U.S. patent application number 15/693772 was filed with the patent office on 2018-04-05 for compositions and methods for enhanced fluorescence.
The applicant listed for this patent is Life Technologies Corporation, Pierce Biotechnology, Inc.. Invention is credited to Robert AGGELER, Matthew BAKER, Surbhi DESAI, Kyle GEE, Shih-Jung HUANG, Marie Christine NLEND, Aleksey RUKAVISHNIKOV, Scott SWEENEY.
Application Number | 20180092993 15/693772 |
Document ID | / |
Family ID | 59846737 |
Filed Date | 2018-04-05 |
United States Patent
Application |
20180092993 |
Kind Code |
A1 |
DESAI; Surbhi ; et
al. |
April 5, 2018 |
COMPOSITIONS AND METHODS FOR ENHANCED FLUORESCENCE
Abstract
This disclosure relates to the field of fluorescent dyes, and in
particular, compositions and methods for increasing fluorescent
signals and the reduction of fluorescent quenching.
Inventors: |
DESAI; Surbhi; (Rockford,
IL) ; NLEND; Marie Christine; (Rockford, IL) ;
GEE; Kyle; (Springfield, OR) ; BAKER; Matthew;
(Rockford, IL) ; AGGELER; Robert; (Eugene, OR)
; SWEENEY; Scott; (Eugene, OR) ; RUKAVISHNIKOV;
Aleksey; (Eugene, OR) ; HUANG; Shih-Jung;
(Eugene, OR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Life Technologies Corporation
Pierce Biotechnology, Inc. |
Carlsbad
Rockford |
CA
IL |
US
US |
|
|
Family ID: |
59846737 |
Appl. No.: |
15/693772 |
Filed: |
September 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62382594 |
Sep 1, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/18 20130101;
G01N 33/533 20130101; G01N 33/582 20130101; G01N 33/68 20130101;
A61K 49/0058 20130101; A61K 47/10 20130101; A61K 49/0015
20130101 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61K 47/18 20060101 A61K047/18; A61K 47/10 20060101
A61K047/10; G01N 33/533 20060101 G01N033/533; G01N 33/68 20060101
G01N033/68 |
Claims
1. A composition comprising a first antibody, wherein two or more
fluorescent labels and two or more spacer molecules are covalently
attached to the first antibody, and wherein the fluorescent label
and spacer molecules are not covalently attached to each other.
2. The composition of claim 1, wherein the first antibody exhibits
higher fluorescent emission levels than a second antibody prepared
with an equivalent amount of fluorescent label but without the
spacer molecule.
3. The composition of claim 1, wherein the first antibody exhibits
higher fluorescent emission levels than a second antibody, wherein
the first antibody and the second antibody each have the same
number of covalently bound fluorescent labels, and wherein the
second antibody does not have a covalently bound spacers
molecule.
4. The composition of claim 1, wherein the spacer molecule reduces
quenching of the fluorescent labels as compared to the quenching in
the absence of the spacer molecule.
5. The composition of claim 1, wherein the spacer molecule is
conjugated to the antibody to a reactive group.
6. The composition of claim 5, wherein the reactive group is an
amine group.
7. The composition of claim 6, wherein the amine group is on a
lysine residue.
8.-13. (canceled)
14. The composition of claim 1, wherein the spacer molecule is
selected from acetate and polyethylene glycol (PEG).
15.-23. (canceled)
25. The composition of claim 1, wherein the ratio of fluorescent
label to antibody is from 1 to 50.
26. The composition of claim 25, wherein the ratio of fluorescent
label to antibody is from 5 to 30.
27.-38. (canceled)
39. A method for increasing fluorescence of a fluorescently labeled
biomolecule, the method comprising: (a) conjugating a spacer
molecule to a biomolecule; and (b) conjugating a fluorescent label
to the biomolecule; wherein steps (a) and (b) can be done
simultaneously or in any order, and wherein the spacer and
fluorescent label are not conjugated to each other.
40. The method of claim 39, wherein the spacer molecule reduces
quenching of the fluorescent labels as compared to the quenching in
the absence of the spacer molecule.
41. The method of claim 39, wherein the spacer molecule is
conjugated to the antibody to a reactive group.
42. The method of claim 41, wherein the reactive group is an amine
group.
43. The method of claim 42, wherein the amine group is on a lysine
residue.
44.-122. (canceled)
123. A method for preparing a fluorescently labeled biomolecule,
the method comprising: (a) conjugating a reactive group and two or
more fluorescent labels to a spacer molecule, thereby forming a
fluorescently labeled spacer molecule, and (b) conjugating the
fluorescently labeled spacer molecule to the biomolecule, thereby
forming the fluorescently labeled biomolecule, and wherein the
individual fluorescent labels of the fluorescently labeled
biomolecule have a Fluorescent Ratio on a per fluorescent label
basis that is equal to or greater than 0.5.
124. The method of claim 123, wherein an average of from one to ten
fluorescently labeled spacer molecules are conjugated to each
biomolecule.
125. The method of claim 123, wherein an average of from three to
ten fluorescently labeled spacer molecules are conjugated to each
biomolecule.
126. The method of claim 123, wherein the fluorescently labeled
spacer molecule is a multi-armed polymer.
127. The method of claim 126, wherein the multi-armed polymer is a
branched polyethylene glycol molecule.
128-131. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/382,594, filed Sep. 1, 2016, the disclosure is
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] This disclosure relates to the field of fluorescent dyes,
and in particular, compositions and methods for increasing
fluorescent signals and the reduction of fluorescent quenching.
BACKGROUND
[0003] Fluorescently labeled biomolecules are widely used in
various methods, including those related to quantitation assays and
cellular imaging. Biomolecules such as antibodies, antigens, DNA,
and RNA are fluorescently labeled and used in applications such as
immunofluorescence (IFC), flow cytometry, Fluorescence-Activated
Cell Sorting (FACS), immunohistochemistry (IHC), Western blotting,
drug-binding studies, enzyme kinetics, imaging (immunocytochemistry
ICC) including HCA, in vivo imaging, as well as in nucleic acid
hybridizations. Fluorescent dyes continue to be chosen for these
applications because they are easy to use and are not only specific
and sensitive but also provide multiplexing options. However, not
all applications have been amenable to fluorescent labeling. For
example, in many applications the intensity of the fluorescence may
be decreased as increased quantities of fluorescent label (high dye
to protein ratios) are added to biomolecules (quenching). Moreover,
fluorescence may also be decreased through steric hindrance caused
by fluorescent labeling of the biomolecule. There is a need for
fluorescent labels and conjugation methods that exhibit enhanced
signal strength.
SUMMARY
[0004] Disclosed herein are compositions and methods for increasing
intensity of a fluorescent dye conjugated to a biomolecule through,
for example, modified linkers, chemistries and specified protocols.
One aspect of the invention is thus to improve the performance of
dyes. It has been found that fluorescence intensity may be
increased when a biomolecule is labeled with both a dye and a
spacer molecule as compared to the corresponding conjugate made
with the dye alone. Conjugates made with these spacer molecules
show fluorescence enhancement in Western blotting, dot blot assays,
plate assays, flow cytometry and immunoassay applications (e.g.,
immunofluorescence imaging applications).
[0005] Further, enhanced fluorescence is seen even when fewer dye
molecules are associated with (covalently or non-covalently) each
biomolecule. This effect is believed to be associated with the
attachment of spacer groups to the biomolecules that exhibit
enhanced fluorescence. While not wishing to be bound to theory, it
is believed that the enhanced fluorescence results from decreased
quenching of dye emissions. In some aspects, the invention is
directed, in part, to compositions comprising a first biomolecule
(e.g., a first antibody), wherein two or more fluorescent labels
and two or more spacer molecules are covalently attached to the
first biomolecule, and wherein the fluorescent and spacer molecules
are not covalently attached to each other. In some instances, the
first biomolecule (e.g., a first antibody) exhibits higher
fluorescent emission levels than a second biomolecule (e.g., a
second antibody) prepared with an equivalent amount of fluorescent
label but without the spacer. In additional instances, the first
biomolecule exhibits higher fluorescent emission levels than the
second biomolecule, wherein the first biomolecule and the second
biomolecule each have the same number of covalently bound
fluorescent labels, and wherein the second biomolecule does not
have covalently bound spacers. In specific embodiments, the spacer
molecules reduce quenching of the fluorescent labels as compared to
the quenching in the absence of spacer.
[0006] In some aspects of the invention, the spacer molecules are
conjugated to the biomolecule through a reactive group. Such
reactive groups may be amine reactive groups (e.g. NHS esters, the
amine group or groups may be at the amine terminus of a polypeptide
and/or lysine side chains), sulfhydryl groups, carboxylic acid
groups, etc. Additional groups to which spacer molecules may be
conjugated include cysteine residues, aspartic acid residues,
glutamic acid residues and/or the carboxy terminus of a
polypeptide. Further, fluorescent labels may be conjugated to the
biomolecule by a conjugation arm. These fluorescent labels may be
positively charged, neutral, and/or negatively charged.
[0007] In some embodiments, fluorescent labels used in the practice
of the invention (both compositions and methods) may be cyanine,
benzo-rhodamne, bodipy, fluorescein, benzopyrillium derivatives and
further include ALEXA FLUOR.RTM. dye and/or DYLIGHT.TM. dyes. Such
dyes may be selected from the group consisting of ALEXA FLUOR.RTM.
350, ALEXA FLUOR.RTM. 405, ALEXA FLUOR.RTM. 430, ALEXA FLUOR.RTM.
488, ALEXA FLUOR.RTM. 500, ALEXA FLUOR.RTM. 514, ALEXA FLUOR.RTM.
532, ALEXA FLUOR.RTM. 546, ALEXA FLUOR.RTM. 555, ALEXA FLUOR.RTM.
568, ALEXAFLUOR.RTM. 594, ALEXA FLUOR.RTM. 610-X, ALEXA FLUOR.RTM.
633, ALEXA FLUOR.RTM. 647, ALEXAFLUOR.RTM. 660, ALEXA FLUOR.RTM.
680, ALEXA FLUOR.RTM. 700, ALEXA FLUOR.RTM. 750, ALEXAFLUOR.RTM.
790, AMCA-X, BODIPY.RTM. 630/650, BODIPY.RTM. 650/665, BODIPY.RTM.
FL, BODIPY.RTM. TMR, BODIPY.RTM. TR, BODIPY.RTM. TR-X, CASCADE
BLUE.RTM., Dinitrophenyl, Fluorescein, HEX, JOE, MARINA BLUE.RTM.,
OREGON GREEN.RTM. 488, OREGON GREEN.RTM. 514, PACIFIC BLUE.TM.,
PACIFIC ORANGE.TM., RHODAMINE GREEN.TM., QSY.RTM. 7, QSY.RTM. 9,
QSY.RTM. 21, QSY.RTM. 35, ROX, RHODAMINE RED.TM., TET, TAMRA,
tetramethyl rhodamine, FAM, TEXAS RED.RTM.,
7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl
ester (DDAO-SE), DYLIGHT.TM. 350, DYLIGHT.TM. 405, DYLIGHT.TM. 488,
DYLIGHT.TM. 550, DYLIGHT.TM. 594, DYLIGHT.TM. 633, DYLIGHT.TM. 650,
DYLIGHT.TM. 680, DYLIGHT.TM. 755, and DYLIGHT.TM. 800. Of course,
other dyes, as well as modified forms of the above dyes, may be
used in the practice of the invention.
[0008] The spacer may be negatively or neutrally charged may be
used in the practice of the invention. Further, the spacer may be
selected from, for example, acetate and polyethylene glycol (PEG).
Spacers may comprises an acetyl group and may be an acetate
molecule (e.g., sulfo-NHS-acetate). Further, spacers may comprise
or consist of (PEG)n, wherein n is selected from 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14 or 15 and/or MS-(PEG)n, wherein n is
selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or
15.
[0009] Further, spacers used in the practice of the invention may
comprise or consist of one or more group selected from an alkanoyl,
alkenoyl, and alkynoyl (--C(O)C.sub.nH.sub.m), wherein n is 1 to 20
atoms, wherein m>n, wherein the carbon atoms can be connected to
each other by single, double, and/or triple bonds. Alkyl, alkenyl,
and/or alkynyl groups may be further substituted with
--(OCH.sub.2CH.sub.2O).sub.x--(CH.sub.2).sub.y--OR, in which x is 1
to 20, y is 1 to 6, and R is H or C.sub.1-6 alkyl. Additionally,
the alkyl, alkenyl, and/or alkynyl groups may be further
substituted with ammonium (--NH.sub.3.sup.+), quaternary ammonium
(--NR.sub.3.sup.+) groups in which R is C.sub.1-6 alkyl.
[0010] Further, in specific embodiments of the invention, the
fluorescent dye may comprises one or more ALEXA FLUOR.RTM. 350,
ALEXA FLUOR.RTM. 405, ALEXA FLUOR.RTM. 430, ALEXA FLUOR.RTM. 488,
ALEXA FLUOR.RTM. 500, ALEXA FLUOR.RTM. 514, ALEXA FLUOR.RTM. 532,
ALEXAFLUOR.RTM. 546, ALEXA FLUOR.RTM. 555, ALEXA FLUOR.RTM. 568,
ALEXA FLUOR.RTM. 594, ALEXAFLUOR.RTM. 610-X, ALEXA FLUOR.RTM. 633,
ALEXA FLUOR.RTM. 647, ALEXA FLUOR.RTM. 660, ALEXAFLUOR.RTM. 680,
ALEXA FLUOR.RTM. 700, ALEXA FLUOR.RTM. 750, ALEXA FLUOR.RTM. 790,
AMCA-X, BODIPY.RTM. 630/650, BODIPY.RTM. 650/665, BODIPY.RTM. FL,
BODIPY.RTM. TMR, BODIPY.RTM. TR, BODIPY.RTM. TR-X, CASCADE
BLUE.RTM., Dinitrophenyl, Fluorescein, HEX, JOE, MARINA BLUE.RTM.,
OREGON GREEN.RTM. 488, OREGON GREEN.RTM. 514, PACIFIC BLUE.TM.,
PACIFIC ORANGE.TM., RHODAMINE GREEN.TM., QSY.RTM. 7, QSY.RTM. 9,
QSY.RTM. 21, QSY.RTM. 35, ROX, RHODAMINE RED.TM., TET, TAMRA,
tetramethyl rhodamine, FAM, TEXAS RED.RTM., or
7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl
ester (DDAO-SE); and the spacer may comprise one or more of
sulfo-NHS-acetate; (PEG)n, wherein n is selected from 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; MS-(PEG)n, wherein n is
selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;
alkanoyl, alkenoyl, or alkynoyl (--C(O)C.sub.nH.sub.m), wherein n
is 1 to 20 atoms, wherein m>n, wherein the carbon atoms can be
connected to each other by single, double, and/or triple bonds; or
alkyl, alkenyl, or alkynyl groups that are further substituted with
--(OCH.sub.2CH.sub.2O).sub.x--(CH.sub.2).sub.y--OR, in which x is 1
to 20, y is 1 to 6, and R is H or C.sub.1-6 alkyl or wherein the
alkyl, alkenyl, and/or alkynyl groups are further substituted with
ammonium (--NH.sub.3.sup.+), quaternary ammonium
((--NR.sub.3.sup.+) groups in which R is C.sub.1-6 alkyl. Further,
alkyl, alkenyl, and/or alkynyl groups may be further substituted
with phosphonium groups (--PQ.sub.3.sup.+) in which Q is aryl,
substituted aryl, or C.sub.1-6 alkyl.
[0011] Compositions and methods of the invention may contain or use
fluorescently labeled biomolecules (e.g., antibodies), wherein the
ratio of fluorescent label to biomolecule is from 1 to 50, from 5
to 30, or from 1 to 20. Compositions and methods of the invention
may contain or use fluorescently labeled biomolecules (e.g.,
antibodies), wherein the spacer agent to biomolecule ratio is from
1 to 50, from 5 to 30, from 5 to 30, or from 1 to 20.
[0012] Additionally, compositions and methods of the invention may
contain or use fluorescently labeled biomolecules (e.g.,
antibodies), wherein the spacer agent is in molar excess to the
plurality of fluorescent labels in an amount from 0.1 to 25, from 1
to 15, or from 2.5 to 10 fold; wherein the spacer agent is in molar
excess to the plurality of fluorescent labels in an amount of 2.5
fold; wherein the spacer agent is in molar excess to the plurality
of fluorescent labels in an amount of 5 fold; wherein the spacer
agent is in molar excess to the plurality of fluorescent labels in
an amount of 7.5 fold; or wherein the spacer agent is in molar
excess to the plurality of fluorescent labels in an amount of 10
fold.
[0013] Further, compositions and methods of the invention may
contain or use fluorescently labeled biomolecules (e.g.,
antibodies), wherein the percentage of binding sites on the
biomolecule (e.g., accessible amine groups) occupied by the
plurality of fluorescent labels is between 1% and 99%.
[0014] In some embodiments, the presence of the spacer increases
the detectable fluorescence of the fluorescent label by at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 100%, at least
125%, at least 150%, at least 200%, at least 300%, at least 400%,
or at least 500%.
[0015] The invention is also directed, in part, to methods for
increasing fluorescence of a fluorescently labeled biomolecule.
Such methods include those comprising: (a) conjugating a spacer
molecule to a biomolecule; and (b) conjugating a fluorescent label
to the biomolecule; wherein steps (a) and (b) can be done
simultaneously or in any order, and wherein the spacer and
fluorescent label are not conjugated to each other. Further, the
spacer molecules may reduce quenching of the fluorescent labels as
compared to the amount of quenching that occurs in the absence of
spacer.
[0016] The invention is also directed, in part, to methods for
identifying a spacer molecule capable of enhancing the fluorescent
emission fluorescently labeled biomolecule. Such methods may
comprise: (a) conjugating a spacer molecule to a biomolecule,
independent of the plurality of fluorescent labels conjugated to
the biomolecule; (b) testing whether the presence of the spacer
agent in addition to the plurality of fluorescent labels conjugated
to the biomolecule increases the detectable fluorescence of the
plurality of fluorescent labels; and (c) identifying the spacer
agent as one that will reduce quenching of the fluorescent label
conjugated to the protein when the presence of the spacer agent in
addition to the plurality of fluorescent labels conjugated to the
biomolecule increases the detectable fluorescence of the plurality
of fluorescent labels. In some instances, the spacer agent is
conjugated to the biomolecule at primary lysine side chains present
on the biomolecule. Further, the biomolecule is an antibody or
antibody fragment. Also, the plurality of fluorescent labels may be
negatively and/or positively charged. Additionally, the spacer
agents may be negatively and/or positively charged.
[0017] The invention is further directed, in part to methods for
determining the presence of a desired target in a biological
sample. Such methods may comprise: (a) contacting the biological
sample with a composition an antibody, wherein two or more
fluorescent labels and two or more spacer molecules are covalently
attached to the antibody, wherein the fluorescent and spacer
molecules are not covalently attached to each other, (b) detecting
fluorescence emitted by the plurality of fluorescent labels; and
(c) determining the presence of the desired target in the
biological sample when fluorescence emitted by the plurality of
fluorescent labels is detected. Biological samples used in the
practice of the invention may comprise cell lysates, intact cells
(e.g., intact cells in fluid, such as bodily fluids), isolated
proteins, and/or recombinant proteins. Further, the biological
sample may be immobilized on a solid support. Further, biological
sample include live animals, such as mammals.
