In vitro fluorescence polarization assay

G - Physics – 01 – N

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G01N 33/542 (2006.01) G01N 33/566 (2006.01)

Patent

CA 2250067

An in vitro assay method permits the identification of a test substance which inhibits the mutual association of a pair of proteins. The method includes the steps of providing a pair of proteins capable of mutual association, one of said proteins bearing a covalently linked fluorophore; preparing a mixture containing the two proteins and at least one test substance; irradiating the mixture with polarized light of a suitable wavelength permitting excitation of the fluorophore as indicated by emission of polarized light; measuring the degree of polarization of the emission, and determining the effect of the presence or concentration of the test substance in decreasing the observed emission polarization of a mixture of the two proteins alone. Inhibitory activity of in the test substance correlates with decreased depolarization values.

L'invention concerne un procédé de dosage in vitro permettant d'identifier une substance test qui inhibe l'association mutuelle d'une paire de protéines. Ce procédé consiste à produire une paire de protéines aptes à s'associer, l'une des protéins étant porteuse d'une substande fluorescente liée de manière covalente; à préparer un mélange contenant les deux protéins et au moins une substance test; à irradier le mélange avec de la lumière polarisée d'une longueur d'onde appropriée permettant l'excitation de la substance fluorescente telle que l'indique l'émission de lumière polarisée; à mesurer le degré de polarisation de l'émission et à déterminer l'effet dû à la présence ou à la concentration de la substance test en réduisant la polarisation de l'émission observée d'un mélange de deux protéines seules. L'activité inhibitrice de la substance test est en corrélation avec les valeurs réduites de dépolarisation.

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