Gradient method for accurate affinity determinations

Richard B M Schasfoort

doi:10.1016/j.ab.2023.115085

Gradient method for accurate affinity determinations

Anal Biochem. 2023 Apr 15:667:115085. doi: 10.1016/j.ab.2023.115085. Epub 2023 Feb 19.

Author

Richard B M Schasfoort¹

Affiliation

¹ Medical Cell Biophysics Group, University of Twente, PO Box 217, 7500 AE, Enschede, the Netherlands. Electronic address: r.b.m.schasfoort@utwente.nl.

PMID: 36809845
DOI: 10.1016/j.ab.2023.115085

Abstract

The value of the affinity constants (kd, ka, and KD) that are determined by label free interaction analysis methods are strongly affected by the ligand density at the sensor surface [1]. This paper outlines a new SPR-imaging method that applies a ligand density gradient enabling the analyte response to be extrapolated to R_max = 0 μRIU. The mass transport limited region is used to determine the analyte concentration. Cumbersome optimization procedures for tuning the ligand density is prevented and surface dependent effects as rebinding, strong biphasic behavior etcetera are minimized. The method can be fully automated for e.g. accurate determination of the quality of antibodies from commercial sources.

Keywords: Affinity; Biosensor; Kinetics; Label-free; SPR imaging.

MeSH terms

Antibodies / analysis
Biosensing Techniques* / methods
Kinetics
Ligands
Surface Plasmon Resonance* / methods

Substances

Ligands
Antibodies