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Macromolecular binding and kinetic analysis with optically sectioned planar format assays.

Homanaz Ghafari1, Mithun Parambath, Quentin S Hanley

  • 1School of Science and Technology, Nottingham Trent University, UK.

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Summary

Separation-free optically sectioned planar format assays (OSPFAs) enable real-time analysis of molecular binding using fluorescence. This method overcomes background interference, proving effective for drug screening and kinetic studies.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Biophysics

Background:

  • Fluorescence intensity methods struggle with background noise in real-time macromolecular interaction analysis.
  • Separation-free optically sectioned planar format assays (OSPFAs) with confocal detection address solution fluorescence interference.

Purpose of the Study:

  • To report OSPFAs for indicator displacement and kinetic assessment of binding.
  • To demonstrate OSPFAs' suitability for drug screening and characterizing probe-target interactions.

Main Methods:

  • Adaptation of a commercial androgen receptor binding domain indicator displacement assay into an OSPFA.
  • Application of OSPFA to study antibody binding kinetics in a sandwich immunoassay.
  • Utilizing Langmuir fits for kinetic analysis of binding rates.

Main Results:

  • An OSPFA yielded an IC(50) of 6.5 nM for testosterone with Z' = 0.77, indicating suitability for drug screening.
  • Forward rate constants ranged from 2 × 10(3) M(-1) s(-1) to 6 × 10(4) M(-1) s(-1).
  • Reverse constants ranged from 1 × 10(-4) s(-1) to 4 × 10(-3) s(-1), suitable for probe-target interactions.

Conclusions:

  • OSPFAs are effective for investigating kinetics and binding interactions using fluorescence under wash-free conditions.
  • OSPFAs offer a viable alternative to other separation-free methods like SPR and ellipsometry.
  • OSPFAs facilitate practical dynamic binding studies for diverse molecules, even without significant mass changes.