Method for estimating the single molecular affinity

Affinity constants (kd, ka, and KD) can be determined by methods that apply immobilized ligands such as immunoassays and label-free biosensor technologies. This article outlines a new surface plasmon resonance (SPR) array imaging method that yields affinity constants that can be considered as the best estimate of the affinity constant for single biomolecular interactions. Calculated rate (kd and ka) and dissociation equilibrium (KD) constants for various ligand densities and analyte concentrations are extrapolated to the KD at the zero response level (KDR0). By applying this method to an LGR5-exo-Fc¿RSPO1-FH interaction couple, the KDR0 was determined as 3.1 nM.