DIRECT (99)MTC-LABELING OF ANTIBODIES BY SODIUM DITHIONITE REDUCTION,AND ROLE OF ASCORBATE AS A STABILIZER IN CYSTEINE CHALLENGE

A method for the direct Tc-99m-labeling of antibodies by dithionite re duction was developed. Among three murine monoclonal IgG1 and one huma n polyclonal IgG (hIgG) antibodies tested, hIgG was the most quickly r educed by dithionite. These differences may reflect the reactivities o f antibody disulfide bonds toward the oxidation products of dithionite . By optimizing reduction conditions to generate enough free sulfhydry l groups, it was possible to radiolabel human IgG and monoclonal antib ody 170 with Tc-99m with a 90% monomeric antibody efficiency. The proc ess avoided colloid formation. In contrast, about 0.1 sulfhydryl group s per antibody molecule, less than 1% of the possible 36, were detecte d after treatment with ascorbate (up to 35,000:1 molar ratio) at room temperature for 1 h for the antibodies tested. Sulfhydryl groups gener ated in antibodies were estimated using a new method: 5-iodoacetamidof luorescein-labeled antibodies quantitated by size exclusion HPLC. Asco rbate was found to prevent antibody aggregate formation in cysteine-ch allenged samples.