Book Description

Crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (K$\sb{\rm a}$ = 1.7 10$\sp{10}$ M$\sp{-1}$) complexed with fluorescyl ligand defined the six antibody active site contact residues involved in FI binding. For better definition of the relative role of each antigen contact residue in high affinity FI binding, each contact residue was changed to various amino acids in the single chain derivative of Mab 4-4-20 and following expression in Escherichia coli, denaturation, refolding and purification, each SCA (single chain antibody) mutant was characterized in terms of FI binding affinity, Q$\sb{\rm max}$ (maximum fluorescein fluorescence quenching), $\lambda\sb{\rm max}$ (Absorption maxima) and idiotype. Alanine substitutions at the six ligand-contact residues reduced the SCA binding affinities and quenching maxima for all the Ala mutants except L27d which retained wild type binding characteristics. Results of Ala substitution mutagenesis suggested that L32$\sp{\rm Tyr}$, L91$\sp{\rm Ser}$ and H33$\sp{\rm Trp}$ are most important for high affinity FI binding and efficient FI quenching. Substitution of Tyr and Phe at residue H33 resulted in binding affinities that were greater than that obtained from the H33$\sp{\rm Ala}$ mutant and suggested that another aromatic amino acid could substitute for Trp this residue. A phenylalanine substitution at L32$\sp{\rm Tyr}$, which disrupted the Tyr hydrogen bond with fluorescyl ligand, resulted in a decreased the binding affinity ($\sim$30-fold), but did not effect the quenching maxima. Finally, other amino acid substitutions at L34$\sp{\rm Arg}$, L91$\sp{\rm Ser}$ and L27d$\sp{\rm His}$ resulted in SCA mutants that possessed lower binding affinities and quenching maxima than that obtained for the respective Ala mutant.