Gen5 v3

Total anti-SARS-CoV-2 spike antibodies were determined using an indirect ELISA^{29 (link)}, which is based on the Krammer assay^{33 (link)} using a standard curve derived from a pool of SARS-COV-2 convalescent plasma samples on every plate. Standardised EUs were determined from a single dilution of each sample against the standard curve which was plotted using the 4-Parameter logistic model (Gen5 v3.09, BioTek). Each assay plate consisted of samples and controls plated in triplicate, with ten standard points in duplicate and four blank wells.

ELISA was used to quantify levels of IgG and IgA against Spike trimer, RBD and N in serum and BAL samples, as previously described⁶² . Briefly, 96-well plates (U bottom) were coated with 1 μg/ml SARS-CoV-2 antigen and stored at 4^oC overnight for at least 16 h. The next day, plates were washed 3 times with PBS/0.05% Tween-20 and blocked with 2% BSA or 1% casein in PBS for 1 h at room temperature. Sera and BAL diluted in 0.1% BSA-PBS were plated in duplicate and incubated for 2 h at room temperature alongside an internal positive control (dilution of a convalescent serum) to measure plate to plate variation. For the standard curve, a pooled sera of SARS-CoV-2 infected participants was used in a two-fold serial dilution to produce either eight or nine standard points (depending on the antigen) that were assigned as arbitrary units. Goat anti-human IgG (γ-chain specific, A9544, Millipore-Sigma) or IgA (α-chain specific, A9669, Millipore-Sigma or 2050-04, Southern Biotech) conjugated to alkaline phosphatase was used as secondary antibodies, and plates were developed by adding 4-nitrophenyl phosphate in diethanolamine substrate buffer. Optical densities were measured using an Omega microplate reader at 405 nm. Blank-corrected samples and standard values were plotted using the 4-Parameter logistic model (Gen5 v3.09, BioTek).

Standardised total anti-spike IgG ELISA^{65 (link)} and anti-spike subclass and isotype ELISAs^{44 (link),66 (link)} were performed. In brief, ELISA plates were coated with 2 µg/mL of full-length trimerised SARS-CoV-2 spike glycoprotein protein overnight at 4 °C and blocked with casein in PBS. Plasma samples were diluted in PBS and tested in triplicate. Goat anti-human IgG conjugated to alkaline phosphatase was added as the secondary antibody, and plates were developed using 4-nitrophenyl phosphate in diethanolamine substrate buffer. Plates were read at 405 nm, and standardised ELISA units (EU) were determined using a 4-parameter logistic model and various pre-determined control cut-offs (Gen5 v3.09, BioTek). Plate washing in-between each step was undertaken using 0.05% Tween-20 in PBS.Serology for IgG to SARS-CoV-2 nucleocapsid protein was performed using the Abbott Architect i2000 chemiluminescent microparticle immunoassay (Abbott, Maidenhead, UK) and carried out according to manufacturer’s instructions using serum. The manufacturer threshold for confirming detection of antibodies is ≥1.40 arbitrary units. Levels between 0.50-1.39 arbitrary units designate equivocal levels (Abbott Diagnostics Product Information Letter PI1060-2020). Values below 0.5 were set to half the LLOQ (i.e. 0.25).