Fluorescence spectroscopy of monoclonal antibodies produced against the fluorescyl hapten conjugated through the xanthene ring.

Two mouse anti-fluorescyl monoclonal antibodies (mAb), clones FL43.1 and FL55.3, were produced to the fluorescein hapten, which was conjugated to the carrier protein through the 4' position of the xanthene ring. Association constants (K A) and thermodynamic parameters for both mAb were ascertained by monitoring the steady-state intrinsic and fluorescein fluorescence. Both techniques were in good agreement and gaveK A values in the 10(9) M (-1) range. Ligand-induced intrinsic fluorescence quenching showed a hypsochromic shift for mAb FL43.1, but not for FL55.3, suggesting that the ligand interacts with different tryptophan residues in each mAb. Because these mAb are directed toward the phenylcarboxylate portion of fluorescein, the different ionic and structural forms should be useful as indicators of antibody binding site pH and buffering capacity near the binding site.