Multimerized Antigen Probes for B Cell Detection

Antigen-specific B cells were detected using biotinylated proteins in combination with different streptavidin-fluorophore conjugates^{42 (link)}. Biotinylated proteins were multimerized with fluorescently labeled streptavidin for 1 h at 4 °C. Full-length spike protein (R&D Systems) was mixed with streptavidin-Brilliant Violet 421 (BioLegend) at a 10:1 mass ratio (for example, 200 ng spike with 20 ng streptavidin; approximately 4:1 molar ratio). Spike RBD (R&D Systems) was mixed with streptavidin allophycocyanin (BioLegend) at a 2:1 mass ratio (for example, 25 ng RBD with 12.5 ng streptavidin; approximately 4:1 molar ratio). Biotinylated influenza hemagglutinin pools (A/Brisbane/02/2018/H1N1, B/Colorado/06/2017; Immune Technology) were mixed with streptavidin-phycoerythrin (BioLegend) at a 6.25:1 mass ratio (for example, 100 ng hemagglutinin pool with 16 ng streptavidin; approximately 6:1 molar ratio). Streptavidin-Brilliant Violet 711 (BD Biosciences) was used as a decoy probe without biotinylated protein to gate out cells that nonspecifically bind streptavidin. Antigen probes for spike, RBD and hemagglutinin were prepared individually and mixed together after multimerization with 5 µM of free D-biotin (Avidity LLC) to minimize potential cross-reactivity between probes.