Characterization of affinity-labeled fluorescyl ligand to specifically-purified rabbit IgG antibodies

Fluorescein isothiocyanate was reacted with purified rabbit anti-fluorescyl IgG antibodies under defined conditions. Relative to the normal IgG control, significantly more ligand was conjugated to the purified antibody which was then inhibited from further binding with radioactively labeled ligand (³H-N-acetylfluorescein amine). Fluorescyl ligand conjugated to purified antibody preparations exhibited a red spectral shift characteristic of specifically bound fluorophore. Fluorescence of ligand conjugated to antibody was quenched about 95% relative to ~ 90% for ligand reversibly bound. The quantum yield (Φ) of the former is indicative of a thiocarbamyl linkage within the antibody-active site. Fluorescence lifetime (t) measurements by phase and modulation showed a close correlation between liganded and affinity-labeled antibody relative to free ligand and fluorescyl conjugated non-antibody proteins. The fluorescyl-affinity label was not dissociated from the antibody-active site upon denaturation of the protein in 6M guanidine-HCl. Reduction, alkylation and resolution of H and L chains in two different dispersing agents showed that the ligand remained attached to either chain. Fluorescyl ligand bound to high affinity IgG (i.e. non-affinity labeled) was quantitatively released under conditions required for H and L chain production. The fluorescence of affinity labeled ligand was enhanced in high concentrations of deuterium oxide. The latter is characteristic of ligand bound specifically to the antibody active site and is not observed with fluorescein covalently conjugated to non-specific Ig.