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:
This application is a continuation of U.S. patent application Ser. No. 11/927,223, filed Oct. 29, 2007, issued as U.S. Pat. No. 7,943,327 on May 17, 2011, which is a divisional of U.S. patent application Ser. No. 11/136,168, filed May 24, 2005, now abandoned, which claims the benefit of U.S. Provisional Application No. 60/573,783, filed May 24, 2004, each of which are incorporated by reference herein in their entireties. 
    
    
     FIELD OF THE INVENTION 
     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 
     Previous methods of measuring drug activity have been laborious and complex. Typically, a drug target (the protein to which a drug binds in order to achieve its intended effect) is purified and the drug&#39;s effect on target activity over time is measured using increasing concentrations of the drug. Drug kinetics are then determined using standard data manipulations, such as the Scatchard or Lineweaver Burke plots. 
     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&#39;s activity and kinetics. 
     What is needed in the art is a method that allows the simultaneous detection of both total target and target-drug binding that would simplify and improve the accuracy of the determination of drug kinetics. It would be preferred if the method allowed such measurements without the prior purification of targets, for example in whole cells. It would be especially preferred if mixed populations of cells, such as are found in whole blood, could be studied without the need for prior separation of cell populations. 
     SUMMARY OF INVENTION 
     The following definitions are used herein: 
     “Anti-Total” antibody is an antibody that binds to the target at an epitope that is separate or independent from the drug binding site. Thus, such an antibody will detect “total” target level in a given sample. 
     “Anti-Free-Site” antibody is an antibody that binds to the drug binding or interaction site on the target, such that binding of the drug to the target and binding of the anti-free site antibody to the target are exclusive. Thus, such an antibody will detect drug-free target and be inhibited by drug binding. Bound target-drug concentration can thus be determined, as follows:
 
[Total target]=[Free-Site]+[Target-drug]
 
[Target-drug]=[Total target]−[Free-Site]
 
     “Drug” is any pharmaceutical agent. Drugs as used herein also include the use of antibodies and their derivatives as therapeutic agents. 
     By “simultaneous” what is meant is that the measurements are taken at the same time from the same sample, whether or not the cytometer performs the measurements at the same actual instant or performs the measurements sequentially. 
     “Target” is defined as the protein that a given drug interacts with. 
     “Target-drug” is the target as it binds to or otherwise interacts with the drug of interest. 
     The following abbreviations are used in herein: 
     
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Abbreviations 
               
             
          
           
               
                 Abbreviation 
                 Expansion 
               
               
                   
               
               
                 ABC 
                 Antibody Binding Capacity - The ABC is the number of  
               
               
                   
                 monoclonal antibodies a sample will bind, and correlates to  
               
               
                   
                 the number of antigens expressed on the cell surface. 
               
               
                 Eff. F/P 
                 The effective number of fluorochrome molecules  
               
               
                   
                 conjugated per each antibody molecule determined by  
               
               
                   
                 measured fluorescence intensity of antibody capture  
               
               
                   
                 microspheres measured on a flow cytometer calibrated 
               
               
                   
                 in units of fluorochrome specific MESF 
               
               
                 F/P 
                 Number of fluorochrome molecules conjugated per each  
               
               
                   
                 antibody molecule determined by absorbance on a 
               
               
                   
                 spectrophotometer. 
               
               
                 FCS 
                 Fetal Calf Serum 
               
               
                 FSC-H 
                 Forward angle light scatter 
               
               
                 MESF 
                 Molecules of Equivalent Soluble Fluorochrome. Corrects  
               
               
                   
                 for changes in extinction coefficient, quenching, and small  
               
               
                   
                 spectra shifts. Using the appropriate calibration controls,  
               
               
                   
                 MESF and ABC can be calculated directly by the  
               
               
                   
                 software that controls the cytometer. 
               
