U.S. patent application number 11/136168 was filed with the patent office on 2005-12-01 for simultaneous assay of target and target-drug binding.
This patent application is currently assigned to Esoterix, Inc.. Invention is credited to Purvis, Norman B., Stelzer, Gregory T..
Application Number | 20050266503 11/136168 |
Document ID | / |
Family ID | 35425827 |
Filed Date | 2005-12-01 |
United States Patent
Application |
20050266503 |
Kind Code |
A1 |
Purvis, Norman B. ; et
al. |
December 1, 2005 |
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.
Inventors: |
Purvis, Norman B.;
(Brentwood, TN) ; Stelzer, Gregory T.; (Brentwood,
TN) |
Correspondence
Address: |
BAKER & MCKENZIE LLP
711 LOUISIANA
SUITE 3400
HOUSTON
TX
77002-2716
US
|
Assignee: |
Esoterix, Inc.
Austin
TX
|
Family ID: |
35425827 |
Appl. No.: |
11/136168 |
Filed: |
May 24, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60573783 |
May 24, 2004 |
|
|
|
Current U.S.
Class: |
435/7.2 |
Current CPC
Class: |
G01N 33/6854 20130101;
G01N 33/5091 20130101; G01N 2333/70596 20130101; G01N 33/5008
20130101; G01N 33/6872 20130101; G01N 33/6863 20130101; Y10T
436/101666 20150115; G01N 33/94 20130101; G01N 2333/7158 20130101;
G01N 2333/70553 20130101; G01N 2333/70514 20130101 |
Class at
Publication: |
435/007.2 |
International
Class: |
G01N 033/53; G01N
033/567 |
Claims
What is claimed is:
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.degree. 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.degree. 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.
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 order to achieve its intended effect) is
purified and the drug'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 plot or Lineweaver Burke.
[0005] 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.
[0006] 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 THE INVENTION
[0007] The following definitions are used herein:
[0008] "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.
[0009] "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]
[0010] "Drug" is any pharmaceutical agent. Drugs as used herein
also include the use of antibodies and their derivatives as
therapeutic agents.
[0011] 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.
[0012] "Target" is defined as the protein that a given drug
interacts with.
[0013] "Target-drug" is the target as it binds to or otherwise
interacts with the drug of interest.
[0014] The following abbreviations are used in herein:
1TABLE 1 Abbreviations Abbre- viation 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
fluorchrome 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 KH2PO4; 8 g/l NaCl; 2.16 g/l
Na2HPO4.7H2O, pH 7.4) PD Pharmacodynamics PK Pharmacokinetics SSC-H
Right angle light scatter .alpha.AgX anti-antigen X - an antibody
directed against a generic antigen called X
[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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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); Cy5PE; Cy7PE; Texas Red (TR);
Allophycocyanin (APC); Cy5; Cy7APC; Cascade Blue; and the like.
[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.
[0022] 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.
[0023] 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.
[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.
[0025] 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.
[0026] 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
[0027] 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.
[0028] 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
.alpha.AgX-FITC, which is the drug insensitive antibody
(anti-Total). The right panel is .alpha.AgX-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).
[0029] FIG. 3. Total Antibody Binding to Neutrophils: Drug
concentration in ng/ml on the x-axis is plotted against
.alpha.AgX-FITC ABC. The antibody staining is constant, thus this
antibody detects total AgX levels and is not drug sensitive.
[0030] FIG. 4. Drug Sensitive Antibody Binding to Neutrophils: Drug
concentration in ng/ml on the x-axis is plotted against
.alpha.AgX-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.
[0031] FIG. 5. Percent Saturation on Neutrophils: Drug
concentration in ng/ml on the x-axis is plotted against percent
saturation (.alpha.Ag Drug saturation) on the y-axis. Percent
saturation is calculated by: % Saturation=[(.alpha.AgX-FITC
ABC-.alpha.Ag X-PE ABC)/(.alpha.Ag X-FITC ABC)]X100.
[0032] 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.
DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0033] 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
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] The antibodies, target and drugs validated to date for use
in the methods of the inventions include:
2TABLE 2 Exemplary Target-Drug Combinations and Antibody Pairs
Anti-Total Anti-Free Site Target Drug Antibody Vendor (Cat. #)
Antibody Vendor (Cat. #) CD11b Neutrophil anti-human Beckman
Coulter anti-human BD Biosciences inhibitory factor CD11b-FITC
(IM0530) CD11b-PE (30455S) CD11a chimeric anti-human BD Biosciences
anti-human Dako-Cytomation humanized CD11a-PE (555384) CD11a-FITC
(F0712) anti-CD11a CD18 Proprietary anti-human BD Biosciences
anti-human Dako-Cytomation CD18-PE (555924) CD18-FITC (F0839) CD22
Proprietary anti-human Serotec anti-human Immunotech CD22-FITC
(MCA553F) CD22-PE (IM1835) CD25 Proprietary anti-human BD
Biosciences anti-human BD Biosciences CD25-PE (555432) CD25-PE
(341009)
EXAMPLE 2
Additional Antibody Pairs
[0041] 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.
3TABLE 3 Additional Target-Drug Combinations and Antibody Pairs
Target Drug Antibodies CD20 Rituximab, PRO70769, Rituximab, Bexxar
Bexxar (Tositumomab), B1, Immu-106, Ibritumomab, HI47, 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, M.phi.P9 CD30 In
Development cAC10-vcMMAE, SGN-30, Ki-1, K0145-3, HSR-4, BER-H2
HLA-Dr Hu1D10 Hu1D10, L243, BRA30, 1DO9C3, 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- S3504 (CCR1), 48607 (CCR2),
Receptors D, GSK-873, 140 83103 (CCR3), Ig1 (CCR4), (e.g. CCR1
UK-427, 857 140706 (CCR6), 11A9 (CCR6), to CCR7) (CCR5) 3D12
(CCR7), 112509 (CCR9) ESRA fulvestrant 6F11, ER1D5, AER311,
ER88
EXAMPLE 3
Flow Cytometry
[0042] 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.
[0043] General Protocol: For analysis of whole blood by flow
cytometry, the following protocol was employed: 100 .mu.l 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.degree. C.
[0044] 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.degree.
C. for 1 hr. Immediately after incubation, the cells were collected
by centrifugation, and erythrocytes were lysed using a standard
ammonium chloride lysing procedure.
[0045] 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.
[0046] We have used the BECKMAN COULTER.TM. Epics XL or BECTON
DICKINSON.TM. 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.TM.) and Excel (MICROSOFT.TM.) or equivalent. Exemplary
results for the CD11b/Neutrophil Inhibitory Factor experiments are
shown in FIG. 1 through 6.
[0047] 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 CD 14 positive cells (monocytes) and CD14 negative
(neutrophils).
[0048] 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.
[0049] 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 (.alpha.AgX-FITC) and drug sensitive antibody (anti-Free-Site)
was labeled with PE (.alpha.AgX-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.
[0050] The histograms in FIG. 2 show fluorescence intensity at
.about.525 nm (left panel, FITC signal) and .about.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
.alpha.AgX-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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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
[0055] Although the invention has been thus far exemplified with
surface targets, such as CD11 b, 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.TM. or PermiFlow.TM. 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.
[0056] 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..TM. 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.degree. C., and most preferred 43.degree. C.
* * * * *