*4.1. Radionuclides, Radiolabeling, and Quality Control of the Radiolabeled Antibody*

The (1→3)-β-glucan-targeting 400-2 antibody (Biosupplies Australia Pty. Ltd., Parkville, Australia) was first conjugated to the chelating agent *C*-functionalized *trans*-cyclohexyldiethylene-triamine penta-acetic acid derivative (CHXA") (Macrocyclics, San Antonio, TX, USA). Conjugation and labeling were carried out according to a previously published method [13]. To summarize, conjugation was carried out at 37 ◦C for 1.5 h in a sodium carbonate buffer at pH 8.5 that had been previously run through a chelex column to remove any trace metal impurities. After the conjugation, the reaction was exchanged into a chelexed 0.15 M ammonium acetate buffer at pH 6.5. The <sup>213</sup>Bi/ <sup>225</sup>Ac radionuclide generator was purchased from Oak Ridge National Laboratory (Oak Ridge, TN, USA). <sup>213</sup>Bi was eluted from the <sup>213</sup>Bi/ <sup>225</sup>Ac radionuclide generator using 300 µL 0.1 M hydroiodic acid (HI) solution followed by 300 µL milliQ H2O [14]. The elution pH was adjusted to 6.5 with 80 µL of 5 M ammonium acetate buffer (chelexed) prior to the addition of the desired amount of CHXA" conjugated antibody (370:1 kBq/µg specific activity, 55.5 kBq/pmol) and the reaction mixture was heated for 5 min at 37 ◦C with shaking, followed by an addition of 3 µL of 0.05 M EDTA solution. The <sup>213</sup>Bi-labeled antibody was then purified on a 0.5 mL Amicon disposable size exclusion filter (30K MW cut-off, Fisher, Ottawa, ON, Canada. The percentage of radiolabeling was measured by silica gel instant thin-layer chromatography (SG-iTLC, Agilent Technologies, Santa Clara, CA, USA). using 0.15 M ammonium acetate buffer as the eluent. SG-iTLCs were cut in half, and each half was read on a Wizard2 2470 Automatic Gamma Counter (R<sup>f</sup> = 0 containing radiolabeled antibody, R<sup>f</sup> = 1 containing free <sup>213</sup>Bi (Perkin Elmer, Waltham, MA, USA). Only the batches with >98% radiochemical purity were used for administration to the dogs.
