Radioiodination of proteins
Proteins, including OST6Fab fragment, streptavidin, and streptavidin-Fab conjugates, were radioiodinated with 125I using the chloramine T method (17). Forty micrograms of protein in 0.3 M phosphate buffer, VX-809 7.5, and 125I (22.2–29.6 MBq) (Du Pont, Wilmington, DE) were mixed with 2.5 μg of chloramine T (Nacalai Tesque, Kyoto, Japan) dissolved in 0.3 M phosphate buffer. After 5 min, radiolabeled proteins were separated from free iodine by chromatography through PD10 column, and the radiochemical purity was determined as being more than 97% by high-performance liquid chromatography. Specific activities of radiolabeled proteins were 370–740 MBq/mg.
In vitro reactivity
The biotin binding ability of the conjugates was tested using polystyrene beads coated with biotinylated antibody or immobilized D-Biotin gel (Pierce). Polystyrene beads of 6.4 mm in diameter were coated with biotinylated 145-9, a mouse MAb against CA125, as reported previously (15). The radioiodinated conjugates were incubated with the beads or the biotin gel for 30 min at room temperature and the bound percentage of radioactivity was determined.