T-cell responses to SARS-CoV-2 were measured using Human IFN-γ ELISpot PRO kit (Mabtech) 18-24 months after COVID-19 infection or initial evaluation.
Cryopreserved peripheral blood mononuclear cells were rapidly thawed, viability was assessed by 0.4% trypan blue dye exclusion, and cells were rested in 10% human serum-Iscove's Modified Dulbecco Medium with 20 U/mL IL-2 at 37°C in a humidified 5% CO2 incubator for 2 hours before stimulation. 4×10 5 cells/well were added to strip-plate precoated with anti-IFN-γ (mAb 1-D1K) and stimulated with a pool of combined peptides to cover the domains of the entire spike protein 'S' (PepTivator SARS-CoV-2 Prot_S, S1 and S+, Miltenyi) and the nucleocapsid phosphoprotein 'N' (PepTivator SARS-CoV-2 Prot_N, Miltenyi). The final concentration of each peptide in the cell suspension was 1 µg/mL. Anti-CD3 was used as a positive control. All stimulation conditions were performed in the presence of anti-CD28 (1 µg/mL). Plates were incubated for 18 hours at 37°C with 5% CO2. Spot-forming cells were detected according to the antibody manufacturer's instructions and counted using an automated ELISpot Reader System (CTL software). The number of spots was expressed as spots/4×10 5 cells.
Cryopreserved peripheral blood mononuclear cells were rapidly thawed, viability was assessed by 0.4% trypan blue dye exclusion, and cells were rested in 10% human serum-Iscove's Modified Dulbecco Medium with 20 U/mL IL-2 at 37°C in a humidified 5% CO2 incubator for 2 hours before stimulation. 4×10 5 cells/well were added to strip-plate precoated with anti-IFN-γ (mAb 1-D1K) and stimulated with a pool of combined peptides to cover the domains of the entire spike protein 'S' (PepTivator SARS-CoV-2 Prot_S, S1 and S+, Miltenyi) and the nucleocapsid phosphoprotein 'N' (PepTivator SARS-CoV-2 Prot_N, Miltenyi). The final concentration of each peptide in the cell suspension was 1 µg/mL. Anti-CD3 was used as a positive control. All stimulation conditions were performed in the presence of anti-CD28 (1 µg/mL). Plates were incubated for 18 hours at 37°C with 5% CO2. Spot-forming cells were detected according to the antibody manufacturer's instructions and counted using an automated ELISpot Reader System (CTL software). The number of spots was expressed as spots/4×10 5 cells.
