Investigations of ascorbate for direct labeling of antibodies with technetium-99m

Recently, a method for the direct labeling of antibodies with ^99mTc was described in which sulfhydryls were reportedly generated by reduction of antibody disulfides with ascorbic acid. Thereafter, these proteins may be labeled at high efficiency with ^99mTc following reduction of pertechnetate with dithionite. This investigation was initially conducted to evaluate the mechanism of the increased stability towards cysteine challenge reported for the label and subsequently to determine the role of ascorbate in the labeling process. Methods: It was possible to reproduce the reported high labeling efficiencies by increasing the dithionite concentration fivefold, presumably because of variabilities among lots of commercial sodium dithionite. Results: Despite success in labeling, it was not possible to confirm that antibody reduction followed the treatment with ascorbate. Using both Ellman's reagent and 2,2' dithiodipyridine as indicators, we were unable to detect sulfhydryls on one IgG antibody treated at ten times the suggested ascorbate-to-antibody molar ratio. It was estimated that the number of sulfhydryls generated could not have been more than 1% (dithiodipyridine) to 2% (Ellman's). Furthermore, radiolabeling efficiencies for two IgG antibodies and stabilities of the label to cysteine challenge were unchanged when the ascorbate was eliminated. The number of sulfhydryls generated by treatment of the antibody with dithionite at 1-2 times the concentration required for adequate labeling was about 1% (dithiodipyridine) to 5% (Ellman's). Conclusion: For the conditions of this investigation and for the antibodies employed, ascorbate apparently played no more than a minor role at best in the labeling process. If antibody reduction occurred, this most likely was a result of residual dithionite presented to the protein along with the reduced ^99mTc.