SARS-CoV-2 Spike Protein-specific IFN-γ ELISPOT

SARS-CoV-2 S1 and S2 protein-specific IFN-γ releases were measured using the method of enzyme-linked immunosorbent spot (ELISPOT) assays (Cat# 551083, BD Bioscience, USA) according to a previously described procedure.^{62 (link)} Briefly, the 96-well ELISPOT plates were coated with purified anti-mouse IFN-γ monoclonal antibody overnight at 4°C. Then, the plates were blocked and 2 × 10⁵ fresh splenocytes were added into each well and incubated with peptide pools of S1 or S2 for 20 hours at 37°C in a humidified incubator with 5% CO₂. The final concentration for each peptide was 1 μg/ml. After incubation, detecting antibody and Avidin-HRP were added sequentially. Finally, the plates were developed using the BD™ ELISPOT AEC Substrate Set (Cat#551951, BD Bioscience, USA) according to the manufacturer’s manual. Spots representing IFN-γ producing cells were enumerated using an automated ELISPOT plate reader (ChampSpot III Elispot Reader, Saizhi, Beijing, China). At the same time, the supernatants in the wells of ELISPOT plates were also collected for detecting secreted cytokines using a multiplexed cytokine beads array kit (Cat#741054, Biolegend, USA). The concentrations of secreted cytokines were detected using a BD Fortessa flow cytometer (BD Biosciences, USA). Data were analyzed using the LEGENDplex Data Analysis software suit (Biolegend, USA).