SARS-CoV-2 Antibody ELISA Protocol

ELISAs were performed as previously described in Goenka et al^{13 (link)} and Halliday et al¹⁸ , based on the methodology described previously^{1 (link)}. Spike, RBD and N-protein were each diluted in sterile PBS (Sigma) and MaxiSorp plates (NUNC) were coated with either 10 mg/mL (spike) or 20 mg/mL (RBD; N-protein) of protein overnight at 4 °C before use. Plates were blocked with a 1 h incubation in 3% Bovine Serum Albumin (BSA) (Sigma-Aldrich) in PBS with 0.1% Tween-20 (Sigma-Aldrich) (PBS-T) at room temperature. Serum samples were thawed on ice before use, tested in duplicate and diluted to a final volume of 100 µL per well at a pre-optimised dilution, either at 1 in 50 (IgA) or 1 in 450 dilution (IgG), in dilution buffer (1% BSA in PBS-T). All samples were tested on a single plate for each antigen and antibody isotype combination. Secondary antibodies were used as follows with the dilution factor indicated: HRP conjugated anti-human IgG (Southern Biotech: 1 in 25,000) and IgA (Sigma: 1 in 6,000- 10,000). SIGMA FAST ^TM OPD (o-phenylenediamine dihydrochloride) (Sigma-Aldrich) was used to develop plates and reactions were stopped after 30 min with 3 M HCl. ODs were read at 492 nm and 620 nm using the same reader used for salivary ELISAs.