The labeling of high affinity sites of antibodies with 99mTc

Labeling of F(ab')2 with 99mTc was investigated. The best labeling procedure for F(ab')2 was then applied to IgG and Fab. Stannous ion was used as the reducing agent for 99mTcO-4 and free DTPA was used as a competing reagent to prevent colloid formation and loosely bound 99mTc. The competition reactions revealed two 99mTc binding sites for F(ab')2 and IgG. One is a high capacity, low affinity site. This accounts for 76 and 84% of total IgG and F(ab')2 binding sites. The labeling of these sites can be prevented if the antibody is labeled in the presence of free DTPA. The second site is a low capacity, high affinity site. The labeling of these sites cannot be prevented by free DTPA. Fab, unlike IgG and F(ab')2, does not have an appreciable percentage of high affinity sites. The determination of sulfhydryl groups using Ellman's reagent indicates the production of 5.5, 4.2 and 0.9 sulfhydryl groups when IgG, F(ab')2 and Fab were exposed to 56 micrograms/mL SnCl2 X 2H2O. These sulfhydryl groups may be the source of the high affinity binding. Biodistribution in mice for 99mTc labeled F(ab')2 and F(ab')2-DTPA, both prepared in the presence of excess free DTPA, was similar to that of F(ab')2-DTPA-111In.