Direct 99mTc-labeling of antibodies by sodium dithionite reduction, and role of ascorbate as a stabilizer in cysteine challenge

A method for the direct 99mTc-labeling of antibodies by dithionite reduction was developed. Among three murine monoclonal IgG1 and one human polyclonal IgG (hIgG) antibodies tested, hIgG was the most quickly reduced by dithionite. These differences may reflect the reactivities of antibody disulfide bonds toward the oxidation products of dithionite. By optimizing reduction conditions to generate enough free sulfhydryl groups, it was possible to radiolabel human IgG and monoclonal antibody 170 with 99mTc with a 90% monomeric antibody efficiency. The process avoided colloid formation. In contrast, about 0.1 sulfhydryl groups per antibody molecule, less than 1% of the possible 36, were detected after treatment with ascorbate (up to 35,000:1 molar ratio) at room temperature for 1 h for the antibodies tested. Sulfhydryl groups generated in antibodies were estimated using a new method: 5-iodoacetamidofluorescein-labeled antibodies quantitated by size exclusion HPLC. Ascorbate was found to prevent antibody aggregate formation in cysteine-challenged samples.