The aTCRmu-F(ab')2, was functionalized by conjugation with the p-isothiocanatobenzyl derivate of desferrioxamine (DFO-Bz-NCS, Macrocyclics Inc., Richardson, TX) for subsequent labeling with zirconium-89 (89Zr; t/2=3.3 days; Emax β+=0.9 MeV). A 3-fold molar excess of the chelator was added to 2-3 mg protein in a total volume of 500 µl followed by incubation at 37 °C for 30 minutes. Purification of the immuno-conjugate from the unbound chelator was performed by size exclusion chromatography (Sephadex G-25 M, PD10 column, cut off >30 kDa, GE Healthcare) according to the protocol described by Perk et al. 9 (link). The immunoconjugate concentration was determined by a Nanophotometer (Implem). The 89Zr-labeling of aTCRmu-F(ab')2 was performed based on the protocol of Vosjan et al. 10 (link) with slight modifications. Briefly, 37.0 to 74.0 MBq of 89Zr in 1 M oxalic acid (BV Cyclotron VU, The Netherland) was adjusted to pH 7.0-7.2 with 2 M sodium carbonate and 0.5 M HEPES (pH 7.0) followed by addition of 100 to 250 µg DFO-aTCRmu-F(ab')2 and 0.5 M HEPES pH 7.0. After incubation of the mixture for 30 min at 37 °C, the 89Zr-Df-immunocomplex was purified using 0.25 M sodium acetate/gentisic acid 5 mg/ml buffer solution (pH 5.5) by size exclusion chromatography (Sephadex G-25M column, GE Healthcare) and radiochemical purity (RCP) was assessed by ITLC and radio-HPLC.