               
                 MFI 
                 Mean Fluorescence Intensity 
               
               
                 PBS 
                 phosphate buffered saline (200 mg/l KCl;  
               
               
                   
                 200 mg/l KH 2 PO 4 ; 8 g/l NaCl;  
               
               
                   
                 2.16 g/l Na 2 HPO 4  7H 2 O, pH 7.4) 
               
               
                 PD 
                 Pharmacodynamics 
               
               
                 PK 
                 Pharmacokinetics 
               
               
                 SSC-H 
                 Right angle light scatter 
               
               
                 αAgX 
                 anti-antigen X - an antibody directed against a generic  
               
               
                   
                 antigen called X 
               
               
                   
               
             
          
         
       
     
     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. 
     The method generally is a cell based, two antibody assay that allows detection of total target in the sample and the simultaneous detection of that percentage of targets that are drug-bound and/or drug-free. Simultaneous detection is achieved by using different labels that can each be detected at the same time. In a preferred embodiment, the method uses flow cytometry to detect the different labels simultaneously. 
     The method generally involves the detection of total target using a labeled antibody that binds to the target. At the same time, target-drug binding pairs are detected with a second labeled antibody that binds to the drug binding site on the target. The principle assumption in the method is that binding of the drug to the target will result in the subsequent inhibition of an antibody directed at drug binding site of the target. Should this assumption hold true, the relationship between the drug blockade of antibody binding and the concentration of drug would form the basis of a very sensitive and specific “inhibition” immunoassay for bound drug. 
     Validation of the assay requires evaluation of several monoclonal antibodies in order to identify suitable antibody pairs, as follows: 1) a first antibody that is not inhibited by drug binding, thus providing measurement of total target level, and 2) a second antibody that is effectively inhibited by drug binding, thus proving the central assumption of competition between antibody and drug binding. If demonstrable antibody inhibition by drug binding is observed, then the assay can be calibrated by performing a drug inhibition standard curve. In this manner, the level of drug binding to target can be calculated based upon the level of antibody staining. 
     After antibody staining, the cells are fixed to allow for cytometric analysis. Both cell surface and internal targets can be studied, provided the fixation method is sufficiently gentle to retain cell morphology together with a good level of staining. The two antibodies are then simultaneously detected using flow cytometry, wherein the cytometer is appropriately gated to allow detection of the two labels at the same time. In a preferred embodiment, additional labels can be used to type the cells according to surface antigens. Thus, the cells need not be separated prior to study and complex samples, such as whole blood, can be studied. 
     The labels should be chosen with the operating characteristics of the cytometer in mind such that there is sufficient separation of signal so as to allow the cytometer to distinguish between the two or more signals. Many such labels are known in the art, including fluorescent isothiocyanate (FITC, aka fluorescein), Phycoerythrin (PE); CY5™ PE; CY7™ PE; TEXAS RED™ (TR); Allophycocyanin (APC); CY5™; CY7™ APC; CASCADE BLUE™; and the like. 
     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. 
     We have exemplified the method with whole blood by first lysing the RBCs in isotonic solution, and then by separating the cells of interest based on gating according to surface antigen staining. For example, using CD14 as a surface antigen, it was possible to separately analyze both monocytes and neutrophils in the same blood sample. The method can be generally applied and other cell populations, such as bone marrow, can also be studied. 
     In this novel approach to cell-based analysis of drug pharmacodynamics (PD), cells taken from subjects exposed to various drug concentrations can be assayed to determine the proportion of total available drug targets per cell that are occupied by drug (% saturation). In addition, should the target be expressed at various levels in different populations of cells within the sample (or a different levels between patients), the method allows for simultaneous and independent quantification of target saturation on each different cell population. 
     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 pharmacokinetic (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. 
     In one embodiment, the invention is a method of measuring drug activity in whole cells by reacting cells with labeled anti-Total antibody (detects total target in the sample) and with a labeled anti-Free-Site antibody (detects only drug free target). The cells are fixed and both antibodies are quantified by flow cytometry. The level of drug bound target is proportional to the level of anti-total antibody staining minus the anti-Free-Site antibody staining. The method can also be applied to mixed populations of cells if the cells are pre-typed by staining with a third antibody. The method is also applicable to both cell surface and internal targets. 
     The method can be used to determine a variety of drug kinetic parameters, including pharmacokinetics and pharmacodynamics. It can also be used to determine circulating free drug levels in serum or plasma if used with a standard curve of anti-Free-Site antibody binding versus drug concentration. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . Flow Cytometry:  FIG. 1A  shows side-scatter (SSC-H) on the x-axis plotted against forward-scatter (FSC-H) on the y-axis. All nucleated cells remaining in the lysed blood sample are indicated. The cytometer is then gated to separate the APC-labeled and non-labeled cells in  FIG. 1B , which shows side-scatter (SSC-H) on the x-axis plotted against the APC labeled anti-CD14 antibody scatter (“CD14-APC”) on the y-axis. The separated monocytes (moderate side-scatter, CD14+) and neutrophils (high side-scatter, CD14−) are indicated. 
         FIG. 2 . Antibody Binding Characteristics: Fluorescence intensity (MESF) on the x-axis is plotted against the total number of cells (Number) on the y-axis. The left panel indicates αAgX-FITC, which is the drug insensitive antibody (anti-Total). The right panel is αAgX-PE, which is the drug sensitive antibody (anti-Free-Site). Varying amounts of drug were added to each sample (0-Top, 50 ng/ml-middle, 100 ng/ml-bottom). Inhibition of the drug sensitive antibody by drug is indicated by a loss of fluorescence intensity in the staining by the anti-Free-Site antibody, shown by the peak shifting to the left (right panel). 
         FIG. 3 . Total Antibody Binding to Neutrophils: Drug concentration in ng/ml on the x-axis is plotted against αAgX-FITC ABC. The antibody staining is constant, thus this antibody detects total AgX levels and is not drug sensitive. 
         FIG. 4 . Drug Sensitive Antibody Binding to Neutrophils: Drug concentration in ng/ml on the x-axis is plotted against αAgX-PE ABC. The antibody staining decreases with increasing drug concentration, thus this antibody binds to the drug binding site and the drug competes with antibody binding. 
         FIG. 5 . Percent Saturation on Neutrophils: Drug concentration in ng/ml on the x-axis is plotted against percent saturation (αAg Drug saturation) on the y-axis. Percent saturation is calculated by: % Saturation=[(αAgX-FITC ABC-αAg X-PE ABC)/(αAg X-FITC ABC)]×100. 
         FIG. 6 . Standard Curve: Standard curve of free drug concentration verses fluorescence (MESF) of the drug sensitive anti-AgX antibody on a target cell line. 
     
