Bindingstests

Binding tests, or binding assays, are laboratory procedures that quantify the interaction between two or more molecules, most commonly a ligand and a molecular target such as a protein, receptor, nucleic acid, or cell surface molecule. They are used to measure affinity, specificity, kinetics, and occupancy, and to characterize molecular interactions for research, diagnostics, and drug development.

Common formats include direct binding assays, where a labeled ligand binds to the target; competitive binding assays, where an unlabeled competitor displaces the labeled ligand; and kinetic or equilibrium assays that yield binding constants. Popular techniques encompass radioligand binding assays, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), fluorescence polarization or anisotropy, FRET-based methods, and enzyme-linked immunosorbent assays (ELISA) used as binding assays.

Key parameters derived from binding data include the dissociation constant (Kd), maximum binding (Bmax), the inhibition constant (Ki) from competition assays, and kinetic constants kon and koff estimated from time-resolved measurements or SPR/ITC data. Data are typically fitted with nonlinear regression to appropriate binding models, such as the one-site or two-site models, or the Hill equation.

Typical workflow begins with preparation and characterization of the target, incubation with a detectable ligand under defined conditions to reach equilibrium, followed by separation of bound from free ligand or real-time monitoring (as in SPR). Non-specific binding is estimated with control samples, and appropriate subtraction is performed. Replicate measurements and proper calibration are essential for accurate quantification.

Applications span drug discovery and pharmacology, where binding affinity informs lead optimization; diagnostics, where binding assays detect antibodies or antigens; and basic biology studies of receptor-ligand or protein–protein interactions. Limitations include non-specific binding, artifacts from labels or immobilization, potential deviations from equilibrium, assay sensitivity, and safety considerations with radioactive labels.