96 well high bind plates

Cloned heavy- and light-chain plasmids derived from RBD-specific IgM⁺ B cells were initially screened in small-scale transfections. 293T cells (ATCC) were plated at 80% confluency and transfected with 0.5 μg each of paired heavy- and light-chain plasmids using polyethylenimine. 16 h later, medium was replaced with serum free-medium, and after 3–4 d, supernatants were harvested and cellular debris was removed by centrifugation. Antibody expression levels were determined using human IgG or IgM ELISA Antibody Pair Kit (Stemcell Technologies) according to the manufacturer’s instructions. RBD specificity was determined by ELISA as previously described (Rodda et al., 2021 (link)). Briefly, 96-well high-bind plates (Corning) were coated with 2 μg/ml SARS-CoV-2 RBD protein overnight at 4°C, washed with PBS and 0.05% Tween 20 (PBS-T), blocked with 3% milk in PBS-T, and incubated with serially diluted culture supernatants for 2 h at room temperature. Plates were washed, and bound antibodies were detected using anti-human IgG-HRP or anti-human IgM-HRP (Jackson ImmunoResearch) at a 1:3,000 dilution followed by 1× 3,3′,5,5″-tetramethylbenzidine (Invitrogen) and 1 M HCl. OD was measured on a spectrophotometer at 450 and 570 nm, and data were analyzed in Prism (v9.01; GraphPad).