Concentration test assays are designed as indirect (inhibition) assays. A known concentration of a relevant binding protein is mixed with the sample and injected over a sensor surface on which a corresponding derivative is immobilized. Any target molecules present in the sample bind to the binding protein and so inhibit it from binding to the sensor surface. The higher the concentration of the target molecule in the sample, the higher the level of inhibition and hence the lower the SPR (see below) response. Concentrations are calculated by interpolation of the binding responses on a calibration curve.
Example calibration curve.
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Concentration can be determined for purified molecules or for molecules in complex mixtures such as 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.
Technology overview
Biacore systems monitor protein interactions in real-time using a label-free detection method. Sample in solution is injected over a sensor surface on which potential interacting partners are immobilized. As the injected sample interacts with the immobilized partners, the refractive index at the interface between the sensor surface and the solution alters to a degree proportional to the change in mass at the surface. The phenomenon of surface plasmon resonance (SPR) is exploited to detect these changes in real time and data are presented in a “sensorgram” (SPR response plotted against time).
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