Simple quantitative fluorescent assay method for determining...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...

Reexamination Certificate

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Reexamination Certificate

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07122329

ABSTRACT:
The invention relates to a simple quantitative fluorescent assay method for determining the activity of transport proteins, more specifically multi-drug resistance associated proteins (MRPs). The method of the invention is performed on a well sealed culture of polarized cells expressing a transport protein of interest grown to confluency on a permeable support, said confluent cell culture forming well separated apical and basolateral compartments. The cells are contacted with a cell permeable non-fluorescent derivative of a fluorescent compound (e.g. calcein AM) in one compartment and, after a certain period of incubation, the fluorescence intensity detected in a sample taken from the opposite compartment is indicative of the activity of the transport protein of interest expressed in the cells. The invention also concerns methods for the qualitative and quantitative determination of inhibitors and activators of transport proteins of interest and methods for assessing basolateral and/or apical localization of plasma membrane bound transport proteins of interest in polarized cells.

REFERENCES:
R. Evers et al. (1998) “Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA,” Journal of Clinical Investigation vol. 101, pp. 1310-1319.
L. Homolya et al. (1996) “A new method for quantitative assessment of P-glycoprotein-related multidrug resistance in tumour cells,” British Journal of Cancer vol. 73, pp. 849-855.
N. Feller et al. (1995) “ATP-dependent efflux of calcein by the multidrug resistance protein (MRP): no inhibition by intracellular glutathione depletion,” FEBS Letters vol. 368, pp. 385-388.

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