Iodination of monoclonal IgG antibodies at a sub-stoichiometric level: Immunoreactivity changes related to the site of iodine incorporation

Thirteen monoclonal antibodies (MAbs) were labeled with 125I to a different degree such as to cover the range from 0.5–20 μCi/μg. By SDS polyacrylamid gel electrophoresis, the amount of iodine incorporated into heavy (h) and light (l) chains was determined. Comparing different MAbs, h:l ratios varied from 0.6–34.6, but virtually no variation was observed with individual MAbs labeled at different levels. Immunoreactivity of labeled MAbs was analyzed with antigen-positive tumor cells according to the Lineweaver Burk method. Immunoreactive fractions were found to decrease with increasing iodine incorporation in 9 12 MAbs, while binding affinities decreased in 5 12 MAbs; only 1 MAb was stable in both respects. Immunoreactivity changes were not linked to preferential h or l chain labeling, nor to the isotype. This result indicated incorporation of the first iodine atom to take place at individually distinct residues, with a minimum estimate of two or four sites, depending on whether preferential chain labeling or random incorporation took place. In cases where increasing labeling led to a gradual decrease of binding affinity, a shift in the spectrum of acceptor residues has to be assumed.