2.5. Antibody and radiolabeling

The selection, production and specificity of the AbN44v6 antibody has been described previously [29]. Direct iodination of AbN44v6 with ¹³¹I was performed by adding 20–40 μL (4 mg/mL) Chloramine-T (CAT) to typically 24–120 μg of AbN44v6 (1.2 mg/mL in PBS) and between 2 and 12 MBq of ¹³¹I with a 60 s incubation on ice before ending the reacting by adding 40–80 μL (4 mg/mL) of Na₂SO₅ (NBS). Iodination with ¹²⁵I was done using the Pierce Iodination Method using Pierce iodination tubes per manufacturer's instructions. Typically, 50 μg of AbN44v6 (1.2 mg/mL in PBS) were labeled with 5 MBq of ¹²⁵I. Instant thin layer chromatography (ITLC) determined the labeling yield using 70 % acetone as the mobile phase and a Bass 1800 II phosphoimaging system (Fuji, Japan) for analysis. The labeling yields of ¹³¹I-AbN44v6 were 88 % ± 7 %, with specific activities of 73 kBq ± 6 kBq per μg. The yields of all ¹²⁵I-iodinations were 100 %, with specific activities of 100 kBq/μg.