title: Kinetic epitope mapping of monoclonal antibodies raised against the Yersinia pestis virulence factor LcrV.
abstract: Five monoclonal antibodies, mAb7.3, mAb29.3, mAb46.3, mAb12.3 and mAb36.3, raised to the LcrV virulence factor from Yersinia pestis were characterised for their Fab affinity against the purified protein and their Fc affinity to Protein A/G as a proxy for the FcγR receptor. Kinetic measurements were performed label-free in a localised particle plasmon array reader. The Fc-ProteinA/G complex first-order half-life was determined for each antibody and fell in the range of 0.8-3.8h. The Fab first-order half-lives had ranged from 3.4 to 9.2h although two antibodies, mAb12.3 and mAb36.3, showed low affinity interactions. Competitive binding studies of mixtures of the Fab-active antibodies were performed to measure the relative binding efficiency of one antibody in the presence of the other. A geometric relative positioning of the epitopes of mAb7.3, mAb29.3 and mAb46.3 was determined based on the footprint locus of the antibody and the percentage of competitive binding. The two known protective antibodies mAb7.3 and mAb29.3 showed greater interference, indicating epitopes close to one another compared to the non-protective mAb46.3 antibody. The Fab-Fc complex half-life screen and epitope mapping are potentially useful tools in the screening of therapeutic antibodies or vaccine candidates.
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Read
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Rouslan V
authors.initials: RV
authors.lastname: Olkhov
authors.affiliation: Biomedical Sciences Department, Defence Science and Technology Laboratory, Porton Down, Salisbury, Wilts. SP4 0JQ, UK.
authors.forename: E Diane
authors.initials: ED
authors.lastname: Williamson
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK. Electronic address: Andrew.m.shaw@exeter.ac.uk.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Shaw
mesh_terms: D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000942:Antigens, Bacterial / Q000276:immunology*; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007700:Kinetics; D010930:Plague / Q000276:immunology* / Q000382:microbiology; D052899:Pore Forming Cytotoxic Proteins / Q000276:immunology*; D037521:Virulence Factors / Q000276:immunology*; D015010:Yersinia pestis / Q000276:immunology*
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; C043908:LcrV protein, Yersinia; D052899:Pore Forming Cytotoxic Proteins; D037521:Virulence Factors
keywords: Antibody; Dissociation lifetime; Epitope mapping; Fab region; Kinetic analysis; Label-free; Steric hindrance; Surface plasmon resonance