Thanks to PureKinetics™, MP-SPR is sensitive platform to determine drug – target interactions even with small molecules. Label-free interactions are measured in real-time revealing affinity and kinetic of the binding. Optimize various analytes (Nucleotide, Peptide, Drug molecule, Antibody, Virus etc.) targeting to different ligands (Protein, Nucleotide, Peptide, Receptor, Membrane receptor, Antibody etc.).
Interaction measurements can be performed in diverse liquids, including complex liquids such as serum, saliva or organic solvents.
Figure 2. Small molecular weight drug tolcapone (7 concentrations) binding to human serum albumin (HSA) measured using KineticTitration. Measured data was fitted using TraceDrawer software to determine affinity and kinetic constants. Dissociation rate was measured after the last sample. The biomolecular interaction is a two state
binding event.
KineticTitration significantly reduces time required to run an assay with different concentrations. It is also useful for interactions that are difficult to regenerate or when regeneration damages the ligand on the surface. In the measurement, analyte samples are flown over the surface in a series from low to high concentration, without dissociation and regeneration between the samples as required in other methods. The dissociation rate is measured
after the last analyte sample.
Here, KineticTitration was utilized to measure binding of small molecular weight drug (tolcapone, 273 Da) to an immobilized protein (human serum albumin, HSA). The results revealed two different binding sites, first with KD = 9.7 × 10-7 M, ka = 1.99 × 103 (M*s)-1 and kd = 1.93 × 10-3 s-1 and second with KD2 = 1.9 × 10-6 M, ka2 = 1.41 × 102 (M*s)-1, kd 2 = 2.67 × 10-4 s-1. The dissociation of Tolcapone from HSA is slow, so the typical multi-step measurement cycle would have taken 3 times more time than using KineticTitration.
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Figure 4. Kinetic evaluation of indomethacin binding. Different concentrations of analyte (coloured curves) and fit (black curves).
The interaction of a small molecular weight drug (indomethacin) to Human serum albumin (HSA) was measured with label-free Multi-Parametric Surface Plasmon Resonance (MP-SPR). HSA was attached to a sensor surface with amine coupling chemistry. Affinity (KD) and kinetic constants (ka and kd) of indomethacin interaction were determined with TraceDrawer™ for MP-SPR Navi™. Premium quality kinetic data was ensured using PureKinetics™ feature that separates bulk effect from binding kinetics.
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Figure 1. MDCKII cell monolayer was deposited on a gold sensor slide. Drug interaction with the cell monolayer was measured in constant flow conditions.
Cell monolayer was deposited on a SPR sensor slide. MultiParametric Surface Plasmon Resonance (MP-SPR) was used to measure drugs (propranolol and D-mannitol) interaction with the cell monolayer in real time and in controlled flow conditions. It was possible to distinguish between paracellular and transcellular drug absorption routes.
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