[0018] The invention is also directed, in part, to compositions
comprising a first nucleic acid molecule, wherein two or more
fluorescent labels and two or more spacer molecules are covalently
attached to the first nucleic acid molecule, and wherein the
fluorescent and spacer molecules are not covalently attached to
each other. In some embodiments, the first nucleic acid molecule
may exhibit a higher fluorescent emission levels than a second
nucleic acid molecule prepared with an equivalent amount of
fluorescent label but without the spacer. Further, in some
embodiments, the first nucleic acid molecule may exhibit a higher
fluorescent emission levels than a second nucleic acid molecule,
wherein the first nucleic acid molecule and the second nucleic acid
molecule each have the same number of covalently bound fluorescent
labels, and wherein the second nucleic acid molecule does not have
a covalently bound spacers.
[0019] The invention also includes conjugated antibodies comprising
antibody each conjugated to a plurality of fluorescent labels
(e.g., and average of from about two to about thirty, from about
two to about twenty, from about three to about thirty, from about
two to about fifteen, from about three to about fifteen, from about
four to about thirty, from about six to about twenty, from about
seven to about thirty, etc.), wherein the conjugated antibodies
comprises one or more of the following characteristics: [0020] (a)
a Fluorescent Ratio on a per fluorescent label basis that is equal
to or greater than 0.5, [0021] (b) at least four fluorescent labels
are conjugated to the antibody, [0022] (c) the total fluorescence
of the antibody is at least 20 percent greater than the
fluorescence of the unconjugated fluorescent molecule, and/or
[0023] (d) an average of from about 3 to about 80 fluorescent
labels attached to each of the antibody molecules.
[0024] Further, the fluorescent labels may be conjugated to
biomolecules (e.g., antibodies) by one or more (e.g., from about
one to about fifteen, from about two to about ten, from about two
to about fifteen, from about two to about eight, from about three
to about ten, from about three to about six, etc.) multi-armed
polymers. Additionally, the arms of the multi-armed polymer may be
composed of a chemical of a type selected from the group consisting
of: (a) a polyethylene glycol, (b) a polysaccharide, and (c) a
polypeptide, as well as other materials. Further, the average brush
distance between the fluorescent labels may be between 200 to 800
Angstroms (e.g., from about 200 to about 700, from about 300 to
about 800, from about 400 to about 800, from about 500 to about
800, from about 200 to about 600, from about 500 to about 800, from
about 300 to about 700, from about 350 to about 800, etc.). Also,
the fluorescent labels conjugated to the antibody (or other
biomolecule) may be separated from the antibody (or other
biomolecule) by at least 16 (e.g., from about 16 to about 800, from
about 25 to about 800, from about 40 to about 800, from about 60 to
about 800, from about 100 to about 800, from about 200 to about
800, from about 250 to about 800, from about 150 to about 600,
etc.) covalent bonds. Additionally, the conjugated antibody (or
other biomolecule) may be conjugated to fluorescent labels which
may be one or more dye selected from the group consisting of ALEXA
FLUOR.RTM. 350, ALEXA FLUOR.RTM. 405, ALEXA FLUOR.RTM. 430, ALEXA
FLUOR.RTM. 488, ALEXA FLUOR.RTM. 500, ALEXA FLUOR.RTM. 514, ALEXA
FLUOR.RTM. 532, ALEXA FLUOR.RTM. 546, ALEXA FLUOR.RTM. 555, ALEXA
FLUOR.RTM. 568, ALEXA FLUOR.RTM. 594, ALEXA FLUOR.RTM. 610-X, ALEXA
FLUOR.RTM. 633, ALEXA FLUOR.RTM. 647, ALEXA FLUOR.RTM. 660, ALEXA
FLUOR.RTM. 680, ALEXAFLUOR.RTM. 700, ALEXA FLUOR.RTM. 750, ALEXA
FLUOR.RTM. 790, AMCA-X, BODIPY.RTM. 630/650, BODIPY.RTM. 650/665,
BODIPY.RTM. FL, BODIPY.RTM. TMR, BODIPY.RTM. TR, BODIPY.RTM. TR-X,
CASCADE BLUE.RTM., Dinitrophenyl, Fluorescein, HEX, JOE, MARINA
BLUE.RTM., OREGON GREEN.RTM. 488, OREGON GREEN.RTM. 514, PACIFIC
BLUE.TM., PACIFIC ORANGE.TM., RHODAMINE GREEN.TM., QSY.RTM. 7,
QSY.RTM. 9, QSY.RTM. 21, QSY.RTM. 35, ROX, RHODAMINE RED.TM., TET,
TAMRA, tetramethyl rhodamine, FAM, TEXAS RED.RTM., or
7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl
ester (DDAO-SE); and fluorescent labels from the group consisting
of DYLIGHT.TM. 350, DYLIGHT.TM. 405, DYLIGHT.TM. 488, DYLIGHT.TM.
550, DYLIGHT.TM. 594, DYLIGHT.TM. 633, DYLIGHT.TM. 650, DYLIGHT.TM.
680, DYLIGHT.TM. 755, and DYLIGHT.TM. 800 and pegylated DYLIGHT.TM.
dyes.
[0025] The invention also includes method for preparing
fluorescently labeled biomolecules, these method may comprising:
(a) conjugating a reactive group and two or more fluorescent labels
to a spacer molecule, thereby forming a fluorescently labeled
spacer molecule, and (b) conjugating the fluorescently labeled
spacer molecule to the biomolecule, thereby forming the
fluorescently labeled biomolecule, wherein the individual
fluorescent labels of the fluorescently labeled biomolecule have a
Fluorescent Ratio on a per fluorescent label basis that is equal to
or greater than 0.5. Further, an average of from one to ten (e.g.,
from about one to about nine, from about two to about ten, from
about three to about ten, from about four to about ten, from about
five to about ten, from about two to about six, from about three to
about six, from about three to about seven, etc.) fluorescently
labeled spacer molecules may be conjugated to each biomolecule.
Additionally, the fluorescently labeled spacer molecule may be a
multi-armed polymer (e.g., a branched polyethylene glycol
molecule). Further, spacer molecules (e.g., multi-armed polymers)
may each be conjugated to an average of between 4 and 20 (e.g.,
from about 4 to about 10, from about 3 to about 8, from about 4 to
about 8, from about 3 to about 9, etc.) fluorescent labels.
Further, spacer molecules (e.g., the multi-armed polymers) may have
a molecular weight between 4,000 and 80,000 daltons (e.g., from
about 4,000 to about 70,000, from about 4,000 to about 60,000, from
about 4,000 to about 50,000, from about 4,000 to about 40,000, from
about 10,000 to about 70,000, from about 15,000 to about 60,000,
etc.).
[0026] The invention further includes method for detecting
fluorescently labeled biomolecules. Such methods may comprise: (a)
exposing the fluorescently labeled biomolecule (e.g., an antibody)
with light that excites fluorescent labels conjugated to the
biomolecule and (2) detecting emitted light produced by the
fluorescent labels conjugated to the biomolecule. In some
instances, the fluorescently labeled biomolecule may be conjugated
to four or more fluorescent labels. Further, the individual
fluorescent labels of the fluorescently labeled biomolecule may
have a Fluorescent Ratio on a per fluorescent label basis that is
equal to or greater than 0.7 (e.g., from about 0.7 to about 1.0,
from about 0.7 to about 0.95, from about 0.7 to about 0.9, from
about 0.7 to about 0.85, from about 0.75 to about 0.95, etc.).
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows a schematic representation of one embodiment of
the invention. In this embodiment, an antibody is conjugated with
NHS fluorescent dyes at two different dye molar excesses and a
Sulfo NHS-Acetate spacer in 50 mM borate buffer pH 8.5. Conjugation
of the dye and spacer result in enhanced sensitivity and reduced
quenching.
[0028] FIG. 2 shows a schematic representation of some embodiments
of the invention. In this embodiment, an antibody is conjugated to
NHS-fluorescent dyes and a methyl-PEG-NHS-ester spacer in 50 mM
borate buffer pH 8.5. The spacers used in this embodiment are
MS(PEG).sub.4, MS(PEG).sub.8 and MS(PEG).sub.12.
[0029] FIG. 3 shows results from a software analysis of a dot blot
captured using an imaging instrument. The dot blot was tested with
GAM antibodies co-labeled with NHS acetate or MS(PEG).sub.4 spacers
and DYLIGHT.TM. 488 fluorescent dye (abbreviated in Table 1
"DYLIGHT.TM. 488"). DYLIGHT.TM. 488-GAM conjugates made with NHS
Acetate or MS(PEG).sub.4 resulted in a 1.2 to 1.8-fold improvement
in fluorescent intensity ranging from 1.2 to 1.8-fold over the base
conjugate (made without the spacer). Lanes of this figures are as
follows:
TABLE-US-00001 TABLE 1 Lane Dye/Spacer 1 DYLIGHT .TM. 488-5X, No
Spacer 2 DYLIGHT .TM. 488-5X, 2.5X NHS Acetate 3 DYLIGHT .TM.
488-5X, 5X NHS Acetate 4 Blank Well 5 DYLIGHT .TM. 488-5X, 3.75X
MS(PEG).sub.4 6 DYLIGHT .TM. 488-5X, 5X MS(PEG).sub.4 7 DYLIGHT
.TM. 488-5X, 10X MS(PEG).sub.4 8 DYLIGHT .TM. 488-7.5X, No Spacer 9
DYLIGHT .TM. 488-7.5X, 2.5X NHS Acetate 10 DYLIGHT .TM. 488-7.5X,
5X NHS Acetate 11 Blank Well 12 DYLIGHT .TM. 488-7.5X, 3.75X
MS(PEG).sub.4 13 DYLIGHT .TM. 488-7.5X, 5X MS(PEG).sub.4 14 DYLIGHT
.TM. 488-7.5X, 10X MS(PEG).sub.4 15 DYLIGHT .TM. 488-10X, No Spacer
16 DYLIGHT .TM. 488-10X, 2.5X NHS Acetate 17 DYLIGHT .TM. 488-10X,
5X NHS Acetate 18 Blank Well 19 DYLIGHT .TM. 488-10X, 3.75X
MS(PEG).sub.4 20 DYLIGHT .TM. 488-10X, 5X MS(PEG).sub.4 21 DYLIGHT
.TM. 488-10X, 10X MS(PEG).sub.4 22 DYLIGHT .TM. 488-15X, No Spacer
23 DYLIGHT .TM. 488-15X, 2.5X NHS Acetate 24 DYLIGHT .TM. 488-15X,
5X NHS Acetate 25 DYLIGHT .TM. 488-15X, 10X NHS Acetate 26 DYLIGHT
.TM. 488-15X, 3.75X MS(PEG).sub.4 27 DYLIGHT .TM. 488-15X, 5X
MS(PEG).sub.4 28 DYLIGHT .TM. 488-15X, 10X MS(PEG).sub.4 29 DYLIGHT
.TM. 488-20X, No Spacer 30 DYLIGHT .TM. 488-20X, 2.5X NHS Acetate
31 DYLIGHT .TM. 488-20X, 5X NHS Acetate 32 DYLIGHT .TM. 488-20X,
10X NHS Acetate 33 DYLIGHT .TM. 488-20X, 3.75X MS(PEG).sub.4 34
DYLIGHT .TM. 488-20X, 5X MS(PEG).sub.4 35 DYLIGHT .TM. 488-20X, 10X
MS(PEG).sub.4
[0030] FIG. 4: Dot Blot--DYLIGHT.TM. 488-GAM detection of Mouse IgG
Fold improvement from NHS Acetate (2.5.times., 5.times.) or
MS(PEG)4 (3.75.times.) over base conjugates at dye various molar
excesses. This figure shows results from a software analysis of a
dot blot captured using an imaging instrument. The dot blot was
tested with an assay using GAM antibodies co-labeled with NHS
acetate or MS(PEG).sub.4 spacers and DYLIGHT.TM. 488 fluorescent
dye. These data confirmed results from FIG. 3 using another source
for the antibody. Both NHS acetate and MS(PEG).sub.4 spacers
provided significant improvement (as much as about a 2.6 increase)
in fluorescent signal intensity as compared to antibody-dye
conjugates alone.
[0031] FIG. 5: Dot Blot--Detection of Mouse IgG with DYLIGHT.TM.
550-2.times.PEG-GAR. Signal/background fold improvement from NHS
Acetate (2.5.times., 5.times.) or MS(PEG)4 (3.75.times.) over base
conjugates at various molar excesses. This shows results from a
software analysis of a dot blot captured using an imaging
instrument. The dot blot was tested with DYLIGHT.TM. 550-GAM
antibodies co-labeled with NHS Acetate or MS(PEG).sub.4 spacers and
DYLIGHT.TM. 550 fluorescent dye. Mouse IgG was serially diluted 1:1
from 1000 ng/dot. All DYLIGHT.TM. 550-2.times.PEG-GAR secondary
antibodies were diluted 1/5000 of 1 mg/ml stock. NHS acetate or
MS(PEG).sub.4 added to the conjugation mix provided an improvement
(as much as about a 1.6 increase) of signal intensity as compared
to the base conjugates at each respective dye molar excesses.
Improvement ranged from 1.2 to 1.6-folds; notably for the following
GAM-DYLIGHT.TM. 550-2.times.PEG: 10.times. Dye+5.times. Acetate,
15.times. Dye+3.75.times. MS(PEG).sub.4, 20.times. Dye+5.times.
Acetate, and 20.times.+3.75.times. MS(PEG).sub.4 that showed an
improvement of greater than 1.3-fold over the respective base
conjugates.
[0032] FIG. 6: Dot Blot--DyLight 650-4.times.PEG-GAM detection of
Mouse IgG Fold improvement from NHS Acetate (2.5.times., 5.times.
and 10.times.) or MS(PEG)4 (3.75.times., 5.times., 10.times.) over
base conjugates at each dye molar excess. This figure shows results
from a software analysis of a dot blot captured using an imaging
instrument. The dot blot was tested with GAM antibodies co-labeled
with NHS Acetate (2.5.times., 5.times. and 10.times.) or
MS(PEG).sub.4 (3.75.times.) and DYLIGHT.TM. 650-4.times.PEG
(abbreviated in Table 2 "DYLIGHT.TM. 650") at
(10.times.-20.times.). Mouse IgG was serially diluted 1:1 from 1000
ng/dot. All DYLIGHT.TM. 650-4.times.PEG-GAR secondary antibodies
were diluted 1/10,000 of 1 mg/ml stock. Conjugates with high dye
substitution tend to perform better in applications such as Dot
Blot and western blotting. Both NHS Acetate and (MS)PEG.sub.4 bring
significant improvement in sensitivity and signal/background (as
much as about a 2.2 increase) over the initial base conjugates. NHS
acetate added at 2.5.times. molar excess to GAM-DYLIGHT.TM.
650-4.times.PEG-15.times. improved intensity by 1.7-fold. The
improvement provided by NHS Acetate showed 1.3-fold better
performance than with the conjugate prepared with the highest molar
excess (20.times.). All MS(PEG).sub.4 added to GAM-DYLIGHT.TM.
650-4.times.PEG-15.times. conjugation improved fluorescence
intensity by 1.8 to 2.2-fold and performed better than the
corresponding highest base conjugate GAM-DYLIGHT.TM.
650-4.times.PEG-20.times.. Lanes of this figures are as
follows:
TABLE-US-00002 TABLE 2 Lane Dye/Spacer 1 DYLIGHT .TM. 650-5X, No
Spacer 2 DYLIGHT .TM. 650-5X, 2.5X NHS Acetate 3 DYLIGHT .TM.
650-5X, 5X NHS Acetate 4 Blank Well 5 DYLIGHT .TM. 650-5X, 3.75X
MS(PEG)4 6 DYLIGHT .TM. 650-5X, 5X MS(PEG)4 7 DYLIGHT .TM. 650-5X,
10X MS(PEG)4 8 DYLIGHT .TM. 650-7.5X, No Spacer 9 DYLIGHT .TM.
650-7.5X, 2.5X NHS Acetate 10 DYLIGHT .TM. 650-7.5X, 5X NHS Acetate
11 Blank Well 12 DYLIGHT .TM. 650-7.5X, 3.75X MS(PEG)4 13 DYLIGHT
.TM. 650-7.5X, 5X MS(PEG)4 14 DYLIGHT .TM. 650-7.5X, 10X MS(PEG)4
15 DYLIGHT .TM. 650-10X, No Spacer 16 DYLIGHT .TM. 650-10X, 2.5X
NHS Acetate 17 DYLIGHT .TM. 650-10X, 5X NHS Acetate 18 Blank Well
19 DYLIGHT .TM. 650-10X, 3.75X MS(PEG)4 20 DYLIGHT .TM. 650-10X, 5X
MS(PEG)4 21 DYLIGHT .TM. 650-10X, 10X MS(PEG)4 22 DYLIGHT .TM.
650-15X, No Spacer 23 DYLIGHT .TM. 650-15X, 2.5X NHS Acetate 24
DYLIGHT .TM. 650-15X, 5X NHS Acetate 25 DYLIGHT .TM. 650-15X, 10X
NHS Acetate 26 DYLIGHT .TM. 650-15X, 3.75X MS(PEG)4 27 DYLIGHT .TM.
650-15X, 5X MS(PEG)4 28 DYLIGHT .TM. 650-15X, 10X MS(PEG)4 29
DYLIGHT .TM. 650-20X, No Spacer 30 DYLIGHT .TM. 650-20X, 2.5X NHS
Acetate 31 DYLIGHT .TM. 650-20X, 5X NHS Acetate 32 DYLIGHT .TM.
650-20X, 10X NHS Acetate 33 DYLIGHT .TM. 650-20X, 3.75X MS(PEG)4 34
DYLIGHT .TM. 650-20X, 5X MS(PEG)4 35 DYLIGHT .TM. 650-20X, 10X
MS(PEG)4
[0033] FIG. 7: Dot Blot--Fold improvement of DYLIGHT.TM.
880-4.times.PEG conjugates from NHS Acetate (2.5.times., 5.times.
and 10.times.) or MS(PEG)4 (3.75.times., 5.times., 10.times.) over
base conjugates at each molar excess. This figure demonstrates the
effect of the addition of NHS Acetate (2.5.times., 5.times. and
10.times.) or MS(PEG).sub.4 (3.75.times., 5.times., 10.times.) on
the detectable fluorescence level of GAM-DYLIGHT.TM.
800-4.times.PEG (abbreviated in Table 3 "DYLIGHT.TM. 800") in a
fluorescent dot blot assay. Mouse IgG was serially diluted 1:2 from
1000 ng/dot. All DYLIGHT.TM. 800-4.times.PEG-GAR secondary
antibodies were diluted to 1/20,000 of 1 mg/ml stock. This dot blot
application shows that the addition of MS(PEG).sub.4 (3.75.times.
and 5.times.) and NHS Acetate (5.times.) significantly enhanced the
fluorescence intensity and sensitivity of base DYLIGHT.TM.
