Bira500 kit

HLA‐E heavy chain (without transmembrane domain and with or without a C‐terminal biotinylation tag, AviTagsequence) and β2m were expressed and refolded with the peptide of interest and subsequently purified, as previously described [38 ]. To biotinylate complexes prior to size exclusion chromatography, complexes were AviTag treated with biotin‐protein ligase (BirA) according to the manufacturer's instructions (Avidity BirA‐500 kit) [39 (link)].

A multiplexed luminex assay was used to measure antigen-specific isotypes and FcR-binding, as previously described (Brown et al., 2017 (link)). Briefly, antigens were covalently linked to carboxyl-modified Magplex © Luminex beads using Sulfo-NHS (Pierce) and EDC (Thermo Fisher). Antigens used for this assay were 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), and SARS-CoV-2 S2 (Sino Biological). Antigen-coupled beads were blocked with PBS-TBN, resuspended in PBS, and maintained at 4°C.
Immune complexes were formed by adding antigen coupled beads to appropriately diluted serum or breastmilk supernatant. Plates were then incubated overnight at 4°C, shaking at 700 rpm. The next day, plates were washed in assay buffer (0.1% BSA, 0.02% Tween in PBS). To detect antigen-specific isotypes, immune complexes were stained with PE-coupled mouse anti-human IgG1, IgA1, IgA2, or IgM (Southern Biotech). To detect FcR-binding, Avi-Tagged FcRs (Duke Human Vaccine Institute) were biotinylated using a BirA500 kit (Avidity). The biotinylated FcRs were then labeled with streptavidin-PE and added to the immune complexes. Fluorescence was acquired using an iQue (Intellicyt). Antigen-specific isotype titer and FcR-binding was reported as the median fluorescence intensity (MFI).

Biotinylation of the HAs was performed using the BirA500 kit (Avidity) following the manufacturer’s instructions. To compensate for the reduced activity in PBS, twice the amount of BirA was added and the reaction was additionally supplied with twice the amount of biotin using the supplied BIO-200. The biotinylation reaction was allowed to proceed for 1.5 hr at 30°C before 0.2 µm filtering and purification over an S200 column (Cytiva). The trimeric HAs were then concentrated and flash-frozen in liquid nitrogen for single-use aliquots. Biotinylated HAs were quality controlled by a gel shift assay. Approximately 2 µg of biotinylated HA was heated in non-reducing Laemmli buffer (Bio-Rad, #1610737) at 95°C for 5 min. Once cooled to room temperature, excess streptavidin was added and allowed to incubate for at least 5 min. As a control, samples were run with PBS added rather than streptavidin. The mixture was then run on a Mini-PROTEAN TGX Stain-Free gel (Bio-Rad, #4568096) and imaged. All biotinylated HAs shifted in the presence of streptavidin, indicating successful biotinylation.

Recombinant SARS-CoV-2 RBD (from the Wuhan-1 strain, which shares an identical S protein to the WA-1 strain) was generated as previously described (25 (link)). For tetramer generation, RBD proteins were biotinylated with the BirA500 kit (Avidity), tetramerized with streptavidin-phycoerythrin (SA-PE) (Agilent, PJRS301-1), and stored in 50% glycerol at –20°C as previously described (45 (link)). Decoy reagents were generated by tetramerizing an irrelevant biotinylated protein with SA-PE previously conjugated to Dylight594 NHS Ester (Thermo Fisher Scientific, 46413) and Dylight650 NHS Ester (Thermo Fisher Scientific, 62266).