Direct Radioiodination of Bispecific Antibodies

The bispecific RmAb158-scFv8D3 was labelled with iodine-125 (¹²⁵I) for ex vivo experiments and iodine-124 (¹²⁴I) for PET experiments using direct radioiodination 27 (link), The method is based on electrophilic attack of the phenolic ring of tyrosine residues by in situ oxidized iodine. Briefly, for ¹²⁵I-labelling, 120 pmoles of antibody or recombinant fusion protein (assumed Mw 210 kDa), ¹²⁵I stock solution (Perkin-Elmer Inc., Waltham, MA, USA) and 5 µg Chloramine-T (Sigma Aldrich, Stockholm, Sweden) were mixed in PBS to a final volume of 110 µl. The reaction was allowed to proceed for 90 s and subsequently quenched by addition of double molar excess of sodium metabisulfite (Sigma Aldrich) and dilution to 500 µl in PBS. For ¹²⁴I-labelling, 60 µl ¹²⁴I stock solution (Perkin-Elmer Inc.) was pre-incubated 15 min with 12 µl 50 µM NaI before addition of 240 pmoles of recombinant fusion proteins and 40 µg Chloramine-T, mixed in PBS to a final volume of 420 µl. The reaction was allowed to proceed for 120 s and subsequently quenched by addition of 80 µl of sodium metabisulfite in PBS (1 mg/ml). The radiolabeled proteins were purified from free iodine and low-molecular weight components with a disposable NAP-5 size exclusion column, Mw cut-off 5 kDa (GE Healthcare AB, Uppsala, Sweden), according to the manufacturer's instructions and eluted in 1 ml of PBS. The yield was calculated based on the added radioactivity and the radioactivity in the purified radioligand solution. Labelling was always performed less than 2 h prior to each study.