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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.

Original publication




Journal article


Biophys J

Publication Date





3503 - 3509


Algorithms, Antibodies, Monoclonal, Antigen-Antibody Complex, Electrophoresis, Polyacrylamide Gel, Hydrogen-Ion Concentration, Mass Spectrometry, Protein Binding, Recombinant Proteins, Spectrometry, Mass, Electrospray Ionization, Yersinia pestis