Streptavidin enzyme conjugate

For IFN-γ enzyme-linked immunospot assay, nitrocellulose-bottomed 96-well plates (MultiScreen HTS-IP, Millipore) were coated overnight at 4°C with anti-IFN-γ mAb (AN18 Mabtech). Plates were blocked with 10% FBS-containing medium for at least 30 min at room temperature. Erythrocyte-depleted spleen cells from immunized or naive mice were plated in triplicates at four different cell concentrations (1×10⁶ to 1.25×10⁵ c/well) including HER-2⁺ cell line lysate as antigen (5:1 responder to stimulator ratio). Plates were then incubated overnight at 37°C, washed five times and incubated with biotinylated anti-IFN-γmAb (R4-6A2-biotinMabtech) for 2 hours at room temperature. After washing, streptavidin-enzyme conjugate (Mabtech) was added to each well and incubated for 1 hour at room temperature, followed by 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Sigma). The reaction was terminated on the appearance of dark spots by washing the plates with tap water. The spots were counted using the 4-Plate ELISPOT Reader V2.1 (Aelvis GmbH).

Anti-IFN-γ pre-coated plates (Mabtech) were first blocked with 10% FBS. Then, 2.5 × 10⁵ mouse splenocytes and 1 µg mL⁻¹ SARS-CoV-2 peptide pools (15-mers overlapping by 11; JPT Peptides) were added and incubated overnight for 24 h at 37 °C and 5% CO₂. After incubation, biotinylated cytokine-specific detection antibodies (Mabtech), streptavidin-enzyme conjugate, and substrate (Mabtech) were added. Finally, each well was observed under an optical microscope (Zeiss, Germany) and the number of secreting cells was calculated ^{18 (link),30 (link)}.
To detect the level of IL-6, a high binding ELISA plate (Biomat, Italy) was separately coated with anti-mouse IL-6 (Southern Biotech) and then diluted samples from supernatant of activated mouse splenocytes were separately added. After 1 h incubation at 37 °C, plates were washed with PBS and then 100 μL of secondary antibody, including anti-mouse IL-6-HRP (Southern Biotech), was added. Then, 50 μL of TMB (Sigma-Aldrich) was added. After 15 min, 100 μL sulfuric acid (Sigma) was added and OD of each well was read by a Spectrophotometer at 450 nm (BioTek Industries) and then the serum level of IL-6 was quantified by standard curve ^{18 (link)}.