title: Identification and fine mapping of a linear B cell epitope of human vimentin.
abstract: Knowledge about antibody-antigen interactions is important for the understanding of the immune system mechanisms and for supporting development of drugs and biomarkers. A tool for identification of these antigenic epitopes of specific antibodies is epitope mapping. In this study, a modified enzyme-linked immunosorbent assay was applied for epitope mapping of a mouse monoclonal vimentin antibody using overlapping resin-bound peptides covering the entire vimentin protein. The minimal epitope required for binding was identified as the LDSLPLVD sequence using N- and C-terminally truncated peptides. The peptide sequence LDSLPLVDTH was identified as the complete epitope, corresponding to amino acids 428-437 in the C-terminal end of the human vimentin protein. Alanine scanning and functionality scanning applying substituted peptides were used to identify amino acids essential for antibody reactivity. In particular, the two aspartate residues were found to be essential for antibody reactivity since these amino acids could not be substituted without a reduction in antibody reactivity. The majority of the remaining amino acids could be substituted without reducing antibody reactivity notably. These results confirm that charged amino acids are essential for antibody reactivity and that the vimentin antibody is dependent on side-chain interactions in combination with backbone interactions.
authors.affiliation: Department of Systems Biology, Technical University of Denmark , Kongens Lyngby , Denmark.
authors.forename: Catharina E
authors.initials: CE
authors.lastname: Dam
authors.forename: Gunnar
authors.initials: G
authors.lastname: Houen
authors.forename: Paul R
authors.initials: PR
authors.lastname: Hansen
authors.forename: Nicole H
authors.initials: NH
authors.lastname: Trier
mesh_terms: D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D018604:Epitope Mapping; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008969:Molecular Sequence Data; D014746:Vimentin / Q000737:chemistry* / Q000276:immunology
chemical_list: D014746:Vimentin
keywords: Epitope mapping; monoclonal antibody; solid-phase peptide synthesis; synthetic resin-bound peptides