Multimers were constructed from AviTag-biotinylated human His-tagged CD19 (Aero Biosystems, CD9-H82E9), AviTag-biotinylated human His-tagged HER2 (Aero Biosystems, HE2-H82E2), site-specifically biotinylated Tn-glycosylated podoplanin G(T*)KPPLEE peptide (glycosylation marked with *)5 (link), and biotinylated bovine serum albumin (BioVision, 7097-5). Biotinylated dodecamer base protein and either Alexa Fluor 647-labeled streptavidin (BioLegend, 405237) or allophycocyanin-labeled streptavidin (BioLegend, 405207) were used for the multimer scaffold.
To generate antigen-tetramers, biotinylated antigen ligand was added to fluorescently-labeled tetrameric streptavidin at a 4:1 molar ratio for 30 minutes at 4°C in the dark. This mixture was diluted with PBS to convenient concentrations for staining.
To generate antigen-dodecamers, biotinylated tetrameric dodecamer base protein was incubated with fluorescently-labeled tetrameric streptavidin at a 1:4 molar ratio for 30 minutes at 4°C in the dark. Subsequently, biotinylated antigen ligand was added at a 12:1 molar ratio for an additional 30 minutes at 4°C in the dark. This mixture was diluted with PBS to convenient concentrations for staining.
To generate antigen-tetramers, biotinylated antigen ligand was added to fluorescently-labeled tetrameric streptavidin at a 4:1 molar ratio for 30 minutes at 4°C in the dark. This mixture was diluted with PBS to convenient concentrations for staining.
To generate antigen-dodecamers, biotinylated tetrameric dodecamer base protein was incubated with fluorescently-labeled tetrameric streptavidin at a 1:4 molar ratio for 30 minutes at 4°C in the dark. Subsequently, biotinylated antigen ligand was added at a 12:1 molar ratio for an additional 30 minutes at 4°C in the dark. This mixture was diluted with PBS to convenient concentrations for staining.