Chloramine t

We selected iodine-125 for radiolabeling
due to its non-residualizing properties that facilitate rapid elimination
of potentially free iodine to assure that the majority of the detected
radioactivity was derived from intact antibody.^{42 (link)} For radioiodination of antibodies, the chloramine-T method
was used^{35 (link)} in which chloramine-T oxidizes
iodine-125 to its cationic form and reacts with the anionic form of
tyrosine to form [¹²⁵I]I-tyrosine.³⁶ On average 102 ± 36.37 μg of antibody was labeled with
42.3 ± 24.91 MBq iodine-125 (PerkinElmer Inc., Waltham, MA, USA)
depending on the experimental setup. chloramine-T (Sigma Aldrich)
was added (5 μg, 200 μM in PBS) and incubated for 90 s
at RT. The reaction was quenched by the addition of sodium-metabisulfite
(10 μg, 440 μM in PBS, Sigma-Aldrich). After purifying
the radiolabeled antibody with disposable Zeba spin desalting columns
(7K MWCO, 0.5 mL, 89882, Thermo Fisher), the final activity was measured
in an ion chamber. For experiments with radiolabeled TCO-modified
antibody (Figure 2B),
radiolabeling was performed before TCO-modification to prevent damage
of TCO induced by chloramine-T.^{37 (link)}