800-4.times.PEG conjugate by 1.5 to 6-fold. Lanes of this figures
are as follows:
TABLE-US-00003 TABLE 3 Lane Dye/Spacer 1 DYLIGHT .TM. 800-5X, No
Spacer 2 DYLIGHT .TM. 800-5X, 2.5X NHS Acetate 3 DYLIGHT .TM.
800-5X, 5X NHS Acetate 4 Blank Well 5 DYLIGHT .TM. 800-5X, 3.75X
MS(PEG)4 6 DYLIGHT .TM. 800-5X, 5X MS(PEG)4 7 DYLIGHT .TM. 800-5X,
10X MS(PEG)4 8 DYLIGHT .TM. 800-7.5X, No Spacer 9 DYLIGHT .TM.
800-7.5X, 2.5X NHS Acetate 10 DYLIGHT .TM. 800-7.5X, 5X NHS Acetate
11 Blank Well 12 DYLIGHT .TM. 800-7.5X, 3.75X MS(PEG)4 13 DYLIGHT
.TM. 800-7.5X, 5X MS(PEG)4 14 DYLIGHT .TM. 800-7.5X, 10X MS(PEG)4
15 DYLIGHT .TM. 800-10X, No Spacer 16 DYLIGHT .TM. 800-10X, 2.5X
NHS Acetate 17 DYLIGHT .TM. 800-10X, 5X NHS Acetate 18 Blank Well
19 DYLIGHT .TM. 800-10X, 3.75X MS(PEG)4 20 DYLIGHT .TM. 800-10X, 5X
MS(PEG)4 21 DYLIGHT .TM. 800-10X, 10X MS(PEG)4 22 DYLIGHT .TM.
800-15X, No Spacer 23 DYLIGHT .TM. 800-15X, 2.5X NHS Acetate 24
DYLIGHT .TM. 800-15X, 5X NHS Acetate 25 DYLIGHT .TM. 800-15X, 10X
NHS Acetate 26 DYLIGHT .TM. 800-15X, 3.75X MS(PEG)4 27 DYLIGHT .TM.
800-15X, 5X MS(PEG)4 28 DYLIGHT .TM. 800-15X, 10X MS(PEG)4 29
DYLIGHT .TM. 800-20X, No Spacer 30 DYLIGHT .TM. 800-20X, 2.5X NHS
Acetate 31 DYLIGHT .TM. 800-20X, 5X NHS Acetate 32 DYLIGHT .TM.
800-20X, 10X NHS Acetate 33 DYLIGHT .TM. 800-20X, 3.75X MS(PEG)4 34
DYLIGHT .TM. 800-20X, 5X MS(PEG)4 35 DYLIGHT .TM. 800-20X, 10X
MS(PEG)4
[0034] FIG. 8 Western blot assay demonstrating the effect of the
addition of NHS Acetate (at 2.5.times., 5.times. and 10.times.
molar excess) or MS(PEG).sub.4 (at 3.75.times. molar excess) on the
fluorescence detection level of GAR-DYLIGHT.TM. 488 conjugated to
the antibody at molar excesses ranging from 5.times. to 20.times.
of dye. A431 cell lysate was diluted 3-fold from 1 .mu.g/well. The
rabbit primary antibodies used were anti-Hsp90 diluted 1/5000 from
1 mg/ml and anti-Cyclophilin B diluted 1/5000 from 1 mg/ml. All
DYLIGHT.TM. secondary antibodies were diluted 1/5000 from 1 mg/ml
stock. These results show that In a Western blot application, there
is noticeable increase of fluorescent intensity over the base
conjugate (made without the spacer) at each dye molar excess from
7.5.times. to 20.times. for DYLIGHT.TM. 488-GAR conjugated with the
addition of NHS Acetate or MS(PEG).sub.4.
[0035] FIG. 9 shows the effect of NHS Acetate (5.times.) or
MS(PEG).sub.4 (5.times.)) on GAM-DYLIGHT.TM. 650-4.times.PEG-GAR
(at 7.5.times. molar excess of dye) in a Western Blot. HeLa cell
lysate was diluted 4-fold from 0.5 .mu.g/well. Primary antibody
mouse anti-PDI was diluted to 1/5000 of 1 mg/ml. All DYLIGHT.TM.
secondary antibodies were diluted to 1/5000 of 1 mg/ml stock. NHS
acetate added at 5.times. molar excess to GAM-DYLIGHT.TM.
650-4.times.PEG-7.5.times. conjugation improved intensity by
1.5-fold. NHS acetate added at 3.75.times. molar excess to
GAM-DYLIGHT.TM. 650-4.times.PEG-7.5.times. conjugation improved
intensity by 1.4-fold.
[0036] FIG. 10 shows results from a Western blot assay testing the
effect of NHS Acetate (2.5.times., 5.times.) or MS(PEG).sub.4 on
the detectable fluorescence level of GAR-DYLIGHT.TM.
800-4.times.PEG (abbreviated in Table 4 "DYLIGHT.TM. 800"). A431
cell lysate was serially diluted 1:1. Primary antibodies rabbit
anti-Hsp90 and anti-Cyclophilin B were both diluted 1/5000. An
DYLIGHT.TM. secondary antibodies were diluted to 1/20,000 of 1
mg/ml stock. The addition of MS(PEG).sub.4 (3.75.times. and
5.times.) and NHS Acetate (2.5-5.times.) significantly enhanced the
fluorescence intensity and sensitivity of base DYLIGHT.TM.
800-4.times.PEG conjugate by 20-100% at different molar excesses of
the dye in this Western blotting application. Lanes of this figures
are as follows:
TABLE-US-00004 TABLE 4 Lane Dye/Spacer 1 DYLIGHT .TM. 800-5x No
Spacer 2 DYLIGHT .TM. 800-5X, 2.5X NHS Acetate 3 DYLIGHT .TM.
800-5X, 5X NHS Acetate 4 DYLIGHT .TM. 800-5X, 5X MS(PEG)4 5 DYLIGHT
.TM. 800-7.5x No Spacer 6 DYLIGHT .TM. 800-7.5X, 2.5X NHS Acetate 7
DYLIGHT .TM. 800-7.5X, 5X NHS Acetate 8 DYLIGHT .TM. 800-7.5X, 5X
MS(PEG)4 9 DYLIGHT .TM. 800-10x No Spacer 10 DYLIGHT .TM. 800-10X,
2.5X NHS Acetate 11 DYLIGHT .TM. 800-10X, 5X NHS Acetate 12 DYLIGHT
.TM. 800-10X, 5X MS(PEG)4 13 DYLIGHT .TM. 800-15x No Spacer 14
DYLIGHT .TM. 800-15X, 2.5X NHS Acetate 15 DYLIGHT .TM. 800-15X, 5X
NHS Acetate 16 DYLIGHT .TM. 800-15X, 5X MS(PEG)4
[0037] FIG. 11 demonstrates the effect of the addition of NHS
Acetate (2.5.times., 5.times. and 10.times.) or MS(PEG).sub.4
(5.times.) and MS(PEG).sub.8 (5.times.) on GAM-DYLIGHT.TM.
550-2.times.PEG (at a 12.5.times. molar excess of dye) in
fluorescent Western blot and dot blot assays. HeLa cell lysate was
diluted 4-fold from 0.5 .mu.g/well and was stained with anti-PDI
primary antibody diluted to 1/5000 of 1 mg/ml. All DYLIGHT.TM.
secondary antibodies were diluted to 1/5000 of 1 mg/ml stock.
Western Blotting and Dot Blot assays showed that the addition of
MS(PEG).sub.4 (5.times.) and NHS acetate (2.5.times. and 5.times.)
significantly enhanced the fluorescence intensity and sensitivity
of base DYLIGHT.TM. 550-2.times.PEG conjugates by at least by
2-fold. Conjugates prepared with longer chain MS(PEG).sub.8 did not
show significant improvement over the base conjugate.
[0038] FIG. 12 shows the effect of NHS Acetate (2.5.times.,
5.times.) or MS(PEG).sub.4 (5.times.) on GAM-DYLIGHT.TM.
680-4.times.PEG-GAR (at 10.times. molar excess of dye) in Western
blot and dot blot assays. For Western blotting, HeLa cell lysate
was diluted 4-fold from 0.5 .mu.g/well and anti-PDI primary
antibody was diluted to 1/5000 of 1 mg/ml. For dot blotting, mouse
IgG was serially diluted 1:2 from 1000 ng/dot. All DYLIGHT.TM.
680-4.times.PEG-GAR secondary antibodies were diluted to 1/20000 of
1 mg/ml stock. Both Western Blots and Dot Blots show that the
addition of MS(PEG).sub.4 (5.times.) and NHS acetate (2.5.times.
and 5.times.) significantly enhanced the fluorescence intensity and
sensitivity of base DYLIGHT.TM. 680-4.times.PEG conjugate by 3 to
4-fold.
[0039] FIG. 13A (IFC with DYLIGHT.TM. 488-GAM 4 .mu.g/ml.
Signal/background fold improvement from NHS Acetate (2.5.times.,
5.times.) or MS(PEG)4 (3.75.times.) over base conjugates at various
molar excesses) and FIG. 13B (IFC--Detection of PDI with
DYLIGHT.TM. 488-GAR 4 .mu.g/ml. Signal/background fold improvement
from NHS Acetate (2.5.times., 5.times.) or MS(PEG)4 (3.75.times.)
over base conjugates at various molar excesses) demonstrates the
effect of NHS acetate (2.5.times. and 5.times.) or MS(PEG).sub.4
(3.75.times.) addition on fluorescence of GAM-DYLIGHT.TM. 488 (13A)
and GAR-DYLIGHT.TM. 488 (13B) at 7.5.times. to 20.times. molar
excess of dye in cellular imaging assays. FIG. 13A: A549 cells were
stained with a pH2Ax primary antibody diluted to 1/1000 of the 1
mg/ml stock. All DYLIGHT.TM. 488 secondary antibodies were diluted
to 1/250 of the 1 mg/ml stock. NHS acetate modified conjugates
provided an improvement of signal/background as compared to the
base conjugates at 15.times. dye molar excesses; ranging from 1.4
to 1.5-fold (GAM) and 1.1 to 1.6-fold (GAR). For GAM conjugates the
most significant improvement was observed with NHS Acetate at
5.times. and with MS(PEG).sub.4 at 3.75.times. for GAR conjugates
the more noticeable improvement was observed with NHS Acetate at
2.5.times. and with MS(PEG).sub.4 at 3.75.times.. FIG. 13B shows a
similar experiment where A549 cells were stained with a pH2Ax
primary antibody.
[0040] FIG. 14: IFC Detection of PDI with DYLIGHT.TM.
550-2.times.PEG (7.5.times. to 20.times.) 4 .mu.g/ml. Fold
improvement from the addition of NHS Acetate (2.5.times., 5.times.
and 10.times.) or MS(PEG)4 (3.75.times., 5.times., 10.times.) over
base conjugates at each dye molar excesses. This figure shows the
effect of NHS Acetate (2.5.times., 5.times. and 10.times.) or
MS(PEG).sub.4 (3.75.times., 5.times. and 10.times.) addition to
GAM-DYLIGHT.TM. 550-2.times.PEG-GAM (at 7.5.times. to 20.times.
molar excess of dye) on fluorescence cellular imaging assays. U2OS
cells were stained with an anti-PDI primary antibody diluted to
1/100 of the 1 mg/ml stock. All DYLIGHT.TM. 550-2.times.PEG-GAM
secondary antibodies (abbreviated in Table 5 "DYLIGHT.TM. 550")
were diluted to 1/250 of the 1 mg/ml stock. In this cellular
imaging application, the addition of 5.times.NHS Acetate generated
about 50% improvement as compared to the base conjugate (made
without the additives) for DYLIGHT.TM. 550-2.times.PEG GAM
conjugate at 12.5.times. dye molar excess, and addition of
3.75.times. MS(PEG).sub.4 resulted in about 50% improvement over
the base conjugates at 20.times. dye molar excess. Lanes of this
figures are as follows:
TABLE-US-00005 TABLE 5 Lane Dye/Spacer 1 DYLIGHT .TM. 550-7.5X, No
Spacer 2 DYLIGHT .TM. 550-7.5X, 2.5X NHS Acetate 3 DYLIGHT .TM.
550-7.5X, 5X NHS Acetate 4 Blank 5 DYLIGHT .TM. 550-7.5X, 3.75X
MS(PEG).sub.4 6 DYLIGHT .TM. 550-7.5X, 5X MS(PEG).sub.4 7 DYLIGHT
.TM. 550-7.5X, 10X MS(PEG).sub.4 8 DYLIGHT .TM. 550-10X, No Spacer
9 DYLIGHT .TM. 550-10X, 2.5X NHS Acetate 10 DYLIGHT .TM. 550-10X,
5X NHS Acetate 11 Blank 12 DYLIGHT .TM. 550-10X, 3.75X
MS(PEG).sub.4 13 DYLIGHT .TM. 550-10X, 5X MS(PEG).sub.4 14 DYLIGHT
.TM. 550-10X, 10X MS(PEG).sub.4 15 DYLIGHT .TM. 550-12.5X, No
Spacer 16 DYLIGHT .TM. 550-12.5X, 2.5X NHSAcetate 17 DYLIGHT .TM.
550-12.5X, 5X NHS Acetate 18 Blank 19 DYLIGHT .TM. 550-12.5X, 3.75X
MS(PEG).sub.4 20 DYLIGHT .TM. 550-12.5X, 5X MS(PEG).sub.4 21
DYLIGHT .TM. 550-12.5X, 10X MS(PEG).sub.4 22 DYLIGHT .TM. 550-15X,
No Spacer 23 DYLIGHT .TM. 550-15X, 2.5X NHS Acetate 24 DYLIGHT .TM.
550-15X, 5X NHS Acetate 25 DYLIGHT .TM. 550-15X, 10X NHS Acetate 26
DYLIGHT .TM. 550-15X, 3.75X MS(PEG).sub.4 27 DYLIGHT .TM. 550-15X,
5X MS(PEG).sub.4 28 DYLIGHT .TM. 550-15X, 10X MS(PEG).sub.4 29
DYLIGHT .TM. 550-25X, No Spacer 30 DYLIGHT .TM. 550-20X, 2.5X NHS
Acetate 31 DYLIGHT .TM. 550-20X, 5X NHS Acetate 32 DYLIGHT .TM.
550-20X, 10X NHS Acetate 33 DYLIGHT .TM. 550-20X, 3.75X
MS(PEG).sub.4 34 DYLIGHT .TM. 550-20X, 5X MS(PEG).sub.4 35 DYLIGHT
.TM. 550-20X, 10X MS(PEG).sub.4
[0041] FIG. 15: IFC Detection of PDI with DYLIGHT.TM.
650-4.times.PEG (7.5.times. to 20.times.) 4 .mu.g/ml. Fold
improvement from the addition of NHS Acetate (2.5.times., 5.times.
and 10.times.) or MS(PEG)4 (3.75.times., 5.times., 10.times.) over
base conjugates at each dye molar excesses This figure shows effect
of adding NHS Acetate (2.5.times., 5.times. and 10.times.) or
MS(PEG).sub.4 (3.75.times., 5.times., 10.times.) on GAM-DYLIGHT.TM.
650-4.times.PEG in a fluorescence cellular imaging assay
GAM-DYLIGHT.TM. 650-4.times.PEG). U2OS cells were stained with
anti-PDI primary antibody diluted to 1/100 of 1 mg/ml stock. All
DYLIGHT.TM. 650-4.times.PEG-GAM secondary antibodies (abbreviated
in Table 6 "DYLIGHT.TM. 650") were diluted to 1/250 of 1 mg/ml
stock. In this application, the addition of NHS Acetate-5.times.
generated about 70% improvement as compared to the base conjugate
(made without the additives) for DYLIGHT.TM. 650-4.times.PEG-GAM
conjugate at 20.times. molar excess, and MS(PEG).sub.4-3.75.times.
showed about 90% improvement over the base conjugates at 20.times.
molar excess. Lanes of this figures are as follows:
TABLE-US-00006 TABLE 6 Lane Dye/Spacer 1 DYLIGHT .TM. 650-5X, No
Spacer 2 DYLIGHT .TM. 650-5X, 2.5X NHS Acetate 3 DYLIGHT .TM.
650-5X, 5X NHS Acetate 4 Blank 5 DYLIGHT .TM. 650-5X, 3.75X
MS(PEG).sub.4 6 DYLIGHT .TM. 650-5X, 5X MS(PEG).sub.4 7 DYLIGHT
.TM. 650-5X, 10X MS(PEG).sub.4 8 DYLIGHT .TM. 650-7.5X, No Spacer 9
DYLIGHT .TM. 650-7.5X, 2.5X NHS Acetate 10 DYLIGHT .TM. 650-7.5X,
5X NHS Acetate 11 Blank 12 DYLIGHT .TM. 650-7.5X, 3.75X
MS(PEG).sub.4 13 DYLIGHT .TM. 650-7.5X, 5X MS(PEG).sub.4 14 DYLIGHT
.TM. 650-7.5X, 10X MS(PEG).sub.4 15 DYLIGHT .TM. 650-10X, No Spacer
16 DYLIGHT .TM. 650-10X, 2.5X NHS Acetate 17 DYLIGHT .TM. 650-10X,
5X NHS Acetate 18 Blank 19 DYLIGHT .TM. 650-10X, 3.75X
MS(PEG).sub.4 20 DYLIGHT .TM. 650-10X, 5X MS(PEG).sub.4 21 DYLIGHT
.TM. 650-10X, 10X MS(PEG).sub.4 22 DYLIGHT .TM. 650-15X, No Spacer
23 DYLIGHT .TM. 650-15X, 2.5X NHS Acetate 24 DYLIGHT .TM. 650-15X,
5X NHS Acetate 25 DYLIGHT .TM. 650-15X, 10X NHS Acetate 26 DYLIGHT
.TM. 650-15X, 3.75X MS(PEG).sub.4 27 DYLIGHT .TM. 650-15X, 5X
MS(PEG).sub.4 28 DYLIGHT .TM. 650-15X, 10X MS(PEG).sub.4 29 DYLIGHT
.TM. 650-20X, No Spacer 30 DYLIGHT .TM. 650-20X, 2.5X NHS Acetate
31 DYLIGHT .TM. 650-20X, 5X NHS Acetate 32 DYLIGHT .TM. 650-20X,
10X NHS Acetate 33 DYLIGHT .TM. 650-20X, 3.75X MS(PEG).sub.4 34
DYLIGHT .TM. 650-20X, 5X MS(PEG).sub.4 35 DYLIGHT .TM. 650-20X, 10X
MS(PEG).sub.4
[0042] FIG. 16: IFC--DYLIGHT.TM. 680-4.times.PEG Fold improvement
from NHS Acetate (2.5.times., 5.times. and 10.times.) or MS(PEG)4
(3.75.times., 5.times., 10.times.) over base conjugates at each
molar excess. This figure shows the effect of the addition of NHS
Acetate (2.5.times., 5.times. and 10.times.) or MS(PEG).sub.4
(3.3.75.times., 5.times., 10.times.) on GAM-DYLIGHT.TM.
