COMPREHENSIVE EPITOPE ANALYSIS OF MONOCLONAL ANTI-PROENKEPHALIN ANTIBODIES USING PHAGE DISPLAY LIBRARIES AND SYNTHETIC PEPTIDES - REVELATION OF ANTIBODY FINE SPECIFICITIES CAUSED BY SOMATIC MUTATIONS IN THE VARIABLE REGION GENES

Filamentous phage libraries, displaying 6, 12 or 20 amino acid residue peptides at the N terminus of coat protein pill were used to define a nd localize the epitopes of 15 monoclonal antibodies raised against hu man proenkephalin, a neuropeptide precursor. Eight monoclonal antibodi es (PE14 to PE19, PE23 and PE25), which inhibit each other's binding t o proenkephalin, recognized phage clones selected by PE14, PE15, PE19, PE23 and PE25. With the peptide sequences DLL(X)(X)LL (12mer library) and DLL(X)(X)L (6mer library) shared by most of the phage clones it w as possible to define the putative antibody epitope 155DLLKELL161 on h uman proenkephalin. For five antibodies (PE13, PE20 to PE22 and PE24) belonging to another inhibition group, a common consensus motif G(X)D( X)E(X)(X)V(X)(X)R could be defined with the help of a 20mer library Th e corresponding minimum epitope sequence has been found to be 175GSDNE EEVSKR185. Antibody PE1, raised in a separate fusion, was able to sele ct phage clones from a 12mer and 20mer library, revealing that the seq uence 187GGFMRG192 is probably the antibody epitope. The assumed local ization of the epitopes was confirmed by screening a set of overlappin g synthetic peptides, covering the region of human proenkephalin thoug ht to contain all antibody binding sites. It was found that antibodies , although recognizing the same epitope, gave different binding patter ns with the selected phage clones. By analysing the V-H chain sequence s of these antibodies it could be shown that a varying number of somat ic mutations is likely to be the reason for the observed differences i n antibody fine specificity.