ELISPOT Assay for Antigen-Specific T Cell Responses

IFN-γ and IL-2-based ELISPOT assays were performed to detect antigen-specific T lymphocyte responses [10 (link)]. Spleens of C57BL/6 mice were removed after one week of booster immunization, and splenocytes were isolated. 96-well flat-bottom plates were pre-coated with 10 μg/ml of anti-mouse IFN-γ Ab or anti-mouse IL-2 Ab (BD Biosciences, USA) and stored at 4°C overnight. RPMI 1640 containing 10% FBS was added with 200 μl/well and incubated for 2 h at room temperature to block the wells. Then, 0.1 MOI of VACV was diluted in RPMI 1640 containing 10% FBS and added 100 μl/well to ELISPOT plate microwells. Meanwhile, the cell suspension was prepared at 1 × 10⁷ cells/ml, and 100 μl/well of cell suspension was added to each well which was incubated in a 37°C, 5% CO₂ incubator. After 24 h of incubation, the cells were removed, and the plates were processed in turn with biotinylated IFN-γ or IL-2 detection antibody (BD Biosciences, USA), streptavidin-HRP conjugate (BD Biosciences, USA), and AEC substrate (BD Biosciences, USA). After waiting 15 min for visible spots, the plates were washed clean with deionized water and dried. The number of spots was counted, and the ELISPOT plate microwells were photographed using an automatic ELISPOT reader and image analysis software (Cellular Technology Ltd.).