Simultaneous assay of target and target-drug binding

Abstract

Whole cell, simultaneous target and drug-target assay using differentially labeled antibodies and flow cytometry. First antibody binds to total target and second antibody binds to the drug binding site of the target, thus drug binding will competitively inhibit the second antibody allowing for a competitive inhibition assay of drug-target binding. The assay allows for whole cell analysis and even analysis of mixed populations of cells, yet provides detailed kinetic assessment of drug activity.

Description

PRIOR RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 60 / 573,783 filed May 24, 2004.FEDERALLY SPONSORED RESEARCH STATEMENT [0002] Not applicable. FIELD OF THE INVENTION [0003] The invention relates to a new cell based method of detecting or measuring drug activity, by simultaneously measuring target and target-drug combinations by flow cytometry. The method uses a pair of antibodies, one that is drug insensitive and will indicate total target level regardless of whether drug is present or not. The second is drug sensitive and will not bind to target at the same time that drug is bound to target. The drug and second antibody thus compete for the same or overlapping binding sites. Both antibodies can be simultaneously quantified when differentially labeled. BACKGROUND OF THE INVENTION [0004] Previous methods of measuring drug activity have been laborious and complex. Typically, a drug target (the protein to which a drug binds in orde...

AI Technical Summary

Benefits of technology

[0015] The invention provides quantitative cell-based measurements of experimental drugs designed to bind to very specific protein “targets.” It allows real time detection of target level and drug-bound target level, thus simplifying and improving on the prior art methods of studying drug kinetics.

[0021] A great many conjugated antibodies are commercially available and these can easily be screened to identify antibody pairs with the requisite binding characteristics (Total and Free-Site binding). Those antibodies that are not conjugated can easily be conjugated with an appropriate dye using one of the many available conjugation kits. Commercial suppliers will also provide custom antibodies on demand.

[0024] An alternative application of this method is for the analysis of free drug in plasma or serum of subjects exposed to various drug dosage regimens. Data from this assay is useful in pharmocokinetic (PK) modeling of functional drug activity. The PK application utilizes a cell line that constitutively expresses the receptor protein target to which the drug binds. Incubation of the cell line with a standard range of drug concentrations followed by staining of the cells with fluorochrome-conjugated drug sensitive monoclonal antibodies allows for the construction of a very specific standard curve of antibody fluorescence as it inversely relates to drug concentration. Subsequent to the construction of the standard curve, it is possible to derive the amount of drug in specimens of unknown concentration by extrapolation of the degree of drug-induced inhibition of antibody fluorescence to drug concentration as defined by the standard curve.

Problems solved by technology

Previous methods of measuring drug activity have been laborious and complex.

However, protein purification and multiple assays make such methods laborious and not conducive to the high throughput generation of data.

Further, purified proteins by definition are outside their normal body environment, and the changes in environment can complicate or change the way a protein behaves.

This can result in misleading or incomplete information about a drug's activity and kinetics.

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