Labeling Antibodies and ADCs for Imaging

Herceptin (trastuzumab, Roche) and Kadcyla (T-DM1, Roche) were obtained from the University of Michigan Pharmacy. Alexa Fluor 680 NHS Ester (AF680, ThermoFisher Scientific, A37567), IRDye 800CW NHS Ester (IRDye, LI-COR, 929-70020), and CellTrace™ Far Red DDAO-SE (DDAO, ThermoFisher Scientific, C34553) were conjugated to the antibodies following the manufacturer's instructions as previously described(15 (link),16 (link)). Antibody/ADC at 2mg/mL supplemented with 10% sodium bicarbonate (v/v) was reacted with dye at molar ratios of 0.5 (AF680, IRDye) and 1.5 (DDAO) for 2 hours at room temperature and purified using P6 Biogel (1g gel/10mL PBS) resulting in dye to protein ratios of approximately 0.3 (AF680, IRDye) and 0.7 (DDAO). Our previous work has shown that the distribution of T-DM1 is unchanged after labeling with AF680 at dye to protein ratio of 0.3 or less(17 (link)). Antibody/ADC dye conjugates were run on SDS-PAGE and scanned on the Odyssey CLx Scanner (LI-COR) to ensure free dye was removed. For fluorescence histology, antimouse CD31 (BioLegend, 102402) was conjugated with Alexa Fluor 555 (ThermoFisher Scientific, A37571), mouse antihuman IgG Fc antibody (BioLegend, 409302) was conjugated with Alexa Fluor 488 (ThermoFisher Scientific, A20000), and trastuzumab was conjugated with Alexa Fluor 750 (ThermoFisher Scientific, A20011) at dye to protein ratios of 1.5.