Method for estimating the single molecular affinity.
Affinity constants (k(d), k(a), and K(D)) 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 (k(d) and k(a)) and dissociation equilibrium (K(D)) constants for various ligand densities and analyte concentrations are extrapolated to the K(D) at the zero response level (K(D)(R0)). By applying this method to an LGR5-exo-Fc-RSPO1-FH interaction couple, the K(D)(R0) was determined as 3.1 nM.
Medical Cell Biophysics Group, MIRA Institute, University of Twente, 7500 AE Enschede, The Netherlands. r.b.m.schasfoort@utwente.nl
Richard B M
RB
Schasfoort
Wim de
Wd
Lau
Alex van der
Av
Kooi
Hans
H
Clevers
Gerard H M
GH
Engbers
D008024:Ligands; D040081:Protein Array Analysis; D060066:Protein Interaction Maps; D020349:Surface Plasmon Resonance
D008024:Ligands