title: Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach.
abstract: Using a combination of nanoflow-electrospray ionization and time-of-flight mass spectrometry we have analyzed the oligomeric state of the recombinant V antigen from Yersinia pestis, the causative agent of plague. The mass spectrometry results show that at pH 6.8 the V antigen in solution exists predominantly as a dimer and a weakly associated tetramer. A monoclonal antibody 7.3, raised against the V antigen, gave rise to mass spectra containing a series of well-resolved charge states at m/z 6000. After addition of aliquots of solution containing V antigen in substoichiometric and molar equivalents, the spectra revealed that two molecules of the V antigen bind to the antibody. Collision-induced dissociation of the antibody-antigen complex results in the selective release of the dimer from the complex supporting the proposed 1:2 antibody:antigen stoichiometry. Control experiments with the recombinant F1 antigen, also from Yersinia pestis, establish that the antibody is specific for the V antigen because no complex with F1 was detected even in the presence of a 10-fold molar excess of F1 antigen. More generally this work demonstrates a rapid means of assessing antigen subunit interactions as well as the stoichiometry and specificity of binding in antibody-antigen complexes.
authors.affiliation: Oxford Centre for Molecular Sciences, New Chemistry Laboratory, Oxford OX1 3QT, United Kingdom.
authors.forename: M A
authors.initials: MA
authors.lastname: Tito
authors.forename: J
authors.initials: J
authors.lastname: Miller
authors.forename: N
authors.initials: N
authors.lastname: Walker
authors.forename: K F
authors.initials: KF
authors.lastname: Griffin
authors.forename: E D
authors.initials: ED
authors.lastname: Williamson
authors.forename: D
authors.initials: D
authors.lastname: Despeyroux-Hill
authors.forename: R W
authors.initials: RW
authors.lastname: Titball
authors.forename: C V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D000465:Algorithms; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D015010:Yersinia pestis / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D011994:Recombinant Proteins