680-4.times.PEG) in a cellular imaging assay. U2OS cells were
stained with mouse anti-PDI primary antibody diluted to 1/100 of 1
mg/ml stock. All DYLIGHT.TM. 680-4.times.PEG-GAM secondary
antibodies (abbreviated in Table 7 "DYLIGHT.TM. 680") were diluted
to 1/250 of 1 mg/ml stock: In this cellular imaging application,
the addition of NHS Acetate-5.times. generated about 70%
improvement for dyes conjugates at both 7.5.times. and 10.times. as
compared to the base conjugate (made without the additives) for
DYLIGHT.TM. 680-4.times.PEG. GAM conjugate at molar excesses of
15.times. molar excess and MS(PEG).sub.4-3.75.times. showed about
80% improvement over the base conjugates at 15.times. molar
excesses. Lanes of this figures are as follows:
TABLE-US-00007 TABLE 7 Lane Dye/Spacer 1 DYLIGHT .TM. 680-5X, No
Spacer 2 DYLIGHT .TM. 680-5X, 2.5X NHS Acetate 3 DYLIGHT .TM.
680-5X, 5X NHS Acetate 4 Blank 5 DYLIGHT .TM. 680-5X, 3.75X
MS(PEG).sub.4 6 DYLIGHT .TM. 680-5X, 5X MS(PEG).sub.4 7 DYLIGHT
.TM. 680-5X, 10X MS(PEG).sub.4 8 DYLIGHT .TM. 680-7.5X, No Spacer 9
DYLIGHT .TM. 680-7.5X, 2.5X NHS Acetate 10 DYLIGHT .TM. 680-7.5X,
5X NHS Acetate 11 Blank 12 DYLIGHT .TM. 680-7.5X, 3.75X
MS(PEG).sub.4 13 DYLIGHT .TM. 680-7.5X, 5X MS(PEG).sub.4 14 DYLIGHT
.TM. 680-7.5X, 10X MS(PEG).sub.4 15 DYLIGHT .TM. 680-10X, No Spacer
16 DYLIGHT .TM. 680-10X, 2.5X NHS Acetate 17 DYLIGHT .TM. 680-10X,
5X NHS Acetate 18 Blank 19 DYLIGHT .TM. 680-10X, 3.75X
MS(PEG).sub.4 20 DYLIGHT .TM. 680-10X, 5X MS(PEG).sub.4 21 DYLIGHT
.TM. 680-10X, 10X MS(PEG).sub.4 22 DYLIGHT .TM. 680-15X, No Spacer
23 DYLIGHT .TM. 680-15X, 2.5X NHS Acetate 24 DYLIGHT .TM. 680-15X,
5X NHS Acetate 25 DYLIGHT .TM. 680-15X, 10X NHS Acetate 26 DYLIGHT
.TM. 680-15X, 3.75X MS(PEG).sub.4 27 DYLIGHT .TM. 680-15X, 5X
MS(PEG).sub.4 28 DYLIGHT .TM. 680-15X, 10X MS(PEG).sub.4 29 DYLIGHT
.TM. 680-20X, No Spacer 30 DYLIGHT .TM. 680-20X, 2.5X NHS Acetate
31 DYLIGHT .TM. 680-20X, 5X NHS Acetate 32 DYLIGHT .TM. 680-20X,
10X NHS Acetate 33 DYLIGHT .TM. 680-20X, 3.75X MS(PEG).sub.4 34
DYLIGHT .TM. 680-20X, 5X MS(PEG).sub.4 35 DYLIGHT .TM. 680-20X, 10X
MS(PEG).sub.4
[0043] FIG. 17: TAMRA-GAM co-conjugates with or without Betaine
This figure exemplifies observed fluorescence levels of TAMRA-goat
anti-mouse antibody (GAM) conjugates, with or without betaine, at
various dye/protein molar ratios. Levels of betaine were tested at
2.5, 5, and 10.times. molar excess as compared to moles of
antibody. These antibodies labeled with NHS-Rhodamine (TAMRA) and
conjugated with a variety of NHS-Betaine concentrations (Betaine
2.5.times., Betaine 5.times., Betaine 10.times. molar excess of
dye) displayed an increase in total fluorescence when the
antibodies were conjugated with Betaine as the spacer agent.
[0044] FIG. 18 shows a schematic representation of the generation
and attachment of branched PEG molecules containing fluorescent
labels to an antibody molecule. In step 1, a reactive group and
fluorescent labels are attached to NH.sub.2 groups on branched PEG
molecules. In step 2, attachment sites are added to the antibody
molecule. In step 3, the fluorescently labeled branched PEG
molecules are covalently linked to the antibody molecule.
[0045] FIG. 19 is an illustration showing one example each of a
single-armed connector (e.g., amylose) and a multi-armed connector
(dextran). BM stands for biomolecule and AP stand for attachment
point, meaning that a fluorescent label is attached at that
point.
[0046] FIG. 20 is an illustration of different types of star
polymers and an example of components of some star polymers and an
example of how star polymers can be prepared. FIG. 20A is an
illustration of a star polymer with a core (the black circle) and
multiple of arms (the black lines). The stars represent fluorescent
labels covalently linked to the arms. Some of the arms are not
fluorescently labeled to indicate that the labeling did not go to
completion. FIG. 20B is an illustration of a similar star polymer
where the arms are of two different types (e.g., polyethylene
glycol and polyvinyl alcohol), the different arm types being
represented by the solid and dashed lines. The left side of FIG.
20C shows a partial representation of a core (the black
semi-circles) with reactive groups (the grey bars) that can be used
to attach other chemical entities or to serve as initiators for
polymerization. A core is represented in the center that has
adapters (solid black lines) attached to reactive groups. Polymer
arms are shown to the right (black dashed lines) attached to the
adapters, and labeled with fluorescent molecules (stars)
[0047] FIG. 21 shows an exemplary polylysine molecule of a type
that can be used in the practice of the invention. The R.sub.1 and
R2 groups are shown as unlabeled. These groups can be used as
attachment points for fluorescent labels and for conjugation to
biomolecules (e.g., antibodies).
[0048] FIG. 22: SK3 mouse anti-human CD4 antibody conjugated with
AF647 branched PEG constructs. SK3 mouse anti-human CD4 antibody
(at 5.5 mg/mL) was modified with ALEXA FLUOR.RTM. 647 succinimidyl
ester at 10 fold molar excess ester to antibody (dashed line). SK3,
which was tagged with 10-fold excess of Azido-SE to antibody, was
click conjugated to 100 .mu.M AF647-HG20K8 PEG-sDIBO at 1 mg/mL
azido-SK3 antibody for 20 hours at 25.degree. C., quenched with 5
mM NaN.sub.3 and conjugate was purified with Millipore AMICON.TM.
Ultra-2 100 kDa centrifugal filter (dotted line). SK3, which was
tagged with 20-fold excess of Azido-SE to antibody, was click
conjugated to 600 .mu.M AF647-HG20K8 PEG-sDIBO at 3 mg/mL azido-SK3
antibody for 3 hours at 37.degree. C., quenched with 5 mM NaN.sub.3
and conjugate was purified with Millipore AMICON.TM. Ultra-2 100
kDa centrifugal filter (solid line). One million Ficoll-isolated
PBMC/well in a 96 well plate were stained with the SK3 conjugates
using a 7 point titration of 1 .mu.g to 0.015 .mu.g of antibody.
Analysis of the stained cells was carried out using the ATTUNE.TM.
NxT Flow Cytometer.
[0049] FIG. 23: SK3 mouse anti-human CD4 antibody conjugated with
AF647 branched PEG constructs. SK3 mouse anti-human CD4 antibody
(at 5.5 mg/mL) was modified with ALEXA FLUOR.RTM. 647 succinimidyl
ester at 10 fold molar excess ester to antibody (AF). SK3, which
was tagged with 20-fold excess of Azido-SE to antibody, was click
conjugated to 600 .mu.M AF647-HG20K8 PEG-sDIBO at 3 mg/mL azido-SK3
antibody for 3 hours at 37.degree. C., quenched with 5 mM NaN.sub.3
and conjugate was purified with Millipore AMICON.TM. Ultra-2 100
kDa centrifugal filter (B1). SK3, which was tagged with 10-fold
excess of Azido-SE to antibody, was click conjugated to 100 .mu.M
AF647-HG20K8 PEG-sDIBO at 1 mg/mL azido-SK3 antibody for 20 hours
at 25.degree. C., quenched with 5 mM NaN.sub.3 and conjugate was
purified with Millipore AMICON.TM. Ultra-2 100 kDa centrifugal
filter (B2). One million Ficoll-isolated PBMC/well in a 96 well
plate were stained with the SK3 conjugates using a 7 point
titration of 1 .mu.g to 0.015 .mu.g of antibody. Analysis of the
stained cells was carried out using the ATTUNE.TM. NxT Flow
Cytometer and compared to allophycocyanin (APC) (Thermo Fisher
Scientific, cat. no. MHCD0405).
DETAILED DESCRIPTION
[0050] Fluorescent labels are widely used for imaging because they
provide direct, quantitative, specific and sensitive detection of
biomolecules including proteins and nucleic acids. Modified
fluorescent labels that are sulfonated and/or PEG modified offer
increased sensitivity compared to the basic unmodified dyes.
However, even these modified fluorescent labels exhibit
fluorescence quenching at certain dye to protein (D/P) ratios.
[0051] Increase in fluorescence is seen with increasing molar
excesses of the labeling dye, however, molar excesses which result
in exceeding optimal dye to protein (D/P) ratios, typically result
in quenching and/or precipitation of the biomolecule especially in
fluorescent imaging application where spatial conformation of
antigen/antibody or DNA/RNA interactions may cause static
quenching.
[0052] In some embodiments, the invention comprises methods of
reducing quenching and/or increasing fluorescence signal with
highly labeled conjugates (e.g., biomolecules with high dye to
protein ratios (D/P)) that in standard conjugation results in
decreased fluorescence. Modifications can be made on proteins,
nucleic acids and other biomolecules (e.g., oligosaccharides). In
some embodiments, the invention comprises compositions comprising
fluorescently labeled biomolecules that exhibit increased
fluorescent signal and/or reduced quenching, wherein the
composition comprises a biomolecule, a spacer, and a fluorescent
label, wherein the spacer and fluorescent label are not directly
conjugated to each other.
[0053] The invention also relates to compositions which exhibit
enhanced fluorescence on a per fluorescent label basis, as well as
methods for producing and using such compositions. By way of
illustration, assume that a single fluorescent label attached to a
biomolecule sets a baseline of 100% fluorescent emission. Further
assume, when two fluorescent labels are attached to the same
biomolecule, that each of the two fluorescent labels exhibits an
average of 80% of the baseline fluorescent emission. The invention
is directed, in part, to compositions and methods for increasing
the average fluorescent emission above the 80% of the baseline.
[0054] In some instances, compositions of the invention, as well as
compositions used in methods of the invention, may be defined by
one or more functional property. Examples of such properties are
the numbers of fluorescent labels associated with a labeled
molecule (e.g., a biomolecule), the average distance (measured in
any of a number of different ways) between fluorescent labels on
the labeled molecule, and/or the quantum yield of fluorescent
labels on the labeled molecule.
[0055] One measure of measuring fluorescent intensity is by
measurement of Quantum Yield. Quantum Yield (.PHI.) for fluorescent
systems is effectively the emission efficiency of a given
fluorophore and may be determined by the equation:
.PHI. = Number of Photons Emitted Number of Photons Absorbed
##EQU00001##
[0056] Quantum Yield may also be used to measure quenching effects,
as set out below in Example 8. Further, instruments, such as the
Hamamatsu Absolute PL Quantum Yield Spectrometer (Hamamatsu Corp.,
Bridgewater N.J. 08807, C11347-11Quantaurus-QY Absolute PL Quantum
Yield Spectrometer), that may be used to measure quantum yield are
commercially available.
[0057] As set out in Example 8 and Table 25, quantum yield of a
fluorescently labeled molecule can be compared to that of the free
fluorescent label. If the quantum yield of a single unit of the
free fluorescent label under conditions where effectively no
quenching occurs is set as one, then this can be used as a
benchmark for comparison of the fluorescence generated by each
fluorescent label attached to the labeled molecule. In many
instances, compositions of the invention include fluorescently
labeled molecules that are labeled with multiple fluorescent labels
where the average amount of fluorescent emission on a per
fluorescent label basis is at least 70% (0.7 Fluorescent Ratio)
(e.g., from about 70% to about 99%, from about 70% to about 90%,
from about 80% to about 99%, from about 85% to about 99%, from
about 87% to about 99%, from about 90% to about 99%, from about 80%
to about 95%, from about 85% to about 96%, etc.) of the fluorescent
emission of the free fluorescent label.
[0058] As set out in Example 8 and Table 25, fluorescent intensity
may be determined is by the measurement of total fluorescence of a
fluorescently labeled molecule compared to the fluorescence of the
free label. If the fluorescence of a single unit of the free
fluorescent label under conditions where effectively no quenching
occurs is set as one, then this can be used as a benchmark for
comparison of the fluorescence generated by each fluorescent label
attached to the labeled molecule. In many instances, compositions
of the invention include fluorescently labeled molecules that are
labeled with multiple fluorescent labels where the average amount
of fluorescent emission on a per fluorescent label basis is at
least 70% (0.7 Fluorescent Ratio) (e.g., from about 70% to about
99%, from about 70% to about 90%, from about 80% to about 99%, from
about 85% to about 99%, from about 87% to about 99%, from about 90%
to about 99%, from about 80% to about 95%, from about 85% to about
96%, etc.) of the fluorescent emission of the free fluorescent
label.
[0059] As set out in Table 25, the brightness of a fluorescently
labeled molecule compared to the free fluorescent label can be
determined. Brightness is proportional to the product of quantum
yield (.PHI.), extinction coefficient (.epsilon.) and number of
dyes per molecule (N) as given in the equation:
B=.PHI..times..epsilon..times.N
Thus the ratio of the brightness of the free fluorescent label to
that of the labeled molecule can be used to describe the total
fluorescence enhancement.
[0060] As an example, the data in Table 25 sets as a benchmark
ALEXA FLUOR.RTM. 647 in deionized water. Further, this sets a
benchmark of 100% quantum yield of free dye and a brightness ratio
of 1.0. Amongst samples, the molecule AF647-20K8 had 73% of the
quantum yield of the free dye but showed a fluorescent enhancement
of 5.8.times. over the free dye. It is also shown that the sample
AF647-10K4 had the highest percent quantum yield of the free dye
(89%) but had a fluorescent enhancement of only 3.6.times. versus
the free dye. These data show that the degree of fluorescent
enhancement seen for these molecules can be directly correlated
with the length of the arms. The data also show that when arm
length is held constant and more fluorescently labeled arms are
added to a polymer, then the fluorescent enhancement tends to
increase.
[0061] The invention thus includes compositions and methods for
linking multiple fluorescent labels to individual molecules (e.g.,
biomolecules) such that the fluorescent labels are spaced in a
manner that enhances fluorescent signal. This may be done by the
reduction of quenching. One method for enhancing fluorescent signal
is to spatially separate fluorescent labels present in a sample.
This is especially useful when multiple fluorescent labels are
attached to the same molecule (e.g., biomolecule) that is to be
detected.
[0062] In some aspects, the invention comprises methods of
producing an antibody conjugated to a spacer and a fluorescent
label, wherein a spacer agent is used to conjugate a spacer to an
antibody, and wherein the spacer is not conjugated to the
fluorescent label. Also encompassed are compositions comprising a
spacer, an antibody, and a fluorescent label, wherein the spacer is
not conjugated to the fluorescent label.
[0063] In some embodiments, the spacer may be capable of reducing
quenching of a plurality of fluorescent labels conjugated to an
antibody.
[0064] In some embodiments, a method of producing a nucleic acid
conjugated to a spacer agent is encompassed, wherein the spacer
agent is not directly conjugated to the fluorescent label. Such
spacer agents may be capable of reducing quenching of a fluorescent
label conjugated to a nucleic acid.
[0065] In some embodiments, the invention includes compositions and
methods related to the spatial separation fluorescent labels from
the point on a molecule (e.g., biomolecule) to which they are
conjugated to. In many instances, this will be done by connection
for one or more fluorescent labels to a spacer and connection of
the spacer to the molecule (e.g., biomolecule). Example of such
compositions and methods are shown in FIG. 18.
Definitions
[0066] This description and exemplary embodiments should not be
taken as limiting. For the purposes of this specification and
appended claims, unless otherwise indicated, all numbers expressing
quantities, percentages, or proportions, and other numerical values
used in the specification and claims, are to be understood as being
modified in all instances by the term "about," to the extent they
are not already so modified. Accordingly, unless indicated to the
contrary, the numerical parameters set forth in the following
specification and attached claims are approximations that may vary
depending upon the desired properties sought to be obtained. At the
very least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of the claims, each numerical
parameter should at least be construed in light of the number of
reported significant digits and by applying ordinary rounding
techniques.
[0067] It is noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the," and any
singular use of any word, include plural referents unless expressly
and unequivocally limited to one referent. As used herein, the term
"include" and its grammatical variants are intended to be
non-limiting, such that recitation of items in a list is not to the
exclusion of other like items that can be substituted or added to
the listed items.
[0068] As used herein, a "biomolecule" refers to any molecule that
may be included in a biological system, including but not limited
to, a synthetic or naturally occurring protein or fragment thereof,
glycoprotein, lipoprotein, amino acid, nucleoside, nucleotide,
nucleic acid, oligonucleotide, DNA, RNA, carbohydrate, sugar,
lipid, fatty acid, hapten, antibody, and the like.
[0069] The terms "protein" and "polypeptide" are used herein in a
generic sense to include polymers of amino acid residues of any
length. The term "peptide" as used herein refers to a polymer in
which the monomers are amino acids and are joined together through
amide bonds, alternatively referred to as a polypeptide. When the
amino acids are .alpha.-amino acids, either the L-optical isomer or
the D-optical isomer can be used. Additionally, unnatural amino
acids, for example, .beta.-alanine, phenylglycine and homoarginine
are also included. Commonly encountered amino acids that are not
gene-encoded may also be used in the present invention. All of the
amino acids used in the present invention may be either the D- or
L-isomer. The L-isomers are generally used. In addition, other
peptidomimetics are also useful in the present invention. For a
general review, see, Spatola, A. F., in Chemistry and Biochemistry
of Amino Acids, Peptides and Proteins, B. Weinstein, eds., Marcel
Dekker, New York, p. 267 (1983).
[0070] The term "antibody" as used herein refers to a protein of
the immunoglobulin (Ig) superfamily that binds noncovalently to
certain substances (e.g., antigens and immunogens) to form an
antibody-antigen complex, including but not limited to antibodies
produced by hybridoma cell lines, by immunization to elicit a
polyclonal antibody response, by chemical synthesis, and by
recombinant host cells that have been transformed with an
expression vector that encodes the antibody. In humans, the
immunoglobulin antibodies are classified as IgA, IgD, IgE, IgG, and
IgM and members of each class are said to have the same isotype.
Human IgA and IgG isotypes are further subdivided into subtypes
IgA.sub.1, and IgA.sub.2, and IgG.sub.1, IgG.sub.2, IgG.sub.3, and
IgG.sub.4. Mice have generally the same isotypes as humans, but the
IgG isotype is subdivided into IgG.sub.1, IgG.sub.2a, IgG.sub.2b,
and IgG.sub.3 subtypes. Thus, it will be understood that the term
"antibody" as used herein includes within its scope (a) any of the
various classes or sub-classes of immunoglobulin (e.g., IgA, IgD,
IgG, IgM, and IgE derived from any animal that produced antibodies)
and (b) polyclonal and monoclonal antibodies, such as murine,
chimeric, or humanized antibodies. Antibody molecules have regions
of amino acid sequences that can act as an antigenic determinant
(e.g. the Fc region, the kappa light chain, the lambda light chain,
the hinge region, etc.). An antibody that is generated against a
selected region is designated anti-[region] (e.g., anti-Fc,
anti-kappa light chain, anti-lambda light chain, etc.). An antibody
is typically generated against an antigen by immunizing an organism
with a macromolecule to initiate lymphocyte activation to express
the immunoglobulin protein. The term antibody, as used herein, also
covers any polypeptide or protein having a binding domain that is,
or is homologous to, an antibody binding domain, including, without
limitation, single-chain Fv molecules (scFv), wherein a VH domain
and a VL domain are linked by a peptide linker that allows the two
domains to associate to form an antigen binding site (Bird et al.,
Science 242:423 (1988) and Huston et al., Proc. Natl. Acad. Sci.
USA 85:5879 (1988)). These can be derived from natural sources, or
they may be partly or wholly synthetically produced.
[0071] Further, VHH antibodies may be used either as obtained from
antigen stimulated cells or as engineered antigen binding
proteins.
[0072] The term "antibody fragments" as used herein refers to
fragments of antibodies that retain the principal selective binding
characteristics of the whole antibody. Particular fragments are
well-known in the art, for example, Fab, Fab', and F(ab').sub.2,
which are obtained by digestion with various proteases and which
lack the Fc fragment of an intact antibody or the so-called
"half-molecule" fragments obtained by reductive cleavage of the
disulfide bonds connecting the heavy chain components in the intact
antibody. Such fragments also include isolated fragments consisting
of the light-chain-variable region, "Fv" fragments consisting of
the variable regions of the heavy and light chains, and recombinant
single chain polypeptide molecules in which light and heavy
variable regions are connected by a peptide linker. Other examples
of binding fragments include (i) the Fd fragment, consisting of the
VH and CH1 domains; (ii) the dAb fragment (Ward et al., Nature
341:544 (1989)), which consists of a VH domain; (iii) isolated CDR
regions; and (iv) single-chain Fv molecules (scFv) described above.
In addition, arbitrary fragments can be made using recombinant
technology that retains antigen-recognition characteristics.
[0073] Exemplary VHH antibodies that may be used are single-domain
antibody that are antibody fragments composed of a single monomeric
variable antibody domain. Such antibody fragments typically have a
molecular weight of only 12-25 kDa and are thus smaller than many
other antibodies (150-160 kDa), which are composed of two heavy
protein chains and two light chains.
[0074] As used herein, an "antigen" refers to a molecule that
induces, or is capable of inducing, the formation of an antibody or
to which an antibody binds selectively, including but not limited
to a biological material. Antigen also refers to "immunogen". An
antibody binds selectively to an antigen when there is a relative
lack of cross-reactivity with or interference by other substances
present.
[0075] The term "reactive group" as used herein refers to a group
that is capable of reacting with another chemical group to form a
covalent bond, i.e., is covalently reactive under suitable reaction
conditions, and generally represents a point of attachment for
another substance. Reactive groups generally include nucleophiles,
electrophiles and photoactivatable groups. Exemplary reactive
groups include, but not limited to, olefins, acetylenes, alcohols,
phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic
acids, esters, amides, cyanates, isocyanates, thiocyanates,
isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo,
diazonium, nitro, nitriles, mercaptans, sulfides, disulfides,
sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals,
ketals, anhydrides, sulfates, sulfenic acids isonitriles, amidines,
imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic
acids, alkynes and azides.
[0076] As used herein, a "spacer," "spacer molecule," or "spacer
agent" refers to a compound (e.g., an organic compound) that when
conjugated to a biomolecule, directly or indirectly, is capable of
enhancing fluorescence emitted from the biomolecule. This is
believed to result from reduction of fluorescent quenching of a
fluorescent label. Any number of compounds can act as spacers,
exemplary compounds include NHS-acetate and various forms of
polyethylene glycol (PEG). As used herein, the term "polyethylene
glycol" or "PEG" refers to an oligomer or polymer of ethylene
oxide. PEG polymer chain lengths may vary greatly but tended to
have a molecular mass as high as 10,000,000 g/mol. PEGs are also
available with different geometries. For example, branched PEGs
typically have three to ten PEG chains emanating from a central
core group. Star PEGs have 3 to 100 PEG chains emanating from a
central core group. Comb PEGs have multiple PEG chains normally
grafted onto a polymer backbone. Most PEGs include molecules with a
distribution of molecular weights (i.e., they are polydisperse).
The size distribution can be characterized statistically by its
weight average molecular weight and its number average molecular
weight, the ratio of which is called the polydispersity index.
Exemplary PEG compounds that may be used in the practice of the
invention include MS(PEG).sub.4, MS(PEG).sub.8, and MS(PEG).sub.12
(Thermo Fisher Scientific, Waltham, Mass., cat. nos. 22341, 22509B,
and 22686, respectively), as well as branched chain PEG compounds,
such as (Methyl-PEG.sub.12)3-PEG.sub.4-NHS Ester (Thermo Fisher
Scientific, Waltham, Mass., cat. no. 22421).
[0077] As used herein, the term "direct spacer" refers to a
molecule that has at least one fluorescent labeled attached thereto
and binds directly to a biomolecule. Direct spacers may be (1) a
single polymer or (2) multiple polymers attached to a core.
Examples of direct spacers include single-armed polymers and
multi-armed polymers.
[0078] As used herein, a "polymer" is a molecule composed of
repeating subunits (typically at least 4 repeating subunits).
Polymers may be synthetic or naturally occurring. The repeating
units of a polymer need not be identical. For example, proteins are
polymers that are composed of different amino acid subunits.
Further, polymers need not be fully linear molecules and, thus, may
be branched like dextrans.
[0079] As used herein, the term "single-armed polymer" refers to an
unbranched molecule that to which at least one fluorescent label is
attached and having an unbranched structure (see FIG. 19). Examples
of single-armed polymers that may be used in the practice of the
invention are "linear" polysaccharides (e.g., amylose),
polyethylene glycols, long-chain carbon molecules (e.g., Ahx), and
polypeptides. In some instances, unbranched/linear polysaccharides
are composed of monomers connected to each other by .alpha.1,4
linkages.
[0080] As used herein, the term "multi-armed polymer" refers to a
branched molecule that to which at least one fluorescent label is
attached and having an unbranched structure (see FIG. 19). Examples
of multi-armed polymers that may be used in the practice of the
invention are branched polysaccharides (e.g., dextrans, glycogen),
polyethylene glycols, branched long-chain carbon molecules (e.g.,
Ahx), and branched polypeptides.
[0081] As used herein, the term "conjugation molecule" or
"conjugation arm" refers linkers through which dyes are connected
(e.g., covalently connected) to molecules (e.g., biomolecules).
Conjugation molecule may be bound to a single dye molecule or
multiple dye molecules (the same dye or different dyes).
[0082] As used herein, the term "fluorescence" refers to an optical
phenomenon in which a molecule absorbs a high-energy photon and
re-emits it as a lower-energy (longer-wavelength) photon, with the
energy difference between the absorbed and emitted photons ending
up as molecular vibrations or heat.
[0083] The term "fluorescent label," "fluorescent dye,"
"fluorophore" or "fluorescent moiety", as used herein, refers to a
compound, chemical group, or composition that is inherently
fluorescent. Fluorophores may contain substituents that alter the
solubility, spectral properties or physical properties of the
fluorophore. Numerous fluorophores are known to those skilled in
the art and include, but are not limited to coumarin, cyanine,
benzofuran, a quinoline, a quinazolinone, an indole, a furan, a
benzazole, a borapolyazaindacene and xanthenes including
fluorescein, rhodamine and rhodol as well as other fluorophores
described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF
FLUORESCENT PROBES AND RESEARCH CHEMICALS (9th edition, CD-ROM,
September 2002). Reactive chemistries such as N-hydroxysuccinimide
(NHS), maleimide and hydrazides as well as click chemistry (e.g.
SITECLICK.TM.) are currently being used for conjugation of
florescent labels to biomolecules.
[0084] As used herein, the term "conjugated" refers to a molecule
being attached to another molecule, either directly or indirectly,
either by covalent or noncovalent linkage.
[0085] The term "dye conjugate" refers to a dye molecule bound
covalently or non-covalently to another carrier molecule, such as
an antibody and, in many instances, the dyes are bound covalently.
The dye conjugate can be directly bound through a single covalent
bond, cross-linked or bound through a linker, such as a series of
stable covalent bonds incorporating 1-20 non-hydrogen atoms
selected from the group consisting of C, N, O, S and P that
covalently attach the fluorescent dye to the antibody or another
moiety such as a chemically reactive group or a biological and
non-biological component. The conjugation or linker may involve a
receptor binding motif, such as biotin/avidin.
[0086] The term "near IR dye" or "near IR reporter molecule" or
"NIR dye" or "NIR reporter molecule" as used herein indicates a dye
or reporter molecule with an excitation wavelength of about 580 nm
to about 800 nm. In many instances, the NIR dyes emit in the range
of about 590 nm to about 860 nm. In many instances, NIR dyes are
excited from about 680 to about 790 nm. In many instances, dyes
include ALEXA FLUOR.RTM. 660 Dye, ALEXAFLUOR.RTM. 680 dye, ALEXA
FLUOR.RTM. 700 dye, ALEXA FLUOR.RTM. 750 dye, and ALEXA FLUOR.RTM.
790 dye. The NIR dyes are particularly advantageous for in vivo
imaging because they can be selectively visualized without exciting
endogenous materials present in living body. Some of the NIR dyes
have a large stokes shift, such that the excitation and emission
wavelengths are separated by at least 20, 30, 40, 50, 60, 70 or 80
nm.
[0087] "Solid support" means a substrate material having a rigid or
semi-rigid surface. Typically, at least one surface of the
substrate will be substantially flat, although it may be desirable
to physically separate certain regions with, for example, wells,
raised regions, etched trenches, or other such topology. Solid
support materials also include spheres (including microspheres),
rods (such as optical fibers) and fabricated and irregularly shaped
items.
[0088] Solid support materials include any materials that are used
as affinity matrices or supports for chemical and biological
molecule syntheses and analyses, such as, but are not limited to:
poly(vinylidene difluoride) (PVDF), polystyrene, polycarbonate,
polypropylene, nylon, glass, dextran, chitin, sand, pumice,
polytetrafluoroethylene, agarose, polysaccharides, dendrimers,
buckyballs, polyacrylamide, Kieselguhr-polyacrylamide non-covalent
composite, polystyrene-polyacrylamide covalent composite,
polystyrene-PEG [poly(ethylene glycol)] composite, silicon, rubber,
and other materials used as supports for solid phase syntheses,
affinity separations and purifications, hybridization reactions,
immunoassays and other such applications. The solid support may be
particulate or may be in the form of a continuous surface, such as
a microtiter dish or well, a glass slide, a silicon chip, a
nitrocellulose sheet, nylon mesh, or other such materials.
[0089] "Kit" means a packaged set of related components, typically
one or more compounds or compositions.
Detectable Biomolecules
[0090] Disclosed herein, in some embodiments, are biomolecules that
are detectably labeled with a plurality of fluorescent labels that
also comprise a spacer agent.
[0091] a. Spacers
[0092] A spacer may be any molecule that is capable of enhancing
fluorescent emissions when conjugated to a biomolecule that results
from excitation of a plurality of fluorescent labels independently
conjugated to the biomolecule. It is believed that this is due to
the reduction of fluorescence quenching of the fluorescent
labels.
[0093] In some embodiments, the spacer comprises an acetyl
(--C(O)CH.sub.3) group. In some embodiments, the spacer is an
acetate molecule. In some embodiments, the acetate molecule is
sulfo-NHS-acetate.
[0094] In some embodiments, the spacer agent comprises polyethylene
glycol (PEG). In some embodiments, the spacer agent comprises
(PEG)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14 or 15. In some embodiments, the spacer agent
comprises MS-(PEG)n.
[0095] In some embodiments, the spacer is selected from an
alkanoyl, alkenoyl, and alkynoyl (--C(O)C.sub.nH.sub.m in which n
is 1 to 20 atoms, m>n, the carbon atoms can be connected to each
other by single, double, and/or triple bonds, and the alkyl,
alkenyl, and/or alkynyl groups can be further substituted. In some
embodiments, specific substitutions include poly(ethylene)glycol
moieties, such as
--(OCH.sub.2CH.sub.2O).sub.x--(CH.sub.2).sub.y--OR in which x is 1
to 20, y is 1 to 6, and R is H or C.sub.1-6 alkyl. In some
embodiments, specific substitutions include ammonium
(--NH.sub.3.sup.+), quaternary ammonium ((--NR.sub.3.sup.+) groups
in which R is C.sub.1-6 alkyl, or phosphonium groups
(.about.PQ.sub.3.sup.+) in which Q is aryl, substituted aryl, or
C.sub.1-6 alkyl.
[0096] In some embodiments, the spacer is selected from alkyl,
alkenyl, or alkynyl groups (--C.sub.nH.sub.m in which n is 1 to 20
atoms, m>n, the carbon atoms can be connected to each other by
single, double, and/or triple bonds, and the alkyl, alkenyl, and/or
alkynyl groups can be further substituted. In some embodiments,
specific substitutions include negatively charged sulfonate groups
(--OSO.sub.3--), carboxylate groups (--CO.sub.2--), phosphate
groups (--OPO.sub.3--), and/or phosphonate groups (--PO.sub.3--).
In some embodiments, other substitutions include
poly(ethylene)glycol moieties, such as
--(OCH.sub.2CH.sub.2O).sub.x--(CH.sub.2).sub.y--OR in which x is 1
to 20, y is 1-6, and R is H or C.sub.1-6 alkyl. In some
embodiments, other specific substitutions include ammonium
(--NH.sub.3.sup.+), quaternary ammonium ((--NR.sub.3.sup.+) groups
in which R is C.sub.1-6 alkyl, or phosphonium groups
(.about.PQ.sub.3.sup.+) in which Q is aryl, substituted aryl, or
C.sub.1-6 alkyl.
[0097] In some embodiments, the spacer is positively charged. In
some embodiments, the spacer agent comprises betaine (i.e.,
trimethylglycine).
[0098] In some embodiments, the spacer agent is negatively
charged.
[0099] b. Fluorescent Label
[0100] The fluorescent dyes described herein function as reporter
molecules to confer a detectable signal, directly or indirectly, to
the sample as a result of conjugation to a functional group on the
protein, including, but not limited to, amine groups or thiol
groups. This results in the ability to detect the total protein in
a sample generally in combination with detection of a subset of the
total protein of the sample. In such instances the total protein
labels are detectable distinguished from the dye that labels a
subset of the total protein in the sample.
[0101] Where the detectable response is a fluorescence response, it
is typically a change in fluorescence, such as a change in the
intensity, excitation or emission wavelength, distribution of
fluorescence, fluorescence lifetime, fluorescence polarization, or
a combination thereof.
[0102] The fluorescent dyes can be any fluorophore known to one
skilled in the art. Typically the dye contains one or more aromatic
or heteroaromatic rings, that are optionally substituted one or
more times by a variety of substituents, including without
limitation, halogen, nitro, sulfo, cyano, alkyl, perfluoroalkyl,
alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or
heteroaryl ring system, benzo, or other substituents typically
present on chromophores or fluorophores known in the art.
[0103] A wide variety of fluorophores that may be suitable for
total protein labeling as described herein are already known in the
art (RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT
PROBES AND RESEARCH PRODUCTS (2002)). A fluorescent dye used in the
methods and compositions described herein is any chemical moiety
that exhibits an absorption maximum beyond 280 nm. Such chemical
moieties include, but are not limited to, a pyrene, sulfonated
pyrenes, sulfonated coumarins, sulfonated carbocyanines, sulfonated
xanthenes, an anthracene, a naphthalene, an acridine, a stilbene,
an indole an isoindole, an indolizine, a benzindole, an oxazole or
benzoxazole, a thiazole or benzothiazole, a
4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a carbocyanine, a
carbostyryl, a porphyrin, a salicylate, an anthranilate, an
azulene, a perylene, a pyridine, a quinoline, an isoquinoline, a
chromene, a borapolyazaindacene, a xanthene, a fluorescein, a
rosamine, a rhodamine, a rhodamine, benzo- or dibenzofluorescein,
seminaphthofluorescein, a naphthofluorescein, a bimane, an oxazine
or a benzoxazine, a carbazine, a phenalenone, a coumarin, a
benzofuran, a benzphenalenone) and derivatives thereof. As used
herein, oxazines include resorufins, aminooxazinones,
diaminooxazines, and their benzo-substituted analogs.
[0104] In one aspect the fluorescent dyes contain one or more
aromatic or heteroaromatic rings, that are optionally substituted
one or more times by a variety of substituents, including without
limitation, halogen, nitro, sulfo, cyano, alkyl, perfluoroalkyl,
alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or
heteroaryl ring system, benzo, or other substituents typically
present on chromophores or fluorophores known in the art. In one
aspect the fluorophore is a xanthene that comprises one or more
julolidine rings.
[0105] In an exemplary embodiment, the dyes are independently
substituted by substituents selected from the group consisting of
hydrogen, halogen, amino, substituted amino, alkyl, substituted
alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
alkoxy, sulfo, reactive group, solid support and carrier molecule.
In another embodiment, the xanthene dyes of this invention comprise
both compounds substituted and unsubstituted on the carbon atom of
the central ring of the xanthene by substituents typically found in
the xanthene-based dyes such as phenyl and substituted-phenyl
moieties. In many instances, dyes are rhodamine, fluorescein,
borapolyazaindacene, indole and derivatives thereof.
[0106] Choice of the reactive group used to attach the total
protein labels or expression tag labels to the protein to be
conjugated typically depends on the reactive or functional group on
the substance to be conjugated and the type or length of covalent
linkage desired. The types of functional groups typically present
on the organic or inorganic substances (biomolecule or
non-biomolecule) include, but are not limited to, amines, amides,
thiols, alcohols, phenols, aldehydes, ketones, phosphates,
imidazoles, hydrazines, hydroxylamines, disubstituted amines,
halides, epoxides, silyl halides, carboxylate esters, sulfonate
esters, purines, pyrimidines, carboxylic acids, olefmic bonds, or a
combination of these groups. In proteins a variety of sites may
occur including, but not limited to, amines, thiols, alcohols and
phenols.
[0107] Amine reactive fluorescent dyes that can be used in the
protein labeling methods described herein include, but are not
limited to, ALEXA FLUOR.RTM. 350, ALEXA FLUOR.RTM. 405, ALEXA
FLUOR.RTM. 430, ALEXA FLUOR.RTM. 488, ALEXA FLUOR.RTM. 500, ALEXA
FLUOR.RTM. 514, ALEXAFLUOR.RTM. 532, ALEXA FLUOR.RTM. 546, ALEXA
FLUOR.RTM. 555, ALEXA FLUOR.RTM. 568, ALEXAFLUOR.RTM. 594, ALEXA
FLUOR.RTM. 610-X, ALEXA FLUOR.RTM. 633, ALEXA FLUOR.RTM. 647,
ALEXAFLUOR.RTM. 660, ALEXA FLUOR.RTM. 680, ALEXA FLUOR.RTM. 700,
ALEXA FLUOR.RTM. 750, ALEXAFLUOR.RTM. 790, AMCA-X, BODIPY.RTM.
630/650, BODIPY.RTM. 650/665, BODIPY.RTM. FL, BODIPY.RTM. TMR,
BODIPY.RTM. TR, BODIPY.RTM. TR-X, CASCADE BLUE.RTM., Dinitrophenyl,
Fluorescein, HEX, JOE, MARINA BLUE.RTM., OREGON GREEN.RTM. 488,
OREGON GREEN.RTM. 514, PACIFIC BLUE.TM., PACIFIC ORANGE.TM.,
RHODAMINE GREEN.TM., QSY.RTM. 7, QSY.RTM. 9, QSY.RTM. 21, QSY.RTM.
35, ROX, RHODAMINE RED.TM., TET, TAMRA, tetramethyl rhodamine, FAM,
TEXAS RED.RTM. and
7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl
ester (DDAO-SE).
[0108] In some embodiments, the fluorogenic reagents/dyes that bind
to tags attached to proteins used in the protein labeling methods
described herein are biarsenical fluorophore, including, a
biarsenical derivative of fluorescein, such as, by way of example
only, FlAsH-EDT2
(4'-5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(2,2-ethanedithiol)2)
(LUMIO.TM. Green, Life Technologies Corp., Carlsbad, Calif.), or a
biarsenical derivative of resorufin such as, by way of example
only, ReAsh-EDT2 (LUMIO.TM. Red, Life Technologies Corp., Carlsbad,
Calif.), or may instead be an oxidized derivative, such as
ChoXAsH-EDT2 or HoXAsH-EDT2. In addition, the biarsenical
fluorophore can be a biarsenical derivative of other known
fluorophores, including, but not limited to, the ALEXA FLUOR.RTM.
series described herein, such as, by way of example only, ALEXA
FLUOR.RTM. 350, ALEXA FLUOR.RTM. 430, ALEXA FLUOR.RTM. 488, ALEXA
FLUOR.RTM. 532, ALEXA FLUOR.RTM. 546, ALEXA FLUOR.RTM. 568, ALEXA
FLUOR.RTM. 594, ALEXAFLUOR.RTM. 663 and ALEXA FLUOR.RTM. 660,
available commercially from Molecular Probes (Eugene, Oreg.).
[0109] In some embodiments, the biarsenical fluorophore can be
present at a concentration of at least about 1 .mu.M, 2 .mu.M, 3
.mu.M, 4 .mu.M, 5 .mu.M, 10 .mu.M, 15 .mu.M, 20 .mu.M, 30 .mu.M, 40
.mu.M, 50 .mu.M, 100 .mu.M or more, and at a concentration of no
more than about 500 .mu.M, 400 .mu.M, 300 .mu.M, 200 .mu.M, 100
.mu.M, 90 .mu.M, 80 .mu.M, 70 .mu.M, 60 .mu.M, 50 .mu.M, 40 .mu.M,
30 .mu.M, 20 .mu.M, 15 .mu.M, 10 .mu.M, 5 .mu.M, 4 .mu.M, 3 .mu.M,
2 .mu.M or 1 .mu.M.
[0110] In some embodiments, the tag attached to a protein to which
such fluorogenic dyes binds is a tetracysteine peptide motif,
cys-cys-Xn-cys-cys (SEQ ID NO: 1), wherein each X is any natural
amino acid, non-natural amino acid or combination thereof, and n is
an integer from 2-100. In certain embodiments, n is an integer from
2-90, while in other embodiments n is an integer from 2-80. In
certain embodiments, n is an integer from 2-70, while in other
embodiments n is an integer from 2-60. In certain embodiments, n is
an integer from 2-50, while in other embodiments n is an integer
from 2-40. In certain embodiments, n is an integer from 2-30, while
in other embodiments n is an integer from 2-20. In certain
embodiments, n is an integer from 2-10, while in other embodiments
n is an integer from 2-5. The natural amino acids if such motifs
include, but are not limited to, alanine, arginine, asparagine,
aspartic acid, cysteine, glutamic acid, glutamine, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine, tryptophan, tyrosine and valine. In
certain embodiments, the tetracysteine tag has the sequence CCPGCC
(SEQ ID NO: 2). In other embodiments a 12 amino acid peptide
containing the tetracysteine motif is used including, but not
limited to, the amino acid sequence, AGGCCPGCCGGG (SEQ ID NO: 3).
In addition, the protein can be labeled with a single tetracysteine
tag or the protein can be labeled with a plurality of tetracysteine
tags including, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 tetracysteine tags. Such tags
may be separated from one another within the primary amino acid
sequence of the protein or directly multimerized in tandem as
concatemers.
[0111] In certain embodiments, the tetracysteine peptide has the
sequence cys-cys-Xn-cys-X-cys-X (SEQ ID NO: 1), wherein each X is
any natural amino acid, non-natural amino acid or combination
thereof, and n is an integer from 2-100. In certain embodiments, n
is an integer from 2-90, while in other embodiments n is an integer
from 2-80. In certain embodiments, n is an integer from 2-70, while
in other embodiments n is an integer from 2-60. In certain
embodiments, n is an integer from 2-50, while in other embodiments
n is an integer from 2-40. In certain embodiments, n is an integer
from 2-30, while in other embodiments n is an integer from 2-20. In
certain embodiments, n is an integer from 2-10, while in other
embodiments n is an integer from 2-5. The natural amino acids if
such motifs include, but are not limited to, alanine, arginine,
asparagine, aspartic acid, cysteine, glutamic acid, glutamine,
glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine and
valine. In certain embodiments, the tetracysteine tag has the
sequence CCGGKGNGGCGC (SEQ ID NO: 4).
[0112] The tetracysteine peptide tag or tags can be recombinantly
fused to the protein desired to be labeled, either at the
N-terminus, C-terminus, or in frame within the protein sequence;
expression vectors for creating tetracysteine-fused recombinant
proteins may readily be constructed using techniques known to one
of skill in the art. In certain embodiments the
tetracysteine-tagged protein is expressed recombinantly in host
cells including, but not limited to, in bacterial host cells, in
fungal host cells, in insect cells, in plant cells, or in mammalian
cells. Such bacterial host cells include, but are not limited to,
gram negative and gram positive bacteria of any genus, including,
by way of example only, Escherichia sp. (e.g., E. coli), Klebsiella
sp., Streptomyces sp., Streptococcus sp., Shigella sp.,
Staphylococcus sp., Erwinia sp., Klebsiella sp., Bacillus sp.
(e.g., B. cereus, B. subtilis and B. megaterium), Serratia sp.,
Pseudomonas sp. (e.g., P. aeruginosa and P. syringae) and
Salmonella sp. (e.g., S. typhi and S. typhimurium). Suitable
bacterial strains and serotypes suitable for the invention can
include E. coli serotypes K, B, C, and W. A typical bacterial host
is E. coli strain K-12. The fungal host cells include, by way of
example only, Saccharomyces cerevisiae cells, while the mammalian
cells include, by way of example only, including human cells. In
such embodiments, the protein sample containing the protein of
interest is a lysate of the host cells, that can be unpurified,
partially purified, or substantially purified prior to labeling and
analysis using the methods described herein.
[0113] In other embodiments, the tetracysteine-tagged protein is
expressed in vitro, wherein the protein sample containing the
protein of interest is the cell-free extract in which translation
(and, optionally, transcription) is performed, or a partially
purified or purified fraction thereof. In embodiments in which the
extract permits coupled transcription and translation in a single
cell-free extract, such as the E. coli-based EXPRESSWAY.TM. or
EXPRESSWAY.TM. Plus systems (Life Technologies Corp., Carlsbad,
Calif.), the sample is the cell-free extract in which transcription
and translation commonly occur, or a fraction thereof.
[0114] Alternatively, GATEWAY.RTM. Technology (Life technologies
Corp., Carlsbad, Calif.) is a universal cloning technology that can
be used to express a gene of interest in E. coli.
[0115] The protein to be labeled using the biarsenical dyes
described herein can be any protein having a tetracysteine motif.
The protein to which the tetracysteine tag or tags is fused or
conjugated can be any protein desired to be labeled, either
naturally-occurring or nonnaturally occurring. Naturally-occurring
proteins may have known biological function or not, and may be
known to be expressed or only predicted from genomic sequence. The
protein, if naturally-occurring, can be a complete protein or only
a fragment thereof. The tetracysteine-tagged protein can thus be an
animal protein, such as a human protein or non-human mammalian
protein, a fungal protein, a bacterial protein, including
eubacterial and archaebacterial protein, a plant protein, an insect
protein or a viral protein.
[0116] In addition to the tetracysteine tag, other protein
sequences can usefully be recombinantly appended to the proteins
desired to be labeled. Among such additional protein sequences are
linkers and/or short tags, usefully epitope tags, such as a FLAG
tag, or a myc tag, or other sequences useful for purification, such
as a polyhistidine (e.g., 6.times.his) tag. Alternatively, the
tetracysteine tag or tags can be chemically conjugated to proteins
to be labeled using art-routine conjugation chemistries.
[0117] In some embodiments, the fluorescent label is positively
charged. In some embodiments, the fluorescent label is negatively
charged.
[0118] In some embodiments, the excitation wavelength of the
fluorescent label is between 350 and 850 nm. In some embodiments,
the excitation wavelength of the fluorescent label is far red. In
some embodiments, the excitation wavelength of the fluorescent
label is near infrared. In some embodiments, the excitation
wavelength of the fluorescent label is ultraviolet (UV).
[0119] In some embodiments, the fluorescent label comprises a
DYLIGHT.TM..RTM. fluor. In some embodiments, the DYLIGHT.TM..RTM.
fluor is selected form DYLIGHT.TM. 350, DYLIGHT.TM. 405,
DYLIGHT.TM. 488, DYLIGHT.TM. 550, DYLIGHT.TM. 594, DYLIGHT.TM. 633,
DYLIGHT.TM. 650, DYLIGHT.TM. 680, DYLIGHT.TM. 755, and DYLIGHT.TM.
800. In some embodiments, the DYLIGHT.TM. fluor is conjugated to a
PEG molecule (e.g., 2.times.PEG, 4.times.PEG, 8.times.PEG, or
12.times.PEG).
[0120] In some embodiments, the fluorescent label comprises an
ALEXA FLUOR.RTM.. In some embodiments, the ALEXA FLUOR.RTM. is
selected from ALEXA FLUOR.RTM. 350, ALEXA FLUOR.RTM. 405, ALEXA
FLUOR.RTM. 430, ALEXA FLUOR.RTM. 488, ALEXA FLUOR.RTM. 532, ALEXA
FLUOR.RTM. 546, ALEXAFLUOR.RTM. 555, ALEXA FLUOR.RTM. 568, ALEXA
FLUOR.RTM. 594, ALEXA FLUOR.RTM. 610, ALEXAFLUOR.RTM. 633, ALEXA
FLUOR.RTM. 635, ALEXA FLUOR.RTM. 647, ALEXA FLUOR.RTM. 660,
ALEXAFLUOR.RTM. 680, ALEXA FLUOR.RTM. 700, ALEXA FLUOR.RTM. 750 and
ALEXA FLUOR.RTM. 790.
[0121] In some embodiments, the fluorescent label comprises a
moiety selected from xanthene; coumarin; cyanine; pyrene; oxazine;
borapolyazaindacene; benzopyrylium; and carbopyronine.
[0122] In some embodiments, the fluorescent label comprises
fluorescein (e.g., Cy2 or FITC). In some embodiments, the
fluorescent label comprises rhodamine (e.g., TRITC or Cy3). In some
embodiments, the fluorescent label comprises MCA, coumarin,
RHODAMINE RED, TEXAS RED, CASCADE BLUE, Cy5, Cy5.5, IR680, IR800
and Cy7.
[0123] In some embodiments, the fluorescent label is a modified
fluorescent label (e.g., the fluorescent label has been sulfonated
or conjugated with PEG).
[0124] In some embodiments, the fluorescent label is a fluorescent
protein. In some embodiments, the fluorescent protein is a
phycobiliprotein. Examples of phycobiliproteins useful in the
present invention are allophycocyanin, phycocyanin, phycoerythrin,
allophycocyanin B, B-phycoerythrin, phycoerythrocyanin, and
b-phycoerythrin. The structures of phycobiliproteins have been
studied and their fluorescent spectral properties are known. See A.
N. Glazer, "Photosynthetic Accessory Proteins with Bilin Prosthetic
Groups," Biochemistry of Plants, Volume 8, M. D. Hatch and N. K.
Boardman, EDS., Academic Press, pp. 51-96 (1981), and A. N. Glazer,
"Structure and Evolution of Photosynthetic Accessory Pigment
Systems with Special Reference to Phycobiliproteins," The Evolution
of Protein Structure and Function, B. S. Sigman and M. A. Brazier,
EDS., Academic Press, pp. 221-244 (1980). In some embodiments, the
fluorescent protein has absorption maxima of at least about 450 nm,
often at least about 500 nm, having Stokes shifts of at least 15
nm, often at least about 25 nm, and has fluorescence emission
maxima of at least about 500 nm, often at least about 550 nm.
[0125] In some embodiments, the fluorescent label is a
dipyrromethene boron difluoride dye, as disclosed in US
2014/0349,893, incorporated by reference herein, in its
entirety.
[0126] The amine reactive fluorogenic reagents used in the protein
labeling methods described herein include, but are not limited to,
aroyl-2-quinoline-carboxaldehyde type reagents. Such reagents have
been described in U.S. Pat. No. 5,459,272 and U.S. Pat. No.
5,631,374, each of which is herein incorporated by reference in
their entirety. In some embodiments, the
aroyl-2-quinoline-carboxaldehyde reagent used is
3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde or
3-(2-furoyl)quinoline-2-carboxaldehyde). In certain embodiments,
the amine reactive fluorogenic reagent is
3-(2-furoyl)quinoline-2-carboxaldehyde), while in other
embodiments, the amine reactive fluorogenic reagent is 3-(4
carboxybenzoyl)-quinoline-2-carboxaldehyde.
[0127] c. Biomolecules
[0128] A biomolecule that can be used in the compositions and
methods disclosed herein include any biomolecule that is useful in
molecular biology applications.
[0129] In some embodiments, the biomolecule is an antibody (e.g., a
primary antibody or a secondary antibody). In some embodiments, the
biomolecule is an antibody fragment. Antibodies used in the
practice of the invention may be antibody is polyclonal, monoclonal
or engineered and may be from any source (e.g., shark, chicken or a
mammal, such as a llama, human mouse, rabbit, goat, rat, etc.).
Further, humanized antibodies may be used.
[0130] In some embodiments, the antibody is chimeric.
[0131] In some embodiments, the biomolecule is a protein or
polypeptide. In some embodiments, the biomolecule is a recombinant
polypeptide.
[0132] In some embodiments, the biomolecule is a nucleic acid
molecule. In some embodiments, the nucleic acid molecule is an
oligonucleotide (e.g., between 15 and 50 nucleotides in length). In
some embodiments, the nucleic acid molecule is greater than 50
nucleotides in length, greater than 100 nucleotides in length,
greater than 500 nucleotides in length, greater than 1 kb in
length, greater than 2 kb in length, or greater than 5 kb in
length.
[0133] d. Conjugation of Fluorescent Dyes to Biomolecules
[0134] After selection of an appropriate dye with the desired
spectral characteristics, typically where the excitation wavelength
is at least 580 nm, the dyes may be conjugated to a targeted
carrier molecule, using methods well known in the art (Haugland,
MOLECULAR PROBES HANDBOOK, supra, (2002)). In many instances,
conjugation to form a covalent bond consists of simply mixing the
reactive compounds of the present invention in a suitable solvent
in which both the reactive compound and the spacer molecule to be
conjugated are soluble. The reaction, in many instances, proceeds
spontaneously without added reagents at room temperature or below.
For those reactive compounds that are photoactivated, conjugation
is facilitated by illumination of the reaction mixture to activate
the reactive compound. Chemical modification of water-insoluble
substances, so that a desired compound-conjugate may be prepared,
is, in many instances, performed in an aprotic solvent such as
dimethylformamide, dimethylsulfoxide, acetone, ethyl acetate,
toluene, or chloroform. Similar modification of water-soluble
materials is readily accomplished through the use of the instant
reactive compounds to make them more readily soluble in organic
solvents.
[0135] Preparation of biomolecule (e.g., proteins) conjugates
typically comprises first dissolving the biomolecule to be
conjugated in aqueous buffer at about. 1-10 mg/mL at room
temperature or below. For example, bicarbonate buffers (pH about
8.3), carbonate and borate buffers (pH about 9) are especially
suitable for reaction with succinimidyl esters, phosphate buffers
(pHs of about 7.2-8) for reaction with thiol-reactive functional
groups and carbonate or borate buffers (pH about 9) for reaction
with isothiocyanates and dichlorotriazines. The appropriate
reactive compound is then dissolved in a nonhydroxylic solvent
(usually DMSO or DMF) in an amount sufficient to give a suitable
degree of conjugation when added to a solution of the biomolecule
to be conjugated. The appropriate amount of compound for any
biomolecule (e.g., protein) or other component is conveniently
predetermined by experimentation in which variable amounts of the
compound are added to the biomolecule, the conjugate is
chromatographically purified to separate unconjugated compound and
the compound-biomolecule conjugate is tested in its desired
application.
[0136] Any number of buffers may be used for conjugation reactions,
as well as for other set out herein. Using Examples 1 and 5 for
purposes of illustration, phosphate buffered saline and borate
buffer can be and are used in the conjugation reactions. It is also
believed that carbonate buffer at pH 9.5 can be used. Along these
lines, it is believed that higher pH conjugations require a lower
molar excess of dye and spacer molecules. The invention thus
includes compositions and methods for performing conjugation
reactions where the pH is from about 4.0 to about 10.0 (e.g., from
about 4.0 to about 10.0, from about 5.0 to about 10.0, from about
6.0 to about 10.0, from about 7.0 to about 10.0, from about 7.5 to
about 10.0, from about 8.0 to about 10.0, etc.). Conjugation
reactions may be performed in Borate Buffer 50 mM, pH 8.5 with a
fluorescent dye, or a mixture of each fluorescent dye and a spacer
selected from NHS-Acetate, NHS-MS(PEG).sub.4, NHS-MS(PEG).sub.8 or
NHS-MS(PEG).sub.12 at various molar excesses. The labeling
reactions may be incubated for approximately 1 hour at room
temperature (RT). The NHS activated dye and the NHS activated
spacer agents may be combined before addition to the antibody so
that both reactions are concurrent, allowing for a random spacing
of the dye substitution and of the spacers.
[0137] Buffers that may be used in the practice of the invention
include 2-(N-morpholino)ethanesulfonic acid (MES), phosphate,
3-(N-morpholino)propanesulfonic acid (MOPS),
tris(hydroxymethyl)aminomethane (TRIS), borate,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and
carbonate buffers as examples.
[0138] The concentration of the fluorogenic reagents used in the
biomolecule (e.g., protein) labeling methods described herein are
in the range from 50 nM to 100 mM (e.g., from about 50 nM to about
25 mM, from about 50 nM to about 10 mM, from about 50 nM to about 5
mM, from about 100 nM to about 10 mM, from about 100 nM to about 5
mM, from about 200 nM to about 5 mM, from about 50 nM to about 100
.mu.M, from about 1 .mu.M to about 100 .mu.M, etc.). In certain
embodiments such concentrations are obtained by dilution of a stock
solution of the fluorogenic reagents having a concentration in the
range from 100 nM to 200 mM (e.g., from about 10 .mu.M to about 500
.mu.M, from about 1 .mu.M to about 100 .mu.M, from about 100 .mu.M
to about 200 .mu.M, etc.). In certain embodiments the concentration
of the stock solution is 100 mM. In certain embodiments the
concentration of the stock solution is 50 mM. In certain
embodiments the concentration of the stock solution is 20 mM. In
certain embodiments the concentration of the stock solution is 10
mM. In certain embodiments the concentration of the stock solution
is 1 mM. In certain embodiments the concentration of the stock
solution is 500 .mu.M. In certain embodiments the concentration of
the stock solution is 10 .mu.M.
[0139] In some embodiments, the concentration of the amine reactive
fluorescent dyes used in the biomolecule (e.g., protein) labeling
methods described herein are in the range from 50 nM to about 100
mM (e.g., from about 50 nM to about 50 mM, from about 50 nM to
about 25 mM, from about 50 nM to about 10 mM, from about 50 nM to
about 5 mM, from about 50 nM to about 5 .mu.M, from about 50 nM to
100 .mu.M, from about 100 nM to about 5 mM, etc.). In certain
embodiments such concentrations are obtained by dilution of a stock
solution of the fluorogenic reagents having a concentration in the
range from 100 nM to 200 mM. In certain embodiments the
concentration of the stock solution is 100 mM. In certain
embodiments the concentration of the stock solution is 50 mM. In
certain embodiments the concentration of the stock solution is 20
mM. In certain embodiments the concentration of the stock solution
is 10 mM. In certain embodiments the concentration of the stock
solution is 1 mM. In certain embodiments the concentration of the
stock solution is 500 .mu.M. In certain embodiments the
concentration of the stock solution is from 10 .mu.M to 500 .mu.M.
In certain embodiments the concentration of the stock solution is
from 1 .mu.M to 100 .mu.M. In certain embodiments the concentration
of the stock solution is 10 .mu.M. In certain embodiments the
concentration of the stock solution is from 100 .mu.M to 200
.mu.M.
[0140] In some embodiments, the concentration of the proteins or
protein fragments (e.g., antibody fragments) labeled using the
methods described herein is in the range from 0.01 mg/mL to 200
mg/mL (e.g., from about 0.1 mg/mL to 100 mg/mL, from about 0.1
mg/mL to about 50 mg/mL, from about 0.1 mg/mL to about 10 mg/mL,
from about 0.2 mg/mL to about 100 mg/mL, from about 0.2 mg/mL to
about 50 mg/mL, from about 0.2 mg/mL to about 10 mg/mL, from about
0.3 mg/mL to 10 mg/mL, from about 0.4 mg/mL to about 10 mg/mL, from
about 0.5 mg/mL to about 10 mg/mL etc.).
[0141] In some instances, more than one dye molecule may be
attached at each location on a biomolecule. One way of attaching
more than one dye molecule at a single location on a biomolecule is
through the use of conjugation molecules that bind to more than one
dye molecule. These conjugation molecules are then connected to the
biomolecule and carry with them multiple dye molecules. As an
example, polymeric dendrimers, such as those set out in U.S. Patent
Publication 2012/0256102, may be used multiple fluorescent dye
molecules conjugated to a single polymeric backbone or core
(referred to as "dendrimers" therein) for the attachment of these
dye molecules to a biomolecule. These dendrimers may have regular
or irregular branched polymeric network structures that allow for
the chemical attachment of multiple dye molecules, multiple color
dyes, and/or multiple functional groups, in a combinatorial
fashion.
[0142] Additional examples of conjugation molecules that may be
used include many of the same molecules used as spacers. Thus, in
some instances, the spacer molecule and the conjugation molecule
will have the same structure with the exception that the
conjugation molecule has dye molecules bound to it. Along these
lines, various forms of PEG molecules may be used as conjugation
molecules. Thus, the spacer molecule may contain the dye molecule
(e.g., the fluorescent label) (see FIG. 18).
[0143] The invention thus contemplates the use of conjugation
molecules that each have, on average, from about two to about fifty
(e.g., from about two to about forty-five, from about two to about
fourty, from about two to about thirty-five, from about two to
about thirty, from about two to about twenty, from about five to
about fourty-five, from about ten to about fourty-five, etc.)
associated dye molecules. In many instances, the standard deviation
in the average number of dye molecules bound to conjugation
molecules will be less than 10%, 15%, and/or 20%.
[0144] The degree of labeling may be measure for labeled
biomolecules. Degree of labeling may be calculated as follows.
First, the molarity of the labeled biomolecule is calculated using,
for example, the formula:
Protein concentration ( M ) = A 280 - ( A max .times. CF ) .times.
dilution factor ##EQU00002##
.epsilon.=protein molar extinction coefficient (e.g., the molar
extinction coefficient of IgG is .about.210,000 M.sup.-1 cm.sup.-1)
A.sub.max=Absorbance (A) of a dye solution measured at the
wavelength maximum (.lamda.max) for the dye molecule CF=Correction
factor; adjusts for the amount of absorbance at 280 nm caused by
the dye (see Table 8) Dilution factor=the extent (if any) to which
the protein:dye sample was diluted for absorbance measurement
[0145] The degree of labeling is then calculated using the
formula:
Moles dye per mole protein = A max of the labeled protein ' .times.
protein concentration ( M ) .times. dilution factor
##EQU00003##
.epsilon.'=molar extinction coefficient of the fluorescent dye
TABLE-US-00008 TABLE 8 Characteristics of Exemplary Dye Wavelength
Extinction Correction Fluorophore Maximum (.lamda..sub.max)
Coefficient ( ') Factor (CF) DyLight .RTM. 350 353 nm 15,000
M.sup.-1 cm.sup.-1 0.1440 DyLight 405 405 nm 30,000 M.sup.-1
cm.sup.-1 0.5640 DyLight 488 493 nm 70,000 M.sup.-1 cm.sup.-1
0.1470 DyLight 550 562 nm 150,000 M.sup.-1 cm.sup.-1 0.0806 DyLight
594 595 nm 80,000 M.sup.-1 cm.sup.-1 0.5850 DyLight 633 627 nm
170,000 M.sup.-1 cm.sup.-1 0.1100 DyLight 650 652 nm 250,000
M.sup.-1 cm.sup.-1 0.0371 DyLight 680 684 nm 140,000 M.sup.-1
cm.sup.-1 0.1280 DyLight 755 754 nm 220,000 M.sup.-1 cm.sup.-1
0.0300 DyLight 800 777 nm 270,000 M.sup.-1 cm.sup.-1 0.0452
Fluorescein isothiocyanate (FITC), 494 nm 68,000 M.sup.-1 cm.sup.-1
0.3000 NHS-Fluorescein, 5-IAF Tetramethyl-rhodamine-5-(and6)- 555
nm 65,000 M.sup.-1 cm.sup.-1 0.3400 isothiocyanate (TRITC)
NHS-Rhodamine 570 nm 60,000 M.sup.-1 cm.sup.-1 0.3400 Texas Red
.RTM. Sulfonyl Chloride 595 nm 80,000 M.sup.-1 cm.sup.-1 0.1800
R-Phycoerythrin 566 nm 1,863,000 M.sup.-1 cm.sup.-1 0.1700
AMCA-NHS, AMCA-Sulfo-NHS 346 nm 19,000 M.sup.-1 cm.sup.-1 0.1900 or
AMCA-Hydrazide
[0146] In some embodiments of the invention, enhanced fluorescence
is observed for biomolecules comprising spacers with lower degree
of labeling (DOL) than biomolecules without spacers. As an example,
assume there is antibody that has been labeled with a fluorescent
dye separately with and without a spacer. On an equivalent DOL
basis, the antibody labeled with the dye that also has spacers
bound to it may exhibit an enhancement in fluorescence of between
1.5 and 3.5 times, where 1 would present the same level of
fluorescence for both antibodies. The point being that the amount
of fluorescent signal on a per dye molecule basis increases for
biomolecules bound to both dye and spacer.
[0147] Following addition of the reactive compound to the component
solution, the mixture is incubated for a suitable period (typically
about 1 hour at room temperature to several hours on ice), the
excess compound is removed by gel filtration, dialysis, HPLC,
adsorption on an ion exchange or hydrophobic polymer or other
suitable means. The compound-conjugate is used in solution or
lyophilized. In this way, suitable conjugates can be prepared from
antibodies, antibody fragments, and other targeting carrier
molecules.
[0148] The incubation temperatures used in the methods described
herein can be room temperature, ambient temperature, or
temperatures above room temperature, such as, by way of example
only, at least about 26.degree. C., 27.degree. C., 28.degree. C.,
29.degree. C., 30.degree. C., 40.degree. C., 50.degree. C.,
60.degree. C., 70.degree. C., 80.degree. C., even as high as
90.degree. C., 95.degree. C., 96 C, 97.degree., 98.degree. C.,
99.degree. C. or 100.degree. C. The first incubation temperature
and the second incubation temperature used in the methods described
herein can be the same or different. In some embodiments, the first
incubation temperature is between 20.degree. C. and 80.degree. C.,
between 25.degree. C. and 30.degree. C., and/or at ambient or room
temperature. In some embodiments, the second incubation temperature
is between 20.degree. C. and 80.degree. C., between 65.degree. C.
and 75.degree. C., and/or at approximately 70.degree. C. In other
embodiments, the second incubation temperature is at ambient or
room temperature.
[0149] The incubation times used in the methods described herein
include, but are not limited to, for at least 30 seconds, at least
1 minute, at least 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6
minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes,
20 minutes, 30 minutes, 40 minutes, 50 minutes, at least 1 hour, or
any range herein. The first incubation time and the second
incubation time used in the methods described herein can be the
same or different. In one embodiment, the first incubation time is
0 to 60 minutes, 5 to 10 minutes, and/or at 5 to 10 minutes at room
temperature. In some embodiments, the second incubation time is 0
to 20 minutes and/or approximately 10 minutes. In specific
instances, the second incubation time is approximately 10 minutes
at approximately 70.degree. C. In other embodiments, the first
incubation time is 1 to 3 hours at 25.degree. C., the second
incubation time is overnight at 25.degree. C. and the third
incubation time is 2 to 3 hours at 37.degree. C.
[0150] Conjugates of polymers, including biopolymers and other
higher molecular weight polymers are typically prepared by means
well recognized in the art (for example, Brinkley et al.,
Bioconjugate Chem., 3:2 (1992)). In these embodiments, a single
type of reactive site may be available, as is typical for
polysaccharides) or multiple types of reactive sites (e.g., amines,
thiols, alcohols, phenols) may be available, as is typical for
proteins. Selectivity of labeling is best obtained by selection of
an appropriate reactive dye. For example, modification of thiols
with a thiol-selective reagent such as a haloacetamide or
maleimide, or modification of amines with an amine-reactive reagent
such as an activated ester, acyl azide, isothiocyanate or
3,5-dichloro-2,4,6-triazine. Partial selectivity can also be
obtained by careful control of the reaction conditions.
[0151] When modifying polymers with the compounds, an excess of
compound is typically used, relative to the expected degree of
compound substitution. Any residual, unreacted compound or a
compound hydrolysis product is typically removed by dialysis,
chromatography or precipitation. Presence of residual, unconjugated
dye can be detected by thin layer chromatography using a solvent
that elutes the dye away from its conjugate. In all cases it is
usually the case that the reagents be kept as concentrated as
practical so as to obtain adequate rates of conjugation.
[0152] In certain embodiments of the methods described herein a
control protein or proteins may be labeled to monitor the
effectiveness of labeling, either in a parallel reaction or, if
readily resolvable from the protein desired to be labeled, by
inclusion in the same reaction.
[0153] In certain embodiments of the methods described herein, the
proteins of the labeled sample can usefully be resolved in parallel
with a series of fluorescent molecular weight standards. Usefully,
the standards are spectrally matched to at least one fluorophore
used to label the proteins. Such spectral matching can be
accomplished, for example, by using tetracysteine-tagged protein
standards that are labeled in parallel with the same biarsenical
fluorophore used to label the protein sample, or by using standards
having a fluorescent moiety that is spectrally matched to the
biarsenical fluorophore or other fluorophore used to label the
sample proteins. Examples of standards useful in the practice of
the present invention include the BENCHMARK.TM. family of protein
standards (Life Technologies Corp., Carlsbad, Calif.) and
MARKL2.TM. Unstained Standard (Life Technologies Corp., Carlsbad,
Calif.).
[0154] The methods and compositions described herein can also be
used to quantitate the amount of fluorescently labeled protein
present in a sample. In certain embodiments, the methods described
herein further comprise quantitating the amount of fluorescence
from the biarsenical fluorophore. In certain embodiments, the
methods described herein further comprise quantitating the amount
of fluorescence from the amine reactive fluorescent dye. In certain
embodiments, the methods described herein further comprise
quantitating the amount of fluorescence from the fluorescent moiety
from the amine reactive fluorogenic reagent. The quantitation can
be done without resolution of the proteins present in the protein
sample or after the proteins have been partially or fully resolved,
as by electrophoresis, such as PAGE, 2D-PAGE, or IEF or
chromatography or combinations thereof.
[0155] e. Conjugation of Spacer Molecules to Biomolecule
[0156] In some embodiments, the spacer molecule is conjugated to
the biomolecule using NHS-ester chemistries are described in the
examples herein, other chemistries such as maleimide, pyridyl
disulfide and hyrazides, as well as the SITECLICK.TM. technology
involving azide/alkyne may also be used for this conjugation
strategy.
[0157] In some embodiments, the spacer molecule is conjugated to a
biomolecule (e.g., an antibody) at primary lysine side chains
present on a protein, such as an antibody.
[0158] In some embodiments, the concentration of a protein, protein
fragment or other biomolecule labeled using the methods described
herein is in the range from about 0.01 mg/mL to about 200 mg/mL
(e.g., from about 0.1 mg/mL to about 100 mg/mL, from about 0.1
mg/mL to about 50 mg/mL, from about 0.1 mg/mL to about 10 mg/mL,
from about 0.2 mg/mL to about 100 mg/mL, from about 0.2 mg/mL to
about 50 mg/mL, from about 0.2 mg/mL to about 10 mg/mL, from about
0.3 mg/mL to about 10 mg/mL, from about 0.4 mg/mL to about 10
mg/mL, from about 0.5 mg/mL to about 10 mg/mL, etc.).
[0159] In some embodiments, the dye to protein ratio of the
fluorescent label to the antibody is between 1 and 50. In some
embodiments, the dye to protein ratio of the fluorescent label to
the antibody is between 5 and 30. In some embodiments, the dye to
protein ratio of the fluorescent label to the antibody is between 1
and 20.
[0160] In some embodiments, the spacer to protein ratio is between
1 and 50. In some embodiments, the spacer agent to protein ratio is
between 5 and 30. In some embodiments, the spacer agent to protein
ratio is between 1 and 20.
[0161] In some embodiments, the spacer agent is added in molar
excess to the plurality of fluorescent labels in an amount between
0.1 to 25, between 1 to 15, or between 2.5 to 10 fold. In some
embodiments, the spacer agent is in molar excess to the plurality
of fluorescent labels in an amount of 2.5 fold. In some
embodiments, the spacer agent is in molar excess to the plurality
of fluorescent labels in an amount of 5 fold. In some embodiments,
the spacer agent is in molar excess to the plurality of fluorescent
labels in an amount of 7.5 fold. In some embodiments, the spacer
agent is in molar excess to the plurality of fluorescent labels in
an amount of 10 fold.
[0162] In some embodiments, the spacer agent is conjugated to a
nucleic acid molecule. Protocols for conjugating moieties (e.g.,
fluorescent labels) to nucleic acids are described in the art (see,
e.g., Rombouts et al., Bioconjugate Chem., 27:280-207 (2016)). One
method for labeling nucleic acid molecules is through the use of
the ARES.TM. ALEXA FLUOR.RTM. 488 DNA Labeling Kit (Thermo Fisher,
cat. no. A21665). Amine-modified nucleotides may also be used to
enable labeling of nucleic acid molecules. For example,
5-aminohexylacrylamido-dUTP (aha-dUTP) and
5-aminohexylacrylamido-dCTP (aha-dCTP) can be used to produce
amine-modified DNA by conventional enzymatic incorporation methods
such as reverse transcription, nick translation, random primed
labeling or PCR. The amine-modified DNA can then be labeled with
any amine-reactive dye or hapten. This two-step technique
consistently produces a uniform and high degree of DNA labeling
that is difficult to obtain by other methods.
[0163] One method for achieving a high DOL, with spatially
separated fluorescent labels, is set out in FIG. 18. FIG. 18 shows
the preparation of fluorescently labeled branched PEG molecules and
the attachment of the resulting PEG molecules (direct spacers) to
an antibody, where the PEG molecules are covalently linked to the
antibody by CLICK-IT.TM. reactions (see Example 7).
[0164] The PEG molecules shown in FIG. 18 are each covalently
linked to seven fluorescent labels. Further, the fluorescent labels
may be attached to the individual PEG molecules in a manner such
that the labels have a specified "brush" length with respect to the
labeled molecule. "Brush length" refers to the extended length of
chemical groups that attach a fluorescent labels to a labeled
molecule. As an example, assuming that the average monomer length
in a PEG molecule is about 3.5 angstroms, when n=1 in FIG. 18, then
the brush length of the fluorescent labels would range from about 5
to about 10 angstroms. In many instances, n in FIG. 18 will not be
1. Further, PEG molecules typically vary in size and are described
based upon an average molecular weight. Thus, using the PEG
molecules set out in FIG. 18 for purposes of illustration, a
population of PEG molecules with an average weight of 10,000 would
have an n of around 30. Also, a population of PEG molecules with an
average weight of 40,000 would have an n of around 120. In view of
the fact that both the arm of a branched PEG molecule that links
this molecule to the labeled molecule and the other arm all have a
repeating region, the brush lengths may range from about 15
angstroms to about 800 angstroms (e.g., from about 25 to about 800,
from about 150 to about 800, from about 450 to about 800, from
about 600 to about 800, from about 65 to about 800, from about 25
to about 700, from about 40 to about 700, from about 28 to about
600, from about 28 to about 500, from about 70 to about 700, etc.).
Thus, the invention includes compositions and methods for producing
and using molecules that are covalently linked to at least one
fluorescent label, wherein the fluorescent label(s) has a brush
length of from about 22 angstroms to about 800 angstroms.
