Immunospot human ifn γ single color enzymatic elispot assay kit

PBMCs were isolated from ethylene diamine tetraaceticacid (EDTA) whole blood using LeucosepTM tubes (Greiner Bio-one International GmbH) and Histopaque®-1077 density gradient cell separation medium (Sigma-Aldrich) according to the manufacturers’ instructions. The ImmunoSpot® Human IFN-γ Single-Color Enzymatic ELISpot Assay Kit was utilized according to the manufacturer’s protocol (Cellular Technology Limited). PBMCs were plated at a concentration of 300,000 cells per well and were stimulated with six contiguous peptide pools spanning the length of the G protein sequence at a concentration of 2 µg/mL per peptide. One peptide (sequence AFNTVIALLGSIVII) was excluded due to false positive results. Analysis was performed using the CTL ImmunoSpot® Analyzer and ImmunoSpot® Software (Cellular Technology Limited). Spot forming units (SFU) per 1.0 × 10⁶ PBMCs were summed across the 6 peptide pools for each animal.

PBMCs were isolated from ethylene diamine tetraacetic acid (EDTA) whole blood using LeucosepTM tubes (Greiner Bio-one International GmbH, Frickenhausen, Germany) and Histopaque^®-1077 density gradient cell separation medium (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturers’ instructions. IFN-γ ELISpot assay of PBMCs was performed using the ImmunoSpot^® Human IFN-γ Single-Color Enzymatic ELISpot Assay Kit according to the manufacturer’s protocol (Cellular Technology Limited, Shaker Heights, OH, USA). PBMCs were plated at a concentration of 300,000 cells per well and were stimulated with two contiguous peptide pools spanning the length of the SARS-CoV-2 S protein sequence at a concentration of 2 µg/mL per peptide (Mimotopes, Mulgrave, Australia). Imaging was performed using the CTL ImmunoSpot^® Software (Cellular Technology Limited, Shaker Heights, OH, USA). Spot forming units (SFU) were hand counted and calculated per 10 [6 (link)] PBMCs as summed across the peptide pools for each animal.

ELIspot assays were performed as described previously (8 (link)). PBMCs were plated at a concentration of 300,000 to 500,000 cells per well and were stimulated with four contiguous peptide pools that spanned the length of the SARS-CoV-2 S protein sequence at a concentration of 2 μg/ml per peptide (Mimotopes). The ImmunoSpot Human IFN-γ Single-Color Enzymatic ELISpot Assay Kit was performed according to the manufacturer’s protocol (Cellular Technology). ELISpot plates were inactivated in formalin overnight before removal from BSL4 facilities before reading. Analysis was performed using the CTL ImmunoSpot Analyzer and ImmunoSpot Software (Cellular Technology). Spot-forming units per 1.0 × 10⁶ PBMCs were summed across the four peptide pools for each animal.