2-(4-Isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (SCN-Bn-DTPA; Macrocyclics, Dallas, TX, USA) was used for labeling NuB2 with 90Y. SCN-Bn-DTPA in dimethylformamide was added to NuB2 at 5 mg/ml in 50 mM borate-buffered saline (pH 8.5) at the molar ratio of 5:1. After incubation at 37 °C for 24 h, DTPA–NuB2 was purified using a Bio-Spin column (Bio-Rad Laboratories, Hercules, CA, USA). For radiolabeling, 25–50 μl of a solution of 90YCl3 (37 MBq, Nuclitec, Braunschweig, Germany) was incubated with 50–100 μl of 0.25 M acetate buffer (pH 5.5) for 5 min at room temperature, followed by incubation with 100 μg of DTPA–NuB2 for 1 h at 40 °C. The 90Y-labeled antibody was purified using a Bio-Spin column or PD-10 column. The radiochemical purity of 90Y-NuB2 was confirmed as >95% by Tec-Control Chromatography Strips (Biodex Medical Systems, Shirley, NY, USA) developed with saline. Iodine-125-labeled antibodies were prepared according to standard protocols for the chloramine-T method.
Briefly, 740 kBq/2 μl of Na125I (PerkinElmer, Waltham, MA, USA) and 1 μg of chloramine-T in 1 μl of 0.3 M phosphate buffer were added to 40 μg of mAb in 100 μl of 0.3 M phosphate buffer. The 125I-labeled mAb was purified using a Bio-Spin column.
Briefly, 740 kBq/2 μl of Na125I (PerkinElmer, Waltham, MA, USA) and 1 μg of chloramine-T in 1 μl of 0.3 M phosphate buffer were added to 40 μg of mAb in 100 μl of 0.3 M phosphate buffer. The 125I-labeled mAb was purified using a Bio-Spin column.