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This version was published on December 1, 2006
Journal of Biomolecular Screening, Vol. 11, No. 8, 949-958 (2006)
DOI: 10.1177/1087057106294841

A Simple Theoretical Model for Fluorescence Polarization Binding Assay Development

Olivier Nosjean

Institut de Recherches Servier, Croissy-sur-Seine, France

Sophie Souchaud

Institut de Recherches Servier, Croissy-sur-Seine, France

Clarisse Deniau

Institut de Recherches Servier, Croissy-sur-Seine, France

Olivier Geneste

Institut de Recherches Servier, Croissy-sur-Seine, France

Nicolas Cauquil

Institut de Recherches Servier, Croissy-sur-Seine, France

Jean A. Boutin

Institut de Recherches Servier, Croissy-sur-Seine, France

Fluorescence polarization is a screening technology that is radioactivity free, homogeneous, and ratiometric. The signal measured with this technology is a weighted value of free and bound ligand. As a consequence, saturation curves are accessible only after calculation of the corresponding concentrations of free and bound ligand. To make this technology more accessible to assay development, the authors propose a simple mathematical model that predicts fluorescence polarization values from ligand and receptor total concentrations, depending on the corresponding dissociation constant. This model was validated using data of Bodipy-NDP-{alpha}MSH binding to MC5, obtained after either ligand saturation of a receptor preparation or, conversely, receptor saturation of a ligand solution. These experimental data were also used to calculate the actual concentration of free and bound ligand and receptor and to obtain pharmacological constants by Scatchard analysis. A general method is proposed, which facilitates the design of fluorescence polarization binding assays by relying on the representation of theoretical polarization values. This approach is illustrated by the application to 2 systems of very different affinities.

Key Words: fluorescence polarization • binding assay • assay development • mathematical model • MC5


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