Anti mouse cd45.1 clone a20

Streptavidin-drug conjugates were mixed in a 1:1 molar ratio with biotinylated antibodies and incubated for 15 minutes at 20°C to produce the indirectly conjugated ADCs used in this study. For conversions between mass and molar concentrations, an approximate molecular weight of 210 kDa was used (150 kDa for IgG plus 60 kDa for SAv-drug conjugates). ADCs were prepared using biotinylated anti-mouse CD45.2 (clone 104; BioLegend), anti-human CD45 (clones T29/33 and BC8 from Leinco Technologies; clones 2D1 and HI30 from BioLegend). Biotinylated isotype control antibodies (BioLegend) were used to produce control ADCs, including anti-mouse CD45.1 (clone A20; control for anti-mouse CD45.2), mouse IgG2b (clone MG2b-57; control for anti-human CD45 clone T29/33), and mouse IgG1 (clone MOPC-21; control for anti-human CD45 antibody clones HI30, BC8, and 2D1).
After the 15 minute conjugation reaction, ADCs were diluted to the desired concentration in culture medium for in vitro assays, or endotoxin-free PBS for in vivo administration. All antibodies formulated with sodium azide were exchanged into PBS with Zeba 7 kDa spin columns prior to ADC production. For some in vitro experiments, cells were treated with nonbiotinylated antibody plus free SAv-drug conjugate to demonstrate that interaction of antibody with payload was required for cytotoxicity.