Antibody conjugation

Antibodies were used as either fluorescence-only or dual-labeled conjugates carrying fluorescence and the radioactive isotope ¹¹¹In. Human IgG1 isotypic control antibody (Alpha Diagnostic Intl. Inc.) was dialyzed against phosphate buffered saline (PBS) using the 10,000 Da molecular weight cutoff Slide-A-Lyzer MINI Dialysis device (Thermo Scientific) to remove azide and other additives. To generate NIR fluorescent conjugates, BIWA (1 mg) was incubated with a 3-fold excess of IRDye800CW-N-hydroxysuccinamide (NHS) in 0.1 M sodium carbonate buffer (pH 8.5) for 1 h at room temperature (RT). For fluorescence guided surgery experiments, BIWA was only labeled with IRDye800CW-NHS. The final concentration of the antibody and the molecular substitution ratio were determined spectrophotometrically (Ultrospec 2000 spectrophotometer, Pharmacia Biotech) yielding an antibody:fluorophore ratio of 1:2. BIWA-IRDye800CW was stored in the dark at 4 °C.

For dual-labeling, IRDye800CW-labeled antibodies were conjugated with the chelator p-isothiocyanatobenzyl-diethylenetriaminepentaacetic acid (ITC-DTPA) with a 10-fold excess in 0.1 M sodium carbonate buffer (pH 9.5) for 1 h at RT. The solution was dialyzed for 1 week against 0.25 M ammonium acetate (pH 5.5) using a Slide-A-Lyzer cassette with a molecular weight cutoff of 20,000 Da (Thermo Scientific). The final concentration of the antibody and the molecular substitution ratio of the fluorescent dye were determined spectrophotometrically (Ultrospec 2000 spectrophotometer, Pharmacia Biotech) yielding a ratio of 1.4. DTPA-BIWA-IRDye800CW and DTPA-IgG1-IRDye800CW were stored in the dark at 4 °C until further use.

For radiolabeling, DTPA-BIWA-IRDye800CW and DTPA-IgG1-IRDye800CW were incubated with 0.05 MBq of ¹¹¹In (Mallinckrodt) per microgram of antibody in two volumes of 0.5 M 2-(N-morpholino)ethansulfonic acid (MES) buffer (pH 5.5). For SPECT/CT studies antibodies were labeled with 1.5 MBq of ¹¹¹In per microgram of antibody. After incubation of 20 min at RT 50 mM EDTA was added to a final concentration of 5 mM to chelate unincorporated ¹¹¹In. Labeling efficiency was determined by instant thin-layer chromatography on silica gel strips (Agilent Technologies) using 0.15 M citrate buffer, pH 6.0 as the mobile phase. For all preparations, the radiochemical purity of ¹¹¹In-DTPA-BIWA-IRDye800CW and ¹¹¹In-DTPA-IgG1-IRDye800CW exceeded 95%.

Unperturbed immunoreactivity of the antibody conjugates was confirmed as described⁴⁰ with minor modifications. A serial dilution of UT-SCC58 cells was incubated with radiolabeled antibody conjugate (333 Bq equivalent to 6.7 ng antibody per dilution in DMEM medium) for 30 min at 37 °C. To determine nonspecific binding, an excess of unlabeled antibody conjugate was added to a duplicate of the lowest cell concentration. Unbound antibody was removed by washing with DMEM and samples were analyzed in a γ-counter (Wizard; Pharmacia-LKB). For all preparations, the immunoreactive fraction of available antibody exceeded 85%.