A simple theoretical model for fluorescence polarization binding assay development.
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-alphaMSH binding to MC(5), 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.
Nosjean
Olivier
O
Institut de Recherches Servier, Croissy-sur-Seine, France. olivier.nosjean@fr.netgrs.com
Souchaud
Sophie
S
Deniau
Clarisse
C
Geneste
Olivier
O
Cauquil
Nicolas
N
Boutin
Jean A
JA
D001665:Binding Sites; D001667:Binding, Competitive; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D008024:Ligands; D008962:Models, Theoretical; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*
D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear