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Rapid Determination of Antibody-Antigen Affinity by Mass Photometry
10:17

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Published on: February 8, 2021

Measuring an antibody affinity distribution molecule by molecule.

Jamshid P Temirov1, Andrew R M Bradbury, James H Werner

  • 1Center for Integrated Nanotechnologies and Bioscience Division, Los Alamos National Laboratories, Los Alamos, New Mexico 87545, USA.

Analytical Chemistry
|October 14, 2008
PubMed
Summary

This study uses single molecule fluorescence microscopy to measure antibody binding affinity. An empirical method corrects for quantum dot blinking, revealing the full distribution of binding affinities on surfaces.

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Area of Science:

  • Biophysics
  • Surface Chemistry
  • Nanotechnology

Background:

  • Single molecule fluorescence microscopy enables precise observation of molecular interactions.
  • Quantum dots (QDs) are fluorescent nanoparticles used as probes in biological imaging.
  • Antibody-antigen interactions are fundamental in biological systems and diagnostics.

Purpose of the Study:

  • To measure the binding affinity of individual antibodies using single molecule fluorescence microscopy.
  • To develop a method for correcting quantum dot blinking artifacts in affinity measurements.
  • To characterize the distribution of binding affinities on a surface.

Main Methods:

  • Utilized single molecule fluorescence microscopy to monitor hapten-decorated quantum dot binding to immobilized antibodies.
  • Analyzed fluorescence time histories from individual antibody sites to determine binding kinetics.
  • Developed and applied an empirical method to correct for quantum dot blinking during affinity calculations.

Main Results:

  • Successfully measured binding and unbinding events at the single molecule level.
  • Quantified antibody binding affinity and demonstrated a method to correct for quantum dot blinking.
  • Revealed the complete surface affinity distribution function, not just the average affinity.

Conclusions:

  • Single molecule fluorescence microscopy is a powerful tool for characterizing antibody binding affinities.
  • The developed blinking correction method improves the accuracy of quantum dot-based affinity measurements.
  • Measuring the full affinity distribution provides deeper insights into surface-analyte interactions than average values alone.