U.S. patent application number 09/425549 was filed with the patent office on 2002-06-06 for fluorescent capture assay for kinase activity employing anti-phosphotyrosine antibodies as capture and detection agents.
Invention is credited to DAMAJ, BASSAM, GLICKMAN, J. FRASER, INGLESE, JAMES.
Application Number | 20020068308 09/425549 |
Document ID | / |
Family ID | 23687045 |
Filed Date | 2002-06-06 |
United States Patent
Application |
20020068308 |
Kind Code |
A1 |
GLICKMAN, J. FRASER ; et
al. |
June 6, 2002 |
FLUORESCENT CAPTURE ASSAY FOR KINASE ACTIVITY EMPLOYING
ANTI-PHOSPHOTYROSINE ANTIBODIES AS CAPTURE AND DETECTION AGENTS
Abstract
A method for determining the level of tyrosine kinase activity
in a biological sample is disclosed. The method employs an
anti-phosphotyrosine antibody as both the capture agent and the
detecting agent. The detecting antibody is labeled with a
lanthanide ion, such as europium, as the signal generating entity.
The method is particularly well suited to high throughput
screening, for example, for compounds which modulate tyrosine
kinase activity.
Inventors: |
GLICKMAN, J. FRASER;
(GARWOOD, NJ) ; INGLESE, JAMES; (DAYTON, NJ)
; DAMAJ, BASSAM; (SAN DIEGO, CA) |
Correspondence
Address: |
HESLIN ROTHENBERG FARLEY & MESITI PC
5 COLUMBIA CIRCLE
ALBANY
NY
12203
US
|
Family ID: |
23687045 |
Appl. No.: |
09/425549 |
Filed: |
October 22, 1999 |
Current U.S.
Class: |
435/7.92 |
Current CPC
Class: |
C12Q 1/48 20130101; G01N
33/573 20130101; G01N 2333/91235 20130101 |
Class at
Publication: |
435/7.92 |
International
Class: |
G01N 033/53 |
Claims
1. A method for measuring protein kinase activity in a biological
sample, said method comprising the steps: (a) coating a solid phase
with a first anti-phosphotyrosine antibody; (b) contacting a
biological sample with said solid phase coated with said first
anti-phosphotyrosine antibody so that phosphorylated
tyrosine-containing molecules in the biological sample are bound by
the antibody to the solid phase; (c) determining the level of
kinase activity in said biological sample by measuring the amount
of a second anti-phosphotyrosine antibody which binds to said
phosphorylated tyrosine-containing molecules.
2. The method of claim 1, wherein said second anti-phosphotyrosine
antibody is labeled.
3. The method of claim 1, wherein said first and second
anti-phosphotyrosine antibodies are the same.
4. The method of claim of 2, wherein label on said labeled
anti-phosphotyrosine antibody is a lanthanide ion.
5. The method of claim 4, wherein said lanthanide ion is
europium.
6. The method of claim 1 wherein said biological sample is a cell
fraction.
7. The method of claim 1, wherein said biological sample is a
lysate of whole cells.
8. The method of claim 1 wherein said biological sample is a
purified homogeneous tyrosine kinase.
9. The method of claim 1 wherein said solid phase is a microtiter
plate.
10. The method of claim 1 wherein said solid phase is a bead.
11. A method for screening a substance for the ability to modulate
tyrosine kinase activity comprising the steps: (a) exposing a
portion of a biological sample to said substance; (b) determining
the level of tyrosine kinase activity in said portion by: (1)
contacting said portion with a solid phase coated with a first
anti-phosphotyrosine antibody so that phosphorylated
tyrosine-containing molecules in the biological sample are bound by
the antibody to the solid phase; and (2) measuring the amount of a
second anti-phosphotyrosine antibody which binds to said
phosphorylated tyrosine-containing molecules.
12. The method of claim 11, wherein a portion of said biological
sample is withheld from exposure to said substance, further
comprising the steps: (c) determining the level of tyrosine kinase
activity in the unexposed portion; and (d) comparing the level of
phosphatase activity in the exposed and unexposed portions of said
biological sample.
13. The method of claim 11, further comprising the step of
comparing the level of tyrosine kinase activity in said biological
sample with a standard having a known level of tyrosine kinase
activity.
14. The method of claim 11, wherein said biological sample is a
cell fraction.
15. A method for screening a substance for the ability to modulate
tyrosine kinase activity comprising the steps: (a) exposing a
portion of a cell population to said substance; (b) preparing cell
fractions from cells of said portion of said cell population; and
(c) determining the level of tyrosine kinase activity in said cell
fractions by: (1) contacting said cell fractions with a solid phase
coated with a first anti-phosphotyrosine antibody so that
phosphorylated tyrosine-containing molecules in the biological
sample are bound by the antibody to the solid phase; and (2)
measuring the amount of a second anti-phosphotyrosine antibody
which binds to said phosphorylated tyrosine-containing
molecules.
16. The method of claim 15, wherein a portion of said cell
population is withheld from exposure to said substance, further
comprising the steps: (d) determining the level of tyrosine kinase
activity in cell fractions from the unexposed portion of said cell
population; and (e) comparing the level of phosphatase activity in
the cell fractions from cells of the exposed and unexposed portions
of said cell population.
17. The method of claim 15, further comprising the step of
comparing the level of tyrosine kinase activity in said cell
fractions with a standard having a known level of tyrosine kinase
activity.
18. The method of claim 15, wherein said first and second
anti-phosphotyrosine antibodies are the same.
19. The method of claim 15, wherein said second
anti-phosphotyrosine antibody is labeled.
20. The method of claim 19, wherein label on said labeled
anti-phosphotyrosine antibody is a lanthanide ion.
21. The method of claim 20, wherein said lanthanide ion is
europium.
22. A kit for use in measuring the level of tyrosine kinase
activity in a biological sample comprising: (a) a solid phase
coated with a first anti-phosphotyrosine antibody; (b) a second
anti-phosphotyrosine antibody, wherein said second
anti-phosphotyrosine antibody is labeled with a lanthanide ion; and
(c) instructions for carrying out a method for using said kit.
23. The kit of claim 22 wherein said lanthanide ion is europium.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for determining
the level of kinase activity in a biological sample and, in
particular, to a method of identifying drugs that inhibit tyrosine
kinases, or drugs that inhibit receptors associated with tyrosine
kinases.
BACKGROUND OF THE INVENTION
[0002] Protein phosphorylation is a common regulatory mechanism
used by cells to selectively modify proteins carrying regulatory
signals from outside the cell to the nucleus. The proteins that
execute these biochemical modifications are a group of enzymes
known as protein kinases. They may further be defined by the
substrate residue that they target for phosphorylation. One group
of protein kinases are the tyrosine kinases (TK's), which
selectively phosphorylate a target protein on its tyrosine
residues. Some tyrosine kinases are membrane-bound receptors and
upon activation by a ligand, can autophosphorylate as well as
modify substrates. The initiation of sequential phosphorylation by
ligand stimulation is a paradigm that underlies the action of such
effectors as, for example, epidermal growth factor (EGF), insulin,
platelet-derived growth factor (PDGF), and fibroblast growth factor
(FGF). The receptors for these ligands are tyrosine kinases and
provide the interface between the binding of a ligand (hormone,
growth factor) to the activation of one or more biochemical
pathways. Ligand binding to a receptor tyrosine kinase activates
its intrinsic enzymatic activity. Tyrosine kinases can also be
cytoplasmic, non-receptor type enzymes and act as a downstream
component of a signal transduction pathway.
[0003] The profound cellular effects mediated by tyrosine kinases,
for example, their putative role in angiogenesis [Giroux, S. et al.
Curr. Biol. 9: 369 (1999)] and lymphoid development [Nosaka, T., et
al. Science 270: 800, (1995)] coupled with the implication that
mutant or defective tyrosine kinase variants may be involved in
tumorigenesis [Jeffers, M., et al. PNAS 94: 11445, (1997)], have
made them attractive targets for the development of new therapeutic
molecules.
[0004] Traditional methods for measuring the phosphorylation state
of cellular proteins have relied on .sup.32P-orthophosphate
incorporation, for example, by exposing cells cultured in the
presence of .sup.32P-orthophosphate to the appropriate ligand or
activator. Alternatively, phosphorylated tyrosine residues can be
detected in immunoassays, for example, immunoprecipitation or
blotting using a radiolabeled antiphosphotyrosine antibody. The
fact that the techniques for detecting radioactive isotope (i.e.
blotting, immunoprecipitation, gel electrophoresis) are very time
consuming, however, minimizes the appeal of these methodologies for
high throughput screening.
[0005] More recent methods utilize a standard enzyme-linked
immunosorbent assay (ELISA) for measuring kinase activity. These
methods utilize purified heterologous substrate protein or
synthetic substrate peptides anchored to a microtiter plate. After
exposure of the substrate molecule to a sample containing the
appropriate kinase, the level of phosphorylation is evaluated with
antiphosphotyrosine antibodies to quantitate the amount of
phosphorylated protein bound to the plate. The obvious limitation
of this type of assay is that the activity of a kinase specific for
the particular substrate used, is the only activity detected.
Additionally, methods such as protein tyrosine kinase enzyme assays
are unable to eliminate as potential drug candidates, inhibitors
which are not cell permeable and, therefore, are not good choices
for therapeutic agents.
[0006] Hirth et al., U.S. Pat. No. 5,763,198, for example,
describes an ELISA-type assay in which a substrate-specific
antibody is used as an anchoring molecule to isolate a protein
substrate from a cell lysate preparation and immobilize it on a
solid phase support. Hirth's method then determines the level of
kinase activity by evaluating the tyrosine phosphorylation state of
the protein substrate bound to the solid phase using an
anti-phosphotyrosine antibody as the detecting molecule. Other
methods for measuring tyrosine kinase activity, particularly
tyrosine kinase receptor activity, are described in WO95/04136, EP
0 730 740 B1, and U.S. Pat. No. 5,599,681.
[0007] The availability of an efficient, high throughput assay of
kinase activity, particularly, one that is cell-based, is highly
desirable because it provides the means, not only to characterize
the phosphorylation status of a cell, but to identify agonists and
antagonists useful as therapeutic agents for the treatment of a
wide variety of human diseases.
SUMMARY OF THE INVENTION
[0008] Because the method of the present invention uses a capture
agent capable of capturing any molecule containing a phosphorylated
tyrosine, it can bind any tyrosine kinase substrate which has been
phosphorylated. The method is, therefore, not limited to the
measurement of a single tyrosine kinase. The method can be used
with any kinase molecule without the necessity of having to use
different capture agents for different kinases. Furthermore, the
method of the present invention has the advantage of detecting
cellular signaling events downstream of the receptor, thereby
providing a means for detecting in a single test a drug's ability
to inhibit more than one therapeutic target.
[0009] In one aspect, the invention relates to a method for
measuring tyrosine kinase activity in a biological sample by
contacting the sample with a solid phase coated with a first
anti-phosphotyrosine antibody to capture phosphorylated proteins
contained in the biological sample, and determining the amount of
phosphorylated protein captured using a second labeled
anti-phosphotyrosine antibody as the detecting molecule. The
biological sample may be a purified homogeneous tyrosine kinase, a
cell fraction, such as a cytosol or cell membrane preparation, cell
or tissue extracts or bodily fluids, such as serum, plasma, urine
or the like.
[0010] In another aspect, the invention relates to a method for
measuring tyrosine kinase activity in a biological sample which
employs a lanthanide ion as the reporter group on the labeled
detecting antibody. By virtue of their unique fluorescence
properties, use of lanthanide ions such as samarium (Sm),
dysprosium (Dy), europium (Eu) and terbium (Tb) as labels make the
method of the invention particularly well suited for highthroughput
screening.
[0011] In yet another aspect, the invention features a method of
screening for molecules, including agonist/antagonist small
molecules, that modulate tyrosine kinase activity in whole cells,
cell fractions, purified tyrosine kinase preparations or biological
fluids. Samples are incubated with potential agonists or
antagonists, and assayed for tyrosine kinase activity in accordance
with the method described herein. Where the biological sample is a
preparation of whole cells, the cells are fractionated, and the
cytosol or cell membrane fractions are assayed for tyrosine kinase
activity in accordance with the method of the present invention.
The method can be used to evaluate cell activation through a
general mechanism or through specific receptor activation by
directly measuring the level of phosphorylated proteins within a
biological sample that results from cell activation.
[0012] In yet another aspect, the invention relates to a kit for
use in measuring the level of tyrosine kinase activity in a
biological sample. The kit provides a solid support, for example a
microtiter plate or beads, which is coated with an
anti-phosphotyrosine antibody which captures phosphorylated
proteins contained within the sample. A labeled
anti-phosphotyrosine antibody is also provided to detect the amount
of phosphorylated protein bound to the solid phase.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows the changes in tyrosine phosphorylation as
detected by the method of the present invention when B-cells are
stimulated with anti-IgM.
[0014] FIG. 2 shows the results of stimulating Jurkat cells with
SDF-1.alpha. for various lengths of time prior to assaying for
tyrosine kinase activity in accordance with the method of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0015] All patents, applications, publications, or other references
that are listed herein are hereby incorporated by reference.
[0016] In the description that follows, certain conventions will be
followed as regards the usage of terminology: The term "solid
phase" refers to the solid support to which a capture agent, for
example, an antibody, is attached and, in one embodiment, may be a
microtiter plate. The method can be carried out in conventional
96-well microtiter plates, or in any other container or on any
surface capable of holding liquid samples and of being scanned by
the appropriate detection device, for example a plate reader or
microscope. Other examples include 6 to 1536-well plates, and
microscope slides.
[0017] In another embodiment, a suspendable solid phase can be
employed, including cellulose beads, controlled pore-glass beads,
silica gels, poly-acrylamide beads, latex beads, dimethylacrylamide
beads, glass particles coated with hydrophobic polymers and other
types of polystyrene beads. The preferred bead size is less than
about 50 .mu.m in diameter, most preferably less than 10 .mu.m but
greater than 1 .mu.m. A preferred suspendable support is a 6.2
.mu.m bead made of polystyrene and commercially available from
Spherotech (Libertyville, Ill.). Such beads are avidin coated or
coated with protein A, typically containing 10.sup.6 binding sites
per bead.
[0018] The term "capture agent" refers to a compound or agent which
is able to adhere to a solid phase and which is selective for
phosphorylated proteins contained in the biological sample to be
assayed and typically is an affinity-purified polyclonal antibody
or a monoclonal antibody. Solid phases used for the immobilization
of phosphorylated molecules may be prepared by coating with the
capture agent (anti-phosphotyrosine), either directly or
indirectly. Methods for immobilizing antibodies are known in the
art and are described in Harlow, E. and D. Lane, Antibodies: A
Laboratory Manual, (1988) Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. which is incorporated herein by reference. In
one embodiment, the anti-phosphotyrosine antibody is immobilized to
the solid phase by an antibody bound to the solid phase which is
specific for the Fc portion of the anti-phosphotyrosine antibody.
In another embodiment, the anti-phosphotyrosine antibody is
adsorbed directly to the solid phase.
[0019] The term "biological sample" refers to any source of protein
tyrosine kinase activity and is intended to encompass purified
kinase preparations, cells, both whole and fractionated, cell and
tissue extracts, and bodily fluids, for example, urine, serum,
plasma and the like.
[0020] Sandwich assays are among the most useful and commonly used
assays and are particularly useful in the present invention. A
number of variations of the sandwich assay technique exist. In a
typical assay, an antibody is brought into contact with a
biological sample containing one or more proteins of interest.
After a suitable period of incubation, for a period of time
sufficient to allow formation of an antibody-protein complex, a
second antibody with specificity for a second distinct epitope on
the molecule bound by the first, labelled with a reporter molecule
capable of producing a detectable signal, is then added and
incubated allowing sufficient time for the formation of a tertiary
complex. Any unreacted material is washed away, and the presence of
the second antibody bound is determined by observation of the
visible signal, and may be quantitated by comparing with a control
sample containing known amounts of hapten. Variations on the assay
include using a first antibody labelled with a reporter molecule.
In addition, the "capture" antibodies may be immobilized on a solid
support.
[0021] Typically, the capture agent and the detecting agent are
different molecules each having specificity for different regions
of the molecule to be captured. Capture agents are usually chosen
so that binding of the capture agent does not sterically block
access to the phosphorylated tyrosines of the molecule of interest.
It has now been unexpectedly found that an anti-phosphotyrosine
antibody can be used as the capture agent and as the detection
agent in the same assay. The method of the instant invention
employs a standard ELISA-like sandwich assay. Unlike other similar
assays, however, its versatility stems from its use of an
anti-phosphotyrosine antibody as both the capture agent and the
detection agent. By doing so, the method is able to capture any
tyrosine-containing protein in the biological sample which is
phosphorylated, thereby providing a mechanism to evaluate
phosphorylation that results from cell activation.
[0022] Activation of a cell most frequently occurs when an
appropriate ligand binds to its receptor on the cell surface. Such
ligands may include growth and differentiation factors such as
epidermal growth factor (EGF); platelet-derived growth factor
(PDGF); chemokines, such as the interleukins; SDF-1 and GM-CSF
(activates JAK2 in a variety of cells). Cells can also be activated
by mitogen stimulation, for example, concanavalin A or
phytohemagglutanin for T-cells, pokeweed mitogen or
lipopolysaccharide for B-cells; or an antibody to a cell surface
marker, such as IgM.
[0023] Because they focus on a specific substrate, current
methodologies are only able to detect phosphorylation by a
particular kinase. The method of the present invention confers an
advantage in that it utilizes a capture agent which can capture any
protein in the biological sample such as a cell lysate which has a
tyrosine residue that is phosphorylated. Because detection is not
limited to a single kinase, a broader perspective of the overall
phosphorylation resulting from an activation event can be obtained.
Furthermore, identification of inhibitors which down-regulate
ligand-induced phosphorylation is possible.
[0024] In a preferred embodiment, method of detection for the
subject method is time resolved fluorescence and the detecting
agent, anti-phosphotyrosine antibody, is labeled with a lanthanide
ion, preferably, Europium. Lanthanide ions have unique fluorescence
properties which make them particularly well suited for high
throughput assays such as the method described herein. While
samarium (Sm) and dysprosium (Dy) are suitable for use in the
instant method, europium (Eu) and terbium (Tb) are preferred, with
europium being most preferred.
[0025] In accordance with the method of the present invention, a
solid support, for example, a 96-well polystyrene microtiter plate
is coated with an anti-phosphotyrosine antibody, for example, pY99
(Santa Cruz Biotech, Inc., Santa Cruz, Calif.), by preparing a
solution having an antibody concentration in the range of 0.01
.mu.g/ml to 25 .mu.g/ml, with a preferred concentration of 2.5
.mu.g/ml, in Tris buffered saline (TBS) having a pH in the range of
7.5-8.5 and adding an aliquot of antibody solution to each well of
the plate. A variety of anti-phosphotyrosine antibodies, available
commercially from a number of sources, are suitable for the method
of the present invention. For example, PY-7E1, PY-1B2, and PY20 are
monoclonal mouse anti-phosphotyrosine antibodies available from
Zymed (San Francisco, Calif.) individually or as a cocktail
(PY-Plus.TM.). Zymed also offers an affinity-purified polyclonal
rabbit anti-phosphotyrosine antibody, Z-PY1. A mouse
anti-phosphotyrosine antibody, clone PT-66 is available from Sigma
(St. Louis, Mo.). Furthermore, polyclonal phosphotyrosine
antibodies may be raised in a variety of species according to
immunization methods well known in the art. A method for the
production of monoclonal phosphotyrosine antibodies is described in
U.S. Pat. No. 4,543,439, the contents of which are hereby
incorporated by reference.
[0026] The plate is then incubated for a period of time sufficient
to allow adsorption of the antibody to the plate. Plates coated in
this manner are then rinsed to remove excess antibody and a
blocking solution, for example, SuperBlock.TM. (Pierce Chemical,
Rockford, Ill.) is added followed by an incubation period
sufficient to allow any regions of the plate not coated with
antibody to be coated with the blocking agent. Other suitable
blocking agents include proteins that do not cross-react with any
of the reagents in the assay, for example, bovine serum albumin
(BSA), gelatin, casein or milk protein. At this point, the plates
may be stored at 4.degree. C. for up to two weeks.
[0027] An aliquot of the biological sample to be evaluated is added
to the capture assay plate. Where the biological sample is a
preparation of whole cells, prior to assay, the cells to be
evaluated are lysed with 50 .mu.l of a standard detergent buffer
containing phosphatase inhibitors, for example, 10% triton X-100,
50 mM sodium fluoride, 2 mM EDTA, 2 mM EGTA. The plate containing
the cell lysates is frozen at -80.degree. C. and may be stored
indefinitely. To assay the cell lysates for kinase activity, the
plate is thawed at 37.degree. C., and 100 .mu.l of tris buffered
saline with 0.1% tween 20 is added to each well. After shaking the
plate for 15 minutes, 180 .mu.l of the cell lysate from each well
is transferred to the capture assay plate described above.
[0028] After addition of the sample, the assay plate is incubated
at room temperature with shaking for a period of time sufficient to
allow the phosphorylated proteins contained in the sample to bind
to the antibody coated plate, for example, anywhere from 15 minutes
to several hours. The plate is then washed to remove unbound
protein. Relative phosphorylated protein levels are detected using
a lanthanide-chelate labeled anti-phosphotyrosine antibody in tris
buffered saline (pH 8.0). An anti-phosphotyrosine antibody which is
different from or the same as the one used for the capture antibody
may be labelled for use as the detecting molecule. Any of the
anti-phosphotyrosine antibodies described above for use as the
capture agent are suitable for use as the detecting antibody as
well. Polyclonal as well as monoclonal antibodies may be labelled
with a lanthanide ion.
[0029] The method of the present invention, employs a
lanthanide-chelate as the label for the detecting antibody. The
long-lived and sharp emission spectrum of certain lanthanide ions
has allowed antibody labeling with detection sensitivities similar
to those obtainable with radioisotopes. Use of europium and terbium
chelators for time-resolved fluorometric assays is described in
U.S. Pat. No. 5,854,008, the contents of which are hereby
incorporated by reference. In a preferred embodiment, an
antibody-Eu.sup.+'conjugate is formed in accordance with
manufacturer's instructions. Approximately 100 .mu.l of labeled
antibody is added to each well and the plate is permitted to
incubate with shaking for a period of time sufficient to allow
binding of the antibody to the phosphorylated protein bound to the
plate. The plate is then washed to remove unbound antibody.
Enhancement Solution for measuring Eu.sup.3+(E.G. & G.Wallac,
Turku, FI) is added and the amount of labeled antibody bound is
measured using a time-resolved fluorescence plate reader (Wallac
Oy, Turku, FI).
[0030] Screening Test Compounds
[0031] The method of the present invention can be used to test for
compounds that modulate tyrosine kinase activity and which act as
tyrosine kinase receptor function agonists or antagonists. In one
embodiment, cells bearing the receptor of interest are exposed to
known agonists, known antagonists, and/or test compounds which may
be, or may contain, agonists or antagonists. An agonist,
antagonist, or test compound may be a chemical compound, a mixture
of chemical compounds, a biological macromolecule, or an extract
made from biological materials such as bacteria, plants, fungi, or
animal cells or tissues. Test compounds are evaluated for potential
activity as agonists or antagonists of receptor activation by
inclusion in screening assays described herein. An "agonist"
enhances the activity of a receptor; an "antagonist" diminishes the
activity of a receptor. The terms "agonist" and "antagonist", as
used herein, do not imply a particular mechanism of function.
[0032] In accordance with the method of the present invention,
cells are incubated with a known agonist, a test compound, or a
combination of the two for a period of time in the range of 1
minute to several hours and a range of temperature from about
4.degree. C. to about 37.degree. C. In a preferred embodiment, the
cells are incubated in the presence of test compound and
ligand/activator for 15 minutes at room temperature. The cells are
then lysed and a sample of the cell lysate assayed in a capture
assay plate as described above
[0033] In a preferred embodiment, the resulting cell lysate is
transferred to a well of a microtiter plate to which an antibody
against the phosphorylated proteins is immobilized. In an
alternative embodiment, a cell membrane fraction, especially a
plasma membrane fraction can be purified from the cells treated
with a test compound, using standard methods (Methods in Enzymology
Vol. 198) and assayed for tyrosine kinase activity in accordance
with the method of the subject invention.
[0034] Time Resolved Fluorescence
[0035] In one embodiment, the detection method employed for
determining the level of anti-phosphotyrosine detecting antibody is
time-resolved fluorescence (TRF). Lanthanide-ion-chelates possess
unique fluorescent properties, making them particularly good
reporter groups for high-throughput applications. For this method,
anti-phosphotyrosine antibodies may be labelled with europium (Eu),
terbium (Tb), dysprosium (Dy) or samarium (Sm), with europium and
terbium being preferred and europium being most preferred. In a
preferred embodiment, anti-phosphotyrosine antibodies, for example,
pY99 (available from Santa Cruz Biotech, Inc., Santa Cruz, Calif.;
Upstate Biotechnology, Inc., Lake Placid, N.Y.; or Transduction
Laboratories,) are labeled with a lanthanide ion, such as Europium
(Eu.sup.3+) according to manufacturer's instructions. Briefly,
anti-phosphotyrosine antibody (1 mg) is dialyzed against 50 mM
sodium bicarbonate pH8.35, 0.9% NaCl and adjusted to a
concentration of 2 mg/ml. 0.5 ml of the antibody solution is mixed
with 300 nmol Europium-DTTA labeling reagent (E.G.& G. Wallac,
Inc., Turku, FI) in bicarbonate buffer (50 mM Sodium Bicarbonate,
150 mM NaCl, pH 8.0) and the reaction mixture is incubated at
16.degree. C. overnight in the dark. Eu.sup.3+-chelate-labeled
antibody is then separated from free Eu.sup.3+-chelate by
gel-filtration chromatography on an S200 FPLC column (Pharmacia,
Bridgewater, N.J.).
EXAMPLE 1
[0036] The following example(s) describes the application of the
assay of the invention using cells expressing T-cell receptor
(T-cells) or IgM (B-cells).
[0037] According to the method of the present invention, cells of
interest were grown under conditions appropriate for growth,
proliferation and for expression of the receptor of interest
according to methods of cell culture known to one of ordinary skill
in the art.
[0038] 96-well polystyrene plates were coated with an
anti-phosphotyrosine antibody, pY99 (Santa Cruz Biotech, Inc.,
Santa Cruz, Calif.), by adding 200 .mu.l of an antibody solution in
Tris buffered saline (TBS) pH 8.0 at a concentration of 2.5
.mu.g/ml. The plates were incubated for three hours at room
temperature. The plates were then rinsed and 300 .mu.l of blocking
solution, SuperBlock.mu. (Pierce Chemical, Rockford, Ill.) was
added and the plates were incubated for two hours. At this point,
the plates may be stored at 4.degree. C. for up to two weeks.
[0039] 50 .mu.l of a cell suspension containing human or animal
cells of interest, for example, Jurkat T cells, Ramos B cells, or
Daudi B cells, at a cell density of 2-5.times.10.sup.5, were added
to each well of a 96-well polypropylene U-bottom plate. 10 .mu.l of
test compound in a physiologic buffer was added to the cells to a
final concentration in the range of 1-10 .mu.M. Control wells
received 10 .mu.l of buffer with no test compound. The appropriate
ligand or activator for the receptor of interest, for example,
anti-CD3 antibody to activate the T-cell receptor, or anti-IgM to
activate the B-cells via cell surface immunoglobulin, was added in
10 .mu.l, so that the final concentration of ligand was 100 ng/ml;
the plate was incubated for 15 minutes. The cells were then lysed
with 50 .mu.l of standard detergent buffer containing phosphatase
inhibitors (10% triton X-100, 50 mM sodium fluoride, 2 mM EDTA, 2
mM EGTA). The plate was frozen at -80.degree. C. and may be stored
indefinitely.
[0040] To assay the cell lysates for kinase activity, the plate was
thawed at 37.degree. C., and 100 .mu.l of tris buffered saline with
0.1% Tween 20 was added to each well. After shaking the plate for
15 minutes, 180 .mu.l of the cell lysate from each well was
transferred to the capture plate described above. The capture plate
was incubated for one hour at room temperature with shaking to
allow the phosphorylated proteins contained in the cell lysate to
bind to the antibody coated plate. The plate was then washed to
remove unbound protein. Relative phosphorylated protein levels were
detected by adding 100 .mu.l of a 0.2 .mu.g/ml solution of
europium-chelate labeled anti-phosphotyrosine antibody in tris
buffered saline (pH 8.0) to each well and allowing the plate to
incubate for one hour with shaking. The plate was then washed to
remove unbound antibody. Enhancement Solution for measuring
Eu.sup.3+(E.G. & G.Wallac, Turku, FI) was added and the amount
of labeled antibody bound was measured using a time-resolved
fluorescence plate reader, for example, a Wallac 1420, or Victor
MLC (Wallac Oy, Turku, FI).
[0041] The results, as shown in FIG. 1, indicate that changes in
tyrosine phosphorylation can be detected by stimulating Daudi or
Ramos B-cells with anti-IgM but not significantly with IL-4. It is
known that an anti-IgM antibody, which stimulates the B-cell
receptor, results in detectable changes in phosphorylation of many
proteins.
EXAMPLE 2
[0042] In this example, Jurkat cells were incubated at 37.degree.
C. with 100 nM SDF-1.alpha. for various time intervals prior to
harvesting of the cells and measurement of tyrosine kinase
activity. The results are shown in FIG. 2. There is a decrease in
kinase activity (as indicated by a decrease in fluorescence) as
time increases suggesting an intracellular dephosphorylation event
following cell activation by SDF-1.alpha..
* * * * *