Method for estimating the single molecular affinity

TY - JOUR

T1 - Method for estimating the single molecular affinity

AU - Schasfoort, R.B.

AU - de Lau, W.B.M.

AU - van der Kooi, A.J.

AU - Clevers, H.

AU - Engbers, G.H.

N1 - Reporting year: 2012

PY - 2012

Y1 - 2012

N2 - 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.

AB - 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.

U2 - 10.1016/j.ab.2011.12.011

DO - 10.1016/j.ab.2011.12.011

M3 - Article

SN - 0003-2697

VL - 421

SP - 794

EP - 796

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 2

ER -