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Rapid Determination of Antibody-Antigen Affinity by Mass Photometry
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Published on: February 8, 2021

Method for estimating the single molecular affinity.

Richard B M Schasfoort1, Wim de Lau, Alex van der Kooi

  • 1Medical Cell Biophysics Group, MIRA Institute, University of Twente, 7500 AE Enschede, The Netherlands. r.b.m.schasfoort@utwente.nl

Analytical Biochemistry
|January 3, 2012
PubMed
Summary
This summary is machine-generated.

A novel surface plasmon resonance (SPR) imaging method provides accurate single biomolecular interaction affinity constants. This technique extrapolates data to determine the dissociation equilibrium constant (KD) at zero response level (KD(R0)), yielding precise results like 3.1 nM for LGR5-exo-Fc-RSPO1-FH.

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08:45

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Published on: September 7, 2011

Area of Science:

  • Biochemistry
  • Biophysics
  • Analytical Chemistry

Background:

  • Affinity constants (rate constants k(d), k(a), and dissociation equilibrium constant K(D)) are crucial for understanding biomolecular interactions.
  • Existing methods like immunoassays and label-free biosensors are used to determine these constants.
  • Accurate determination of affinity constants is essential for drug discovery and diagnostics.

Purpose of the Study:

  • To introduce a new surface plasmon resonance (SPR) array imaging method for determining single biomolecular interaction affinity constants.
  • To establish a method for extrapolating affinity constants to the zero response level (K(D)(R0)) for improved accuracy.
  • To validate the method by determining the affinity constant for a specific protein interaction.

Main Methods:

  • Development of a novel SPR array imaging technique.
  • Calculation of rate (k(d), k(a)) and dissociation equilibrium (K(D)) constants across varying ligand densities and analyte concentrations.
  • Extrapolation of calculated constants to determine the K(D) at the zero response level (K(D)(R0)).

Main Results:

  • The SPR array imaging method provides highly accurate affinity constants for single biomolecular interactions.
  • Extrapolation to K(D)(R0) refines the estimation of the true binding affinity.
  • Application to the LGR5-exo-Fc-RSPO1-FH interaction yielded a K(D)(R0) of 3.1 nM.

Conclusions:

  • The novel SPR array imaging method offers a superior approach for quantifying biomolecular interaction affinities.
  • The K(D)(R0) determination provides a more reliable estimate of binding affinity, minimizing surface effects.
  • This method has significant implications for precise characterization of biomolecular interactions in various research fields.