Serum stability and non-specific binding of technetium-99m labeled diaminodithiol for protein labeling.

Previously we investigated the use of DTPA-coupled proteins to simplify labeling with 99mTc but especially to improve the stability of the label. These investigations have now been extended to include several N2S2 ligands such as N,N'-bis(2-methyl-2-mercaptopropyl)ethylenediamine (DADT) and a novel ligand of similar structure with a propylene bridge between two amines, 2-hydroxy-N,N'-bis(2-methyl-2-mercaptopropyl)propylenediamine++ + (DADT-3C-2OH). The condition of labeling of free ligand (pH, buffer and tin concentration) was optimized to provide 100% chelation with 99mTc at reasonable ligand concentrations (100 micrograms/mL or less). Labeling was determined by paper chromatography, reverse-phase and size-exclusion HPLC. After incubation in fresh serum, 37 degrees C for 24 h, repeat analysis showed less than 5% dissociation of the chelate. By contrast, the DTPA chelate shows instability towards oxidation during this period. DADT derivatized on an ethylene carbon showed almost identical serum stability as DADT itself whereas when derivatized on a nitrogen greater instabilities were apparent. Using identical labeling conditions, free DADT was chelated in the presence of IgG at different ligand: protein molar ratios. Non-specific binding of 99mTc to IgG at a 10:1 DADT-HM:IgG molar ratio was as little as 5% and was essentially zero at a 2:1 DADT:IgG molar ratio when labeling was by transcomplexation from 99mTc-EDTA. The DADT-3C-2OH ligand showed superior performance both in regard to serum stability and the absence of non-specific binding. In conclusion, the N2S2 ligands form more stable chelates with 99mTc than does DTPA with reduced non-specific binding and may therefore represent an attractive alternative for labeling proteins with 99mTc by the bifunctional chelate approach.