IFN-γ ELISpot Assay Protocol

Enzyme-linked immunospot (ELISpot) flat-bottomed, 96-well nitrocellulose plates (MAHA S4510, Millipore) were coated with IFN-γ mAb (2 μg ml⁻¹, 1-D1K; MABTECH) and incubated for 2 h at 37 °C. After washing with PBS, plates were blocked with 10% human AB serum for 2 h at 37 °C. Cells were washed, concentrated, plated into each well titrating down in twofold serial dilutions starting from 400,000 PBMCs, in the presence of peptides, negative control (DMSO) and positive control (phorbol 12-myristate 13-acetate and ionomycin (PMA)/ionomycin). Plates were incubated for 24 h in a CO₂ incubator. After incubation, the plates were semi-automatically washed with PBS and then IFN-γ mAb (0.2 μg ml⁻¹, 7-B6–1-biotin; MABTECH) was added to each well. After incubation for 2 h at 37 °C, plates were washed and incubated with streptavidin-alkaline phosphatase (1 μg ml⁻¹, Roche) for 1 h at room temperature. After washing unbound streptavidin-alkaline phosphatase, substrate (5-bromo-4-chloro-3-indolyl phosphate/NBT; Sigma-Aldrich) was added and incubated for 12–15 min for spots to develop. After incubation, plates were washed to remove substrate and dark-violet spots were evaluated using the CTL Immunospot analyzer and software (Cellular Technology Limited) as routinely performed in the laboratory^{38 (link)}.