IDENTIFICATION OF THE FY6 EPITOPE RECOGNIZED BY 2 MONOCLONAL-ANTIBODIES IN THE N-TERMINAL EXTRACELLULAR PORTION OF THE DUFFY ANTIGEN RECEPTOR FOR CHEMOKINES

The epitope Fy6 recognized by two monoclonal antibodies (i3A and BG6), which inhibit binding of chemokines to the Duffy antigen, was charact erized by means of peptides synthesized on pins (Epitope Scanning Kit) and deletion mutagenesis. Both antibodies showed very similar specifi cities. They recognized a linear epitope, the essential portion of whi ch was the heptapeptide Gln-Leu-Asp-Phe-Glu-Asp-Val comprising amino a cid residues 21-27, located between two glycosylation sites of the Duf fy protein. All the amino acid residues of the epitope, except Glu, we re essential for antibody binding, since they could not be replaced by any other amino acid residues or by only one or two. The Glu residue could be replaced by most other amino acid residues, and its replaceme nt by 10 amino acid residues gave a distinct increase in the antibody binding. The results were in full agreement with the finding that the mutant of the Duffy antigen, lacking amino acid residues 23-25 (-Asp-P he-Glu-), did not bind the i3A antibody, but bound the anti-Fy3 monocl onal antibody similarly to the wild type of the Duffy antigen. The app arent affinity constants of both anti-Fy6 antibodies were determined b y surface plasmon resonance, using immunopurified Duffy protein as a l igand. Copyright (C) 1996 Elsevier Science Ltd