Gradient method for accurate affinity determinations.

Area of Science:

• Biophysical Chemistry
• Analytical Chemistry
• Surface Science

Background:

• Affinity constants (kd, ka, KD) in label-free interaction analysis are significantly influenced by ligand density on sensor surfaces.
• Existing methods often require cumbersome optimization to achieve ideal ligand densities.

Purpose of the Study:

• To introduce a novel SPR-imaging method that utilizes a ligand density gradient.
• To enable extrapolation of analyte response to Rmax = 0 μRIU, thereby minimizing surface-dependent artifacts.

Main Methods:

• Development of a surface plasmon resonance (SPR) imaging technique incorporating a ligand density gradient.
• Utilizing the mass transport-limited region for accurate analyte concentration determination.
• Extrapolation of binding response to zero ligand density (Rmax = 0 μRIU).

Main Results:

• The ligand density gradient method effectively extrapolates analyte response, mitigating issues like rebinding and biphasic kinetics.
• Accurate analyte concentration determination is achieved without extensive ligand density optimization.
• The method demonstrates potential for full automation in applications such as antibody quality control.

Conclusions:

• This novel SPR-imaging approach provides a robust solution for accurate affinity constant determination by controlling ligand density effects.
• The method simplifies experimental procedures and enhances the reliability of interaction analysis data.
• It offers a valuable tool for automated quality assessment of biomolecular interactions, particularly for antibodies.