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.

What is claimed is: 
     
         1 . A method of measuring circulating free drug present in a biological fluid sample from a patient, comprising:
 (a) combining a biological fluid sample from a patient who is taking a drug with a target cell, wherein the biological fluid sample contains circulating free drug, and wherein the target cell expresses a target that interacts with the drug to bind to at least a portion of the drug present in the biological fluid sample,   (b) mixing the target cell of step (a) with a labeled anti-Free-Site antibody, wherein the anti-Free Site antibody is a monoclonal antibody that recognizes and binds to an epitope on the target when the drug is not bound to the target but is competitively inhibited by binding of the drug to the target;   (c) washing away unbound anti-Free-Site antibody from the target cell of step (b);   (d) quantifying the amount of anti-Free-Site antibody binding to the target cell of step (c) by flow cytometry; and   (e) determining the amount of circulating free drug present in the biological fluid sample.   
     
     
         2 . The method of  claim 1 , further comprising fixing the target cell of step (d). 
     
     
         3 . The method of  claim 1 , further comprising reacting the target cell of step (c) with a labeled anti-Total antibody and quantifying the amount of anti-Total antibody bound to the target cell to determine the total amount of target expressed by the target cell. 
     
     
         4 . The method  claim 1 , wherein the anti-Free Site antibody or the anti-Total antibody are conjugated to a dye or fluorescent label. 
     
     
         5 . The method of  claim 4 , wherein the anti-Free-Site antibody and the anti-Total antibody are each conjugated to a different dye or fluorescent label. 
     
     
         6 . The method of  claim 1 , wherein the target cell of step (a) is a permeabilized target cell. 
     
     
         7 . The method of  claim 1 , further comprising combining the biological fluid sample of step (a) with one or more cell type-specific antibodies. 
     
     
         8 . The method of  claim 7 , wherein the anti-Free-Site antibody and the one or more cell type-specific antibodies are each conjugated to a different dye or fluorescent label. 
     
     
         9 . The method of  claim 7 , wherein the Anti-Free-Site antibody, the Anti-Total antibody, and the one or more cell type-specific antibodies are each conjugated to a different dye or fluorescent label. 
     
     
         10 . The method of  claim 1 , wherein the determining step comprises comparing the amount of anti-Free-Site antibody bound to the target cell as quantified in step (d) to a standard curve of pre-determined amounts of Anti-Free-Site antibody bound to target cells previously exposed to known amounts of the drug, wherein the standard curve is generated by the method comprising:
 (i) exposing separate aliquots of unbound target cells to known amounts of the drug;   (ii) combining the aliquots of the target cells of (i) with the labeled Anti-Free-Site antibody;   (iii) washing away the unbound anti-Free-Site antibody from the aliquots of the target cells of (ii);   (iv) fixing the aliquots of the target cells of step (iii);   (v) quantifying the amount of Anti-Free-Site antibody binding to the aliquots of the target cells of step (iv); and   (vi) preparing the standard curve showing the relationship between the drug concentration and the quantified binding of the anti-Free-Site antibody to the aliquots of target cells of step (v).   
     
     
         11 . The method of  claim 10 , wherein the aliquots of target cells of (i) are exposed to a permeabilization agent prior to step (ii). 
     
     
         12 . A method of determining drug binding for a drug administered to a subject, comprising:
 (a) isolating target-expressing cells from a biological sample obtained from a subject to whom the drug has been administered, wherein the drug interacts with the target expressed by the target-expressing cells;   (b) incubating the target-expressing cells with an anti-Total antibody and an anti-Free-Site antibody, wherein the anti-Total antibody and the anti-Free-Site antibody are monoclonal antibodies that bind specifically to different epitopes of the same target;   (c) detecting the amount of the anti-Total antibody bound to the target-expressing cells of step (b), wherein the anti-Total antibody binds to an epitope on the target regardless of whether the target is also interacting with the drug;   (d) detecting the amount of the anti-Free-Site antibody bound to the target-expressing cells of step (b), wherein the anti-Free-Site antibody recognizes and binds to an epitope on the target when the drug is not interacting with the target but is competitively inhibited by the interaction of the drug with the target,   wherein the amount of the anti-Total antibody detected in step (c) corresponds to the total amount of target expressed by the target-expressing cells and the amount of the anti-Free-Site antibody detected in step (d) corresponds to the total amount of drug-free target, and   wherein the amount of drug-bound target is proportional to the amount of the anti-Total antibody detected in step (c) less the amount of the anti-Free-Site antibody detected in step (d).   
     
     
         13 . The method of  claim 12 , further comprising permeabilizing the target-expressing cells of step (a) prior to step (b). 
     
     
         14 . The method of  claim 12 , further comprising fixing the target-expressing cells of step (b) prior to step (c). 
     
     
         15 . The method of  claim 12 , wherein the anti-Free-Site antibody and the anti-Total antibody are differentially labeled. 
     
     
         16 . The method of  claim 12 , wherein the anti-Free-Site antibody and the anti-Total antibody are each conjugated to a different dye or a different fluorescent label. 
     
     
         17 . The method of  claim 12 , further comprising combining the isolated target-expressing cells of step (b) with one or more cell type-specific antibodies. 
     
     
         18 . The method of  claim 17 , wherein the anti-Total antibody, the anti-Free-Site antibody and the one or more cell type-specific antibodies are differentially labeled. 
     
     
         19 . The method of  claim 17 , wherein the anti-Total antibody, the anti-Free-Site antibody and the one or more cell type-specific antibodies are each conjugated to a different dye or a different fluorescent label. 
     
     
         20 . The method of  claim 1 , wherein the biological sample comprises a biological fluid containing cells.