    
    
     DETAILED DESCRIPTION 
     The present invention is exemplified with respect to CD11b and a proprietary drug known by the code name Neutrophil Inhibitory Factor. However, the invention has general applicability to the study of any drug-target combinations for which suitable antibody pairs can be identified. The following examples describe the invention as practiced in additional detail, but should not be construed as limiting. 
     Example 1 
     Antibody Optimization 
     The following method was employed to validate the method of the invention using the targets and antibodies described in Table 2, below. However, it can be applied to any novel combinations of antibodies to find suitable antibody pairs useful for any target-drug combinations of interest. 
     Commercially available conjugated antibodies against various antigens (generically called AgX) were identified and ordered for evaluation in the method of the invention. Non-conjugated, purified antibodies as well as proprietary pharmaceutical grade antibodies can also be obtained for evaluation and potential custom conjugation. 
     All anti-AgX antibodies against were evaluated in triplicate in the presence and absence of saturating concentrations of the appropriate drug (in each instance, the drug is specific for a single epitope on the protein AgX) by performing individual multi-point two-fold serial dilutions of each antibody. Additional cell subtype specific antibodies labeled with different fluorochromes may be added to each tube to allow the study of specific populations of cells as necessary. 
     Antibody titration data was plotted to show 1) staining intensity versus antibody staining concentration, 2) percent positive staining versus concentration and 3) the signal to noise ratio versus concentration. These plots aided in the identification of the optimal staining concentration of each antibody, indicated whether saturation staining was possible for each antibody and indicated whether the particular antibody measured total AgX expression or was inhibited by the presence of drug. 
     If multiple anti-Total AgX antibodies and anti-Free-Site AgX antibodies were identified, the antibodies that provided the best signal to noise ratio were chosen. In cases were the commercially available antibodies are sub-saturating or not available in the ideal fluorochrome conjugate, custom conjugates can be ordered or prepared in-house. The effective F/P (effective fluorescence to probe ratio) was determined for each of the anti-Total and anti-Free-Site antibodies identified for use in the drug saturation assay. 
     Drug specific antibody panels were and will continue to be developed. Anti-Total and anti-Free-Site antibodies were used to allow monitoring of drug occupancy on the cells of interest during PK and PD experiments. Ex-vivo spiking experiments using appropriate biological samples spiked at varied concentrations of the drug were used to verify operational characteristics of the antibody cocktails and proper drug saturation calculations based on anti-Total and anti-Free-Site antibody measurements. 
     The antibodies, target and drugs validated to date for use in the methods of the inventions include: 
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Exemplary Target-Drug Combinations and Antibody Pairs 
               
