Patient blood samples were obtained prior to study drug infusion, 30 min to 1 h post-infusion, at the day of surgery, and at 2-4 weeks follow-up. The blood samples were spun down to collect the plasma, and the antibody-dye complex concentrations in plasma at the different time points were assayed.
The assessment of plasma concentration for the cetuximab-IRDye800CW trial has been previously described 6 (link). Briefly, aliquots of plasma sample were resolved by NuPAGE 4-12% Bis-Tris gel (Invitrogen Corporation; Carlsbad, CA), assessed by gel electrophoresis (35 min at 150 V), imaged at 800 nm (Pearl Impulse, LI-COR Biosciences; Lincoln, Nebraska, United States), and quantified by calculating regions-of-interest (ROIs) (Image Studio, LI-COR Biosciences).
For the panitumumab-IRDye800CW trial, the same process was used to verify panitumumab-IRDye800CW at the 150 kDa protein marker. The antibody-dye complex in the patient plasma was assayed with the Spark multimode microplate reader (Tecan Group Ltd., Männedorf, Zürich, Switzerland) with an excitation of 775 nm and emission of 805 nm at room temperature. Aliquots of plasma sample (3 µL) were diluted in UltraPure distilled water (Invitrogen, Thermo Fisher Scientific, Carlsbad, California, United States) (45 µL) and measured in Greiner Bio-One FLUOTRAC black, 96-well half-area clear-bottom microplates (Thermo Fisher Scientific) against a set of panitumumab-IRDye800CW standards. Fluorescence measurements were done in triplicate in separate microplates, and the plasma concentration was determined through comparison with the standards. Total plasma values (mg) of both antibody-dye complexes at each time point were calculated based on patient dose and estimated total body plasma (calculated based on patient weight).
The assessment of plasma concentration for the cetuximab-IRDye800CW trial has been previously described 6 (link). Briefly, aliquots of plasma sample were resolved by NuPAGE 4-12% Bis-Tris gel (Invitrogen Corporation; Carlsbad, CA), assessed by gel electrophoresis (35 min at 150 V), imaged at 800 nm (Pearl Impulse, LI-COR Biosciences; Lincoln, Nebraska, United States), and quantified by calculating regions-of-interest (ROIs) (Image Studio, LI-COR Biosciences).
For the panitumumab-IRDye800CW trial, the same process was used to verify panitumumab-IRDye800CW at the 150 kDa protein marker. The antibody-dye complex in the patient plasma was assayed with the Spark multimode microplate reader (Tecan Group Ltd., Männedorf, Zürich, Switzerland) with an excitation of 775 nm and emission of 805 nm at room temperature. Aliquots of plasma sample (3 µL) were diluted in UltraPure distilled water (Invitrogen, Thermo Fisher Scientific, Carlsbad, California, United States) (45 µL) and measured in Greiner Bio-One FLUOTRAC black, 96-well half-area clear-bottom microplates (Thermo Fisher Scientific) against a set of panitumumab-IRDye800CW standards. Fluorescence measurements were done in triplicate in separate microplates, and the plasma concentration was determined through comparison with the standards. Total plasma values (mg) of both antibody-dye complexes at each time point were calculated based on patient dose and estimated total body plasma (calculated based on patient weight).
