrate solution (BCIP/NBT-plus). Reaction was stopped by washing extensively in tap water until distinct spots emerged, and spots were counted using an ELISPOT Image Analyzer and software (Cell Technology Inc. Jessup, MD). The spots forming cells (SFC) are defined as: SFC = experimental group- negative control group.