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 (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 l 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. (author)