de Strasbourg, Pôle API

The possibility of obtaining from any antibody a fluorescent conjugate which responds to the binding of the antigen by a variation of its fluor-escence, would be of great interest in the analytical sciences and for the construction of protein chips. This possibility was explored with antibody mAbD1.3 directed against hen egg white lysozyme. Rules of design were developed to identify the residues of the antibody to which a fluorophore could be chemically coupled, after changing them to cysteine by muta-genesis. These rules were based on: the target residue belonging to a topo-logical neighbourhood of the antigen in the structure of the complex between antibody and antigen; its absence of functional importance for the interaction with the antigen; and its solvent accessibility in the struc-ture of the free antibody. Seventeen conjugates between the single-chain variable fragment scFv of mAbD1.3 and an environment-sensitive fluoro-phore were constructed. For six of the ten residues which fully satisfied the design rules, the relative variation of the fluorescence intensit