Probing molecular interactions in intact antibody: Antigen complexes, an electrospray time-of-flight mass spectrometry approach

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 spe ctrometry results show that at pH 6.8 the V antigen in solution exists pred ominantly as a dimer and a weakly associated tetramer. A monoclonal antibod y 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 aliqu ots of solution containing V antigen in substoichiometric and molar equival ents, the spectra revealed that two molecules of the V antigen bind to the antibody. Collision-induced dissociation of the anti body-antigen complex r esults in the selective release of the dimer from the complex supporting th e proposed 1:2 antibody:antigen stoichiometry. Control experiments with the recombinant F1 antigen, also from Yersinia pestis, establish that the anti body 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 generall y this work demonstrates a rapid means of assessing antigen subunit interac tions as well as the stoichiometry and specificity of binding in antibody-a ntigen complexes.