SARS-CoV-2 Antibody ELISA Protocol

ELISAs for SARS-CoV-2 antibodies were performed as described previously [13 (link)]. Briefly, 96-well plates were coated overnight at 4°C with purified SARS-CoV-2 antigens in phosphate-buffered saline (PBS). Wells were blocked for 1 hr at room temperature in blocking buffer consisting of PBS with 0.05% Tween 20 (PBS/Tween) and 1X casein (Vector labs., Peterborough, UK). Plates were then washed 3x in PBS/Tween prior to incubation with 50μL of each serum sample diluted 1:100 in blocking buffer. Each plate included two pooled negative controls and two pooled positive controls. Sera were incubated for 1 hour at room temperature. Plates were then washed 3x with PBS/Tween, before incubation for 1 hour with horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG (Bethyl labs., Cambridge Bioscience, Cambridge, UK) diluted 1:2500 in blocking buffer. Plates were washed a further 3x in PBS/Tween before addition of the 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate (Sigma Aldrich, Merck, Dorset, UK). Colour development was allowed to proceed for 10 minutes before the addition of 1M H₂SO₄ stop solution, at which point the absorbance was determined at 450nm on a Multiskan FC plate reader. Full validation of the S1 and RBD ELISA has been described previously [13 (link)].