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Interpolation method for accurate affinity ranking of arrayed ligand-analyte interactions.

Richard B M Schasfoort1, Kiki C Andree2, Niels van der Velde3

  • 1Medical Cell Biophysics Group, MIRA Institute, University of Twente, 7500 AB Enschede, The Netherlands; IBIS Technologies, 7521 PR Enschede, The Netherlands.

Analytical Biochemistry
|February 17, 2016
PubMed
Summary
This summary is machine-generated.

Label density impacts affinity constant measurements in label-free interaction analysis. A surface plasmon resonance (SPR) imaging method provides high-throughput affinity ranking, interpolating values to a standardized response level for reliable comparison.

Keywords:
AffinityBiosensorKineticsLabel freeSPR imaging

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Biotechnology

Background:

  • Label-free interaction analysis methods are crucial for determining binding kinetics and affinity.
  • Ligand density on sensor surfaces can significantly influence the accuracy of measured affinity constants (kd, ka, KD).
  • Standardized methods are needed for reliable, high-throughput affinity ranking across different experimental conditions.

Purpose of the Study:

  • To develop and validate a surface plasmon resonance (SPR) imaging method for high-throughput affinity ranking.
  • To address the impact of varying ligand densities on affinity constant determination.
  • To establish a method for interpolating affinity data to a standardized response level (KD(R100)) for consistent comparisons.

Main Methods:

  • Utilized a 96-plex SPR imaging array for parallel analysis.
  • Employed a kinetic titration experiment without a regeneration step.
  • Applied global fitting analysis to kinetic data from various coupled antibodies binding to a single antigen.
  • Developed an exponential interpolation method to normalize dissociation equilibrium constants (KD) to a response level of 100 Response Units (Rmax = 100 RU).

Main Results:

  • The SPR imaging method enabled high-throughput determination of affinity constants.
  • Globally fitted rate constants (kd, ka) and dissociation equilibrium constants (KD) were obtained for different ligand densities and analyte concentrations.
  • Exponential interpolation successfully normalized KD values to KD(R100), providing a standardized metric for affinity ranking.
  • The method demonstrated reliable affinity ranking for antibody-antigen interactions.

Conclusions:

  • The described SPR imaging method offers a robust approach for high-throughput, density-normalized affinity ranking.
  • Standardizing affinity constants to KD(R100) mitigates the effects of varying ligand densities, enhancing data comparability.
  • This method is valuable for applications requiring efficient screening and ranking of molecular interactions, such as drug discovery and diagnostics.