The kinetics of an interaction, i.e. the rates of complex formation (ka) and dissociation (kd), can be determined from the information in a sensorgram.
If binding occurs as sample passes over a prepared sensor surface, the response in the sensorgram increases. If equilibrium is reached a constant signal will be seen.
Replacing sample with buffer causes the bound molecules to dissociate and the response decreases.
Biacore evaluation software generates the values of ka and kd by fitting the data to interaction models.
Kinetics, rapid association and dissociation / slow association and dissociation | 25sec Requires Flash Player 7
The affinity of an interaction is determined from the level of binding at equilibrium (seen as a constant signal) as a function of sample concentration. Affinity can also be determined from kinetic measurements. For a simple 1:1 interaction, the equilibrium constant KD is the ratio of the kinetic rate constants, kd/ka.
Concentration is determined by monitoring the interaction of a molecule with a prepared sensor surface in the presence of a target molecule in solution (solution inhibition) or excess analyte (surface competition). Concentrations are calculated by interpolation of the binding responses on a calibration curve.
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Concentration can be determined for purified molecules or for molecules in complex mixtures such as serum or food samples.
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A variety of concentration assay formats are used in the Qflex Kits of Biacore Q, a system designed for rapid determination of food quality and safety.
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Concentrations in the nanomolar range can be measured.
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The response reflects the concentration of the molecule in relation to the amount that can interact with the immobilized interaction partner. Values can therefore differ from results achieved by UV absorbance or general protein assays that measure only total amounts.
Multiple interactions during complex formation
Complex formation can be monitored as each component is incorporated into a multimolecular complex. For example:
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The ability of different antibodies to bind simultaneously to an antigen can be used to map epitopes on the antigen.
Multiple Binding, changes in response | 7sec Requires Flash Player 7
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