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Assay Method Information

Assay Name:  Surface Plasmon Resonance (SPR)-Based Biosensor Measurements (KD) and Enzyme Inhibition Assay (IC50)
Description:  SPR experiments where performed with a Biacore A100 using Series S sensor chip CM5 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The proteins were amine coupled to the sensor. The immobilization levels were 5000-6000 RU (resonance units) for wt-thrombin and blocked thrombin (thrombin-DFP). The response used for affinity calculations was the differential steady-state binding signal between wt-thrombin and the blocked thrombin (the wt-thrombin signal minus that of the blocked thrombin). For equilibrium binding responses of compounds binding to thrombin (wt minus blocked), the affinity (KD) was calculated by fitting to the equation of a single-site binding model. The thrombin inhibitor potency (IC50) was measured with a chromogenic substrate method in a robotic microplate processor, using 96-well, half-volume microtiter plates. The linear absorbance increase values were determined by measurements at 405 nm during 40 min with Melagatran as control substance. The IC50 values were calculated by fitting the data to a three parameter equation by Microsoft XLfit.
Affinity data for this assay
 

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Last update November 1, 2007
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