Architect i2000 chemiluminescent microparticle immunoassay

Frozen aliquots of plasma or serum were used to conduct study reference testing, in accordance with local laboratory standard operating procedures. The primary reference test performed on the Abbott Architect i2000 chemiluminescent microparticle immunoassay (Architect) was for SARS-CoV-2 IgG (Abbott Diagnostics, IL, USA; Architect) which detects IgG against the SARS-CoV-2 nucleocapsid (N) protein. The clinical performance of the immunoassay is described in the Supplementary Section. Antibody levels ≥1.4 (manufacturer's arbitrary units; Architect Index: ratio between the sample to the internal calibrator absorbance; S/C or S/CO) were considered positive (22 (link)). The Architect Index result was used as a semi-quantitative measure of antibody positivity (3 (link)).
All 228 samples from the ME study were tested on the Architect™. Whilst in the HCW study, based on the Panbio™ high sensitivity (99.1%, 95% CI:95.3, 100.0) only the samples that gave a positive enrolment Panbio™ test reading were analysed on the Architect™. At the 3-month HCW study follow-up, all samples that were newly Panbio™ test positive as well as samples that were Panbio™ test positive at enrolment (irrespective if they gave Panbio™ negative results after 3-months) were further tested on the Architect™.

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).