In order to assess the applicability of MS to study antibody-antigen interactions we used a model system comprising FAB fragments of anti-digoxigenin antibodies. Digoxin and digoxigenin are ligands having approximately the same affinity for the anti-digoxigenin antibodies. Both compounds can therefore be used as either analyte or reporter ligand. The same MS-based biochemical assay set-up was used as for the streptavidin/biotin system. Because the interaction between anti-digoxigenin antibodies and digoxin is weaker (Ka = approx. 109 L mol-1) with a relatively slower association rate and dissociation rate than streptavidin/ biotin, a longer reaction time is preferred. Therefore a reaction coil volume of 65 mL was chosen for reaction I resulting in a reaction time of 39 s.
The interaction of digoxigenin with the anti-digoxigenin antibodies was measured by incubating various concentrations of digoxigenin with 200 nM anti-digoxigenin antibodies and subsequent injection into the FI-MS system. The interaction was monitored by observing the response of digoxigenin in MS at m/z 408.4. In comparison with the calibration line obtained by injection of digoxigenin in the absence of antibodies, a significant decrease of the digoxigenin response was observed for all digoxigenin concentrations injected. In order to demonstrate that the decrease of the free digoxigenin concentration upon incubation with anti-digoxigenin antibodies is based on specific interactions, the same experiment was repeated, but the anti-digoxigenin antibodies were first incubated with a large excess (2 mM) of digoxin, i.e. a competing ligand. The resulting calibration curve is almost identical with the calibration for digoxigenin measured in the absence of antibodies indicating that digoxigenin is prevented from binding to the antibody due to an excess of competing ligand. A similar behavior was observed when digoxin instead of digoxigenin was used as reporter ligand.
These experiments clearly demonstrate that ESI-MS is suitable for monitoring antibody-antigen interactions by selectively detecting free ligand molecules in the presence of antibody-ligand complexes. Moreover, the development of MS-based biochemical assays is rather straightforward since any detectable analyte can principally be used as reporter ligand. The sensitivity of the biochemical assay depends mainly on the detection sensitivity of the reporter ligand and its binding affinity for the affinity protein. Since digoxin and digoxigenin have similar binding affinities for the anti-digoxigenin antibodies, similar assay sensitivities are obtained when using both compounds as reporter ligands.
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