High‐affinity rat anti‐fluorescein monoclonal antibody with unique fine specificity properties including differntail recognition of dynamic ligand analogues

The ability of antibodies to specifically select and stabilize through binding one or more isomers of highly dynamic ligands remains a relatively unexplored immunochemical problem. The experimental strategy employed in this study was to elicit homogeneous antibodies to polyaromatic fluorescein which exists in one isomeric form. The binding properties of a monoclonal rat antifluorescein antibody specific to a given isomer were quantitatively studied to determine the capacity to bind dynamic analogues of fluorescein which exists in multiple isomers. To generate monoclonal anti-fluorescein antibodies that reacted with specific dynamic analogues of fluorescein possessing unconjugated aromatic ring systems, immune spleenocytes from Lou/M rats immunized with FITC(I)-KLH were fused with Balb/c SP2/0-Ag14 murine myeloma cells forming rat-mouse hybridomas. Cell line P2A12-1-C8 was selected for further characterization from the original 23 stable rat hybrids, since it produced a monoclonal antibody with a binding affinity 2.0 x 10(10)/M for fluorescein based on dissociation rate measurements. P2A12-1-C8 exhibited significant reactivity with HPF and phenol red, which are dynamic structural analogues of the homologous fluorescein ligand. No reactivity was demonstrated with phenolphthalein, which based on relative chemical structures was expected to be more reactive than phenol red. Computer-based molecular modeling and energy minimization studies of fluorescein, HPF, phenol red, and phenolphthalein showed that in terms of the most energetically favorable orientation of the three aromatic rings, phenol red more closely simulated fluorescein than phenolphthalein. The results were analyzed in terms of the mechanisms of dynamic ligand stabilization and binding involving accommodation of specific ligand isomers by energetically permissible conformational states exhibited by an antibody active site. Thus, antibody reactivity of an anti-fluorescein antibody with phenol red and phenolphthalein was dictated more by ligand dynamics and aromatic orientation than by chemical structure similarities.