2.5.5. Preparation of p-SCN-Bz-DFO Conjugated Antibody

Conjugation of *p-SCN-Bz*-DFO to trastuzumab (DFO-trastuzumab) was performed following published procedures with slight modification [3,19]. Briefly, trastuzumab (5 mg/mL) in PBS was buffer exchanged in 0.1 M NaHCO<sup>3</sup> (pH 9) using centrifugal filters and concentrated to 10 mg/mL trastuzumab in the bicarbonate solution. A sixteen-fold mole excess of *p-SCN-Bz*-DFO (16 µg) in DMSO was added dropwise to the trastuzumab (2 mg) solution. The reaction mixture was incubated at 37 ◦C on a shaker at 650 RPM for an hour. The reaction mixture was cooled to room temperature and the unreacted DFO was removed by centrifugations using a spin-cap column (size 10–12 kDa). The buffer was exchanged with PBS using the same centrifugal filters.

#### 2.5.6. Radiolabeling of DFO-Trastuzumab and Determination of Specific activity of [ <sup>89</sup>Zr]Zirconium Oxalate

Zirconium-89 in 1 M oxalic acid was neutralized by diluting with 1 M HEPES pH 7.4 followed by adding 2 M NaCO<sup>3</sup> (pH 11) dropwise until the solution was neutralized (pH 7 ± 0.2). Radiolabeling was performed using 15 different concentrations of DFO-trastuzumab and was prepared by 1:2 serial dilution in HEPES (100 µL) to give final DFO-trastuzumab masses in the range of 200 µg to 0.024 µg. Approximately 3.3 MBq of the neutralized [89Zr] solution was added to each reaction tube. The reaction mixture was incubated at 37 ◦C in a shaker at 650 RPM for two hours. The reaction mixture was cooled to room temperature and 1 µL aliquots were analysed using iTLC with 0.15 M sodium citrate as a mobile phase. iTLC was measured using radioTLC scanner. The results of DFO-trastuzumab titration were plotted and fit with a sigmoidal dose–response curve to create an EC<sup>50</sup> value and used to calculate specific activity in MBq/ug.
