Characterization of monoclonal antibodies to proteinase 3 (PR3) as candidate tools for epitope mapping of human anti-PR3 autoantibodies.
Anti-neutrophil cytoplasmic antibodies directed against PR3 (PR3-ANCA) in patients with Wegener's granulomatosis are supposedly involved in the pathophysiology of this disease as different functional characteristics of the autoantibodies correlate with disease activity. However, little is known about the epitopes of PR3 that are recognized by PR3-ANCA and how epitope specificity may relate to functional characteristics of PR3-ANCA. As candidate tools for epitope mapping we studied 13 anti-PR3 MoAbs, including nine widely used and four newly raised MoAbs, for their mutual binding characteristics to PR3 using biosensor technology. Antigen specificity was confirmed by indirect immunofluorescence, immunoblotting, FACS analysis and antigen-specific ELISA. Competition between anti-PR3 MoAbs in binding to PR3 was investigated in a capture system set up in a BIAcore. In this system grouping of 12 of the 13 anti-PR3 MoAbs based on their mutual recognition patterns was achieved. Four MoAbs, from different research groups, namely 12.8, PR3G-2, 6A6 and Hz1F12, recognized comparable epitopes (group 1). Group 2 MoAbs including PR3G-4 and PR3G-6 bound to overlapping regions on PR3. The MoAbs PR3G-3, 4A5 and WGM2 recognized similar epitopes as they inhibited binding of each other (group 3). The fourth group of related MoAbs consisted of MC-PR3-2, 4A3 and WGM3. Because of its binding characteristics MoAb WGM1 could not be grouped. These results demonstrate that eight well-established anti-PR3 MoAbs produced by different research groups and four newly produced anti-PR3 MoAbs recognize four separate epitope areas on PR3, including one area detected with newly raised MoAbs only.
Van Der Geld
Y M
YM
Department of Clinical Immunology, University Hospital Groningen, The Netherlands. Y.M.van.der.geld@med.rug.nl
Limburg
P C
PC
Kallenberg
C G
CG
D019268:Antibodies, Antineutrophil Cytoplasmic / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D000918:Antibody Specificity / Q000276:immunology; D001323:Autoantibodies / Q000378:metabolism*; D001324:Autoantigens / Q000276:immunology*; D001667:Binding, Competitive / Q000276:immunology; D015374:Biosensing Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D014890:Granulomatosis with Polyangiitis / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D053146:Myeloblastin; D012697:Serine Endopeptidases / Q000276:immunology*
D019268:Antibodies, Antineutrophil Cytoplasmic; D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D000939:Epitopes; D012697:Serine Endopeptidases; D053146:Myeloblastin