Studies of 125I trace labeling of immunoglobulin G by chloramine-T

Abstract

Detailed studies of the parameters which effect the trace iodination of IgG with 125I, at levels of 1 atom/4000–10,000 moles IgG, using chloramine-T, are described. The following observations were noted: (1) The reactions, (a) the formation of ‘active iodine’ (H 2OI +) and, (b) the incorporation of ‘active iodine’ into protein, that comprise the over-all iodination of proteins have been studied separately and together. (2) With IgG, pH, 7·0 and 2° were optimal and reproducible for both reactions (a) and (b), and for the over-all reaction. Among the parameters which were found to influence the individual and over-all reactions and which were studied to establish optimal conditions were the concentrations of oxidant, iodide, and protein as well as pH, time, temperature, ionic strength, and particular batch of protein. (3) Different proteins and separate lots of the same protein show variable amounts of residues which are oxidized prior to the incorporation of iodine. (4) No differences in the immunochemical precipitation characteristics were detected between untreated IgG and IgG iodinated by the present method. (5) Calculations of the distribution of IgG species, iodinated at a level of 1 atom iodine per 4000–10,000 moles IgG, shows the population of iodinated molecules to be essentially homogeneous and in all likelihood, monosubstituted. (6) Typical conditions for labeling IgG at a level of approximately 1 atom iodine/5000 molecules IgG are described in detail.