             
          
           
               
                   
                   
                   
                   
                 Anti-Free  
                   
               
               
                   
                   
                 Anti-Total 
                 Vendor 
                 Site  
                 Vendor 
               
               
                 Target 
                 Drug 
                 Antibody 
                 (Cat. #) 
                 Antibody 
                 (Cat. #) 
               
               
                   
               
               
                 CD11b 
                 Neutrophil 
                 anti-human 
                 Beckman 
                 anti-human 
                 BD 
               
               
                   
                 inhibitory 
                 CD11b- 
                 Coulter 
                 CD11b-PE 
                 Biosciences 
               
               
                   
                 factor 
                 FITC 
                 (IM0530) 
                   
                 (30455S) 
               
               
                 CD11a 
                 chimeric 
                 anti-human 
                 BD 
                 anti-human 
                 Dako- 
               
               
                   
                 humanized 
                 CD11a-PE 
                 Biosciences 
                 CD11a- 
                 Cytomation 
               
               
                   
                 anti- 
                   
                 (555384) 
                 FITC 
                 (F0712) 
               
               
                   
                 CD11a 
                   
                   
                   
                   
               
               
                 CD18 
                 Proprietary 
                 anti-human 
                 BD 
                 anti-human 
                 Dako- 
               
               
                   
                   
                 CD18-PE 
                 Biosciences 
                 CD18- 
                 Cytomation 
               
               
                   
                   
                   
                 (555924) 
                 FITC 
                 (F0839) 
               
               
                 CD22 
                 Proprietary 
                 anti-human 
                 Serotec 
                 anti-human 
                 Immunotech 
               
               
                   
                   
                 CD22- 
                 (MCA553F) 
                 CD22-PE 
                 (IM1835) 
               
               
                   
                   
                 FITC 
                   
                   
                   
               
               
                 CD25 
                 Proprietary 
                 anti-human 
                 BD 
                 anti-human 
                 BD 
               
               
                   
                   
                 CD25-PE 
                 Biosciences 
                 CD25-PE 
                 Biosciences 
               
               
                   
                   
                   
                 (555432) 
                   
                 (341009) 
               
               
                   
               
             
          
         
       
     
     Example 2 
     Additional Antibody Pairs 
     Additional antibody pairs that can be used in the general method of the invention for particular target and drug combinations can be identified using the method described generally in Example 1. Some possible combinations are listed in Table 3. However, the combinations are unlimited and additional combinations can identified by searching MEDLINE, ATCC or the web. 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Additional Target-Drug Combinations and Antibody Pairs 
               
             
          
           
               
                 Target 
                 Drug 
                 Antibodies 
               
               
                   
               
               
                 CD20 
                 Rituximab, Bexxar 
                 PRO70769, Rituximab, Bexxar 
               
               
                   
                   
                 (Tositumomab), B1, Immu-106, 
               
               
                   
                   
                 Ibritumomab, H147, L27 
               
               
                 CD52 
                 Campath-1H  
                 Campath-1H 
               
               
                   
                 (Alemtuzumab) 
                   
               
               
                 CD33 
                 In Development 
                 HB-10306, Mab 251, M195, 
               
               
                   
                   
                 huM195, PC251, L4F3 
               
               
                 CD4 
                 In Development 
                 OKT4, TNX-355, HuMax-CD4, 
               
               
                   
                   
                 GK1.5, W3/25, YTS177.9, 
               
               
                   
                   
                 ORTHOCLONE OKTcdr4a 
               
               
                 CD3 
                 In Development 
                 HB231, HB10166, UCHT1, PS1, 
               
               
                   
                   
                 OKT3 
               
               
                 CD14 
                 In Development 
                 HB-247, HB-246, UCHM-1, 
               
               
                   
