Gradient method for accurate affinity determinations.

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 Rmax = 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.