U.S. patent application number 11/392061 was filed with the patent office on 2006-07-27 for assay system for simultaneous detection and measurement of multiple modified cellular proteins.
This patent application is currently assigned to Bio-Rad Laboratories, Inc.. Invention is credited to Quan Nguyen, Yong Song.
Application Number | 20060166298 11/392061 |
Document ID | / |
Family ID | 23046907 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060166298 |
Kind Code |
A1 |
Nguyen; Quan ; et
al. |
July 27, 2006 |
Assay system for simultaneous detection and measurement of multiple
modified cellular proteins
Abstract
A method and kit for simultaneous detection and/or determination
of a plurality of modified proteins in a sample. The method
comprises: a) contacting the sample under mild protein denaturation
conditions with a plurality of first antibodies capable of binding
to a specific target protein, the first antibodies being
immobilized on solid support material, each first antibody being
differentiable from others by a differentiation parameter, whereby
the first antibodies bind to respective target proteins present in
the sample; b) removing unbound materials from the locus of the
first antibodies; c) contacting the materials from step (b) with
one or more second antibodies, each of which is specific to a class
or subclass of modified proteins or with a plurality of second
antibodies, each of which is specific to a modified protein, so as
to bind the second antibody or antibodies to modified proteins in
the sample; and d) detecting and/or determining a plurality of
modified proteins in the sample. The kit comprises a plurality of
primary antibodies immobilized on the above-mentioned solid support
material, one or more buffers for lysing and for washing cellular
material samples to be assayed, an assay buffer for conducting the
assay, the buffer containing a sulfate or sulfonate detergent, and
one or more second antibodies specific to the modified proteins.
Also disclosed is a process for mild denaturation of modified
proteins for use in such a procedure.
Inventors: |
Nguyen; Quan; (Pleasant
Hill, CA) ; Song; Yong; (San Pablo, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Bio-Rad Laboratories, Inc.
1000 Alfred Nobel Drive
Hercules
CA
94547
|
Family ID: |
23046907 |
Appl. No.: |
11/392061 |
Filed: |
March 28, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10092926 |
Mar 6, 2002 |
7049151 |
|
|
11392061 |
Mar 28, 2006 |
|
|
|
60274130 |
Mar 7, 2001 |
|
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Current U.S.
Class: |
435/7.94 |
Current CPC
Class: |
G01N 33/6854 20130101;
Y10S 435/973 20130101; G01N 33/6842 20130101; G01N 33/6803
20130101 |
Class at
Publication: |
435/007.94 |
International
Class: |
G01N 33/537 20060101
G01N033/537 |
Claims
1-23. (canceled)
24. A kit for simultaneous detection and/or determination of a
plurality of modified proteins in a sample, comprising: (a) a
plurality of first antibodies, each capable of binding to a
specific target protein, each first antibody being immobilized on a
solid support material and each first antibody being differentiable
from others by a differentiation parameter; (b) one or more buffers
for lysing and for washing cellular material samples to be assayed
(c) an assay buffer for conducting the assay, said buffer
containing from about 1 -10 mM of a sulfate or sulfonate detergent;
and (d) one or more second antibodies specific to classes or
subclasses of modified proteins or to specific individual modified
proteins.
25. A kit according to claim 24 wherein the solid support material
comprises a series of subsets of solid particles, each subset being
distinguishable from other subsets in accordance with a particular
property or characteristic.
26. A kit according to claim 25 in which the solid particles are
differentiable by specific color or emission spectra.
27. A kit according to claim 25 in which the solid particles
comprise spherical particles formed from non-porous glass,
polystyrene or latex.
28. A kit according to claim 24 in which the solid support material
is a microchip, a plate having a multiplicity of wells, or a
slide.
29. A kit according to claim 24 wherein the modified proteins are
selected from phosphorylated proteins, glycosylated proteins,
acetylated proteins, methylated proteins, ubiquinated proteins, and
prenylated proteins.
30. A kit according to claim 24 wherein the modified proteins are
phosphorylated proteins.
31. A kit according to claim 24 wherein the second antibodies
comprise one or more antibodies that are specific to classes of
modified proteins.
32. A kit according to claim 24 wherein the second antibodies
comprise one or more antibodies that are specific to subclasses of
modified proteins.
33. A kit according to claim 24 wherein the second antibodies are
specific to phosphorylated proteins.
34. A kit according to claim 24 wherein the second antibodies
comprise a plurality of antibodies, each of which is specific to an
individual modified protein.
35. A kit according to claim 24 further comprising a labeled
moiety.
36 and 37. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority of U.S. provisional
application No. 60/274,130 filed Mar. 7, 2001.
BACKGROUND AND PRIOR ART
[0002] This invention relates to improvements in processes for
conducting "sandwich" assays of proteins and, in particular to an
improved method for simultaneously detecting and/or determining, a
plurality of modified proteins, especially modified cellular
proteins, in a sample. In a typical "sandwich" assay for proteins,
a sample (generally a biological fluid or cell extract) comprising
a mixture of materials, including one or more proteins, is
contacted with a series of microparticles or beads associated with
antibodies that bind to the respective target proteins. Each
combination of microparticles with a particular antibody has a
feature, for instance color, that can distinguish it from the
others. After washing away unbound materials, the microparticles,
now containing proteins bound to the antibodies, are contacted with
a second antibody that binds specifically to a particular protein
to be detected. The second antibody typically includes a means for
its detection, such as a fluorescent marker. The target protein is
detected, and the quantity determined, by means suitable to the
situation (e.g. scanning for fluorescence).
[0003] Most cellular proteins require modifications in order to
transfer from their silent state to the active form. Such
modifications include phosphorylation (for instance, on tyrosine,
threonine and/or serine residues), glycosylation (on membrane
proteins), and acetylation, prenylation and methylation (on lysine
residues). Detection and measurement of such modified proteins, as
currently carried out, is limited to processes that involve
multiple steps but that can determine only a single modified
protein in each assay. For instance, to investigate phosphorylation
of proteins, current procedures use .sup.32P-labeled inorganic
phosphate in combination with immunoprecipitation and SDS-PAGE
autoradiography or Western blot analyses specific for the
phosphorylated form of the target protein. However Western blot
processes for this purpose are slow and time-consuming and can only
determine one analyte at a time.
[0004] A method for simultaneously detecting and, if desired,
determining, two or more modified proteins in a sample, in a single
operation, would be desirable.
SUMMARY OF THE INVENTION
[0005] This invention provides a method and kit for simultaneous
detection and, if desired, quantitative determination, of a
plurality of modified proteins in a given biological sample. These
methods and kits are considered to be a satisfactory replacement
for Western Blot determination of modified proteins.
[0006] In one aspect the invention comprises a method for
simultaneous detection and/or determination of a plurality of
modified proteins in a sample, comprising: [0007] a) contacting the
sample under mild protein denaturation conditions with a plurality
of first antibodies capable of binding to a specific target
protein, the first antibodies being immobilized on solid support
material, each first antibody being differentiable from others by a
differentiation parameter, whereby the first antibodies bind to
respective target proteins present in the sample; [0008] b)
removing unbound materials from the locus of the first antibodies;
[0009] c) contacting the materials from step (b) with one or more
second antibodies, each of which is specific to a class or subclass
of modified proteins or with a plurality of second antibodies, each
of which is specific to a modified protein, so as to bind the
second antibody or antibodies to modified proteins in the sample;
and [0010] d) detecting and/or determining a plurality of modified
proteins in the sample.
[0011] In another aspect, the invention involves a kit for carrying
out such procedures, comprising [0012] (a) a plurality of first
antibodies each capable of binding to a specific target protein,
each first antibody being immobilized on a solid support material
and each first antibody being differentiable from others by a
differentiation parameter; [0013] (b) one or more buffers for
lysing and for washing cellular material samples to be assayed
[0014] (c) an assay buffer for conducting the assay, said buffer
containing from about 1-10 mM of a sulfate or sulfonate detergent;
and [0015] (d) one or more second antibodies specific to classes or
subclasses of modified proteins or to specific individual modified
proteins.
[0016] In yet another aspect, this invention involves a process for
denaturation of modified proteins in an overall process for
simultaneously analyzing a sample for a plurality of modified
proteins, comprising contacting the sample with a sulfate or
sulfonate detergent, preferably in a concentration of about 1-10
mM, at a temperature of between about 4 and about 37.degree. C.,
and for a time of from about 2 to about 72 hours.
DESCRIPTION OF THE DRAWING
[0017] FIG. 1 depicts a comparison of analyses for the modified
protein phospho-Erk2 using a method according to this invention and
a Western blot analysis.
DETAILED DESCRIPTION OF THE INVENTION
[0018] In carrying out the process of this invention, a sample of
material is selected for analysis. The material in general is a
biological fluid or cell extract, for instance, a cell lysate. The
lysate may be obtained from various sources, including tissue
culture cells, cells extracted from blood, or in vivo solid tissue
samples. The lysate is contacted, under mild protein denaturation
conditions, with a plurality of primary antibodies that are
specific to the non-modified portion of the target proteins, i.e.
proteins whose presence and/or quantity in the sample is to be
detected or determined.
[0019] The primary antibodies are immobilized on a solid support
material, each primary antibody being differentiable from others by
a differentiation parameter. The solid support material may be a
plurality of beads or other particles that are differentiable, and
to which the antibodies are immobilized, for example via
conjugation. Alternatively, the antibodies may be immobilized on a
solid substance or device such as a microchip, plate (e.g. a
96-well plate) or slide.
[0020] For particulate supports, the capability to distinguish
between combinations of particles with different antibodies (and
thus between different antibodies) is accomplished by providing a
plurality of particles of different types and immobilizing each
primary antibody on particles of one type. That is, the particles
may be divided into subsets, with each subset being distinguishable
from other subsets according to a particular property,
characteristic or characteristics. Each such subset is conjugated
to a different primary antibody; thus each combination of particles
with a particular antibody is distinguishable from combinations of
particles with other antibodies. For example, the particles may be
divided into subsets where each subset is capable of being
distinguished by a specific color or emission spectra, which may be
provided by the presence of a fluorochrome or combinations of
fluorochromes incorporated within or on it, for example, as
described in U.S. Pat. No. 5,981,180. The coupling of the
antibodies to the beads or particles is accomplished by covalent
coupling or adsorption methods well known to those familiar with
the art and described in the patent and scientific literature (see,
for instance, Immunochemistry of Solid-Phase Immunoassay, John E.
Butler, CRC Press, 1991 and Immobilized Enzymes, Antigens,
Antibodies, and Peptides, edited by Howard H. Weetall, Marcel
Dekker, Inc. N.Y., 1975).
[0021] The particles themselves are typically spherical (i.e.,
"beads" or "microspheres"), with either a rough or a smooth
surface, and are prepared as known in the art. They are made
various materials, usually non-porous glass, polystyrene, latex or
other polymeric materials, and are generally 0.05 micron to 90
micron diameter, preferably 0.5 to 10 micron in diameter, with
densities ranging from about 1 to 2 g/mL, preferably close to the
density of water.
[0022] Once the combinations of primary antibodies and particle
subsets are made, the subsets are combined for use in the assay
procedure and/or kits of the invention.
[0023] If solid supports other than particles are used, for
instance, glass, polymeric or silica chips, plates, slides, etc.,
the primary antibodies are immobilized on the surface of the
support as known in the art at specific locations (e.g. in specific
wells of a plate), and thus are differentiable and may be
identified by their location on the support.
[0024] After lysing the cells, a buffer that contains a sulfate or
sulfonate detergent (preferably in a concentration of 1-10 mM) is
added. The detergent is preferably SDS (sodium dodecyl sulfonate);
however analogous detergents such as alkyl sulfates or alkane
sulfonates may be used. The sample is then contacted with the
immobilized primary antibodies under mild denaturation conditions
(for instance, the above amount of SDS, a temperature of between
about 4 and about 37.degree. C., and for a time of from about 2 to
about 72 hours).
[0025] U.S. Pat. No. 4,658,022 describes a prior art protein assay
of this general type (but used only for determination of individual
proteins) in which the denaturation of the proteins is conducted at
higher temperatures (above 50.degree. C.) and for shorter times
(one minute or less). According to that patent, denaturation of
proteins in general can enable better detection of proteins,
particularly those where the target epitope is hidden. Furthermore,
that patent states that denaturation at higher temperature is
advantageous because it can be done quickly, thus shortening the
overall procedure. However, we have found that denaturation under
such conditions is detrimental to obtaining a proper determination
of proteins in a process for the simultaneous determination of
multiple proteins, such as this invention.
[0026] Consequently, in the process of this invention, mild
denaturation, as defined herein, is used. In that connection, it
should be noted that the fact that a detergent such as SDS would be
useful in mild denaturation is surprising. SDS is generally a
strong detergent that can potentially denature both the target
protein and the capturing antibodies. For instance, SDS can
partially denature the antibodies so as to render them
non-specific, making it possible for them to bind to other targets
than the desired protein. For that reason its use is generally
avoided in protein sandwich assays.
[0027] The denaturation process itself constitutes another aspect
of this invention, and may be used in this process or in another
process for simultaneous assay of multiple modified proteins in a
sample.
[0028] Following the mild denaturation and binding of primary
antibodies to the modified proteins, the material is then contacted
with one or more second antibodies that are specific to, and bind
to, the modified portion of proteins in the sample. The second
antibodies may on the one hand be chosen from antibodies that are
each specific to a certain modified protein for which detection or
determination is sought, for instance, a modified Erk2, JNK or Akt.
In that case a multiplicity of such second antibodies is utilized
so as to enable a simultaneous detection and/or determination of a
multiplicity of such proteins.
[0029] In another embodiment, however, the second antibody or
antibodies are chosen from those that are specific to a class or
subclass of modified proteins. By "class" is meant a type of
protein modified at any position or positions, any number of times,
by a modifying group. Such classes include, for instance,
phosphorylated, glycosylated, ubiquinated, methylated, acetylated
or prenylated proteins in general.
[0030] The term"subclasses" is meant to refer to a subgroup of such
a class, in which the modification occurs at a certain position or,
more typically, on a certain amino acid, irrespective of its
position or positions in the protein. The subclass is not limited
to any specific unmodified protein. Examples of subclasses of
phosphorylated proteins, for instance, include phosphotyrosines,
phosphoserines, and phosphothreonines. In the case of
phosphorylated proteins, the second antibody is preferably a
biotinylated antibody.
[0031] Examples of second antibodies for specific modified proteins
include, for instance, rabbit monoclonal Anti-phospho-ERK2 (Cell
Signaling Technology) and Anti-phospho Lck (BioSources)
(phosphorylation).
[0032] Second antibodies that are specific to a subclass of
proteins include, for instance, antiphosphortyrosine (SIGMA
Chemical, clone PT66 cat 3 B1531 or Transduction Lab, Clone
PY20-cat # P11123), acetylated-lysine polyclonal antibody (Daiichi)
(acetylation), rabbit anti-ubiquitin antibodies (Institute Pasteur)
(ubiquination) and mouse monoclonal anti-methylated arginine
(Abcam) (methylation).
[0033] Use of a second antibody that is specific to a class of
proteins, i.e. to phosphorylated or ubiquinated proteins, enables
simultaneous determination of multiple modified proteins of that
class using a single second antibody. In a further embodiment, a
plurality of such second antibodies is utilized, each being
selective to a different class of modified proteins. That is, a
mixture of second antibodies is used, one (or more) of which is
selective to phosphorylated proteins, another to ubiquitins,
another to methylated proteins, etc. This enables detection and/or
(quantitative) determination of modified proteins in general in a
sample, or of several classes of modified proteins, according to
the second antibodies that are used.
[0034] In the case of phosphorylated proteins, presently available
antibodies selective to this class tend to be less accurate than
desired. Consequently, if the aim of the assay is to detect or
determine modified phosphorylated proteins in general, a
combination of antibodies for subclasses, i.e. antibodies to
phosphorylation on serine, threonine and tyrosine would be used in
preference to a single antibody for the class as a whole.
[0035] Antibodies selective to acetylated proteins include those
selective to specific acetylated proteins and those that detect
acetylated lysine resides in general (i.e. are class-selective for
acetylation). Antibodies selective to glycosylated proteins include
those selective to specific glycosylated proteins and biotinylated
lectin, which is selective to the class of glycosylated
proteins.
[0036] Those familiar with the art will recognize that binding of
analytes to antibodies is influenced by incubation conditions such
as time, temperature, pH, ionic strength of reagents, and the like,
and the conditions of a given assay will be chosen as known in the
art to optimize the sensitivity and specificity of the test and
generally suit the ease of use of the protocol and its adaptability
to automation.
[0037] After contact with the second antibody or mixture of
antibodies, the "sandwiched" materials may then be detected and/or
quantitatively determined, for instance by contact with a labeled
moiety having a component which binds to the second antibodies,
such as streptavidin, and which is labeled with phycoerythrin (PE)
or with another label, which may be a dye or other type of label
such as a radioactive label and determination of the latter using
standard techniques. In some instances the detection may be carried
out by directly binding the labeled moiety to the second
antibodies.
[0038] Covalent attachment of fluorescent labels to streptavidin
may be effected by a variety of techniques previously described in
patent and scientific literature (Haugland, R. P., Bhalagat, M. K.,
Preparation of avidin conjugates, Methods Mol. Biol. 1998;
80:185-96). Typical fluorescent moieties are described in Chapter 3
of the Manual of Clinical Laboratory Immunology, supra.
Alternatively the conjugates may be obtained from a commercial
sources. Fluorescent dyes such as fluorescein, the arylsulfonate
cyanine dyes, phycobiliprotein dyes, bodipy dyes and the like, may
be used. If the particle subsets are distinguished from one another
on the basis of incorporation of fluorochromes, then the dyes used
in the labeled moieties are selected so as to have fluorescent
emissions that are distinct from, and do not interfere with, the
emission spectra of the particle subsets. A preferred type of
fluorescent material is a class of compounds known as
phycobiliproteins, more particularly the phycoerytherins, the
phycocyanins, and the allophycocyanins, most preferably the
phycoerytherins.
[0039] The materials are incubated under appropriate conditions for
binding of the streptavidin to the second antibodies. In this
overall process, the fluorescently labeled streptavidin binds to
the particles through the primary and second antibodies, and
through the binding of primary antibodies to the modified proteins.
The modified proteins thus can be detected and measured by
application of excitation energy having a wavelength selected to
excite the chosen fluorescent label, where the emission spectra
that is generated is distinct from the emission spectra
incorporated in the particles.
[0040] Detection of the modified proteins alternatively may be
carried out without the use of a label, for instance by use of SPR
(Surface-Plasmon Resonance) technology.
[0041] By using the methods and kits of this invention,
simultaneous determination of a multiplicity of modified proteins
in a sample can be carried out. The process is capable of
simultaneously detecting and/or determining as many as 100 or more
modified proteins in a single sample, using as few as 50 cells. It
should be noted, however, that the cell concentration should be
about 0.2-900 .mu.g/ml.
[0042] In order to prevent the dephosphorylation of proteins by
phosphatase, a phosphatase inhibitor, such as NaF or
Na.sub.3VO.sub.4, should be present during the overall process,
from capturing through detection steps.
[0043] Another aspect of this invention is a kit for carrying out
the process. The kit comprises: [0044] (a) a plurality of first
antibodies, each capable of binding to a specific target protein,
each first antibody being immobilized on a solid support material
and each first antibody being differentiable from others by a
differentiation parameter; [0045] (b) one or more buffers for
lysing and for washing cellular material samples to be assayed
[0046] (c) an assay buffer for conducting the assay, said buffer
containing from about 3-10 mM of a sulfate or sulfonate detergent;
and [0047] (d) one or more second antibodies specific to modified
proteins, i.e., either to classes or subclasses of modified
proteins or to specific individual modified proteins.
[0048] As described above, the primary antibodies may either be
bound to a plurality of differentiable beads or immobilized at
different locations on a solid surface or device such as a chip,
slide or plate.
[0049] The kit may have additional components, as described below,
including the labeled moiety for detecting and/or determining the
modified proteins.
EXAMPLE 1.
[0050] The following represents an example of a kit of this
invention, and of a process for simultaneously determining multiple
phosphorylated proteins in a sample.
[0051] A kit is prepared containing the following:
[0052] Phosphoprotein Assay (Detection Modules) [0053] Capturing
antibody-conjugated beads (50.times., 250 .mu.l)
[0054] (Each analyte =2.5.times.10.sup.6 beads/mL) [0055]
Phosphoprotein Detection Antibody (antiphosphortyrosine from SIGMA
Chemical-clone PY54)
[0056] 50.times.for Premixed Multi-Plex Assays (120 .mu.l)
[0057] 100.times.for Unmixed Multi-Plex Assays (70 .mu.l each
analyte)
[0058] (each Detection Antibody=0.2 mg/mL) [0059] Positive Controls
for phospho-JNK and phospho-p38MAPK
[0060] (250 .mu.l/vial at 200 .mu.g/ml) [0061] Positive control for
phosphoproteins (250 .mu.l/vial at 200 .mu.g/ml, per protein).
[0062] Negative control (250 .mu.l/vial at 200 .mu.g/ml
protein)
[0063] Cell Lysis Kit A [0064] Cell Wash Buffer A (1.times., 150
ml) (20 mM Tris-HCI, pH 7.35-7.45, 0.9% NaCl) [0065] Cell Lysis
Buffer A (1.times., 25 ml) (20 mM Tris-HCI, pH 7.8-8.2, 50-500 mM
NaCl, 50-100 mM NaF, 0.02-0.08% NaN.sub.3, 0.5-3% NP40 or
Triton.times.-100, 4-8 mM EDTA) [0066] Cell Lysis Buffer A, Factor
1 (250.times., 100 .mu.l) (500-1000 mM Na.sub.3VO.sub.4) [0067]
Cell Lysis Buffer A, Factor 2 (500.times., 50 .mu.l) (1-2 mg/ml
Leupeptin) [0068] It should be noted that the cell wash and cell
lysis buffers do not contain SDS or any other sulfate or sulfonate
detergent.
[0069] Reagent Kit B [0070] Assay Buffer B (1.times., 10 ml) (20 mM
Tris-HCl, pH 7.8-8.2, 50-500 mM NaCl, 50 mM NaF, 0.5-3% SDS,
0.02-0.08% NaN.sub.3) [0071] Wash Buffer B (1.times., 150 ml)
[0072] Detection Antibody Diluent B (1.times., 10 ml) [0073] Bead
Resuspension Buffer B (1.times., 40 ml) [0074] Streptavidin-PE
(100.times., 70 .mu.l) [0075] Filter Plate (96-well) [0076]
Adhesive Plate Sealers [0077] Phosphoprotein Assay Instruction
Manual Additional items
[0078] The following additional items are not necessarily provided
with such a kit, but are recommended in using the kit to conduct
the process. These items are available from Bio-Rad, Hercules,
Calif.
Bio-Plex Protein Array System
[0079] Bio-Rad catalog # 171-000001, 171-000003, or 171-000005
Bio-Plex Protein Array System Accessories [0080] Bio-Plex
validation kit, Bio-Rad catalog # 171-203000 [0081] Bio-Plex
calibration kit, Bio-Rad catalog # 171-203060 Bio-Rad DC Protein
Assay Kit II [0082] Bio-Rad catalog # 500-0112 (with BSA standard)
Cell Lysate Preparation for Adherent Cells
[0083] Note that optimal performance of this particular
phosphoprotein assay calls for 150 .mu.l of cell lysate (protein
concentrate, 300-900 .mu.g/ml) per well. An addition of 2 mM PMSF
(phenylmethylsulfonyl fluoride) in the cell lysis buffer is also
required. To prepare a 250.times.stock solution (500 mM PMSF),
dissolve 0.436 g PMSF in 5 ml DMSO, aliquot to 0.2-0.5 ml per tube
and store at -20.degree. C.
[0084] The cells are cultured in a 96-well culture plate or other
suitable culture vessel. The final cell lysis buffer is prepared by
adding Factors 1 and 2 and a solution of 500 mM PMSF in DMSO to the
Cell Lysis Buffer A, but no more than 5-10 minutes prior to lysing
the cells. (For a total of 10 ml final cell lysis buffer, add 40
.mu.l of Factor 1, 20 .mu.l of Factor 2 and 40 .mu.l of 500 mM PMSF
to 9.9 ml of 1.times.Cell Lysis Buffer A. Mix well by vortexing.)
Then, the reaction is stopped by quickly rinsing the cells with 200
.mu.l 1.times.Cell Wash Buffer A after aspirating the culture
medium.
[0085] Lysis buffer is then immediately added to each well or
culture vessel. The amount of cell lysis buffer added depends on
the cell concentration in each well. The plate is placed on ice.
Pipetting is carried out up and down for 5 times with a
multi-channel pipette set at 50 .mu.l, followed by agitation of the
plate on a plate shaker at 300 rpm for 20 min at 4.degree. C. (For
other types of culturing vessels, the vessel is kept on ice and the
cell monolayer scraped with a cell scraper; the lysate is
transferred to a 15-ml centrifuge tube then rotated end-over-end
for 20 min at 4 .degree. C.)
[0086] Cellular debris is then removed, for example, by
centrifugation at 2,000 g for 30 min or 4500 g for 15 min at
4.degree. C. The supernatants are collected for the assay. If the
assay is not performed immediately, the cell lysates should be
stored frozen at -70.degree. C.
Cell Lysate Preparation for Suspension Cells
[0087] The cells are cultured and treated as above. The reaction is
stopped and the materials centrifuged at 300 g for 5 min at
4.degree. C. During the centrifugation, the cell lysis buffer is
prepared as above. The supernatant is aspirated, and the ice-cold
lysis buffer is immediately added to each well or culture vessel.
The amount of lysis buffer added depends on the cell concentration
in each well. The plate is placed on ice, pipetted and agitated as
above; cellular debris is removed and the supernatants collected
for the assay as above.
Assay Procedure for Premixed Multi-Plex Assays
Capturing Antibody-Conjugated Beads (Premixed)
[0088] If using a 96-well plate, any section not being used should
be sealed with an adhesive sealer. The premixed capturing
antibody-conjugated beads (25.times.) are vortex mixed vigorously
for 30 seconds, then diluted to a 1.times.concentration using Wash
Buffer B. Each well requires 50 .mu.L of 1.times.capturing antibody
conjugated beads (i.e. 1 .mu.L of capturing antibody conjugated
beads (50.times.)) and 49 .mu.L of 1.times.Wash Buffer B.
Assay Procedure (pre-mixed)
[0089] Capturing antibody-conjugated beads (1.times.) are prepared
as directed above. The Assay Buffer B is brought up to room
temperature or alternately to 37.degree. C. prior to use and vortex
mixed to make sure all components are well dissolved. The cell
lysate samples are thawed on ice if they have been stored frozen.
Then the apparatus is calibrated (a suitable apparatus for
conducting the assay is the Millipore MultiScreen.RTM. Separations
System).
[0090] The desired number of wells of a 96-well filter plate is
pre-wet with 100 .mu.L of 1 .times.Wash Buffer B. The filter plate
is placed on a calibrated filter plate vacuum manifold. and the
buffer is removed by vacuum filtration. The removal of buffer is
carried out between steps of the overall assay procedure, per
common practice.
[0091] The 1 .times.capturing antibody conjugated beads are vortex
mixed for 15 seconds and 50 .mu.L added to each well. 100 .mu.L of
the 1.times.Wash Buffer B is added to each well. 50 .mu.L of
positive controls and the negative control are added to the well
designated for controls. (The positive and negative cell lysate
controls are premixed with 1.times.Assay Buffer B.)
[0092] Then, 25 .mu.L of 1.times.Assay Buffer B is added per well
(except for the controls). 25 .mu.L of unknown cell lysate samples
is added; the plate is sealed and covered and shaken overnight at
room temperature. The 1.times.detection antibody mix is prepared
5-10 minutes before the next step. Premixed Phospho-Protein
Detection Antibody (50.times.) is diluted to a
1.times.concentration with 1.times.Detection Antibody Diluent B.
The total volume of 1.times.Phospho-Protein Detection Antibody
required is based on the number of wells used, allowing for 50
.mu.L per well. The sealing tape is then removed from the plate,
and the buffer is removed by vacuum filtration. The plate is washed
with 1.times.Wash Buffer B, with removal of buffer.
[0093] The detection antibody is vortex mixed gently and 50 .mu.L
added to each well. The plate is sealed and covered, than shaken at
room temperature.
[0094] The Streptavidin-PE conjugate (100.times.) is then diluted
to a 1.times.concentration with 1.times.Wash Buffer B, 5-10 minutes
prior to use. The plate is washed and unsealed and buffer is
removed. The 1.times.Streptavidin-PE conjugate is vortex mixed
vigorously and 50 .mu.L is added to each well. The plate is covered
and sealed, then shaken; then uncovered and washed as before.
[0095] The beads in each well are resuspended with 125 .mu.L
1.times.Bead Resuspension Buffer B. Determination of the content of
modified proteins is done using a Bio-Plex System (available from
Bio-Rad), a specialized microtiter plate reader.
EXAMPLE 2.
Assay for Multiple Modified Proteins
[0096] The above procedure was used to simultaneously analyze a
sample of cell lysates containing five phosphorylated proteins:
phosphorylated p38MAPK, I.kappa.B.alpha., Erk2, JNK and Akt1. The
proteins were phosphorylated at the following sites:
p38MAPK-Thr.sup.180, Tyr.sup.182; I.kappa.B.alpha.-Ser.sup.32,
Ser.sup.36; Erk2-Thr.sup.202, Tyr.sup.204; JNK-Thr.sup.183,
Tyr.sup.185; Akt1-Ser.sup.473. Cell lysates were HeLa and HEK-293
simulated with UV, EGF, Fetal Bovine Serum (FBS) or TNF-alpha. The
uninduced HeLa cell lysate was also prepared and used as a negative
control. All procedures were done on a 96-well filter plate. The
cell lysate samples were analyzed by the above procedure and
verified for expression of phosphorylated proteins via Western
blotting. The result demonstrates: (1) a simultaneous detection of
all five phosphorylated proteins above; (2) only 5-10 .mu.g of cell
lysate per well was needed for the analysis; (3) an inter-plate and
intra-plate coefficient of variation less than 10% and (4) a tight
quantitative and qualitative correlation with Western blotting
data.
[0097] For example, in the detection of phosphorylated
I.kappa.B.alpha., the above assay detected the expression of
phospho-IkB in HeLa cell lysate that was stimulated with TNF-alpha
and not in HEK293 stimulated UV, EGF or FBS. The Western blot probe
for I.kappa.B.alpha.also showed a strong band in TNF-alpha
stimulated HeLa cell lysate and no visible bands for all other
stimulated HEK cell lysates. In another experiment, a dilution of
phospho-Erk stimulated cell lysate ranging from 50-5000 ng per
analysis was assayed using the above procedure and by Western blot.
The increasing band signal of phospho-Erk was observed with
increasing concentration of cell lysate. Similarly, the signal for
the assay according to this invention also increased with
increasing cell lysate concentration. As shown in FIG. 1, when the
signals derived from Western blotting (x-axis) and the above assay
for multiple modified proteins (y-axis), were plotted and linear
curve-fitted, a correlation coefficient (R.sup.2) of 0.9804 was
obtained. This demonstrates a tight quantitative correlation
between the results obtained from (prior art) Western blotting for
a single modified protein and those obtained using the novel
multiplex analysis of the invention.
* * * * *