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.

1 ) a method of measuring drug activity in whole cells, comprising: 
 a) reacting whole cells with a labeled anti-Total antibody and with a labeled anti-Free-Site antibody;    b) fixing said cells;    c) simultaneously quantifying both anti-Total antibody and anti-Free-Site antibody by flow cytometry, 
 i) wherein the anti-Total antibody detects total target level and the anti-Free-Site antibody detects drug-free target level; and  
 ii) wherein the level of drug-free target is proportional to the level of anti-total antibody staining minus the anti-Free-Site antibody staining.  
   
     
     
         2 ) The method of claim ( 1 ), wherein the whole cells are a mixed population of cells and the cells are further typed by staining with a third antibody specific for a particular cell type.  
     
     
         3 ) The method of claim ( 1 ), wherein the target is on the cell surface.  
     
     
         4 ) The method of claim ( 1 ), where the target is inside the cell and the fixation is sufficient to preserve cell morphology.  
     
     
         5 ) The method of claim ( 4 ), wherein the fixative comprises the ingredients 0.75-0.85% formaldehyde, 25-30 mM DNBS, 6.8-7% DMSO and 0.08-0.1% Tween 20 detergent and fixation is performed at about 43° C.  
     
     
         6 ) The method of claim ( 1 ), wherein the anti-Total antibody and anti-Free-Site antibody are selected from the group consisting of those antibody pairs shown in Table 2.  
     
     
         7 ) A method of assessing drug kinetics in a subject, comprising: 
 a) administering a drug to a subject, wherein the drug binds to a target;    b) collecting whole cells from the subject;    c) incubating the whole cells with a labeled anti-Total antibody and with a labeled anti-Free-Site antibody;    d) fixing said cells;    e) simultaneously quantifying both anti-Total antibody and anti-Free-Site antibody by cytometry, 
 i) wherein the anti-Total antibody detects total target and the anti-Free-Site antibody detects drug-free target; and  
 ii) wherein drug-free target is proportional to anti-total antibody minus anti-Free-Site antibody.  
   
     
     
         8 ) The method of claim ( 7 ), wherein the whole cells are a mixed population of cells and the cells are further typed by staining with a third antibody specific for a particular cell type and sorted according to cell type before quantifying anti-Total antibody and anti-Free-Site antibody.  
     
     
         9 ) The method of claim ( 7 ), wherein the target is on the cell surface.  
     
     
         10 ) The method of claim ( 7 ), where the target is inside the cell and the fixation is sufficient to preserve cell morphology.  
     
     
         11 ) The method of claim ( 10 ), wherein the fixative comprises the ingredients 0.75-0.85% formaldehyde, 25-30 mM DNBS, 6.8-7% DMSO and 0.08-0.1% Tween 20 detergent and fixation is performed at about 43° C.  
     
     
         12 ) The method of claim ( 7 ), wherein the anti-Total antibody and anti-Free-Site antibody are selected from the group consisting of those antibody pairs shown in Table 2.  
     
     
         13 ) A method of measuring circulating free drug in a patient, comprising: 
 a) collecting blood from a patient, taking a drug, and purifying serum or plasma from the blood;    b) reacting the serum or plasma with a homogeneous population of cells which express the drug target antigen;    c) reacting the target cell with a labeled anti-Free-Site antibody, 
 wherein the anti-Free-Site antibody detects drug-free target, but is competitively inhibited by drug-target binding;  
   d) quantifying anti-Free-Site antibody by flow cytometry;    e) comparing the result in step d) against a standard curve wherein the level of free drug in serum is inversely proportional to the level of anti-free-site antibody staining, 
 wherein the standard curve is prepared by reacting a cell with known amounts of said drug, reacting said cells with labeled anti-Free-Site antibody; fixing said cells and quantifying anti-Free-Site antibody by flow cytometry, and preparing said standard curve from the results.