Aid elispot reader software

Spleens from immunized animals were removed at Day 51 for the mice and at Day 110 for the hamsters and splenocytes were isolated for protein S-specific T-cell detection with the ELISpotPLUS mouse IFN-γ or ELISpotPLUS hamster IFN-γ kits (Mabtech) according to the manufacturer’s instructions. Briefly, 3–5 × 10⁵ splenocytes/well and 4 μg/well SARS-CoV-2 spike protein B.1.617.2 (Nanjing Vazyme Biotech Co., Ltd) were mixed and incubated at 37 °C. After 48 h followed by 5 washes with PBS, the probe antibodies (anti mouse IFN-γ, Mabtech, 3321-4APW-2; anti hamster IFN-γ, Mabtech, 3102-4APW-2) were added at a concentration of 1 μg/μl and incubated at room temperature for 2 h. After washing 5 times with PBS, alkaline phosphatase labelled streptavidin was added at a dilution of 1 in 1000 and incubated for 1 h at room temperature. After washing 5 times with PBS, alkaline phosphatase labelled streptavidin was added at a dilution of 1 in 1000 and incubated for 1 h at room temperature. After washing 5 times with PBS, the chromogenic solution (BCIP/NBT-plus) was added and incubated at room temperature for 10 min. Colour development was then stopped with deionized water. The number of dots in the wells of the ELISpot plate was analyzed using an ELISpot reader system (AID ELISpot Reader Classic and AID ELISpot Reader Software, Autoimmun Diagnostika GmbH).

Assessment of the IFN-γ T cell response was performed using the Mouse IFN-γ ELISpot^PLUS kit (Mabtech) following the manufacturer’s instructions. Briefly, anti-IFN-γ pre-coated plates were blocked with DMEM + 10% FBS for at least 30 min, then cells were added at 2.5 × 10⁵ cells per well for negative control (media only) and SARS-CoV-2 peptide pools (15-mers overlapping by 11; JPT Peptides) (1 µg mL⁻¹) in 200 µL final volume per well. The positive control wells contained 5 × 10⁴ cells per well in 200 µL final volume per well with 5 µg mL⁻¹ of ConA. Plates were incubated overnight at 5% CO2, 37 °C incubator and developed as per the manufacturer’s protocol. Once dried, plates were read using the AID ELISpot reader ELR03 and AID ELISpot READER software (Autoimmun Diagnostika GmbH).