EPITOPE MAPPING BY NEGATIVE SELECTION OF RANDOMIZED ANTIGEN LIBRARIESDISPLAYED ON FILAMENTOUS PHAGE

Since most antibodies directed against protein antigens recognize epit opes composed of several discontinuous segments of the polypeptide cha in, attempts to delineate the amino acids constituting these epitopes with the use of linear peptides have generally been unsuccessful. Here , a method is described leased on error-prone PCR, phage display and n egative selection, whereby amino acid residues constituting the functi onal epitope are identified in tile context of the native protein. Fir st a library of randomized antigen variants containing most single, do uble and triple amino acid mutants generated by single nucleotide subs titutions is produced by error-prone PCR amplification of the DNA sequ ence encoding the protein antigen. The phage-displayed library is then negatively selected for epitope loss mutants by passing through an af finity matrix derivatized with a specific antibody and positively sele cted for retention of function. This method was applied to the mapping of the epitopes of two murine monoclonal antibodies (MA-7H11 and MA-3 G10) on staphylokinase, a 136 amino acid plasminogen activator secrete d by some strains of Staphylococcus aureus. After two negative/positiv e selection cycles, DNA sequencing of several clones revealed preferen tial amino acid mutations at positions 35 and 130 (with Mn-7H11), and at positions 62, 66 and 136 (with MA-3G10), Affinity measurements of s taphylokinase variants carrying single amino acid mutations at these p ositions confirmed their contribution to the free energy of binding to MA-7H11 and MA-3G10. This approach may be useful for isolating mutant s with altered antigenic or functional properties and in general to ma p critical regions for protein-protein interactions. (C) 1997 Academic Press Limited.