                   
                 IC14, M5E2, MΦP9 
               
               
                 CD30 
                 In Development 
                 cAC10-vcMMAE, SGN-30, Ki-1, 
               
               
                   
                   
                 K0145-3, HSR-4, BER-H2 
               
               
                 HLA-Dr 
                 Hu1D10 
                 Hu1D10, L243, BRA30, 
               
               
                   
                   
                 1D09C3, TNB-211, 1D10, LN3 
               
               
                 MAP Kinase 
                 In Development 
                 SC-154 
               
               
                 (e.g.,  
                   
                   
               
               
                 MAPK1-18) 
                   
                   
               
               
                 RAF-1 
                 In Development 
                 Ab-2 
               
               
                 AKT 
                 In Development 
                 SKB-1 
               
               
                 BCL-2 
                 In Development 
                 YTH-10C4, 124, 10C4 
               
               
                 Chemokine  
                 Sch-417690/Sch-D,  
                 S3504 (CCR1), 48607 (CCR2), 
               
               
                 Receptors 
                 GSK-873, 140 UK-427,  
                 83103 (CCR3), Ig1 (CCR4), 
               
               
                 (e.g. CCR1 to  
                 857 (CCRS) 
                 140706 (CCR6), 11A9 (CCR6), 
               
               
                 CCR7) 
                   
                 3D12 (CCR7), 112509 (CCR9) 
               
               
                 ESRA 
                 fulvestrant 
                 6F11, ER1D5, AER311, ER88 
               
               
                   
               
             
          
         
       
     
     Example 3 
     Flow Cytometry 
     Although the invention has been exemplified with respect to several antibody pair-drug combinations, as indicated in Table 2, only the data from a single study (CD11b) are presented herein for simplicity. These results are representative, although the details for each study will vary. The protocols described are also exemplary, but cell harvesting, antibody staining, fixation, and gating parameters should be (and were) optimized for each experiment. 
     General Protocol: For analysis of whole blood by flow cytometry, the following protocol was employed: 100 μl of whole blood was sterilely collected for each data point. For each sample, 2 ml of standard culture media plus or minus drug was added to the blood and the tubes vortexed briefly to mix. The samples were then incubated to allow drug binding for 1 hr at 37° C. 
     Next the cells were stained with the appropriate antibody or antibodies. A saturating amount (as determined in the titration evaluation of each antibody against the target) of desired antibody was added to each tube, and the tubes were incubated at 37° C. for 1 hr. Immediately after incubation, the cells were collected by centrifugation, and erythrocytes were lysed using a standard ammonium chloride lysing procedure. 
     The cells were collected by centrifugation, and 2 ml of cold PBS+2% FCS was added to each tube. The tubes were placed in the dark at room temperature for at least 10 minutes and up to 1 hour to allow RBC lysis upon resuspension of the fixed whole blood samples in this isotonic solution. The cells were collected again and washed once or twice with PBS+2% FCS with a final resuspension in 1% paraformaldehyde in PBS. The samples were thus ready for data collection. 
     We have used the BECKMAN COULTER™ Epics XL or BECTON DICKINSON™ FACS Calibur Flow cytometer herein, but any properly quality controlled flow cytometer that satisfies established windows of analysis can be used, provided the laser excitation lines and filter configurations are correct for the excitation and detection of all fluorescence labels used in the assay. The analysis that is actually performed will vary depending on the experiment being performed, which of the cellular parameters are of interest, and the actual labels employed. In general, we collect 30,000 ungated events. Data was analyzed in WinList (VERITY SOFTWARE™) and Excel (MICROSOFT™) or equivalent. Exemplary results for the CD11b/Neutrophil Inhibitory Factor experiments are shown in  FIGS. 1 through 6 . 
     Separation of Monocytes and Neutrophils: In the first experiment, whole blood was stained with anti-CD14-APC, the cells were fixed, the RBCs lysed, and the remaining nucleated cells analyzed by cytometer.  FIG. 1A  shows light scatter patterns of the nucleated cells (leukocytes) stained with anti-CD14-APC. In  FIG. 1B , an electronic gating window was constructed around the nucleated cells to separate the cells based on the expression of the CD14 marker. The APC signal was used to separate the leukocytes into CD14 positive cells (monocytes) and CD14 negative (neutrophils). 
     Additional electronic gating windows can be applied to these two cell populations for simultaneous and independent analysis of the binding of the drug sensitive antibody (anti-Free-Site antibody) and the drug insensitive antibody (anti-Total) on each cell type respectively. This type of analysis is shown in  FIG. 2 . 
     Antibody Pair Binding: As above, whole blood was incubated with or without drug, stained with the appropriate antibodies, fixed, lysed and analyzed. This particular experiment was designed so that drug insensitive antibody (anti-Total) was labeled with FITC (αAgX-FITC) and drug sensitive antibody (anti-Free-Site) was labeled with PE (αAgX-PE). However, other labels could be used. Blood was treated with three different concentrations of drug and only the neutrophil results are shown in  FIG. 2 . 
     The histograms in  FIG. 2  show fluorescence intensity at ˜525 nm (left panel, FITC signal) and ˜575 nm (right panel, PE signal) on the x-axis plotted against the number of cells counted (Number) on the y-axis. As the drug level increases, the αAgX-PE peak shifts to the left indicating a decrease in fluorescence intensity. Thus, anti-Free-Site antibody binding decreases due to the competition by the drug for the drug sensitive epitope. In contrast, the fluorescence intensity of the anti-AgX-FITC stain was invariant at all drug concentrations, indicating constant binding of this antibody to the drug insensitive epitope. 
     Target-Drug Binding: In the next two experiments, broader ranges of drug concentrations were investigated. Whole blood was incubated with various concentrations of drug ranging from 0-200 ng/ml, followed by antibody staining, fixing, lysis and analysis as described above. The data in  FIG. 3  demonstrates constant binding of drug insensitive (anti-Total) antibody over a broad range of drug concentration. In contrast, the data in  FIG. 4  demonstrates a dose dependent inhibition of the binding of the drug sensitive anti-AgX-PE (anti-Free-Site) to neutrophils. 
     The data from  FIGS. 3 and 4  were used to calculate percent saturation as shown in  FIG. 5 . Percent Saturation was calculated as the anti-AgX-FITC signal minus the anti-Ag X-PE signal divided by the anti-AgX-FITC signal times  100 . The data demonstrates that the drug causes a dose dependent inhibition of drug sensitive antibody binding to neutrophils. 
     Standard Curve: A target cell line (HL-60) was exposed to different drug concentrations ranging from 0 to 150 ng/ml. Subsequent to drug incubation, the cells were stained with drug sensitive anti-AgX and binding of the fluorescent antibody was quantified on the cytometer as described above and used to prepare this standard curve. Note: This curve was performed using monoclonal antibody binding intensity (MESF). However, the same curve may be constructed using monoclonal ABC. 
     Unknown concentrations of drug in patient specimens can be derived by extrapolation of quantitative fluorescence of the anti-AgX antibody to the respective drug concentration on this standard curve. 
     Example 4 
     Intracellular Targets 
     Although the invention has been thus far exemplified with surface targets, such as CD11b, it is also possible to study internal targets using the methods of the invention because fixatives are now available that allow the detection of internal epitopes. Cells are permeabilized with agents such as 0.05% TRITON X-100™ in PBS, stained with antibody and then fixed. For example, PERMEAFIX™ or PERMIFLOW™ at mild denaturation temperatures preserves cell morphology and thus allows subsequent analysis by flow cytometry. Such methods are described in more detail in co-pending application 60/512,834 filed Oct. 20, 2003. 
     Acceptable fixatives are defined herein as fixatives that allow both cell fixation and permeation, while retaining cell surface morphology and DNA and RNA content, sufficient to allow separation of cells based on light scatter, surface epitopes and/or nucleic acid content. An acceptable fixative contains 0.75-0.85% formaldehyde, 25-30 mM DNBS, 6.8-7% DMSO and 0.08-0.1% TWEEN 20™ detergent. A preferred fixative is PERMIFLOW™. Mild denaturing temperatures are those temperature that improve access to internal antigens, without compromising cell morphology, surface antigens or nucleic acid content. Preferred temperatures range from 39 to 43° C., and most preferred 43° C.