title: Epitope mapping of monoclonal antibodies directed to aminopeptidase A and their relevance for albuminuria in mice.
abstract: We have shown previously that injection of specific combinations of anti-aminopeptidase A monoclonal antibodies induces an acute massive albuminuria in mice. This albuminuria is neither dependent on systemic mediators of inflammation nor angiotensin II. In this study, we examined the contribution of two individual antibodies, the enzyme-inhibiting antibody ASD-37 and the non-enzyme-inhibiting antibody ASD-41, in the induction of albuminuria as well as the interactions between these two monoclonals. In addition, we have mapped the epitopes of both antibodies using in vitro coupled transcription/translation of specifically designed cDNA fragments followed by immunoprecipitation, and using peptide enzyme-linked immunosorbent assay in case of a continuous epitope. A single intravenous injection of 4 mg of either ASD-37 or ASD-41 did not induce albuminuria. This dose of ASD-37 did not completely inhibit enzyme activity. The combination of 4 mg ASD-37/41 (1:1 weight ratio) induced albuminuria and almost completely inhibited enzyme activity. Similar results were obtained with a combination of ASD-37/41 in a 1:39 or 39:1 weight ratio. Administration of 2 mg ASD-41 24 h before injection of 2 mg ASD-37 significantly enhanced albuminuria. The epitope of ASD-37 is located at the C-terminal end of aminopeptidase A, whereas the ASD-41 epitope is mapped near the enzyme active site. Our data suggest that ASD-41 modulates the binding of ASD-37 to its epitope and/or vice versa. As a consequence, ASD-37 and ASD-41 act synergistically, not only in inhibiting enzyme activity but also in inducing albuminuria.
authors.affiliation: Department of Pathology, Division of Nephrology, University Medical Centre Nijmegen, Nijmegen, The Netherlands.
authors.forename: Miriam E
authors.initials: ME
authors.lastname: Gerlofs-Nijland
authors.forename: Karel J M
authors.initials: KJ
authors.lastname: Assmann
authors.forename: Jacco P H F
authors.initials: JP
authors.lastname: van Son
authors.forename: Henry B P M
authors.initials: HB
authors.lastname: Dijkman
authors.forename: Nathalie A J M
authors.initials: NA
authors.lastname: te Loeke
authors.forename: Ruurd
authors.initials: R
authors.lastname: van der Zee
authors.forename: Jack F M
authors.initials: JF
authors.lastname: Wetzels
authors.forename: Patricia J T A
authors.initials: PJ
authors.lastname: Groenen
mesh_terms: D000419:Albuminuria / Q000139:chemically induced*; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000032:analysis* / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000009:adverse effects* / Q000032:analysis* / Q000378:metabolism; D004334:Drug Administration Schedule; D018604:Epitope Mapping / Q000379:methods*; D043384:Glutamyl Aminopeptidase; D007074:Immunoglobulin G / Q000009:adverse effects / Q000737:chemistry; D007275:Injections, Intravenous; D007678:Kidney Glomerulus / Q000201:enzymology / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010446:Peptide Fragments; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase