U.S. patent application number 17/067569 was filed with the patent office on 2021-04-08 for fluorescent dyes, fluorescent dye kits, and methods of preparing labeled molecules.
The applicant listed for this patent is Biotium, Inc.. Invention is credited to Ching-Ying CHEUNG, Hye Eun HOOVER, Wai-Yee LEUNG, Fei MAO.
Application Number | 20210102873 17/067569 |
Document ID | / |
Family ID | 1000005287526 |
Filed Date | 2021-04-08 |
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United States Patent
Application |
20210102873 |
Kind Code |
A1 |
MAO; Fei ; et al. |
April 8, 2021 |
Fluorescent Dyes, Fluorescent Dye Kits, and Methods of Preparing
Labeled Molecules
Abstract
The present invention provides methods, compositions, and kits
useful in preparing labeled molecules, which are useful in the
detection of binding partners.
Inventors: |
MAO; Fei; (Fremont, CA)
; LEUNG; Wai-Yee; (San Ramon, CA) ; CHEUNG;
Ching-Ying; (San Ramon, CA) ; HOOVER; Hye Eun;
(Alameda, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biotium, Inc. |
Fremont |
CA |
US |
|
|
Family ID: |
1000005287526 |
Appl. No.: |
17/067569 |
Filed: |
October 9, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15815035 |
Nov 16, 2017 |
10837881 |
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17067569 |
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14218439 |
Mar 18, 2014 |
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15815035 |
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13423092 |
Mar 16, 2012 |
8709830 |
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14218439 |
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61454484 |
Mar 18, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09B 23/06 20130101;
C07K 1/13 20130101; G01N 33/533 20130101; G01N 33/6854 20130101;
C09B 57/02 20130101; G01N 1/30 20130101; G01N 33/53 20130101; C09B
57/001 20130101; G01N 33/532 20130101; C07K 17/06 20130101; C09B
11/24 20130101; C09B 23/083 20130101; G01N 33/582 20130101 |
International
Class: |
G01N 1/30 20060101
G01N001/30; G01N 33/532 20060101 G01N033/532; G01N 33/58 20060101
G01N033/58; C09B 11/24 20060101 C09B011/24; C09B 57/00 20060101
C09B057/00; C09B 23/08 20060101 C09B023/08; C09B 57/02 20060101
C09B057/02; C09B 23/06 20060101 C09B023/06; G01N 33/533 20060101
G01N033/533; G01N 33/53 20060101 G01N033/53 |
Claims
1-104. (canceled)
105. A method of preparing a labeled protein, comprising: (a)
providing an amine-reactive detectable dye or an amine-reactive
detectable label; (b) combining said amine-reactive detectable dye
or said amine-reactive detectable label, in a buffer, with a sample
solution comprising a target protein, said target protein
comprising an amine; and (c) allowing said amine reactive
detectable dye or said amine-reactive detectable label to react
with said amine in said buffer for about 60 minutes or less;
thereby producing said labeled protein, wherein said labeled
protein is labeled with said amine-reactive detectable dye or said
amine-reactive detectable label.
106. The method of claim 105, wherein the reaction of step (c) is
completed in about 45 minutes or less.
107. The method of claim 105, wherein the reaction of step (c) is
completed in about 30 minutes or less.
108. The method of claim 105, wherein the reaction of step (c) is
completed in about 20 minutes or less.
109. The method of claim 105, wherein said amine-reactive dye or
said amine-reactive detectable label comprises one or more
activated esters.
110. The method of claim 109, wherein said one or more activated
esters is an N-hydroxy succinimidyl ester, N-hydroxy
sulfosuccinimidyl ester, p-sulfo-tetrafluorophenol ester, or a
combination thereof
111. The method of claim 110, wherein said one or more activated
esters is an N-hydroxy succinimidyl ester.
112. The method of claim 105, wherein said amine-reactive
detectable dye or said amine-reactive detectable label comprises
one or more sulfonate groups.
113. The method of claim 105, wherein said sample solution is not
pretreated before steps (a) or (b), wherein said pretreatment
comprises purification of said target protein away from one or more
other components of said sample solution.
114. The method of claim 113, wherein said purification step
comprises protein A-based chromatography, size-exclusion
chromatography, or ultra membrane filtration, or a combination
thereof.
115. The method of claim 105, wherein said target protein is an
antibody or a fragment thereof.
116. The method of claim 105, wherein said amine-reactive
detectable dye or said amine-reactive detectable label is directly
observable.
117. The method of claim 105, wherein said amine-reactive
detectable dye or said amine-reactive detectable label is a
fluorescent dye.
118. The method of claim 105, wherein said amine-reactive
detectable dye or said amine-reactive detectable label comprises a
biotin.
119. The method of claim 105, wherein said amine-reactive
detectable dye is a CF dye, an Alexa Fluor dye, a DyLight Dye, a Cy
dye, an IRDye, a HiLyte dye, a sulfonated or pegylated coumarin
dye, a sulfonated or pegylated xanthene dye, a sulfonated or
pegylated cyanine dye, or a sulfonated or pegylated pyrene dye.
120. The method of claim 105, wherein said sample solution further
comprises one or more stabilizers or preservatives.
121. The method of claim 120, wherein at least one of said one or
more stabilizers or preservatives is bovine serum albumin, gelatin,
or glycerol.
122. The method of claim 121, wherein at least one of said one or
more stabilizers or preservatives is bovine serum albumin.
123. The method of claim 121, wherein at least one of said one or
more stabilizers or preservatives is glycerol.
124. The method of claim 120, wherein at least one of said one or
more stabilizers or preservatives is sodium azide.
Description
CROSS-REFERENCE
[0001] The present application is a continuation of U.S. patent
application Ser. No. 15/815,035, filed on Nov. 16, 2017, which is a
continuation of U.S. patent application Ser. No. 14/218,439, filed
on Mar. 18, 2014, now abandoned, which is a continuation of U.S.
patent application Ser. No. 13/423,092, filed on Mar. 16, 2012, now
U.S. Pat. No. 8,709,830, issued on Apr. 29, 2014, which claims the
benefit of U.S. Provisional Application No. 61/454,484, filed on
Mar. 18, 2011, all of which are incorporated herein by reference in
their entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] Fluorescent dyes are widely used in biological research and
medical diagnostics. Fluorescent dyes are superior to conventional
radioactive materials because fluorescent dyes are less expensive
and less toxic, and can typically be detected with sufficient
sensitivity. In particular, a diversity of fluorophores with a
distinguishable color range has made it more practical to perform
multiplexed assays capable of detecting multiple biological targets
at the same time. The ability to visualize multiple targets in
parallel is often required for delineating the spatial and temporal
relationships amongst different biological targets in vitro and in
vivo. In addition, the generation of a wide range of fluorescent
dyes has opened a new avenue for conducting high-throughput and
automated assays, thus dramatically reducing the unit cost per
assay. Moreover, the low toxicity of fluorescent dyes provides ease
of handling in vitro, and also renders it safer for imaging
biological activities in vivo.
[0003] Fluorescent dyes for applications as described above
generally derive from joining a dye and an agent that is capable of
binding to a given target or binding partner. Current processes for
joining dyes to such binding agents, antibodies being one example,
typically require purification of the antibody away from standard
buffer components, long reaction times, and purification of the
labeled antibody once the reaction is complete. Thus, current
processes consume valuable time and resources, and there is a need
for improvements in the labeling of binding agents.
SUMMARY OF THE INVENTION
[0004] In various embodiments, a method for preparing a labeled
protein comprises: (a) providing i) a reactive dye, such as an
amine-reactive dye, or a reactive detectable label, such as an
amine-reactive detectable label, and ii) a first amine; (b)
combining the amine-reactive dye and the first amine, in a buffer,
with a sample solution comprising a target protein, the target
protein comprising a second amine, to form a combined solution; and
(c) allowing the combined solution to react, thereby producing the
labeled protein. For example, the amine-reactive detectable label
reacts more strongly with the second amine than the first amine. In
various embodiments, the combined solution reacts for less than 3,
2, 1, 0.5 or fewer hours. In various embodiments, the reaction is
essentially complete in about 30 minutes or less. The reactive
detectable label may be a fluorescent dye, biotin, digoxin, a
hapten or an epitope. In some embodiments, the reactive detectable
label is not a dye. The reactive group of the reactive detectable
label may be an amine-reactive group. In some embodiments, the
amine-reactive detectable dye or amine-reactive detectable label
has a molecular weight of less than about 10000, 5000, 3000, 2000,
or 1000 Da. For example, the amine-reactive detectable dye or
amine-reactive detectable label has a molecular weight of less than
about 5000 Da.
[0005] In various embodiments, the target protein is an antibody,
for example an antibody fragment, a recombinant antibody, a
non-human antibody, a chimeric antibody, a humanized antibody, or a
fully human antibody.
[0006] The buffer may be selected from the group consisting of a
sodium carbonate buffer, a sodium bicarbonate buffer, a borate
buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, or a
combination thereof. The buffer may be alkaline. For example, the
pH of the buffer may be in the range from 7 to 10 or the pH of the
combined solution may be greater than 7.8.
[0007] In various embodiments, the first amine is a primary or
secondary amine, such as an aliphatic amine capable of competing in
the labeling reaction. For example, the first amine may be
tris(hydroxymethyl)aminomethane (Tris), a weak primary aliphatic
amine. The amine, such as Tris, may be present in the buffer at a
concentration of greater than 20 mM, or may be formulated to be
present in the combined solution at a concentration of greater than
1 mM, for example formulated to about 10 mM.
[0008] The reactive dye may comprises one or more activated esters,
such as a N-hydroxy succinimidyl ester, N-hydroxy sulfosuccinimidyl
ester, p-sulfo-tetrafluorophenol ester, or a combination thereof.
The reactive dye may comprise one or more sulfonate groups and/or
one or more water-soluble polymer groups such as polyethylene
glycol. In various embodiments, the water-soluble polymer group has
a molecular weight of 300-10000 Daltons, or 450-5000 Daltons.
Reactive dyes may be selected from the group consisting of: a CF
dye, an Alexa Fluor dye, a DyLight Dye, a Cy dye, an IRDye, a
HiLyte dye, a sulfonated and/or pegylated coumarin dye, a
sulfonated and/or pegylated xanthenes dye, a sulfonated
or/pegylated cyanine dye, and a sulfonated and/or pegylated pyrene
dye. In various embodiments, the dye has a chemical structure
according to any of Compounds 1-128.
[0009] In various embodiments, a fixed amount of reactive label is
used to label a target protein in any amount within a certain
range, resulting in a labeled target protein that avoids being
either under labeled or over labeled. For example, the sample
solution comprises between about 1 .mu.g and about 1000 .mu.g, or
between about 5 .mu.g and about 200 .mu.g, or between about 5 .mu.g
and about 20 .mu.g, or between about 20 .mu.g and about 50 .mu.g,
or between about 50 .mu.g and about 100 .mu.g, or between about 50
.mu.g and about 200 .mu.g, or between about 100 .mu.g and about 200
.mu.g of said target protein. The ratio of amount of protein in
microgram (.mu.g) to the amount of reactive dye in nanomole (nmol)
may be from 30:1 to 1:1. The sample solution may further comprise
one or more non-target proteins. For example, the ratio of the
combined weight of target and non-target protein in microgram
(.mu.g) to the amount of reactive dye in nanomole (nmol) may be
from 30:1 to 1:1. In various embodiments, the ratio of the weight
in microgram (.mu.g) of target protein to non-target protein is
from about 10:1 to about 1:10.
[0010] In various embodiments, a method of preparing a
protein-protein conjugate is provided comprises:
[0011] (a) providing i) an amine-reactive bifunctional crosslinker
additionally comprising a hydrolysis-resistant functional group;
and optionally ii) a first amine; (b) combining, in a first buffer,
the amine-reactive bifunctional crosslinker and, if present, the
first amine, with a sample solution comprising a target protein,
the target protein comprising a second amine, to form a first
combined solution; (c) allowing the combined solution to react,
thereby producing a target protein functionalized with a
hydrolysis-resistant functional group; (d) in a second buffer,
combining the target protein functionalized with the
hydrolysis-resistant functional group with a reporter protein
comprising a hydrolysis-resistant reactive group, wherein said
hydrolysis-resistant functional group reacts with said
hydrolysis-resistant reactive group, thereby forming a
protein-protein conjugate. For example, the the amine-reactive
detectable label reacts more strongly with the second amine than
the first amine; and step (d) is performed without purification of
the combined solution from step (c).
[0012] In various embodiments, the first buffer is any aqueous
buffer having a pH from about 7 to about 10. The buffer may
comprise one or more primary or secondary aliphatic amine. For
example, the buffer may be a bicarbonate buffer comprising
Tris.
[0013] The amine-reactive group of the bifunctional crosslinker may
be an activated ester, such as succinimidyl ester, and the
hydrolysis-resistant functional group of the crosslinker may be
selected from an aldehyde, a ketone, an azide, an alkyne, a
phosphine participating in Staudinger conjugation, a diene, a
dienophile, a protected hydrazino, a protected aminooxy. For
example, the crosslinker may be a bifunctional molecule comprising
a succinimidyl ester and an aldehyde group.
[0014] In various embodiments, as with the target protein labeling
using a reactive SDL above, the ratio of amount of protein in
microgram (.mu.g) to the amount of the crosslinker in nanomole
(nmol) may be from 30:1 to 1:1. The sample solution may further
comprise one or more non-target proteins. For example, the ratio of
the combined weight of target and non-target protein in microgram
(.mu.g) to the amount of crosslinker in nanomole (nmol) may be from
30:1 to 1:1. In various embodiments, the ratio of the weight in
microgram (.mu.g) of target protein to non-target protein is from
about 10:1 to about 1:10.
[0015] In various embodiments, the reporter protein is generally a
protein molecule that can produce a detectable signal either by
itself or on interacting with another molecule. For example,
protein tags may include but are not limited to fluorescent
proteins, tandem dyes and enzymes. In some embodiments, the
fluorescent proteins are phycobiliproteins, such as
R-phycoerythrine, tandem dyes are fluorescent resonance energy
transfer (FRET) dyes typically prepared by covalently labeling
fluorescent proteins with one or more synthetic organic dyes, such
as the ones mentioned above. In some embodiments, the enzymes are
horseradish peroxidase or alkaline phosphatase. The
hydrolysis-resistant reactive group on the reporter protein is
generally a reactive group capable of reacting with the
hydrolysis-resistant functional group of the crosslinker to form a
covalent bond. Non-limiting suitable hydrolysis-resistant reactive
groups include an aldehyde, a ketone, an azido, an alkyne, a
phosphine participating in Staudinger conjugation, a diene, a
dienophile, a hydrazone and an oxime.
[0016] In various embodiments, the second buffer may be a buffer
having a pH from about 4 to about 10. According to some
embodiments, the second buffer may comprise one or more catalyst
that facilitates the conjugation of the protein tag to the
functionalized target protein. For example, the second buffer may
be an acidic buffer having a pH from about 4 to about 6.8 and
comprising aniline or an aniline derivative for catalyzing an
aminooxy to an aldehyde conjugation to form an oxime linkage.
[0017] In various embodiments, a pretreatment purification step is
excluded. For example, in various embodiments, protein A-based
chromatography, size-exclusion chromatography, and ultra membrane
filtration are not performed.
[0018] Methods of staining one or more biological targets are also
disclosed. Such methods may comprise (a) preparing one or more
labeled proteins according to the methods described herein, wherein
each of said one or more labeled proteins comprises a targeting
moiety that binds to a binding partner associated with one or more
of said biological targets; and, (b) exposing said one or more
biological targets to said one or more labeled proteins, such that
said labeled protein binds to said binding partner thereby staining
said one or more biological targets. In other embodiments, methods
are provided of staining a biological target comprising (a)
preparing one or more labeled proteins according to the methods
described herein, and (b) exposing said one or more biological
targets to said one or more labeled proteins, such that said
labeled protein binds to said one or more biological targets
thereby staining said one or more biological targets. In various
embodiments, step (b) is performed without purification of said one
or more labeled proteins from step (a). In various embodiments, the
use of a quencher is excluded. Staining may take place in vivo or
in vitro, as desired. In various embodiments, two or more labeled
proteins comprise different dyes, such that said exposing step (b)
renders said different binding partners optically
distinguishable.
[0019] The products of staining may be analyzed by, for example,
flow cytometry, western blot, or by microscopy. For example, the
products may be analyzed using a fluorescence activated cell
sorter.
[0020] In various embodiments, diagnosing a condition of a subject
based on analysis of stained biological targets is performed.
Alternatively, a method for evaluating the efficacy of a test
compound is disclosed.
[0021] A kit for preparing a labeled protein is provided which may
comprise (a) a buffer comprising a first amine, for example a
primary or secondary aliphatic amine; (b) one or more reactive dyes
or reactive detectable labels for labeling one or more target
proteins; (c) a storage buffer; and, (d) instructions in one or
more than one language. The kit comprises components according to
the methods described herein. For example, one or more of dyes may
be present as an amine reactive dye. Buffer may be selected from
the group consisting of: a sodium carbonate buffer, a sodium
bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer,
a HEPES buffer, or a combination thereof. In various embodiments,
the buffer is alkaline, for example, with a pH in the range of 7 to
10. The amine may be tris(hydroxymethyl)aminomethane (Tris),
present in a concentration, for example, of greater than 20 mM, or
in a concentration such that upon addition to a protein, the
concentration would be greater than 20 mM.
[0022] Activated esters are encompassed, such as N-hydroxy
succinimidyl ester, N-hydroxy sulfosuccinimidyl ester,
p-sulfo-tetrafluorophenol ester, or a combination thereof.
[0023] A kit for preparing reporter protein-labeled proteins is
also provided which may comprise (a) an alkaline buffer comprising
a first amine, such as a primary or secondary aliphatic amine; (b)
a bifunctional crosslinker comprising an amine-reactive group and a
hydrolysis-resistant functional group; (c) a reporter protein
functionalized with a pairing hydrolysis-resistant reactive group;
(d) a second buffer suitable for the hydrolysis-resistant
functional group and hydrolysis-resistant reactive group to react
to form a covalent linkage; (e) a storage buffer; and (f)
instructions in one or more than one language.
[0024] In various embodiments, buffer and dye are present in an
amount sufficient to permit labeling of 5 .mu.g-200 .mu.g of a
target protein. Storage buffer may be present, for example, with a
stabilizer. Such stabilizers may include bovine serum albumin,
gelatin, glycerol, sodium azide, tris, or a combination thereof
[0025] In various embodiments, the kit includes a separation device
for purifying a labeled target protein. For example, the separation
device may be a size exclusion chromatography column. In various
embodiments, a stain stabilizing reagent for enhancing dye
fluorescence is present in the kit.
INCORPORATION BY REFERENCE
[0026] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference. The practice of the present invention
employs, unless otherwise indicated, conventional techniques of
immunology, biochemistry, chemistry, molecular biology,
microbiology, cell biology, genomics and recombinant DNA, which are
within the skill of the art. See Sambrook, Fritsch and Maniatis,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds.,
(1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.):
PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.
R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A
LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed.
(1987)), each of which are incorporated by reference for their
teachings of conventional techniques.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0028] FIG. 1 shows the results of a flow cytometry analysis of
cells stained with secondary antibodies labeled according to
methods of the invention.
[0029] FIG. 2 shows the results of a flow cytometry analysis of
cells stained with secondary antibodies labeled according to
methods of the invention.
[0030] FIG. 3 shows the results of a flow cytometry analysis of
cells stained with secondary antibodies labeled according to
methods of the invention.
[0031] FIG. 4 shows the results of a flow cytometry analysis of
cells stained with primary antibodies labeled according to methods
of the invention.
[0032] FIG. 5 shows the results of a flow cytometry analysis of
cells stained with primary antibodies labeled according to methods
of the invention.
[0033] FIG. 6 shows images from a microscopic analysis of cells
stained with primary antibodies labeled according to methods of the
invention.
[0034] FIG. 7 shows images from a microscopic analysis of cells
stained with primary antibodies labeled according to methods of the
invention, in addition to other stains.
[0035] FIG. 8 shows the results of a flow cytometry analysis of
cells stained with primary antibodies labeled according to methods
of the invention.
DEFINITIONS
[0036] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear, cyclic, or branched. The polymer
may comprise modified amino acids, and it may be interrupted by
non-amino acids. The terms also encompass amino acid polymers that
have been modified, for example, via sulfonation, glycosylation,
lipidation, acetylation, phosphorylation, iodination, methylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, transfer-RNA mediated addition of
amino acids to proteins such as arginylation, ubiquitination, or
any other manipulation, such as conjugation with a labeling
component. As used herein the term "amino acid" refers to either
natural and/or unnatural or synthetic amino acids, including
glycine and both the D or L optical isomers, and amino acid analogs
and peptidomimetics.
[0037] A "binding agent" is a molecule that exhibits binding
selectivity towards a binding partner or a target molecule to which
it binds. A binding agent may be a biomolecule such as a
polypeptide such as an antibody or protein, polypeptide-based
toxin, amino acid, nucleotide, polynucleotides including DNA and
RNA, lipids, and carbohydrates, or a combination thereof. A binding
agent may also be a hapten, drug, ion-complexing agent such as
metal chelators, microparticles, synthetic or natural polymers,
cells, viruses, or other fluorescent molecules including the dye
molecule according to the invention.
[0038] A "targeting moiety" is the portion of the binding agent
that binds to a binding partner. A targeting moiety may be, without
limitation, a nucleotide sequence within a polynucleotide that
selectively binds to another polynucleotide or polypeptide. Another
non-limiting example of a targeting moiety may be a polypeptide
sequence within a larger polypeptide sequence which binds
specifically to a polynucleotide sequence or a second polypeptide
sequence. A targeting moiety may be a small molecule or structural
motif which will bind to a protein receptor, another small molecule
motif, or complexing agent, without limitation. The selective
binding may be a specific binding event.
[0039] A "binding partner" is a molecule or particle which is bound
by the targeting moiety. It can be a cell, virus, fragment of a
cell, antibody, fragment of an antibody, peptide, protein,
polynucleotide, antigen, small molecule, lipid, carbohydrate, or a
combination thereof. It may be bound selectively or specifically by
the binding agent. In general, binding is considered "specific"
when binding between a binding partner and the target of the
binding partner (a binding pair) is at least two times greater,
such as more than 10, 1000, 1000, 10000 times greater, than
background resulting from binding to non-target molecules in a
sample.
[0040] One skilled in the art will realize that such a functional
group may also be called a reactive group; As used herein, the
terms "reactive group" and "functional group" refer to chemical
groups that can undergo chemical reactions with other chemical
moieties under certain conditions. When a first reactive group
reacts with such a chemical moiety (for example a second reactive
group), one or more covalent bonds is formed. In such cases, the
second reactive group will generally be termed a "functional
group".
[0041] As used herein, the term "about" generally means within 10%,
5%, 1%, or 0.5% or a given value or range. Alternatively, the term
"about" refers to an acceptable standard error of the mean, when
considered by one of ordinary skill in the art.
[0042] The term "aliphatic" refers to an organic compound or
radical characterized by a straight chain or branched chain
structure, or closed ring structure, any of which contain saturated
carbon bonds and optionally one or more unconjugated carbon-carbon
unsaturated bonds, such as a carbon-carbon double bond.
DETAILED DESCRIPTION OF THE INVENTION
[0043] In some aspects, the present invention provides methods for
labeling target proteins with either a reactive dye, a reactive
detectable label, or with a reporter protein. The resulting labeled
target protein can be used, for example, in subsequent biological
staining or other experiments. In some embodiments, the resulting
labeled target protein is used without further purification.
[0044] In some embodiments, target proteins that are labeled by the
methods of the invention are members of a binding pair, such as a
binding agent and a target. In some embodiments, the binding agent
is an antibody and the target is an antigen. The term "antibody" as
used herein refers to immunoglobulin molecules and immunologically
active portions of immunoglobulin molecules, i.e., molecules that
contain an antigen-binding site which specifically binds
("immunoreacts with") an antigen. Non-limiting examples include an
antibodies, antibody fragments, recombinant antibodies, non-human
antibodies, chimeric antibodies, humanized antibodies, or fully
human antibodies. Structurally, the simplest naturally occurring
antibody (e.g., IgG) comprises four polypeptide chains, two heavy
(H) chains and two light (L) chains inter-connected by disulfide
bonds. The immunoglobulins represent a large family of molecules
that include several types of molecules, such as IgD, IgG, IgA, IgM
and IgE. The term "immunoglobulin molecule" includes, for example,
hybrid antibodies, or altered antibodies, and fragments thereof. It
has been shown that the antigen binding function of an antibody can
be performed by fragments of a naturally-occurring antibody. These
fragments are collectively termed "antigen-binding units". Antigen
binding units can be broadly divided into "single-chain" ("Sc") and
"non-single-chain" ("Nsc") types based on their molecular
structures.
[0045] Also encompassed within the terms "antibodies" are
immunoglobulin molecules of a variety of species origins including
invertebrates and vertebrates. The term "human" as applies to an
antibody or an antigen binding unit refers to an immunoglobulin
molecule expressed by a human gene or fragment thereof. The term
"humanized" as applies to a non-human (e.g. rodent or primate)
antibodies are hybrid immunoglobulins, immunoglobulin chains or
fragments thereof which contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, rabbit or primate having the desired
specificity, affinity and capacity. In some instances, Fv framework
region (FR) residues of the human immunoglobulin are replaced by
corresponding non-human residues. Furthermore, the humanized
antibody may comprise residues which are found neither in the
recipient antibody nor in the imported CDR or framework sequences.
These modifications are made to further refine and optimize
antibody performance and minimize immunogenicity when introduced
into a human body. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody may also comprise
at least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin.
[0046] Detectable labels, such as amine-reactive detectable labels,
are generally molecules that can directly or indirectly produce or
result in a detectable signal either by themselves or upon
interaction with another molecule. In some embodiments, detectable
labels have a molecular weight of less than about 5000 dalton.
Non-limiting examples of detectable labels include labels
comprising fluorescent dyes, biotin, digoxin, haptens and
epitopes.
[0047] Dyes useful in labeling proteins are known in the art. In
general, a dye is a molecule, compound, or substance that can
provide an optically detectable signal, such as a colorimetric,
luminescent, bioluminescent, chemiluminescent, phosphorescent, or
fluorescent signal. In some embodiments, the dye is a fluorescent
dye. Non-limiting examples of dyes, some of which are commercially
available, include CF dyes (Biotium, Inc.), Alexa Fluor dyes
(Invitrogen), DyLight dyes (Thermo Fisher), Cy dyes (GE
Healthscience), IRDyes (Li-Cor Biosciences, Inc.), and HiLyte dyes
(Anaspec, Inc.). Other non-limiting example of dyes include any of
Compounds 1-128, listed in Tables 1-4 and the Examples. In some
embodiments, the excitation and/or emission wavelengths of the dye
are between 350 nm to 900 nm, or between 400 nm to 700 nm, or
between 450-650 nm.
[0048] In some embodiments, the dye is water soluble. In some
embodiments, the dye comprises about, less than about, or more than
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfonate groups. In
some embodiments, the dye comprises about, less than about, or more
than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more water-soluble
polymer groups, such as polyethylene glycol (PEG). A water soluble
polymer group can have any suitable molecular weight, such as
300-10000 Daltons, including 450-5000 Daltons. In some embodiments,
a water soluble polymer group has a molecular weight of about, more
than about, or less than about 200, 300, 400, 450, 500, 600, 700,
800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000,
9000, 10000, 20000, 30000, 40000, 50000, or more Daltons, or any
range between any two such weights. In some embodiments, the dye is
sulfonated and/or pegylated, such as in a sulfonated and/or
pegylated coumarin dye, a sulfonated and/or pegylated xanthene dye,
a sulfonated and/or pegylated cyanine dye, or a sulfonated and/or
pegylated pyrene dye.
[0049] In some embodiments the dye is an amine reactive dye,
comprising one or more groups that react to form an amide bond with
an amine. In some embodiments, the amine reactive dye comprises
about, less than about, or more than about 1, 2, or 3
amine-reactive groups. In some embodiments, the amine reactive
group is an activated ester. Examples of activated esters include,
but are not limited to, N-hydroxy succinimidyl ester, N-hydroxy
sulfosuccinimidyl ester, p-sulfo-tetrafluorophenol ester,
pentafluorophenyl esters, tetrafluorophenyl esters, p-nitrophenyl
esters, 2,4-dinitrophenyl ester, 4-nitrophenyl ester,
3-Hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt),
carboxylic acids activated using common carbodiimides such as but
not limited to diisopropylcarbodiimide (DIPCDI),
N,N'-dicyclohexylcarbodiimide (DCC),
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDC);
and carboxylic acids activated with an uronium salt or a
phosphonium salt, such as but not limited to
O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
(HBTU), O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU),
2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HCTU), (2-(6-Chloro-1H-benzotriazole-1
-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate) (TCTU),
2-(1H-7-Azabenzotriazol-1 -yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate (HATU),
1-benzotriazolyoxytris-(dimethylamino)phosphonium
hexafluorophosphate (BOP)
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
(PYBOP); or combinations thereof.
[0050] Bifunctional crosslinkers for use in the invention generally
comprise two reactive groups capable of reacting with target and
reporter proteins. For example, at least one of the two reactive
groups is an amine-reactive group. In some embodiments, a
bifunctional crosslinker comprises an amine-reactive group and a
hydrolysis-resistant functional group. The amine-reactive group can
react with an amine group within a target protein, thereby
conjugating the crosslinker to the amine group of the target
protein via an amide bond. The amine-reactive group can be an
activated ester as described herein. Any of the activated esters
described as suitable for use with the amine-reactive dyes or
amine-reactive detectable labels are also suitable for use with the
amine-reactive group of the crosslinker. As an example, the
activated ester may be an N-hydroxy succinimidyl ester.
[0051] Hydrolysis-resistant functional groups can react with the
reporter protein, thereby attaching the other end of the
crosslinker to the reporter protein. In some embodiments, the
functional group is hydrolysis-resistant. For example, a
hydrolysis-resistant functional group or hydrolysis-resistant
reactive group is at least 90% intact in a buffer, such as the
first buffer of the methods of the invention, for at least 24
hours, or for at least 1 month, or for at least 3 months.
Generally, hydrolysis-resistant functional groups are compatible
with the amine-reactive group of the crosslinker such that that
they do not react with each other. Since the amine-reactive group
is an electrophile, the hydrolysis-resistant functional group of
the crosslinker is generally not a nucleophile. Non-limiting
examples of functional groups that are hydrolysis-resistant and
non-nucleophilic include aldehydes, ketones, azido, alkyne,
phosphines participating in Staudinger conjugation, dienes,
dienophiles, protected hydrazine and protected aminooxy. Examples
of pairs of hydrolysis-resistant functional groups and reactive
groups that can react to form a covalent bond are listed below:
TABLE-US-00001 Functional Group Reactive Group Bond formation
reaction aldehyde aminooxy oxime formation aldehyde hydrazino
hydrazone formation ketone aminooxy oxime formation ketone
hydrazino hydrazone aldehyde acetone protected aminooxy oxime
formation aldehyde acetone protected hydrazino hydrazone formation
ketone acetone protected aminooxy oxime formation ketone acetone
protected hydrazino hydrazone formation azido alkyne Click
chemistry diene dienophile Diels-Alder reaction Azido phosphine
Staudinger reaction
[0052] In some embodiments, the hydrolysis-resistant functional
group excludes maleimide or disulfide linker-forming groups such as
pyridyldisulfide. In one embodiment, the bifunctional crosslinker
comprises an N-hydroxy succinimidyl ester and an aldehyde
functional group.
[0053] Buffers useful for labeling the target protein with an
amine-reactive dye, an amine-reactive detectable label, or with a
bifunctional crosslinker (i.e., a first reaction buffer) in the
methods of the invention include any that support a labeling
reaction as described herein. In some embodiments, the buffer is an
alkaline buffer, having a pH greater than about 7, for example
having a pH from about 7 to about 14, such as a pH of about, more
than about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0,
8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14. In some
embodiments the buffer has a pH of about, more than about, or less
than about 4, 4.5, 5, 5.5, 6, or 6.5. Non-limiting examples of
useful buffers include sodium carbonate buffers, sodium bicarbonate
buffers, borate buffers, tris buffers, MOPS buffers, HEPES buffers,
and combinations thereof. In some embodiments, the buffer is of a
concentration such that the combined solution has a pH from about 6
to about 11, such as from about 7.9 to about 9.8, or from about 8
to about 9. In some embodiments, the pH of the combined solution
about, more than about, or less than about 6, 6.5, 7, 7.5, 7.6,
7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,
9.0, 10, 10.5, 11, or more. In some embodiments, the buffer has a
starting concentration that is about, more than about, or less than
about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,
or more than the concentration of the buffer in the combined
solution. In one example, a 10.times. reaction buffer comprises 500
mM sodium bicarbonate and 100 mM Tris.
[0054] In some embodiments, the buffer used in the invention
comprises a first amine, for example an aliphatic amine, capable of
competing with a second amine group of the target protein in
reacting with the reactive dye or crosslinker described above. In
some embodiments, the first amine is a primary or secondary amine,
e.g. a primary or secondary aliphatic amine. In general, a first
amine is at least 50% less reactive than the w amine of lysine in
an amide formation reaction with an activated carboxylic acid
ester, such as 60%, 70%, 80%, 90%, or less reactive. Reactivity can
be measured, for example, as a reaction rate in an aqueous
solution. A non-limiting example of a first amine includes
tris(hydroxymethyl)aminomethane (Tris). In various embodiments, a
first amine with the specified reactivity is a primary or secondary
amine. In some embodiments, the amine acts as a regulating agent of
the labeling reaction such that the fraction of labeled to
unlabeled amine groups in the target protein and/or the number of
labels per target protein are kept within a desired range. For
example, when the amount of antibody is relatively high, most of
the dye, label, or crosslinker molecules react with the antibody
molecules while a side-reaction between the first amine and the
dye, label, or crosslinker is minimal, such that the antibody is
sufficiently labeled. In another example, when the amount of target
protein (e.g. antibody) is relatively low, the side-reaction
between the first amine and the dye, label, or crosslinker is
relatively increased, such that consumption of the dye, label, or
crosslinker by the first amine reduces the likelihood that the
small amount of antibody will be overly labeled. In some
embodiments, more than about 95%, 96%, 97%, 98%, 99%, 99.5%, or
more of the target protein in a sample is labeled. In some
embodiments, each target protein is labeled with about, or more
than about 1, or with about, more than about, or less than about,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 dye, label or crosslinker
molecules. In some embodiments, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, 99.5%, or more of labeled target protein in a sample have the
same number of dye molecules. While not wishing to be bound by
theory, it is believed that in circumstances where a target protein
is mixed with a non-target protein (such as an antibody stabilized
with bovine serum albumin (BSA), and both the target protein and
non-target protein are labeled equally in terms of labeling
efficiency per amine basis on each protein, a staining process may
still tolerate the more or less equivalent labeling. For example,
in one embodiment, non-target protein may not have affinity for a
subsequent binding target, allowing the non-target protein to be
removed by washing during a subsequent step of binding to a
biological target.
[0055] In various embodiments, a suitable dye is highly
water-soluble by possessing water soluble groups, such as sulfonate
group and/or a water-soluble polymer as defined herein. While not
wishing to be bound by theory, it is believed that the labeling
method can tolerate up to 4 to 5 times of a non-target protein
(relative to the amount of target protein) as described herein. In
various embodiments, up to 10 fold non-target protein may be
present without sacrificing the performance of the labeled target
protein. In general, labeling comprises linking a dye to a target
protein by way of covalent interactions. In some embodiments,
labeling comprises formation of a covalent bond, such as an amide
bond.
[0056] In some embodiments, the amount of target protein in a
sample solution is between about 0.1 .mu.g-10000 .mu.g, such as
between 1 .mu.g-1000 .mu.g, 5 .mu.g-200 .mu.g, 5 .mu.g-20 .mu.g, 20
.mu.g-50 .mu.g, or 50 .mu.g-100 .mu.g. In some embodiments, the
amount of target protein in a sample is about, less than about, or
more than about 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25, 50, 100, 150, 200, 250, 500, 1000, 1500, 2000, 3000,
4000, 5000, 6000, 7000, 8000, 9000, 10000, or more micrograms
(.mu.g). In some embodiments, m1 and m2 (both in .mu.g) define the
lower and upper limits of an amount of target protein in a sample
labeling reaction. In some embodiments, the relationship between m1
and m2 for a target protein to be labeled by a specified amount of
reactive dye is expressed as m2/m1, where m2/m1 has a specified
value or range of values. In some embodiments, m1 is from about 0.1
.mu.g to about 500 .mu.g, and m2/m1 is from about 1 to about 20. In
some embodiments, m1 is from about 1 .mu.g to about 200 .mu.g, and
m2/m1 is from about 2 to about 5. In some embodiments, m1 is from
about 5 .mu.g to about 50 .mu.g, and m2/m1 is from about 2 to about
4. In some embodiment, m1 is about 5 .mu.g and m2/m1 is about 4. In
some embodiment, m1 is about 20 .mu.g and m2/m1 is about 2.5. In
some embodiment, m1 is about 50 .mu.g and m2/m1 is about 2.
[0057] In some embodiments, the target protein is dissolved in a
sample buffer. A target protein dissolved in a sample buffer may
have any concentration suitable for a labeling reaction, such as
about, less than about, or more than about 0.001, 0.01, 0.1, 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more mg/mL, or any
range therebetween, such as from about 0.01-10 mg/mL, 0.1-5 mg/mL,
or 0.5-1 mg/mL. A buffer for dissolving a target protein, such as
an antibody, may be any of a number of commonly used biological
buffers, including but not limited to phosphate buffered saline
(PBS), Tris, MOPS, HEPES, and combinations thereof. In some
embodiments, the concentration of Tris is less than about 1000 mM,
100 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 5 mM, or less.
[0058] In some embodiments, a sample solution comprising a target
protein also comprises an amount of one or more stabilizers and/or
one or more preservatives. Common stabilizers include, but are not
limited to, bovine serum albumin (BSA), gelatin and glycerol. In
some embodiments, the amount of BSA or gelatin is about, more than
about, or less than about 50-fold, 40-fold, 30-fold, 20-fold,
10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold,
2-fold, 1-fold, or less than the amount of the target protein by
weight. In some embodiments, the amount of glycerol is about, more
than about, or less than about 50%, 25%, 20%, 15%, 10%, 5%, 4%, 3%,
2%, 1% or less of the buffer. Common preservatives include, but are
not limited to, sodium azide, thimerosal, and other antimicrobial
agents. A sample solution may or may not be pretreated to purify a
target protein away from one or more other sample solution
components. In some embodiments, the sample solution is not
pretreated to purify the target protein away from one or more other
components of the sample solution. Methods for removing one or more
excess sample solution components, such as preservatives and/or
stabilizers, are known in the art, non-limiting examples of which
include protein A-based affinity chromatography, size-exclusion
chromatography, and ultra membrane filtration. Commercial kits for
protein purification, such as antibody cleaning kits, may also be
used for removing one or more excess sample solution components.
Size-exclusion column chromatography and ultra membrane filtration
are useful for removing stabilizers/preservatives of relatively
small molecules, such as glycerol, Tris, and amino acids. Ultra
membrane filtration may also be used to adjust target protein
concentration to a desired value. Ultra membrane filtration can be
conveniently and rapidly carried out on a centrifuge using a
commercial ultra membrane filtration vial. The membranes in such
ultra filtration devices typically have different pore sizes, or
so-called Molecular Weight Cut-off sizes (MWCO), permitting
relatively small molecules to go through while retaining bigger
molecules, such as proteins. In some embodiments, a membrane with a
MWCO of about, less than about, or more than about 1, 5, 10, 15,
20, 25, 50, 100, or more kD is used to purify a target protein
before labeling. Purification methods may be used to purify labeled
protein.
[0059] In some embodiments, the labeling reaction with a dye,
detectable label or crosslinker takes place at temperature of
about, less than about, or more than about 4.degree. C., 10.degree.
C., 15.degree. C., 16.degree. C., 17.degree. C., 18.degree. C.,
19.degree. C., 20.degree. C., 21.degree. C., 22.degree. C.,
23.degree. C., 24.degree. C., 25.degree. C., 26.degree. C.,
27.degree. C., 28.degree. C., 29.degree. C., 30.degree. C.,
37.degree. C., 40.degree. C., 50.degree. C., or higher. In some
embodiments, the labeling reaction takes place a room temperature,
such as between 15.degree. C. -30.degree. C. In some embodiments,
the labeling reaction is completed in less than about 5, 4, 3, 2,
1, or fewer hours; or less than about 60, 45, 30, 25, 20, 15, 10,
9, 8, 7, 6, 5, or fewer minutes. In some embodiments a completed
labeling reaction containing a target protein labeled with a
detectable label such as a dye, is used, such as in a detection
process, without further manipulation, such as without the addition
of other reagents, such as a quenching agent, and/or without
purification, isolation, or concentration of the labeled target
protein. Likewise, in some embodiments, a completed labeling
reaction containing a target protein labeled with a crosslinker can
be used directly in a subsequent protein-protein conjugation
reaction without any purification of the crosslinker-labeled target
protein as described herein.
[0060] A second reaction buffer used in the protein-protein
conjugation method of the invention can be any buffer that
facilitates the reaction between the hydrolysis-resistant
functional group of the crosslinker and the pairing
hydrolysis-resistant reactive group on the reporter protein
described further in details below. In some embodiments, the second
buffer comprises a catalyst that can accelerate the conjugation
reaction. Similar to the reaction buffer used in detectable
label-target protein conjugation or the first reaction buffer used
in conjugating the crosslinker to the target protein, in some
embodiments the second reaction buffer is provided as a
concentrated solution that is about, more than about, or less than
about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,
or more than the concentration of the buffer in the second combined
solution. In some embodiments, the second buffer is a buffer that
facilities an oxime or hydrazone bond formation, such as the
reaction between an aldehyde functional group and an aminooxy or
hydrazino reactive group. Oxime and hydrazone formation generally
prefers a relatively acidic buffer, such as a buffer with a pH from
about 3 to about 7. Generally, aryl amines, such as aniline, are
known to catalyze oxime and hydrazone formation. Thus, according to
one embodiment, the second buffer is a phosphate buffer with a pH
between about 3 and about 4, further comprising between about 1 and
about 1000, or about 10 and about 200, or about 50 and about 150,
or about 75 and about 125 mM aniline. In some embodiments, the
second buffer comprises between about 95 and 105 mM aniline, for
example about 100 mM aniline.
[0061] A reporter protein suitable for the protein-protein
conjugation method is generally a protein that can produce a
detectable signal either by itself or upon interaction with another
molecule. According to one embodiment, reporter proteins are
enzymes that upon interaction with a colorogenic or fluorogenic
enzyme substrate produce a colored or fluorescent product. For
example, horseradish peroxidase (HRP) and alkaline phosphatase (AP)
both are commonly used as reporter proteins that interact with a
substrate to produce a detectable colored or fluorescent product.
Some enzyme reporter proteins can also interact with a substrate to
produce detectable luminescence or bioluminescence. According to
one embodiment, reporter proteins are fluorescent proteins, which
are natural or recombinant protein molecules that bear one or more
fluorophores. According to one preferred embodiment, fluorescent
proteins are phycobiliproteins. Phycobiliproteins are a family of
fluorescent proteins found in cyanobacteria and eukaryotic red
algae. In nature, these highly colored proteins function as
light-harvesting antenna molecules to collect photo energy for
biochemical synthesis. Phycobiliproteins are considered to be the
brightest fluorescent dyes due to their large extinction
coefficient (700,000-2,410,000) and high fluorescence quantum yield
(0.68-0.98). As a result, phycobiliproteins are widely used as
fluorescent labels for biological detection, offering sensitivity
many times higher than small synthetic dyes.
Phycobiliprotein-labeled probes are extensively used in bead-based
detection, microarrays and flow cytometry. In some preferred
embodiments, phycobiliproteins are R-phycerythrine (R-PE),
allophycocianin (APC) and Peridinin-chlorophyll-protein (PerCP).
R-PE and PerCP are both excitable by the common 488 nm argon laser,
but emit at .about.578 nm and .about.675 nm, respectively. APC, on
the other hand, is excitable by the red He--Ne laser with emission
at .about.660 nm. The variety of emission wavelengths by
phycobiliproteins can be further extended by the use of so-called
tandem dyes. Tandem dyes are energy transfer dyes prepared by
covalently labeling phycobiliproteins with a suitable synthetic dye
that acts as energy receptor. In a tandem dye, the phycobiliprotein
acts an antenna dye to efficiently absorb excitation light and then
transfer its emission energy to the acceptor synthetic dye, which
re-emits at the emission wavelength of the acceptor dye. By using
appropriate acceptor dyes, the original emission wavelengths of
phycobiliproteins can be extended to virtually anywhere between
about 580 nm and 800 nm. Thus, by using a combination of regular
synthetic dyes, phycobiliproteins and tandem dyes, it is possible
for one to detect many biological targets in the same sample by
using only a limited number of excitation sources. For example, by
using a combination of dyes, it is possible to detect over 10
targets in the same sample by flow cytometry.
[0062] In some embodiments of the methods of the invention, the
reporter proteins are pre-functionalized with a
hydrolysis-resistant reactive group capable of reacting with the
hydrolysis-resistant functional group of the crosslinker-modified
target protein to form a covalent linkage. The pre-functionalized
reporter protein is usually purified such that it is substantially
free of any unattached small molecule comprising the same reactive
group that may interfere with the protein-protein conjugation
reaction. The pre-functionalized reporter protein may be prepared
by reacting any of the above mentioned reporter protein with a
crosslinker comprising a first reactive group capable of reacting
with an amine or thiol on the reporter protein to form a covalent
linkage and a second hydrolysis-resistant reactive group capable of
reacting with the functional group on the crosslinker-modified
target protein. The functionalization reaction of the reporter
protein may be carried out in any of the buffers mentioned
previously herein. The functionalized reporter protein may be
purified by size exclusion column, ultramembrane filtration or
dialysis to remove any unattached crosslinker. In some cases, the
hydrolysis-resistant reactive group may not be a pairing reactive
group to the hydrolysis-resistant functional group on the
crosslinker-modified target protein; that is, the initial
hydrolysis-resistant reactive group on the reporter protein may not
react with the hydrolysis-resistant functional group on the target
protein. In such a case, the initially functionalized reporter
protein is usually reacted with a second crosslinker which, upon
reacting with the initially functionalized reporter protein,
provides a suitable hydrolysis-resistant reactive group for
conjugation to the target protein.
[0063] According to one embodiment, the invention provides a
reporter protein prefunctionalized with an aminooxy or hydrazino
group, wherein the reporter protein comprises 1 to about 15
aminooxy or hydrazino groups and the wherein the functionalized
reporter protein is purified to remove unreacted aminooxy or
hydrazino crosslinker. The aminooxy or hydrazino groups herein are
usually "free" reactive group, i.e. they are not in a protected
form, such as aminooxy or hydrazino groups protected by acetone in
the form of an oxime or hydrazone. Free aminooxy or hydrazino
groups possess much higher reactivity toward aldehyde or ketone
groups in conjugation reaction than their protected forms. In one
embodiment, the reporter protein comprises 1 to about 6 aminooxy or
hydrazino groups. According to some embodiments, the
pre-functionalized reporter proteins are aminooxy-functionalized
reporter proteins selected from the list consisting of
phycobiliproteins, tandem dyes, horseradish peroxidase and alkaline
phosphatase. In some embodiments, the functionalized reporter
protein may be provided as lyophilized solid or as a solution.
[0064] Provided herein is also a method of preparing an aminooxy-
or hydrazino-functionalized reporter protein, comprising the steps
of: (a) reacting a reporter protein with a bifunctional crosslinker
comprising an amine-reactive group and an aldehyde or ketone group
in a buffer for a time sufficient to form an aldehyde or ketone
functionalized reporter protein; (b) reacting the aldehyde- or
ketone-functionalized reporter protein with an excess of a
bis-aminooxy, a bis-hydrazino or a aminooxy-hydrazino crosslinker
in a buffer for a time sufficient to form an aminooxy- or
hydrazino-functionalized reporter protein; and (c) purifying said
functionalized reporter protein.
[0065] The conjugation of a reporter protein to the
crosslinker-modified target protein can be carried out at any
temperature from above 0.degree. C. to about 90.degree. C.,
depending on the stability of the protein. More typically, the
reaction is carried out from about 4.degree. C. to about 45.degree.
C. In some embodiments, the reaction is carried out at room
temperature, such as from about 15.degree. C. to about 30.degree.
C. The reaction time may be longer than that for functionalizing
the target protein with the crosslinker because of the relatively
large size of the two proteins. When the reaction is carried out at
room temperature, the reaction time may be greater than 15 minutes,
or greater than 30 minutes. In some embodiments, the reaction time
is about 1 hour at room temperature. In some embodiments a
completed labeling reaction containing a target protein labeled
with a reporter protein is used, such as in a detection process,
without further manipulation, such as without the addition of other
reagents, such as a quenching agent, and/or without purification,
isolation, or concentration of the labeled target protein.
[0066] In some embodiments, one or more portions of an amount of
labeled protein produced by the labeling reaction are used directly
in one or more processes, and unused labeled protein is stored for
later use. In some embodiments, a storage buffer is added to a
labeled protein to produce a stored protein solution. In general, a
storage buffer is effective in increasing the time following a
labeling reaction during which a labeled protein remains stable. In
general, a labeled protein is considered "stable" when it retains
its activity, or a substantial portion thereof, such as for use in
a detection process. In some embodiments, a stored protein is
considered stable when about or more than about 30%, 40%, 50%, 60%,
70%, 80%, 85%, 90%, 95%, 97.5%, 99%, 99.5%, 99.9%, or more of the
pre-storage activity remains. In some embodiments, the stored
labeled protein is stable for about or more than about 1, 2, 3, 4,
5, 6, 7, 8, or more weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
or more months; or 1, 2, 3, or more years. In some embodiments, a
storage buffer comprises one or more stabilizers and/or one or more
preservatives. Non-limiting examples of stabilizers include bovine
serum albumin, gelatin, and glycerol. Non-limiting examples of
preservatives include sodium azide, thimerosal, and other
antimicrobial agents. In general, a storage buffer can have either
a lower pH or higher pH than the buffer used in the labeling
reaction, such that addition of the storage buffer lowers or raises
the pH of the combined solution containing the labeled protein. In
some embodiments, the pH of a solution containing a stored, labeled
protein is about, less than about, or more than about 5.5, 6, 6.5,
7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.5, 9, or
higher. In some embodiments, the pH of the stored protein solution
is between about 6.0-9.0, such as 6.5-8.0, or 7.2-7.5.
[0067] Labeled proteins of the present invention can be used as
indicators of the presence, absence, relative abundance, and/or
amount of a biological target, such as may be found in a biological
sample or organism, for example by use in a detection process. In
one aspect, the invention provides a method for staining one or
more biological targets. In one embodiment, the method comprises
(a) preparing one or more labeled proteins according to a method of
the present invention, wherein each of said one or more labeled
proteins comprises a targeting moiety that binds to a binding
partner associated with one or more of said biological targets;
and, (b) exposing said one or more biological targets to said one
or more labeled proteins, such that said labeled protein binds to
said binding partner thereby staining said one or more biological
targets. In some embodiments, step (b) is performed without
purification of the one or more labeled proteins. In some
embodiments, step (a) is not terminated by the addition of a
quencher.
[0068] In general, a biological sample refers to any biological
tissue or fluid. In some embodiments, samples include, but are not
limited to, cells in culture, bone marrow; blood; blood cells
(e.g., white blood cells, red blood cells, etc.); ascites; tissue
or fine needle biopsy samples; cell-containing body fluids; free
floating nucleic acids; sputum; urine; cerebrospinal fluid,
peritoneal fluid; pleural fluid; washings or lavages such as a
ductal lavages or broncheoalveolar lavages; aspirates; scrapings;
bone marrow specimens; tissue biopsy specimens; surgical specimens;
other body fluids, secretions, and/or excretions; and/or cells
therefrom. In some embodiments, a sample comprises cells obtained
from a patient. The cells may be, for example, from blood, bone
marrow, and/or from tissue derived from solid organs, such as
brain, spleen, bone, heart, vascular, lung, kidney, liver,
pituitary, endocrine glands, lymph node, dispersed primary cells,
tumor cells. Biological samples may include sections of tissues,
including but not limited to frozen or fixed sections taken for
histological purposes. In some embodiments, a sample may be a body
fluid, including, but not limited to, blood fluids, lymph, ascitic
fluids, gynecological fluids, and urine. Samples may be obtained
from a subject by any of a wide variety of methods known in the
art, including without limitation biopsy (e.g., fine needle
aspiration or tissue biopsy), surgery, and collection of body fluid
(e.g., blood, lymph, etc.). Biological samples also include any
material derived by processing any of the above samples. Derived
samples may, for example, include nucleic acids or proteins
extracted from the biological sample, or obtained by subjecting the
sample to techniques such as amplification or reverse transcription
of mRNA, or isolation and/or purification of certain
components.
[0069] A wide variety of cell lines for tissue culture are known in
the art. Examples of cell lines include, but are not limited to,
C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7,
HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R,
Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620,
SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat,
J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,
MRCS, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A,
BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast,
3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse
fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172,
A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B,
bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO,
CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COR-L23,
COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1,
CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1,
EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,
Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,
KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A,
MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R,
MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20,
NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer,
PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3,
T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells,
WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof.
Cell lines are available from a variety of sources known to those
with skill in the art (see, e.g., the American Type Culture
Collection (ATCC) (Manassus, Va.)).
[0070] In some embodiments, staining of the one or more biological
target is performed in vivo or in vitro. Many methods for staining
or otherwise detecting a biological target using binding partners
are known in the art, and include without limitation
immunohistochemistry, immunosorbent assays (e.g. ELISA, with or
without a linked enzyme), flow cytometry, fluorescence activated
cell sorting (FACS), microarray (e.g. protein array), and other
binding partner assays whereby the presence of a binding partner is
indicated by retention by an organism, sample, or portion thereof
of the one or more labeled protein (e.g. Western blot). In some
embodiments, the label carried by the labeled protein is directly
observable, such as in a fluorescent dye detectable by exposure to
light of a particular frequency or range of frequencies. Typically,
excess labeled protein is washed away prior to detection, such that
labeled protein that remains is indicative of the presence,
absence, relative abundance, and/or quantity of the biological
target. Examples of biological targets include, but are not limited
to, amino acids, polypeptides, nucleotides, polynucleotides (e.g.
DNA or RNA), carbohydrates, lipids, metabolites, cell signaling
molecules, cluster of differentiation proteins, hormones, cell
surface proteins, intracellular proteins, fragments thereof, and
combinations or complexes thereof In some embodiments, two or more
different labeled proteins, each recognizing a different binding
partner or set of binding partners are used to detect two or more
different biological targets in a single sample or organism. The
different labeled proteins typically comprise different targeting
moieties, and may or may not comprise the same dye. In some
embodiments, each of two or more different labeled proteins are
labeled with different dyes, such that exposing two or more
biological targets to the two or more differently labeled proteins
renders the two or more biological targets optically
distinguishable. In some embodiments, one or more biological
targets are exposed to a primary binding agent, such as an
antibody, that is unlabeled, before exposure to a labeled protein
that specifically binds the primary binding agent.
[0071] In some embodiments, stained biological targets are analyzed
by flow cytometry. Methods and devices for carrying out flow
cytometry on cells and other particles are known in the art. Flow
cytometry is a technique for counting, examining, and/or sorting
microscopic particles suspended in a stream of fluid. It allows
simultaneous multiparametric analysis of the physical and/or
chemical characteristics of single cells flowing through an
optical/electronic detection apparatus. A description of a typical
flow cytometry process follows. One or more beams of light, usually
laser light, of one or more frequencies are directed onto a
hydrodynamically focused stream of fluid. A number of detectors are
aimed at the point where the stream passes through the light beam;
one in line with the light beam (Forward Scatter or FSC) and
several perpendicular to it (Side Scatter (SSC) and one or more
fluorescent detectors. Each suspended particle passing through the
beam scatters the light in some way, and fluorescent chemicals in
the particle may be excited into emitting light at a lower
frequency than the light source. This combination of scattered and
fluorescent light is picked up by the detectors, and by analyzing
fluctuations in brightness at each detector (one for each
fluorescent emission peak), it is possible to deduce various facts
about the physical and chemical structure of each individual
particle. FSC correlates with the cell size and SSC depends on the
inner complexity of the particle, such as shape of the nucleus, the
amount and type of cytoplasmic granules or the membrane roughness.
Subsequent to detection, cells may be routed to a receptacle based
on one or more characteristics measured by said detection so as to
sort and physically group cells based on such characteristics, such
as in fluorescence activated cell sorting (FACS). Each of a
plurality of receptacles may be designated to contain a different
set of cells, each set having a different characteristic or set of
characteristics. Receptacles can be any suitable container,
including but not limited to wells of a multi-well plate; tubes;
and ordered tubes, such as in a rack or an array.
[0072] In some embodiments, stained biological targets are analyzed
by western blot. Methods and systems for carrying out Western blots
are known in the art. Briefly, this method involves separation of a
substrate from other protein by means of a separation medium, such
as an acrylamide gel, followed by transfer of the substrate to a
membrane (e.g., nylon or PVDF). Presence of the substrate is then
detected by labeled binding partners specific for the substrate,
such as antibodies, which may in turn be detected by a further
binding reagent. Where this first binding partner is a labeled
protein, use of further binding reagents may not be necessary. This
method enables both quantitation of an amount of substrate and
determination of its identity by a relative position on the
membrane which is indicative of a migration distance in the
separation medium (e.g. during electrophoresis).
[0073] In some embodiments, stained biological targets are analyzed
by immunohistochemistry. Methods and systems for
immunohistochemical analysis are known in the art. In general,
detections involving cells include sample fixation and
permeabilization, sample blocking with a blocking solution,
incubation of the sample with one or more labeled primary binding
agents, washing of the stained sample, and detection. Typically,
sections of a tissue sample are adhered to a microscope slide. The
sample is then exposed to one or more binding agents, each with
specificity for one or more targets. Where the one or more binding
agents comprise a detectable label, such as a dye, staining is
evaluated by detecting the detectable label. Where the one or more
binding agents do not comprises a detectable label, the sample are
exposed to one or more second binding agents, each with specificity
to one or more of the first binding agents and comprising a
detectable label, such as a dye. In some embodiments, stained
samples are analyzed under a microscope. The combination of
exposure to unlabeled binding agent followed by exposure to a
second, labeled binding agent can be used in combination with any
detection process, for example to amplify a signal.
[0074] In some embodiments, the method further comprises
visualizing fluorescence of the stained one or more biological
targets. Visualization may be by eye, or may be performed by a
device, such as a camera, whereby an image is produced. Typically,
visualization is performed after a washing step to remove excess
labeled protein. In some embodiments, visualization is performed
immediately after washing. In some embodiments, visualization is
performed at about, before about, or after about 1, 5, 10, 15, 20,
30, 45, 60, 90, 180, or more minutes after washing; 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 24, or more hours after washing; or 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 60, or more days after
washing. In some embodiments, a biological samples stained by the
methods of the invention are stored for later visualization. In
general, visualization involves detecting the presence and
optionally level of one or more dyes used to stain one or more
biological targets in a biological sample, which in turn is
indicative of the presence and optionally level of the one or more
biological targets in a biological sample. Where the dye is
fluorescent dye, visualization may comprise exposure to an
excitation frequency, following by detecting frequency and/or
intensity of fluorescent light emitted by one or more dyes used to
stain a biological sample. In general, the wavelength of excitation
for a given dye is shorter than the wavelength emitted by the dye.
In some embodiments, the excitation wavelength is about, less than
about, or more than about 250 nm, 300 nm, 350 nm, 400 nm, 450 nm,
500 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm,
615, 620 nm, 625, 630 nm, 635, 640 nm, 645, 650 nm, 655, 660 nm,
665, 670 nm, 675, 680 nm, 685, 690 nm, 695, 700 nm, 705, 710 nm,
715, 720 nm, 725, 730 nm, 735, 740 nm, 745, 750 nm, 755, 760 nm,
765, 770 nm, 775, 780 nm, 790 nm, 800 nm, 810 nm, 820 nm, 830 nm,
840 nm, 850 nm, 860 nm, 870 nm, 880 nm, 890 nm, 900 nm, 910 nm, 920
nm, 930 nm, 940 nm, 950 nm, 960 nm, 970 nm, 980 nm, 990 nm, 1000
nm, 1020 nm, 1040 nm, 1060 nm, 1080 nm, 1100 nm, 1120 nm, 1140 nm,
1160 nm, 1180 nm, 1200 nm, or any range including two such
wavelengths as endpoints, such as from about 350 nm to about 1200
nm, and from about 450 nm to about 750 nm. In some embodiments, the
emission wavelength is about, less than about, or more than about
250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 560 nm, 570
nm, 580 nm, 590 nm, 600 nm, 610 nm, 615, 620 nm, 625, 630 nm, 635,
640 nm, 645, 650 nm, 655, 660 nm, 665, 670 nm, 675, 680 nm, 685,
690 nm, 695, 700 nm, 705, 710 nm, 715, 720 nm, 725, 730 nm, 735,
740 nm, 745, 750 nm, 755, 760 nm, 765, 770 nm, 775, 780 nm, 790 nm,
800 nm, 810 nm, 820 nm, 830 nm, 840 nm, 850 nm, 860 nm, 870 nm, 880
nm, 890 nm, 900 nm, 910 nm, 920 nm, 930 nm, 940 nm, 950 nm, 960 nm,
970 nm, 980 nm, 990 nm, 1000 nm, 1020 nm, 1040 nm, 1060 nm, 1080
nm, 1100 nm, 1120 nm, 1140 nm, 1160 nm, 1180 nm, 1200 nm, 1220 nm,
1240 nm, 1250 nm, or any range including two such wavelengths as
endpoints, such as from about 360 nm to about 1250 nm.
[0075] In some embodiments, a diagnosis of a condition in a subject
is made based on the results of the staining process. Conditions
that may be diagnosed according to these methods include any that
are associated with a detectable marker or characteristic that can
be detected by exposure to a labeled protein of the present
invention. For example, a large number of antibodies against
various disease targets, including but not limited to
tumor-associated antigens, have been deposited at the American Type
Culture Collection (ATCC) and/or have published variable region
sequences and are available for use in the claimed methods and
compositions. Antibody sequences or antibody-secreting hybridomas
against almost any disease-associated antigen may be obtained by a
simple search of the ATCC, NCBI and/or USPTO databases for
antibodies against a selected disease-associated target of
interest. The antigen binding domains of the cloned antibodies may
be amplified, excised, ligated into an expression vector,
transfected into an adapted host cell and used for protein
production, using standard techniques well known in the art.
Examples of diseases and conditions that may be identified using
the methods and compositions of the invention include, but are not
limited to, inherited diseases, infectious diseases, diseases
arising from genetic mutation, poisoning, and cancer.
[0076] In one aspect, the invention provides a method for
evaluating the efficacy of a test compound. In one embodiment, the
method comprises (a) exposing a system comprising a drug target to
a test compound; (b) staining one or more biological targets in
said system according to a method of the invention; and evaluating
the efficacy of said test compound based on the results of step
(b). The drug target and the biological target may be the same or
different. In general, efficacy is measured by comparing the degree
of an effect in a system receiving the test compound with the
degree of an effect in a system receiving no compound or a
reference compound. In general, an effect is measured by detecting
the stained biological target, such as by visualizing a fluorescent
dye. Examples of effects on biological targets as may be detected
using a labeled protein of the invention include, but are not
limited to, changes in absolute abundance, changes in relative
abundance, changes in localization (such as within a cell, within a
tissue, within an organ, or within an organ system), and any effect
that can be observed or quantified by visualizing the labeled
protein. Systems that can be exposed to a drug target include in
vtiro systems and in vivo systems, such as reactions in solution,
cultured cells, and whole organisms. Staining may be performed on
the system itself, or on a sample therefrom. For example, a test
compound may be delivered to a subject, such as a human, and an
effect on a tissue of the subject may be evaluated in a sample of
the tissue removed from the subject by staining the sample
according to the methods of the invention.
[0077] Examples of test compounds include, but are not limited to,
drugs, small molecules, activators, inhibitors, antitumor agents,
anti-miotics, steroids, sympathomimetics, local anesthetics,
antimicrobial agents, antihypertensive agents, antihypertensive
diuretics, cardiotonics, coronary vasodilators, vasoconstrictors,
.beta.-blockers, antiarrhythmic agents, calcium antagonists,
anti-convulsants, agents for dizziness, tranquilizers,
antipsychotics, muscle relaxants, respiratory agents, non-steroidal
hormones, antihormones, vitamins, herb medicines, antimuscarinic,
muscarinic cholinergic blocking agents, mydriatics, psychic
energizers, humoral agents, antispasmodics, antidepressant drugs,
anti-diabetics, anorectic drugs, anti-allergenics, decongestants,
antipyretics, antimigrane, anti-malarials, anti-ulcerative,
peptides, anti-estrogen, anti-hormone agents, antiulcer agents,
anesthetic agent, drugs having an action on the central nervous
system, growth factors, mitogens, cytokines, adhesion molecules,
hormones, polynucleotides, antibodies, natural compounds, lactones,
chemotherapeutic agents, immune modulators, carbohydrates,
proteases, ions, reactive oxygen species, protein fragments, or
combinations thereof. In some embodiments, a physical parameter is
used to modulate the system instead of a test compound having a
drug target. Examples of physical parameters include, but are not
limited to radiation, heat, cold, UV radiation, changes in
pressure, and combinations thereof.
[0078] The test compounds may be found and/or isolated from a
variety of custom and commercially available combinatorial
libraries. In one embodiment, the pool of test compounds may
include libraries of antitumor agents such as, chemotherapeutic
agent is selected from the group consisting of adriamycin,
5-fluorouracil (5FU), etoposide (VP-16), camptothecin,
actinomycin-D, mitomycin C, cisplatin (CDDP), doxorubicin,
etoposide, verapamil, podophyllotoxin, carboplatin, procarbazine,
mechlorethamine, cyclophosphamide, ifosfamide, melphalan,
chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin,
bleomycin, plicomycin, mitomycin, tamoxifen, taxol, transplatinum,
vincristin, vinblastin, methotrexate, pilocarpine, mixtures and
combinations thereof and the like.
[0079] Analgesic anti-inflammatory agents such as, acetaminophen,
aspirin, salicylic acid, methyl salicylate, choline salicylate,
glycol salicylate, 1-menthol, camphor, mefenamic acid, fluphenamic
acid, indomethacin, diclofenac, alclofenac, ibuprofen, ketoprofen,
naproxene, pranoprofen, fenoprofen, sulindac, fenbufen, clidanac,
flurbiprofen, indoprofen, protizidic acid, fentiazac, tolmetin,
tiaprofenic acid, bendazac, bufexamac, piroxicam, phenylbutazone,
oxyphenbutazone, clofezone, pentazocine, mepirizole, and the like.
Drugs having an action on the central nervous system, for example
sedatives, hypnotics, antianxiety agents, analgesics and
anesthetics, such as, chloral, buprenorphine, naloxone,
haloperidol, fluphenazine, pentobarbital, phenobarbital,
secobarbital, amobarbital, cydobarbital, codeine, lidocaine,
tetracaine, dyclonine, dibucaine, cocaine, procaine, mepivacaine,
bupivacaine, etidocaine, prilocaine, benzocaine, fentanyl,
nicotine, and the like. Local anesthetics such as, benzocaine,
procaine, dibucaine, lidocaine, and the like.
[0080] Antihistaminics or antiallergic agents such as,
diphenhydramine, dimenhydrinate, perphenazine, triprolidine,
pyrilamine, chlorcyclizine, promethazine, carbinoxamine,
tripelennamine, brompheniramine, hydroxyzine, cyclizine, meclizine,
clorprenaline, terfenadine, chlorpheniramine, and the like.
Anti-allergenics such as, antazoline, methapyrilene,
chlorpheniramine, pyrilamine, pheniramine, and the like.
Decongestants such as, phenylephrine, ephedrine, naphazoline,
tetrahydrozoline, and the like.
[0081] Antipyretics such as, aspirin, salicylamide, non-steroidal
anti-inflammatory agents, and the like. Antimigrane agents such as,
dihydroergotamine, pizotyline, and the like. Acetonide
anti-inflammatory agents, such as hydrocortisone, cortisone,
dexamethasone, fluocinolone, triamcinolone, medrysone,
prednisolone, flurandrenolide, prednisone, halcinonide,
methylprednisolone, fludrocortisone, corticosterone, paramethasone,
betamethasone, ibuprophen, naproxen, fenoprofen, fenbufen,
flurbiprofen, indoprofen, ketoprofen, suprofen, indomethacin,
piroxicam, aspirin, salicylic acid, diflunisal, methyl salicylate,
phenylbutazone, sulindac, mefenamic acid, meclofenamate sodium,
tolmetin, and the like. Muscle relaxants such as, tolperisone,
baclofen, dantrolene sodium, cyclobenzaprine.
[0082] Steroids such as, androgenic steroids, such as,
testosterone, methyltestosterone, fluoxymesterone, estrogens such
as, conjugated estrogens, esterified estrogens, estropipate,
17-.beta. estradiol, 17-.beta. estradiol valerate, equilin,
mestranol, estrone, estriol, 17.beta. ethinyl estradiol,
diethylstilbestrol, progestational agents, such as, progesterone,
19-norprogesterone, norethindrone, norethindrone acetate,
melengestrol, chlormadinone, ethisterone, medroxyprogesterone
acetate, hydroxyprogesterone caproate, ethynodiol diacetate,
norethynodrel, 17-.alpha. hydroxyprogesterone, dydrogesterone,
dimethisterone, ethinylestrenol, norgestrel, demegestone,
promegestone, megestrol acetate, and the like.
[0083] Respiratory agents such as, theophilline and
.beta.2-adrenergic agonists, such as, albuterol, terbutaline,
metaproterenol, ritodrine, carbuterol, fenoterol, quinterenol,
rimiterol, solmefamol, soterenol, tetroquinol, and the like.
Sympathomimetics such as, dopamine, norepinephrine,
phenylpropanolamine, phenylephrine, pseudoephedrine, amphetamine,
propylhexedrine, arecoline, and the like.
[0084] Antimicrobial agents including antibacterial agents,
antifungal agents, antimycotic agents and antiviral agents;
tetracyclines such as, oxytetracycline, penicillins, such as,
ampicillin, cephalosporins such as, cefalotin, aminoglycosides,
such as, kanamycin, macrolides such as, erythromycin,
chloramphenicol, iodides, nitrofrantoin, nystatin, amphotericin,
fradiomycin, sulfonamides, purrolnitrin, clotrimazole, miconazole
chloramphenicol, sulfacetamide, sulfamethazine, sulfadiazine,
sulfamerazine, sulfamethizole and sulfisoxazole; antivirals,
including idoxuridine; clarithromycin; and other anti-infectives
including nitrofurazone, and the like.
[0085] Antihypertensive agents such as, clonidine,
.alpha.-methyldopa, reserpine, syrosingopine, rescinnamine,
cinnarizine, hydrazine, prazosin, and the like. Antihypertensive
diuretics such as, chlorothiazide, hydrochlorothrazide,
bendoflumethazide, trichlormethiazide, furosemide, tripamide,
methylclothiazide, penfluzide, hydrothiazide, spironolactone,
metolazone, and the like. Cardiotonics such as, digitalis,
ubidecarenone, dopamine, and the like. Coronary vasodilators such
as, organic nitrates such as, nitroglycerine, isosorbitol
dinitrate, erythritol tetranitrate, and pentaerythritol
tetranitrate, dipyridamole, dilazep, trapidil, trimetazidine, and
the like. Vasoconstrictors such as, dihydroergotamine,
dihydroergotoxine, and the like. .beta.-blockers or antiarrhythmic
agents such as, timolol pindolol, propranolol, and the like.
Humoral agents such as, the prostaglandins, natural and synthetic,
for example PGE 1 , PGE 2.alpha., and PGF 2.alpha., and the PGE 1
analog misoprostol. Antispasmodics such as, atropine,
methantheline, papaverine, cinnamedrine, methscopolamine, and the
like.
[0086] Calcium antagonists and other circulatory organ agents, such
as, aptopril, diltiazem, nifedipine, nicardipine, verapamil,
bencyclane, ifenprodil tartarate, molsidomine, clonidine, prazosin,
and the like. Anti-convulsants such as, nitrazepam, meprobamate,
phenytoin, and the like. Agents for dizziness such as,
isoprenaline, betahistine, scopolamine, and the like. Tranquilizers
such as, reserprine, chlorpromazine, and antianxiety
benzodiazepines such as, alprazolam, chlordiazepoxide,
clorazeptate, halazepam, oxazepam, prazepam, clonazepam,
flurazepam, triazolam, lorazepam, diazepam, and the like.
Antipsychotics such as, phenothiazines including thiopropazate,
chlorpromazine, triflupromazine, mesoridazine, piperracetazine,
thioridazine, acetophenazine, fluphenazine, perphenazine,
trifluoperazine, and other major tranqulizers such as,
chlorprathixene, thiothixene, haloperidol, bromperidol, loxapine,
and molindone, as well as, those agents used at lower doses in the
treatment of nausea, vomiting, and the like.
[0087] Respiratory agents such as, codeine, ephedrine,
isoproterenol, dextromethorphan, orciprenaline, ipratropium
bromide, cromglycic acid, and the like. Non-steroidal hormones or
antihormones such as, corticotropin, oxytocin, vasopressin,
salivary hormone, thyroid hormone, adrenal hormone, kallikrein,
insulin, oxendolone, and the like. Vitamins such as, vitamins A, B,
C, D, E and K and derivatives thereof, calciferols, mecobalamin,
and the like for dermatologically use. Enzymes such as, lysozyme,
urokinaze, and the like. Herb medicines or crude extracts such as,
Aloe vera, and the like.
[0088] Mydriatics such as, atropine, cyclopentolate, homatropine,
scopolamine, tropicamide, eucatropine, hydroxyamphetamine, and the
like. Psychic energizers such as 3-(2-aminopropy)indole,
3-(2-aminobutyl)indole, and the like. Antidepressant drugs such as,
isocarboxazid, phenelzine, tranylcypromine, imipramine,
amitriptyline, trimipramine, doxepin, desipramine, nortriptyline,
protriptyline, amoxapine, maprotiline, trazodone, and the like.
[0089] Anti-diabetics such as, insulin, and the like and anticancer
drugs such as, tamoxifen, methotrexate, and the like. Anorectic
drugs such as, dextroamphetamine, methamphetamine,
phenylpropanolamine, fenfluramine, diethylpropion, mazindol,
phentermine, and the like. Anti-malarials such as, the
4-aminoquinolines, alphaaminoquinolines, chloroquine,
pyrimethamine, and the like. Anti-ulcerative agents such as,
misoprostol, omeprazole, enprostil, and the like. Antiulcer agents
such as, allantoin, aldioxa, alcloxa, N-methylscopolamine
methylsuflate, and the like. The drugs mentioned above may be used
in combination as required. Moreover, the above drugs may be used
either in the free form or, if capable of forming salts, in the
form of a salt with a suitable acid or base. If the drugs have a
carboxyl group, their esters may be employed.
[0090] In general, a biological target is any target comprising a
binding partner to which a labeled protein of the current invention
specifically binds. The methods and compositions of the invention
may be employed to examine and profile the status of any biological
target in a cellular pathway, or collections of such biological
targets. Single or multiple distinct pathways may be profiled
(sequentially or simultaneously), or subsets of biological targets
within a single pathway or across multiple pathways may be examined
(again, sequentially or simultaneously).
[0091] A wide variety of activation events can be detected by
labeled proteins of the present invention. In general, the basic
requirement is that the change in activation results in a change in
the biological target that is detectable by altered binding to a
labeled protein. What is important is to differentiate, using a
labeled protein that specifically binds to a binding partner that
is indicative of an activation state, thus distinguishing between
two or more activation states (e.g. "off" and "on"). Typically, the
binding partner is specific for an activation state of an
activatable element.
[0092] The activation state of an individual activatable element is
either in the on or off state. As an illustrative example, and
without intending to be limited to any theory, an individual
phosphorylatable site on a protein can activate or deactivate the
protein. The terms "on" and "off," when applied to an activatable
element that is a part of a biological target, are used here to
describe the state of the activatable element, and not necessarily
the overall state of the biological target of which it is a part.
Typically, a cell possesses a plurality of a particular protein or
other constituent with an activatable element and this plurality of
proteins or constituents usually has some proteins or constituents
whose individual activatable element is in the on state and other
proteins or constituents whose individual activatable element is in
the off state. Since the activation state of each activatable
element is measured through the use of a labeled protein that
recognizes a specific activation state, only those activatable
elements in the specific activation state recognized by the binding
element, representing some fraction of the total number of
activatable elements, will be bound by the labeled protein to
generate a measurable signal. The measurable signal corresponding
to the summation of individual activatable elements of a particular
type that are activated in a sample is the "activation level" for
that activatable element in that sample. In some embodiments,
activation level is measured at the level of single cells, such as
a plurality of cells in a sample.
[0093] Activation levels for a particular activatable element may
vary among individual cells so that when a plurality of cells is
analyzed, the activation levels follow a distribution. The
distribution may be a normal distribution, also known as a Gaussian
distribution, or it may be of another type. Different populations
of cells may have different distributions of activation levels that
can then serve to distinguish between the populations. In some
embodiments, the basis for classifying cells is that the
distribution of activation levels for one or more specific
activatable elements will differ among different phenotypes. A
certain activation level, or more typically a range of activation
levels for one or more activatable elements seen in a cell or a
population of cells, is indicative that that cell or population of
cells belongs to a distinctive phenotype. Other measurements, such
as cellular levels (e.g., expression levels) of biomolecules that
may not contain activatable elements, may also be used to classify
cells in addition to activation levels of activatable elements; it
will be appreciated that these levels also will follow a
distribution, similar to activatable elements. Thus, the activation
level or levels of one or more activatable elements, optionally in
conjunction with levels of one or more levels of biomolecules that
may not contain activatable elements, of a cell or a population of
cells may be used to classify a cell or a population of cells into
a class. Once the activation level of intracellular activatable
elements of individual single cells is known they can be placed
into one or more classes, e.g., a class that corresponds to a
phenotype. A class encompasses a class of cells wherein every cell
has the same or substantially the same known activation level, or
range of activation levels, of one or more intracellular
activatable elements, or other binding partners detected by the
labeled proteins of the invention. For example, if the activation
levels of five intracellular activatable elements are analyzed,
predefined classes that encompass one or more of the intracellular
activatable elements can be constructed based on the activation
level, or ranges of the activation levels, of each of these five
elements. It is understood that activation levels can exist as a
distribution and that an activation level of a particular element
used to classify a cell may be a particular point on the
distribution but more typically may be a portion of the
distribution.
[0094] In addition to activation levels of intracellular
activatable elements, expression levels of intracellular or
extracellular binding partners can be used alone or in combination
with activation states of activatable elements and/or expression
levels to classify cells. In some embodiments, other
characteristics that affect the status of a biological target may
also be used to classify a cell. Examples include the translocation
of binding partners or changes in their turnover rates and the
formation and disassociation of complexes of comprising a binding
partner. Such complexes can include multi-protein complexes,
multi-lipid complexes, homo- or hetero-dimers or oligomers, and
combinations thereof Other characteristics include proteolytic
cleavage, e.g. from exposure of a cell to an extracellular protease
or from the intracellular proteolytic cleavage of a binding
partner.
[0095] In some embodiments, the physiological status of one or more
cells is determined by examining and profiling the activation level
of one or more activatable elements in a cellular pathway. In some
embodiments, a cell is classified according to the activation level
of a plurality of activatable elements. In some embodiments, the
activation level of one or more activatable elements in a sample is
correlated with a condition. In some embodiments, the activation
level of one or more activatable elements in single cells within
the sample is determined. Cellular constituents that may include
activatable elements include without limitation, proteins,
carbohydrates, lipids, nucleic acids and metabolites. Upon
activation, a change occurs to the activatable element, such as
covalent modification of the activatable element (e.g., binding of
a molecule or group to the activatable element, including but not
limited to, phosphorylation, acetylation, methylation,
ubiquitination) or a conformational change. Such changes generally
contribute to changes in particular biological, biochemical, or
physical properties of the biological target that contains the
activatable element. The state of the biological target that
contains the activatable element is often determined to some
degree, though not necessarily completely, by the state of
activation of a particular activatable element of the biological
target. For example, a complex may comprise a protein having
multiple activation sites, and the particular activation states of
these sites may overall determine the activation state of the
protein and/or complex. Additional factors, such as the binding of
other proteins, pH, ion concentration, interaction with other
cellular constituents, and the like, can also affect the state of
the biological target. In some embodiments, the activation levels
of a plurality of intracellular activatable elements in single
cells are determined. In some embodiments, at least about 2, 3, 4,
5, 6, 7, 8, 9, 10 or more than 10 intracellular activatable
elements are determined.
[0096] Activation states of activatable elements may result from
chemical additions or modifications of biomolecules and include
biochemical processes such as glycosylation, phosphorylation,
acetylation, methylation, biotinylation, glutamylation,
glycylation, hydroxylation, isomerization, prenylation,
myristoylation, lipoylation, phosphopantetheinylation, sulfation,
ISGylation, nitrosylation, palmitoylation, SUMOylation,
ubiquitination, neddylation, citrullination, amidation, and
disulfide bond formation, disulfide bond reduction. Other possible
chemical additions or modifications of biomolecules include the
formation of protein carbonyls, direct modifications of protein
side chains, such as o-tyrosine, chloro-, nitrotyrosine, and
dityrosine, and protein adducts derived from reactions with
carbohydrate and lipid derivatives. Other modifications may be
non-covalent, such as binding of a ligand or binding of an
allosteric modulator. In general, the activation level is
determined by detecting binding of a labeled protein that is
specific for either the modified activatable element or the
unmodified activatable element, such as a labeled antibody specific
to a protein phosphorylated at a particular site, which antibody
does not specifically bind to the unphosphorylated protein.
[0097] Examples of proteins that may include activatable elements
include, but are not limited to kinases, phosphatases, lipid
signaling molecules, adaptor/scaffold proteins, cytokines, cytokine
regulators, ubiquitination enzymes, adhesion molecules,
cytoskeletal/contractile proteins, heterotrimeric G proteins, small
molecular weight GTPases, guanine nucleotide exchange factors,
GTPase activating proteins, caspases, proteins involved in
apoptosis (e.g. PARP), cell cycle regulators, molecular chaperones,
metabolic enzymes, vesicular transport proteins, hydroxylases,
isomerases, deacetylases, methylases, demethylases, tumor
suppressor genes, proteases, ion channels, molecular transporters,
transcription factors/DNA binding factors, regulators of
transcription, and regulators of translation. Examples of
activatable elements, activation states and methods of determining
the activation level of activatable elements are described in US
Publication Number 20060073474 entitled "Methods and compositions
for detecting the activation state of multiple proteins in single
cells" and U.S. Pat. No. 7,393,656 entitled "Methods and
compositions for risk stratification" the content of which are
incorporate here by reference.
[0098] In some embodiments, the binding partner is selected from
the group consisting of HER receptors, PDGF receptors, Kit
receptor, FGF receptors, Eph receptors, Trk receptors, IGF
receptors, Insulin receptor, Met receptor, Ret, VEGF receptors,
TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr,
Yes, Csk, Ab1, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim
kinase, ILK, Tpl, ALK, TGF.beta. receptors, BMP receptors, MEKKs,
ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK,
Bub, Myt 1, Wee1, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1,
Akt2, Akt3, p90Rsks, p70S6Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2,
Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs, Chk1, Chk2, LKB-1,
MAPKAPKs, Pim1, Pim2, Pim3, IKKs, Cdks, Jnks, Erks, IKKs, GSK3a,
GSK3p, Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class 3, mTor,
SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR, Receptor protein
tyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptor
tyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases
(MKPs), Dual Specificity phosphatases (DUSPs), CDC25 phosphatases,
Low molecular weight tyrosine phosphatase, Eyes absent (EYA)
tyrosine phosphatases, Slingshot phosphatases (SSH), serine
phosphatases, PP2A, PP2B, PP2C, PP1, PP5, inositol phosphatases,
PTEN, SHIPs, myotubularins, phosphoinositide kinases,
phospholipases, prostaglandin synthases, 5-lipoxygenase,
sphingosine kinases, sphingomyelinases, adaptor/scaffold proteins,
Shc, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP), SLAP,
Dok, KSR, MyD88, Crk, CrkL, GAD, Nck, Grb2 associated binder (GAB),
Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-Cell
leukemia family, IL-2, IL-4, IL-8, IL-6, interferon .gamma.,
interferon .beta., suppressors of cytokine signaling (SOCs), Cbl,
SCF ubiquitination ligase complex, APC/C, adhesion molecules,
integrins, Immunoglobulin-like adhesion molecules, selectins,
cadherins, catenins, focal adhesion kinase, p130CAS, fodrin, actin,
paxillin, myosin, myosin binding proteins, tubulin, eg5/KSP, CENPs,
.beta.-adrenergic receptors, muscarinic receptors, adenylyl cyclase
receptors, small molecular weight GTPases, H-Ras, K-Ras, N-Ras,
Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam, Sos, Dbl, PRK,
TSC1,2, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2, Caspase
3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, Bcl-2, Mcl-1,
Bcl-XL, Bcl-w, Bcl-B, Al, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf,
Hrk, Noxa, Puma, IAPs, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7,
Cyclin D, Cyclin E, Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP,
p21CIP, molecular chaperones, Hsp90s, Hsp70, Hsp27, metabolic
enzymes, Acetyl-CoAa Carboxylase, ATP citrate lyase, nitric oxide
synthase, caveolins, endosomal sorting complex required for
transport (ESCRT) proteins, vesicular protein sorting (Vsps),
hydroxylases, prolyl-hydroxylases PHD-1, 2 and 3, asparagine
hydroxylase FIH transferases, Pin1 prolyl isomerase,
topoisomerases, deacetylases, Histone deacetylases, sirtuins,
histone acetylases, CBP/P300 family, MYST family, ATF2, DNA methyl
transferases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, VHL,
WT-1, p53, Hdm, PTEN, ubiquitin proteases, urokinase-type
plasminogen activator (uPA) and uPA receptor (uPAR) system,
cathepsins, metalloproteinases, esterases, hydrolases, separase,
potassium channels, sodium channels, multi-drug resistance
proteins, P-Gycoprotein, nucleoside transporters, Ets, Elk, SMADs,
Rel-A (p65-NFKB), CREB, NFAT, ATF-2, AFT, Myc, Fos, Spl, Egr-1,
T-bet, .beta.-catenin, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr-1, FOXO
STAT1, STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-1, HMGA, pS6,
4EPB-1, eIF4E-binding protein, RNA polymerase, initiation factors,
elongation factors.
[0099] In some embodiments, the classification of a cell according
to activation level of an activatable element, e.g., in a cellular
pathway comprises classifying the cell as a cell that is correlated
with a clinical outcome. In some embodiments, the clinical outcome
is the prognosis and/or diagnosis of a condition. In some
embodiments, methods and compositions are provided for the
classification of a cell according to the activation level of an
activatable element, e.g., in a cellular pathway wherein the
classification comprises classifying a cell as a cell that is
correlated to a patient response to a treatment. In some
embodiments, methods and compositions are provided for the
classification of a cell according to the activation level of an
activatable element, e.g., in a cellular pathway wherein the
classification comprises selecting a method of treatment.
[0100] In one aspect, the invention provides kits containing any
one or more of the elements disclosed in the above methods and
compositions. In some embodiments, the kit comprises one or more
elements for carrying out a method of the invention, the elements
in one or more containers. In one embodiment, a kit for labeling a
target protein with a dye or detectable label comprises a buffer
comprising an amine; one or more reactive dye or detectable label
for labeling one or more target proteins; a storage buffer; and
instruction in one or more languages, for example in more than one
language. The reactive dye or detectable label may be a reactive
dye, a reactive biotin, a reactive digoxin or a reactive epitope.
The dye can be any of the dyes described herein, including but not
limited to an amine reactive dye. The buffer can be any of the
buffers described herein, including but not limited to a sodium
carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a
Tris buffer, a MOPS buffer, a HEPES buffer, and combinations
thereof. In some embodiments, the buffer is alkaline. In some
embodiments, the buffer has a pH from about 7 to about 10. The
amine can be any amine described herein, including but not limited
to Tris. In some embodiments, the dye comprises one or more
activated esters, one or more sulfonates, and/or one or more
water-soluble polymers, as described herein. In some embodiments,
the buffer and dye are sufficient in amount to permit labeling of 5
.mu.g to 200 .mu.g of a target protein, such as more than about
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more of the
target protein in a sample is labeled. The storage buffer can be
any of the storage buffers described herein, and may comprise a
stabilizer and/or a preservative as described herein.
[0101] In another aspect, a kit for conjugating a reporter protein
to a target protein is provided, the kit comprising a first buffer
comprising a first amine as described herein; a second buffer
comprising an aryl amine; a bifunctional crosslinker comprising an
amine-reactive group and a hydrolysis-resistant functional group;
one or more reporter proteins functionalized with a pairing
hydrolysis-resistant reactive group for labeling one or more target
proteins; a storage buffer; and instruction in one ore more
languages, for example in more than one language. In some
embodiments, the bifunctional crosslinker comprises a succinimidyl
ester and an aldehyde group, and the one or more reporter proteins
are functionalized with aminooxy groups.
[0102] In some embodiments, the kit further comprises a separation
device for purifying a target protein before labeling, and/or for
purifying a labeled target protein. Methods for purifying protein
are known in the art, non-limiting examples of which include
protein A-based affinity chromatography, size-exclusion
chromatography, and ultra membrane filtration. Commercial kits for
protein purification, such as antibody cleaning kits, may also be
used for target protein purification. Size-exclusion column
chromatography and ultra membrane filtration are useful for
removing stabilizers/preservatives of relatively small molecules,
such as glycerol, Tris, and amino acids. Ultra membrane filtration
may also be used to adjust target protein concentration to a
desired value. Ultra membrane filtration can be conveniently and
rapidly carried out on a centrifuge using a commercial ultra
membrane filtration vial. The membranes in such ultra filtration
devices typically have different pore sizes, or so-called Molecular
Weight Cut-off sizes (MWCO), permitting relatively small molecules
to go through while retaining bigger molecules, such as proteins.
In some embodiments, a membrane with a MWCO of about, less than
about, or more than about 1, 5, 10, 15, 20, 25, 50, 100, or more kD
is provided to purify a target protein before and/or after
labeling. Columns for size exclusion chromatography typically
comprise particles or beads having pores of a particular MWCO.
Particles larger than the MWCO pass through the column faster than
particles at or below the MWCO. In some embodiments, a column with
a MWCO of about, less than about, or more than about 1, 2, 10, 15,
20, 25, 50, 100, or more kD is provided to purify a target protein
before and/or after labeling.
[0103] In some embodiments, the kit further comprises a stain
stabilizing reagent for enhancing dye fluorescence, such as by
enhancing fluorescent intensity or reducing a rate of decrease in
fluorescent intensity. Stain stabilizing reagents are known in the
art, non-limiting examples of which include EverBrite (Biotium),
Vectashield (Vector Laboratories), and SlowFade Gold (Invitrogen).
In some embodiments, fluorescent intensity of a dye in the presence
of the stain stabilizing reagent is increased by at least about
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,
300%, 400%, 500% or more above the intensity of the dye in the
absence of the stain stabilizing reagent. In some embodiments,
fluorescent intensity of a dye in the presence of the stain
stabilizing reagent is maintained above a threshold level for a
time that is at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, or more minutes. In some
embodiments, the threshold level is at least about 10%, 20%, 30%,
40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,
99.5%, or more of the starting fluorescent intensity.
[0104] Tables 1-4 provide examples of dyes useful in the methods,
compositions, and kits of the present invention.
TABLE-US-00002 TABLE 1 Compound No. Structure* 1 ##STR00001## 2
##STR00002## 3 ##STR00003## 4 ##STR00004## 5 ##STR00005## 6
##STR00006## 7 ##STR00007## 8 ##STR00008## 9 ##STR00009## 10
##STR00010## 11 ##STR00011## 12 ##STR00012## 13 ##STR00013## 14
##STR00014## 15 ##STR00015## 16 ##STR00016## 17 ##STR00017## 18
##STR00018## *Wherein when a dye comprise a carboxylic acid group,
it is understood that the carboxylic acid group can be readily
converted to an activated ester via the use of any of a number of
reagents, such as DCC, EDC, and TSTU, so that the carboxylic acid
group can react with amine group to form an amide linkage.
TABLE-US-00003 TABLE 2 Exemplary compounds of the invention are
shown. For simplicity, counter ions are not shown in some cases.
Absorption/Emission spectra were measured in PBS unless otherwise
indicated. Compound Absorption/Emission No. Structure Wavelengths
(nm) 19 ##STR00019## 548/571 20 ##STR00020## 559/579 21
##STR00021## 529/548 22 ##STR00022## 603/626 23 ##STR00023##
622/649 24 ##STR00024## 622/649 25 ##STR00025## 638/660 26
##STR00026## 27 ##STR00027## 622/648 28 ##STR00028## 622/649 29
##STR00029## 30 ##STR00030## 31 ##STR00031## 32 ##STR00032## 33
##STR00033## 34 ##STR00034## 35 ##STR00035## 36 ##STR00036## 37
##STR00037## 38 ##STR00038## 599/~620 39 ##STR00039## 40
##STR00040## 592/615 41 ##STR00041## 627/650 42 ##STR00042##
642/662 43 ##STR00043## 660/681 44 ##STR00044## 683/700 45
##STR00045## 680/700 46 ##STR00046## 593/614 47 ##STR00047## 578/
48 ##STR00048## 49 ##STR00049## 50 ##STR00050## 51 ##STR00051##
533/550 52 ##STR00052## 53 ##STR00053## 642/662 54 ##STR00054##
TABLE-US-00004 TABLE 3 .lamda.abs/.lamda.em (nm) Compound No.
Structure (in pH 7.4 PBS) 55 ##STR00055## 488/516 56 ##STR00056##
488/516 57 ##STR00057## 488/516 58 ##STR00058## 488/516 59
##STR00059## 488/516 60 ##STR00060## 488/516 61 ##STR00061## 62
##STR00062## 63 ##STR00063## 529/ 64 ##STR00064## 65 ##STR00065##
66 ##STR00066## 490/520 67 ##STR00067## 490/520 68 ##STR00068##
490/520
TABLE-US-00005 TABLE 4 .lamda.abs/ Com- .lamda.em pound (nm) No.
Structure (H.sub.2O) 69 ##STR00069## 457/ 70 ##STR00070## 550/ 570
71 ##STR00071## 550/ 570 72 ##STR00072## 550/ 570 73 ##STR00073##
550/ 570 74 ##STR00074## 550/ 570 75 ##STR00075## 550/ 570 76
##STR00076## 550/ 570 77 ##STR00077## 650/ 665 78 ##STR00078## 650/
665 79 ##STR00079## 650/ 665 80 ##STR00080## 650/ 665 81
##STR00081## 650/ 665 82 ##STR00082## 660/ 680 83 ##STR00083## 650/
665 84 ##STR00084## 663/ 690 85 ##STR00085## 752/ 778 86
##STR00086## 750/ 775 87 ##STR00087## 675/ 694 88 ##STR00088## 750/
775 89 ##STR00089## 90 ##STR00090## 768/ 788 91 ##STR00091## 92
##STR00092## 635/ 642 93 ##STR00093## 94 ##STR00094## 497/ 513 95
##STR00095## 555/ 565 96 ##STR00096## 650/ 665 97 ##STR00097## 750/
770 98 ##STR00098## 660/ 675 99 ##STR00099## 770/ 790 100
##STR00100## 680/ 700 101 ##STR00101## 790/ 810 102 ##STR00102##
494/ 520 103 ##STR00103## 104 ##STR00104## 501/ 524 105
##STR00105## 106 ##STR00106## 107 ##STR00107## 520/ 546 108
##STR00108## 109 ##STR00109## 110 ##STR00110## 540/ 565 111
##STR00111## 112 ##STR00112## 113 ##STR00113## 114 ##STR00114## 115
##STR00115## 116 ##STR00116## 488/ 515 117 ##STR00117## 494/ 520
118 ##STR00118## 353/ 442 119 ##STR00119## 346/ 442 120
##STR00120## 416/ 465 121 ##STR00121## 430/ 545 122 ##STR00122##
350/ 440 123 ##STR00123## 124 ##STR00124## 125 ##STR00125## 400/
424
EXAMPLES
[0105] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion. The present
examples, along with the methods described herein are presently
representative of preferred embodiments, are exemplary, and are not
intended as limitations on the scope of the invention. Changes
therein and other uses which are encompassed within the spirit of
the invention as defined by the scope of the claims will occur to
those skilled in the art.
Example 1: Analysis of Cells Stained with Secondary Antibody
Labeled with Compound No. 126
[0106] This example describes the labeling of an antibody with a
green fluorescent sulfonated xanthene dye, and the use of such
antibodies for intracellular staining and flow cytometry analysis.
The dye used in this example, Compound No. 126, absorbs light at
about 491 nm and emits light at about 525 nm. The structure of
Compound No. 126 is as follows:
##STR00126##
[0107] Solutions of goat anti-mouse (GAM) labeled with Compound No.
126 were prepared using different dye-to-antibody ratios and
different antibody concentrations in a reaction buffer comprising
10 mM Tris and 50 mM NaHCO.sub.3 (pH 8.3). Detailed labeling
reaction conditions are listed in Table 5 below. Labeling reactions
were carried out by combining antibody and dye in reaction buffer
followed by 30 minutes of incubation. Reaction buffer comprised 10
mM Tris and 50 mM NaHCO.sub.3 with pH .about.8.3. Each labeled GAM
solution was diluted in a storage buffer and then used in cell
staining. Storage buffer (.about.20 mL) was prepared by mixing 10
mL 75 mM Tris pH7 buffer, 200 mg BSA, 150 uL 5% NaN.sub.3, 10 mL
glycerol and 10mg lysine.
TABLE-US-00006 TABLE 5 Labeling condition Antibody Amount Labeled
Amount concen- Antibody- of GAM IgG of GAM tration Amount to-dye
storage antibody IgG in reaction of dye ratio buffer solution
(.mu.g) (mg/mL) (nmoles) (ug/nmole) (uL) #1 100 1 11.25 8.9:1 300
#2 50 2 11.25 4.4:1 300 #3 20 2 5.6 3.6:1 150 #4 20 1 2.8 7.2:1 60
#5 5 1 2.8 1.8:1 60 #6 100 1 9.4 10.6:1 300 #7 50 2 9.4 5.3:1 300
#8 100 1 7.5 13.3:1 300 #9 50 2 7.5 6.7:1 30
[0108] Purified dye-GAM IgG conjugate was also prepared for cell
staining as a control. The purified antibody conjugate was prepared
using a standard antibody conjugation method. Briefly, Compound No.
126 (180 nmoles) in 50 uL dimethylformamide (DMF) was added to 1 mg
of goat anti-mouse at 1 mg/mL in 0.1 mM NaHCO3 (pH 8.3), followed
by incubation at room temperature for 1 hour. The reaction solution
was subject to G-25 Sephadex column equilibrated in and eluted with
1.times. PBS to result in the purified antibody conjugate. This is
referred to as "purified Dye No. 1-GAM" in FIG. 1.
[0109] One million Jurkat cells per sample were stained with 0.25
.mu.g mouse anti-human CD3 (BD Biosciences) followed by 1 .mu.g of
a dye-GAM IgG conjugate prepared according to the above table or 1
.mu.g of the purified GAM conjugate. About 10,000 cells from each
sample were analyzed on a BD FACSCalibur flow cytometer (BD
Biosciences), and fluorescence was detected in the FL1 channel
using 488 nm excitation. Noise represents the average intensity
from cells stained with only goat anti-mouse secondary conjugates.
Signal represents average fluorescence intensity from cells stained
with CD3 and labeled goat anti-mouse IgG. The data are plotted in
FIG. 1. The results show that cells stained with antibodies labeled
according to the invention show high relative fluorescence (white
bars) with low background fluorescence (black bars), and perform
comparably to the purified antibody. FIG. 1 also demonstrates a
lack of correlation between relative fluorescence and the staining
conditions tested, indicating that cell staining is independent of
the conditions under which the labeled antibodies are prepared
(i.e., the antibody-to-dye ratio and antibody concentration).
Example 2: Analysis of Cells Stained with Secondary Antibody
Labeled with Compound No. 42
[0110] This example describes labeling of an antibody with a
far-red fluorescent sulfonated xanthene dye (Compound No. 42), and
the use of such antibodies for intracellular staining and flow
cytometry analysis.
[0111] Solutions of goat anti-mouse (GAM) labeled with Compound No.
42 were prepared using different dye-to-antibody ratios and
different antibody concentrations in a reaction buffer comprising
10 mM Tris and 50 mM NaHCO.sub.3 (pH 8.3). Detailed labeling
reaction conditions are listed in Table 6 below. Labeling reactions
were carried out by combining antibody and dye in reaction buffer
followed by 30 minutes of incubation. Reaction buffer comprised 10
mM Tris and 50 mM NaHCO.sub.3 with pH .about.8.3. Each labeled GAM
solution was diluted in a storage buffer and then used in cell
staining. A typical storage buffer (.about.20 mL) was prepared by
mixing 10 mL 75 mM Tris pH7 buffer, 200 mg BSA, 150 uL 5%
NaN.sub.3, 10 mL glycerol and 10mg lysine.
TABLE-US-00007 TABLE 6 Labeling condition Antibody Amount Labeled
Amount concen- Antibody- of GAM IgG of GAM tration Amount to-dye
storage antibody IgG in reaction of dye ratio buffer solution
(.mu.g) (mg/mL) (nmoles) (ug/nmole) (uL) #1 100 1 11.25 8.9:1 300
#2 50 2 11.25 4.4:1 300 #3 20 2 5.6 3.6:1 150 #4 20 1 2.8 7.2:1 60
#5 5 1 2.8 1.8:1 60 #6 100 1 9.4 10.6:1 300 #7 50 2 9.4 5.3:1 300
#8 100 1 7.5 13.3:1 300 #9 50 2 7.5 6.7:1 30
[0112] Purified Dye-GAM IgG conjugate was also prepared for cell
staining as a control. The purified antibody conjugate was prepared
using standard antibody conjugation method. Briefly, Compound No.
42 (130 nmoles) in 50 uL DMF was added to 1 mg of goat anti-mouse
at 1 mg/mL in 0.1 mM NaHCO.sub.3, followed by incubation at room
temperature for 1 hour. The reaction solution was subject to G-25
Sephadex column equilibrated in and eluted with 1.times. PBS to
result in the purified antibody conjugate. This is referred to as
"purified Dye No. 2-GAM conjugate" in FIG. 2.
[0113] Cell staining was performed as in Example 1. Stained samples
were analyzed on BD FACS Calibur using 635 nm excitation and FL4
detection channel. Relative fluorescence signals are shown in FIG.
2. The data show that cell immunostaining according to the
invention and that according to the conventional method using a
purified antibody conjugate produced comparable results.
Example 3: Analysis of Cells Stained with Secondary Antibody
Labeled with One of Two Compounds
[0114] This example describes labeling of an antibody with either a
near-IR fluorescent sulfonated xanthene dye (Compound No. 45) or a
near-IR sulfonated cyanine dye (Compound No. 127) , and the use of
such antibodies for intracellular staining and flow cytometry
analysis. Compound No. 127, absorbs light at about 680 nm and emits
light at about 698 nm. The structure of Compound No. 127 is as
follows:
##STR00127##
[0115] Solutions of goat anti-mouse (GAM) labeled with Compound No.
45 or Compound No. 127 were prepared using different
dye-to-antibody ratios and different antibody concentrations in a
reaction buffer comprising 10 mM Tris and 50 mM NaHCO.sub.3 (pH
8.3). Detailed labeling reaction conditions are listed in Table 7
below. Labeling reactions were carried out by combining antibody
and dye in reaction buffer followed by 30 minutes of incubation.
Reaction buffer comprised 10 mM Tris and 50 mM NaHCO.sub.3 with pH
.about.8.3. Each labeled GAM solution was diluted in a storage
buffer and then used in cell staining. A typical storage buffer
(.about.20 mL) was prepared by mixing 10 mL 75 mM Tris pH7 buffer,
200 mg BSA, 150 uL 5% NaN.sub.3, 10 mL glycerol, and 10 mg
lysine.
TABLE-US-00008 TABLE 7 Labeled Labeling condition GAM Antibody
Amount IgG Amount concentration Amount of Antibody-to- of storage
antibody Compound of GAM in reaction dye dye ratio buffer solution
No. IgG (.mu.g) (mg/mL) (nmoles) (.mu.g/nmole) (.mu.L) #16 45 50 1
3.8 13.2:1 300 #17 45 100 0.5 3.8 26.3:1 300 #18 45 50 1 5.6 8.9:1
300 #19 45 100 0.5 5.6 17.9:1 300 #20 45 50 1 7.5 6.7:1 300 #21 45
100 0.5 7.5 13.3:1 300 #22 45 50 1 9.4 5.3:1 300 #23 45 100 0.5 9.4
10.6:1 300 #24 127 50 1 3.8 13.2:1 300 #25 127 100 0.5 3.8 26.3:1
300 #26 127 50 1 5.6 8.9:1 300 #27 127 100 0.5 5.6 17.9:1 300 #28
127 50 1 7.5 6.7:1 300 #29 127 100 0.5 7.5 13.3:1 300 #30 127 50 1
9.4 5.3:1 300 #31 127 100 0.5 9.4 10.6:1 300
[0116] Purified GAM IgG conjugates to either Compound No. 54 or
Compound No. 127 were also prepared for cell staining as a control.
The purified antibody conjugates were prepared using standard
antibody conjugation method as described in Example 2. Purified
conjugates with Compound No. 54 are referred to as "purified Dye
No. 3-GAM" in FIG. 3. Purified conjugates with Compound No. 127 are
referred to as "purified Dye No. 4-GAM" in FIG. 4.
[0117] Cell staining experiments were conducted similarly to
Example 1. Stained samples were analyzed on BD FACS Calibur using
635 nm excitation and FL4 detection channel. Relative fluorescence
signals were plotted in FIG. 3. The data show that cell
immunostaining according to the invention and that according to the
conventional method using a purified antibody conjugate produced
comparable results.
Example 4: Analysis of Cells Stained with Secondary Antibody
Labeled with Compound No. 128
[0118] This example describes labeling of an antibody with a
far-red fluorescent sulfonated xanthene dye (Compound No. 128), and
the use of such antibodies for intracellular staining and flow
cytometry analysis. Compound No. 128, absorbs light at about 630 nm
and emits light at about 650 nm. The structure of Compound No. 128
is as follows:
##STR00128##
[0119] Solutions of mouse anti-human CD3 antibody (BD Biosciences)
labeled with Compound No. 128 were prepared using different
dye-to-antibody ratios and different antibody concentrations in a
reaction buffer comprising 10 mM Tris and 50 mM NaHCO.sub.3 (pH
8.3). Detailed labeling reaction conditions are listed in Table 8
below. Labeling reactions were carried out as described in Example
1. Labeled antibody solutions were used directly in cell
staining.
TABLE-US-00009 TABLE 8 Labeling condition Antibody Labeled mouse
Amount concen- Antibody- anti-human of tration Amount to-dye CD3
antibody antibody in reaction of dye ratio solution (.mu.g) (mg/mL)
(nmoles) (ug/nmole) #32 100 0.5 5.6 17.8:1 #33 50 0.5 5.6 8.9:1 #34
20 0.5 2.8 7.2:1 #35 20 0.5 1.4 14.3:1 #36 5 0.5 1.4 3.6:1
[0120] One million Jurkat cells per sample were fixed,
permeabilized, and stained with 0.25 .mu.g of one of the labeled
mouse anti-human CD3 (BD Biosciences) conjugates prepared according
to Table 8. For comparison, commercially available mouse anti-human
CD3 antibody pre-labeled with spectrally similar Alexa Fluor 647
(BD Biosciences) was also used to stain the cells. About 10,000
cells from each sample were analyzed on a BD FACS Calibur flow
cytometer using 635 nm excitation and fluorescence was detected in
the FL4 channel Signal represents average fluorescence intensity of
the stained cells (FIG. 4). The bars in FIG. 4 represent the
average of the fluorescence intensity of duplicate samples. The
results show that cells stained with antibodies labeled according
to the invention show fluorescence signals comparable to the
commercially available control, and the cell staining intensity is
substantially independent of the conditions under which the labeled
antibodies are prepared, such as the antibody-to-dye ratio and
antibody concentration.
Example 5: Analysis of Cells Stained with Primary Antibody Labeled
with Compound No. 42
[0121] This example describes labeling of an anti-.beta.-tubulin
antibody with a far-red fluorescent sulfonated xanthene dye
(Compound No. 42), and the use of such antibodies for intracellular
staining and flow cytometry analysis. Mouse anti-.beta.-tubulin IgM
primary antibody (BD Biosciences) was labeled with Compound No. 42
according to the conditions listed in Table 9 below. Reaction
procedure and storage buffer preparation are as described in
Example 1.
TABLE-US-00010 TABLE 9 Labeling condition Labeled b- Antibody
Amount tubulin Amount concen- Antibody- of IgM of GAM tration
Amount to-dye storage antibody IgG in reaction of dye ratio buffer
solution (.mu.g) (mg/mL) (nmoles) (ug/nmole) (uL) #37 10 0.5 0.56
17.9:1 60 #38 10 0.5 1.3 7.7:1 60 #39 10 0.5 1.4 7.2:1 60 #40 10
0.5 0.75 13.4:1 60 #41 10 0.5 1.5 6.7:1 60 #42 10 0.5 1.88 5.3:1
60
[0122] One million Jurkat cells per sample were stained with 0.25
.mu.g of one of the labeled anti-.beta.-tubulin IgM antibodies
prepared according to Table 9. For comparison, commercially
available anti-.beta.-tubulin IgM pre-labeled with spectrally
similar Alexa Fluor 647 (BD Biosciences) was also used to stain the
cells. About 10,000 cells from each sample were analyzed on a BD
FACS Calibur flow cytometer using 635 nm excitation and
fluorescence was detected in the FL4 channel Signal represents
average fluorescence intensity of the stained cells (FIG. 5). The
results show that cells stained with antibodies labeled according
to the invention show improved fluorescence signal over the
commercially available control, and the cell staining intensity is
substantially independent of the conditions under which the labeled
antibodies are prepared, such as the antibody-to-dye ratio and
antibody concentration.
Example 6: Microscopy Analysis of Cells Stained with Primary
Antibody Labeled with Cmpd. No. 42
[0123] This examples describes the use of antibodies as prepared in
Example 5 for cell staining of .beta.-tubulin and analysis under a
microscope, as compared to a commercially available antibody. Hela
cells were cultured on glass coverslips, fixed with formaldehyde,
permeabilized and blocked with 2% serum/PBS (blocking buffer).
Cells were subsequently stained with solution #37 of Table 9 or
with a purified mouse .beta.-tubulin IgM primary antibody labeled
with Alexa Fluor.RTM. 647 purchased from BD Biosciences. Each
coverslip was labeled with 1 .mu.g antibody conjugates in 200 uL
blocking buffer for 1 hr. Cells were extensively washed with
1.times. PBS and mounted onto glass slides. Images were taken on an
Olympus mercury arc lamp microscope immediately after staining
(t=0) and at 1 minute and 3 minutes after staining. Example images
are shown in FIG. 6. The results show that cells stained with
antibody conjugate of this invention are comparable to the purified
IgM primary antibody labeled with Alexa Fluor.RTM. 647. Cells
stained with Compound No. 42 showed comparable specific staining
and intensity compared to cells stained with commercially purchased
B-tubulin antibody labeled with Alexa Fluor.RTM. 647. In addition,
the labeled antibodies of the invention demonstrate an improved
resistance to photobleaching compared to the commercially available
control.
Example 7: Multi-Color Imaging of Cells Labeled with Compound No.
42
[0124] Hela cells were cultured on glass coverslips, fixed and
stained with Compound No. 42 conjugated with anti-.beta.-tubulin
IgM as in Example 5, CF488A-phalloidin, and DAPI. The cells were
stained with 1 .mu.g IgM antibody conjugate in 200 uL serum
blocking buffer. Stainings with CF488A-phalloidin and DAPI were
carried according to the manufacture's product information sheets
(Biotium). The images were taken on a Zeiss LSM 510 Meta Confocal
system. Example images are shown in FIG. 7 in greyscale. The images
show the dye-antibody labeling technology is compatible with
multi-color imaging with other fluorescent probes.
Example 8: Preparation of Aminooxy-Functionalized R-PE
[0125] A R-phycoerythrin (R-PE) suspension containing 22.4 mg of
R-PE (ProZyme, Hayward, Calif.) was dialyzed against PBS buffer to
remove ammonium sulfate and then concentrated to about 7 mg/mL
(.about.3.2 mL) via ultramembrane filtration. The resulting R-PE
solution was combined with 0.32 mL of a pH 8.4 reaction buffer
comprising 0.5 M sodium bicarbonate and 0.1 M Tris and 78 uL (0.46
umole) of 4-formylbenzoic acid succinimidyl ester in DMF at 6 mM
(prepared by mixing equal amount of 4-formylbenzoic acid,
triethylamine and O-succinimido-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TSTU) and incubing the combined solution for 15
minutes). The combined solution was incubated at room temperature
for about 1 hour. The aldehyde-functionalized R-PE was purified
with a G-25 column eluting with PBS. The purified protein was
concentrated to about 6 mg/mL The functionalized R-PE (3.4 mL, 85
nmoles), 300 uL (8.4 umoles) of 1,3-bis(aminooxy)propane
dihydrochloride dissolved in water and 370 uL of a pH 4 phosphate
buffer comprising 100 mM aniline were mixed. The combined solution
was incubated at room temperature for about 1 hour. The resulting
solution was run through a G-25 size exclusion column eluting with
PBS buffer. The purified aminooxy-functioned R-PE was concentrated
to about 5 mg/mL.
Example 9. Preparation of Goat Anti-Mouse IgG Labeled with R-PE
[0126] Goat anti-mouse (50 .mu.g, 50 uL at 1 mg/mL), 5 uL of pH 8.4
reaction buffer comprising 0.5 M sodium bicarbonate and 100 mM
Tris, and 4-formylbenzoic acid succinimidyl ester prepared above in
Example 8 (1.7 nmoles) in DMF were combined and then incubated for
about 30 minutes. About 25 uL of pH 3.7 phosphate buffer comprising
100 mM aniline and about 150 .mu.g of the aminooxy-R-PE from
Example 8 are combined and incubated at room temperature for about
1 hour. The resulting solution was used for cell staining without
further purification (Example 10).
Example 10: Flow Cytometry Analysis of Intracellular Staining with
R-PE Secondary Antibody Conjugates Prepared Under Various
Conjugation Conditions
[0127] Approximately one million Jurkat cells per sample were
stained with 0.25 .mu.g mouse anti-human CD3 (BD Biosciences)
followed by 1 .mu.g of R-PE goat anti-mouse IgG conjugate. The
fluorescence of 10,000 cells from each sample was analyzed on a BD
FACSCalibur flow cytometer in the FL2 channel (488 nm excitation
laser, 564-606 nm band-pass emission filter). Signal represents
average fluorescence intensity from cells stained with CD3 and goat
anti-mouse IgG. Noise represents the average intensity from cells
stained with only goat anti-mouse secondary conjugates, which
reflects the level of non-specific secondary antibody binding. The
data were plotted in FIG. 8. The results show that cells stained
with antibodies labeled according to the invention show high
specific fluorescence signal and low non-specific antibody
binding.
[0128] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *