A multiplexed Luminex assay was used to determine relative titer of antigen-specific isotypes, subclasses, and FcR binding, as previously described (Brown et al., 2017). For this assay, the following antigens were used: SARS-CoV-2 RBD (kindly provided by Aaron Schmidt), SARS-CoV-2 S (kindly provided by Eric Fischer), SARS-CoV-2 N (Aalto Bio Reagents). SARS-CoV-2 S1 (Sino Biological), SARS-CoV-2 S2 (Sino Biological), pertussis pertactin (List Reagents) and a mix of HA A/Michigan/45/2015 (H1N1), HA A/Singapore/INFIMH-16-0019/2016 (H3N2), B/Phuket/3073/2013 (Immunetech). Antigens were covalently linked to carboxyl-modified Magplex© Luminex beads using Sulfo-NHS (N-hydroxysulfosuccinimide, Pierce) and ethyl dimethylaminopropyl carbodiimide hydrochloride (EDC). Antigen-coupled microspheres were blocked, washed, resuspended in PBS, and stored at 4C.

To form immune complexes, appropriately diluted plasma (1:100 for IgG2/3, 1:500 for IgG1 and FcRn, 1:1000 for all other FcRs) was added to the antigen-coupled microspheres, and plates were incubated overnight at 4°C, shaking at 700 rpm. The following day, plates were washed with 0.1% BSA 0.02% Tween-20. PE-coupled mouse anti-human detection antibodies (Southern Biotech) were used to detect antigen-specific antibody binding. For the detection of FcR binding, Avi-Tagged FcRs (Duke Human Vaccine Institute) were biotinylated using BirA500 kit (Avidity) per manufacturer’s instructions. Biotinylated FcRs were tagged with PE and added to immune complexes. Fluorescence was acquired using an Intellicyt iQue, and relative antigen-specific antibody titer and FcR binding is reported as Median Fluorescence Intensity (MFI).

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