Improved radioiodination of biomolecules using exhaustive chloramine-t oxidation

To improve standardization in analytical reagents we investigated Chloramin e-T radioiodination (I-125) of several biomolecules based on the use of a s ingle amount of the oxidizing agent Chloramine-T as the limiting reagent be ing exhausted during the course of the reaction. Whenever the labeling yiel d resulted in less than one atom I-125/molecule, a second amount of the oxi dizing agent was added. Thereafter, the integrity of the various biomolecul es was assessed using radioimmunoassays, radioreceptor binding assays, or r adioimmunometric assays. Purification yields were done by gel permeation (5 6%+/- 19%, n=230) or by precipitation with trichloroacetic acid (59%+/- 19% , n=230). Specific activity (117 +/- 61 MBq/nmol) and the degree of iodine incorporation (1.4 +/-0.8 atoms of I-125/molecule) were achieved after 300 sec of incubation. A second addition of Chloramine-T resulted in an increas ed labeling yield of all biomolecules tested by a mean factor of 1.8 +/-0.9 . After the second addition of Chloramine-T, we observed for some biomolecu les a significant (p<0.001) decreased effect in biological performance. In conclusion, the use of Chloramine-T as a limiting reagent resulted in molec ules with appropriate immunological and biological performance. In general, tracers were minimally damaged and assessment of the shelf life as well as storing conditions showed the usefulness of the standardization of biomole cule labeling. (C) 2001 Elsevier Science Inc. All rights reserved.