[0165] As an alternative, compositions of the invention may be
described by the number of covalent bonds between the molecules and
the fluorescent label to which they are linked. Again using the
branched PEG molecule set out in FIG. 18 for purposes of
illustration, the number of intervening covalent bonds would be
between about 24 and about 32, depending on where the fluorescent
label is attached to the branched PEG molecule. The invention
includes compositions and methods for producing and using molecules
that are covalently linked to at least one fluorescent label,
wherein the fluorescent label(s) are connected to the molecule by
from about 16 to about 800 (e.g., from about 16 to about 700, from
about 32 to about 800, from about 60 to about 800, from about 100
to about 800, from about 150 to about 800, from about 200 to about
800, from about 250 to about 800, from about 250 to about 700, from
about 250 to about 650, from about 250 to about 600, from about 350
to about 800, etc.) intervening covalent bonds.
[0166] The invention also relates, in part, to spacing out of
multiple fluorescent labels attached to the same location on
individual labeled molecules. Similar to what is set out above with
respect to the separation of fluorescent labels from labeled
molecules, multiple fluorescent labels attached to the same
location on individual labeled molecules may be separated from each
other by distances of from about 15 angstroms to about 800
angstroms (including the ranges set out above) and/or by from about
16 to about 800 (including the ranges set out above) intervening
covalent bonds.
[0167] As noted herein, aspects of the invention relate to the
spacing of fluorescent labels. Table 9 shows estimated
characteristics of a series of branched PEG molecules. It should be
understood that these PEG molecules, as well as other molecules,
exist in multiple formats at different times. For example, arms of
a branch PEG molecule may be fully extended (brush) or fully coiled
(mushroom), as well as effectively all conformations in between.
Further, each arm may be in a different state of extension or
coiling independent of other arms at any one time. Thus, in Table
9, the term "brush" refers to fully extended PEG arms and the term
"mushroom" refers to fully coiled PEG arms. The mushroom state is
modeled upon a very large Flory radius (distance between adjacent
PEG arms) of 80 angstroms to get the smallest number.
[0168] F-F distances in Table 9 are distances between two
fluorophores on the termini of different arms, assuming all of the
fluorophores are equidistant from each other. The maximum distance
is double the arm length. Further, the nearest neighbor value
assumes tetrahedral arrangement for 4 arms and spherical/cubic for
8 arms. Of course, the actual value of F-F distances at any one
time point will typically be somewhere in between the brush
distances and the mushroom distance.
TABLE-US-00009 TABLE 9 Branched PEG Arm Length (Estimated
Fluorophore to Fluorophore (F-F) Distance) F-F F-F Arm Arm Max F-F
Nearest Max F-F Nearest Length Length Dist. Neighbor Dist. Neighbor
Arm EG/ (.ANG.) (.ANG.) (.ANG.) Dist. (.ANG.), (.ANG.), Dist.
(.ANG.), MW Arms mw Arm* (BAL)** (MAL)** BAL (3) Brush (3) MAL (4)
MAL (4) 10000 4 2500 60 208 26 417 340 52 42 10000 8 1250 30 104 13
208 120 26 15 20000 8 2500 60 208 26 417 241 52 30 40000 8 5000 119
417 52 833 481 104 60 * EG/Arm = Arm MW/EG MW ~ Arm MW/42 (MW =
Molecule Weight) **Brush Arm Length (BAL) = EG/Arm * monomer length
~ EG/Arm * 3.5 **Mushroom Arm Length (MAL), as determined by the
equation below (L = . . . ) (3) Fully extended, end of arms
equidistant from each other (Dist. = Distance) (4) Fully coiled,
end of arms equidistant from each other These calculations are
based upon the JenKem following products: 4ARM-NH2HCl and
8ARM-NH2HCl L = Na 5 / 3 D 2 / 3 ##EQU00004## a = 0.35 nm (3.5 A) N
= 30, 60, 120 D = 80 A
[0169] Dextrans are another suitable material for connecting
fluorescent labels to molecules. Dextrans are hydrophilic
polysaccharides characterized by their moderate to high molecular
weight, good water solubility and low toxicity. Dextrans tend to be
biologically inert due to their uncommon
poly-(.alpha.-D-1,6-glucose) linkages. These linkages render them
resistant to cleavage by most endogenous cellular glycosidases.
They also usually have low immunogenicity and are generally
branched molecules.
[0170] Dextrans are commercially available with nominal molecular
weights (MW) varying between 3000 daltons and 2,000,000 daltons.
Dextrans suitable for use in the practice of the invention may be
of any number of different molecule weights, including 3000,
10,000, 40,000, 70,000, 500,000, and 2,000,000 daltons (e.g., from
about 4,000 to about 150,000, from about 6,000 to about 150,000,
from about 8,000 to about 150,000, from about 15,000 to about
150,000, from about 10,000 to about 80,000, from about 12,000 to
about 70,000, etc.).
[0171] Dextrans usually have a degree of substitution (e.g., a DOL)
of 0.2 to 2 of dye molecules per dextran molecule for dextrans in
the 10,000 MW range. Further, dextrans typically contain 0.2-0.7
dyes per dextran in the 3000 MW range, 0.4-2 dyes per dextran in
the 10,000 MW range, 1-4 dyes in the 40,000 MW range and 2-6 dyes
in the 70,000 MW range. Thus, dextrans, as well as other labeled
polymers, used in the practice of the invention may have a DOL
ranging from about 0.03 to 0.3 fluorescent labels per 1,000 MW
(e.g., from about 0.03 to about 0.25, from about 0.08 to about 0.3,
from about 0.09 to about 0.3, from about 0.1 to about 0.3, from
about 0.05 to about 0.2, from about 0.07 to about 0.25, etc. per
1,000 MW).
[0172] Dextrans, as well as other polymers, may be labeled in a
number of ways. For example, fluorescently labeled dextrans may be
prepared by the reaction of a water-soluble amino dextran with a
fluorescent label having a succinimidyl ester group. Fluorescently
labelled dextran may also be prepared by the reaction of a native
dextran with an isothiocyanate derivative of a fluorescent label
such as FITC. Where appropriate, once the fluorescent label has
been added, unreacted amines on the dextran may be capped to yield
a neutral or charged dextran (i.e., negatively or positively
charged). Further, even where capping is not performed, charged
fluorescent labels may be used which can render dextrans anionic or
cationic.
[0173] Another type of polysaccharide that can be useful in the
practice of the invention is amylose. Amylose is a linear
polysaccharide composed of .alpha.-D-glucose units linked by
.alpha.-1,4-glycosidic bonds. Due to hydrogen bonding, amylose
tends to form spiral structures containing six glucose units per
turn. Molecules of this type provide structure regularity that can
be used space out fluorescent labels in consciously designed
manner. The invention thus includes compositions, as well as
methods for making and using such compositions, with fairly static
structural features that may be employed for maintaining a uniform
distance between fluorescent labels. In many instances, the
distance between two fluorescent labels associated with such a
molecule in a manner and under conditions that would result in two
fluorescent labels would not vary in distance from each other by
more than 30% (e.g., from about 5% to about 30%, from about 10% to
about 30%, from about 15% to about 30%, from about 20% to about
30%, from about 10% to about 20%, etc.).
[0174] Polypeptides may also be used in the practice of the
invention. One exemplary such molecule is the branched polylysine
molecule shown in FIG. 21. This molecule may be made according to
methods set out in U.S. Patent Publication No. 2010/0278750. A
number of "R groups" are shown in FIG. 21, representing locations
where fluorescent labels may be attached. In such a molecule the R
groups may be the same or different (e.g., R.sub.1 and R.sub.2).
Further, when a number of R groups are the same, these groups may
act as fluorescent label attachments points where the R groups are
not labeled to completion.
[0175] By way of example, FIG. 21 shows a polymer with 33 R groups,
32 R.sub.1 groups and 1 R.sub.2 group. Assume that all of the R
groups are of the same type. These R groups may be partially
fluorescently labeled in a semi-random manner. By this is meant
that conditions may be provided such that only a percentage of the
R group receive a fluorescent label. Further, the labeling would be
semi-random because some R group, due to their position in the
polymer, would be more prone to receive a fluorescent label. Thus,
the invention includes the design of fluorescently labeled polymers
where the positioning and DOL are adjusted for a desired
fluorescent effect. In some instances, the average number of
available attachment site on a per polymer basis that receive
labels is in the range of 10% to 90% (e.g., from about 10% to about
85%, from about 15% to about 85%, from about 20% to about 85%, from
about 20% to about 75%, from about 30% to about 75%, from about 30%
to about 80%, from about 40% to about 80%, etc.).
[0176] As noted above, FIG. 21 shows a polymer with R.sub.1 and
R.sub.2 groups. If "directional" linkage of the polymer to a
biomolecule (e.g., an antibody) is desired, the R.sub.2 group may
be different from the R.sub.1 groups. If the R groups are the same,
then attached would be "non-directional" in the sense that any R
group in a suitable location on the polymer could serve as a
conjugation point to the biomolecule. Of course, conditions would
be adjusted to achieve a high level of fluorescence, while
maintaining a high level of biological activity for the biomolecule
(e.g., antigen binding).
[0177] Maximization of biomolecule fluorescence may be partially
independent of the number of fluorescent labels on the biomolecule
of interest. Assume for example that there are seven fluorescent
labels on a particular antibody, when labeled under a first set of
conditions, and ten fluorescent labels on the same antibody, when
labeled under the second set of conditions. Further assume that the
amount of total fluorescence of the antibody labeled under the
first set of conditions is greater than the amount of total
fluorescence of the antibody labeled under the second set of
conditions. In this instance, fewer fluorescent labels resulted in
more fluorescence. Thus, other factors being equal (e.g.,
functional activity of the biomolecule), the first set of
conditions would be preferred over the second set of
conditions.
[0178] Any number of linking groups may be used to attach
fluorescent labels to biomolecules. These attachments may be
non-covalent or covalent. Further, non-covalent or covalent can
refer to one or all of (1) the linking of the fluorescent label to
a polymer, (2) the linking of the polymer to a core (when present),
and/or (3) the linking of the polymer or the core, when present, to
the biomolecule.
[0179] In many instances, polymers (with or without cores), as well
as other types of spacers set out herein, may serve the function of
increasing the fluorescent intensity of fluorescent labels attached
to a biomolecule.
[0180] One category of molecules that are useful in the practice of
the invention are referred to as star polymers (see Ren et al.,
Star Polymers, Chemical Reviews, 116:6743-6836 (2016)). Star
polymers are multi-arm molecules that contain a core and a series
of linear polymers (referred to as "arms"). These arms typically
contain terminal functionality to facilitate attachment of other
molecules. Star polymers may be classified as homo-arm (containing
arms of only one composition) or mikto-arm (containing arms of more
than one composition, molecular weight or terminal functionality).
Synthesis of star polymers is typically carried out using either
core first, arm first or grafting onto approaches.
[0181] The core effectively serves as a branching point for the
arms. Any number of molecules can serve as cores for compositions
of the invention. Examples of suitable cores include oligoglycerols
(e.g. hexaglycerol), oligoerythritols (e.g., pentaerythritol,
dipentaerythritol, tripentaerythritol), sorbitol,
trimethylolpropane, silanes (e.g. 1,2-bis(methylsilyl)ethane),
adamantanes, PAMAM (1-, 2-, and 3-generation (G-1-3)
poly(amidamine)) dendrimers, polyethylene imine (PEI) branched
polymers, peptides (e.g., polylysine, polyaspartic acid, etc.).
[0182] The core may already have functional groups for initiation
of polymerization or linking onto of arms or, as shown in FIG. 20,
may require chemical modification in order to facilitate the
attachment of arms.
[0183] Using hexaglycerol as an example, this compound is available
commercially and may be used in the core first synthesis of
star-shaped PEG polymers. StarPEGs with hexaglycerol cores may be
used for the controlled release of drugs and for wound sealing.
StarPEGs with hexaglycerol cores may be produced by the controlled
polymerization of ethylene oxide from the hexaglycerol core. In
this instance, the core is hexaglycerol and the arms are
polyethylene glycol.
[0184] The arms may be composed any number of linear polymers and
will typically function as attachment points for fluorescent labels
and for spacing these labels out from each other, as well as
spacing the labels out from other fluorescent labels attached to
the same labeled molecule (e.g., a biomolecule). Examples of
suitable arms include polyethylene glycol, poly(vinyl pyrrolidone),
polyglycerol, and polyvinyl alcohol, zwitterionic polymers (e.g.
polysuflo betaines) as well as water soluble polymers. In many
instances, polymers suitable for use in the invention will be
non-charged.
[0185] One area of antibodies that is particularly useful for the
attachment of fluorescent labels, when present, is the Fc (fragment
crystallizable) region. The Fc region is at the far end of
antibodies with respect to the antigen bind site(s). This region of
the antibody interacts with cell surface receptors called Fc
receptors and proteins of the complement system. Typically,
attachment of chemical entities at or near the Fc region has little
impact on antigen binding by the antibody. However, interference
with antigen binding events tends to increase with the size of
chemical entities attached to the antibody. Further, large attached
chemical entities tend to interfere with each other, due to steric
hindrance, with respect to the ability to attach to a biomolecule.
Thus, a series of factors may need to be balanced in order to
produce a biomolecule (e.g., an antibody) with both a high level of
fluorescence and a high level of functional activity (e.g., antigen
binding ability). Some of these factors are as follows: (1) Size of
the biomolecule, (2) location of attachment points on the
biomolecule for fluorescent labeling, and (3) the size(s) and three
dimensional structures of the fluorescent labeled molecules being
attached to the biomolecules.
[0186] With respect to antibodies, the invention includes
antibodies that contain one or more of the following features:
[0187] Fluorescently labeled polymers bound (e.g., covalently
bound) at from an average 2 to 10 different locations on the
antibody molecules. [0188] An average of from 3 to 80 fluorescent
labels attached to each of the antibody molecules. [0189] Antigen
binding affinity (K.sub.D) of the fluorescently labeled antibody
molecule is decreased by no more than two (e.g., from about 0.5 to
about 2.0, from about 1.0 to about 2.0, from about 0.5 to about
1.5, from about 0.75 to about 1.5, etc.) orders of magnitude as
compared to the unlabeled form of the antibody. [0190] The average
amount of fluorescent emission on a per fluorescent label basis
bound to the antibody molecules is at least 60% (e.g., from about
60% to about 98%, from about 70% to about 98%, from about 80% to
about 98%, from about 85% to about 98%, from about 80% to about
93%, etc.) that of the free fluorescent label.
[0191] f. Biomolecule/Fluorescent Label/Spacer Combinations
[0192] The invention is based, in part, on combinations of three
components: Biomolecules, fluorescent labels, and spacers. With
respect to proteins (e.g., antibodies), groups that may be used as
attachments sites for fluorescent labels and spacers may not always
be accessible for attachment, especially when the protein has not
be denatured. Further, in many instances, it will be desirable to
maintain proteins in undenatured form.
[0193] Enhancement in fluorescent emission have been found to
relate to various ratios of fluorescent labels and spacers used to
label biomolecules (e.g., antibodies). Further, each biomolecule
has the potential for requiring different ratios of components in
the conjugation process to yield specified enhanced fluorescence
levels. This may be due to different structure (e.g., primary,
secondary, tertiary and quaternary structure) of biomolecules, such
as antibodies, as well as characteristics of the particular
fluorescent label and the particular spacer.
[0194] In some instances, ratios may be based upon the respective
weights of components. In other instances, ratios may be based upon
molar ratios. A number of the figures and example of this
application relate to molar ratios. In some instances (e.g., where
the biomolecule is large and with a large number of conjugation
sites), the use of component weights may be more suitable.
[0195] For antibodies, as well as other biomolecules, the following
ratios of biomolecule to fluorescent label to spacer used in the
conjugation process may vary greatly but in most instances the
amount of biomolecule will be lower than the amount of both
fluorescent label and spacer.
[0196] Further, the density and/or spacing of conjugation sites on
a biomolecule is one factor that will often determine the optimum
ratio of fluorescent label to spacer. This is so because, assuming
enhanced fluorescence is due to reduced quenching, the lower the
total or regional density of conjugations sites, the lower the
amount of quenching would be expected to be. In any event, ratios
of biomolecule to fluorescent label to spacer that may be used can
be described as follows: B.sub.1:FL.sub.2-30:S.sub.2-20, where B is
biomolecule, FL is fluorescent label and S is spacer. Specific
ranges of ratios that may be used in the practice of the invention
include ratios that fall within 1:2-25:2-20 (e.g., from about 1:2:2
to about 1:25:20, from about 1:5:2 to about 1:25:5, from about
1:10:2 to about 1:25:5, from about 1:5:2 to about 1:15:10, from
about 1:10:5 to about 1:25:20, from about 1:10:5 to about 1:15:20,
from about 1:10:5 to about 1:20:20, etc.), wherein the first number
is the amount (e.g., moles) of biomolecule, the second number is
the amount of fluorescent label, and the third number is the amount
of spacer.
[0197] In some instances, fluorescent label and spacer
concentrations used for conjugation will be such that available
attachments sites on biomolecules will be effectively saturated
(e.g., at least 95% of available attachments sites will have bound
thereto either a fluorescent label or a spacer). In such instances,
the fluorescent label and spacer ratio may be a determining factor
in the level for fluorescent enhancement. In many instances the
ratio of fluorescent label to spacer will be between 10:1 to 10:50
(e.g., from about 10:1 to about 10:25, from about 10:1 to about
10:10, from about 10:1 to about 10:5, from about 10:5 to about
10:50, from about 10:5 to about 10:20, from about 10:3 to about
10:30, from about 10:5 to about 10:30, from about 10:10 to about
10:25, etc.).
[0198] Spacers and dyes may be conjugated to a biomolecule at the
same time or sequentially, with either the spacer or the dye being
conjugated to the biomolecule first. In many instances, when the
spacer and dye connect to the biomolecule at the same loci, they
will be conjugated to the biomolecule at the same time. However,
sequential conjugation could be used when the first conjugation
reaction (e.g., of the spacer) is done under conditions where the
binding sites are the biomolecule are not saturated, thus leaving
binding sites available for a second conjugation reaction (e.g., of
the dye).
[0199] g. Buffers
[0200] In some embodiments, the composition of the invention
comprises one or more spacer, one or more fluorescent label in a
buffer. Any of the fluorescent and spacer molecules disclosed
herein may be used together with any buffer known in the art.
[0201] In some embodiments, the compositions disclosed herein
comprise any suitable buffer for a molecular biology
application.
[0202] In some embodiments, the buffer is a suitable storage buffer
(e.g., borate buffer, phosphate buffer or carbonate buffer).
[0203] In some embodiments, the buffer is suitable for buffering a
detectable biomolecule, as disclosed herein, during use in a
detection assay.
Methods
[0204] The instant invention has useful applications in basic
research, high-throughput screening, immunohistochemistry,
fluorescence in situ hybridization (FISH), microarray technology,
diagnostics, and medical therapeutics. The invention can be used in
a variety of assay formats for diagnostic applications in the
disciplines of microbiology, immunology, hematology and blood
transfusion, tissue pathology, forensic pathology, and veterinary
pathology.
[0205] In some embodiments, the compositions described herein can
be used in any molecular biology application wherein fluorescently
labeled molecules are detected. For example, the detectable
biomolecules, as disclosed herein, can be used in Western blotting,
ELISA, flow cytometry, flow cytometry and applications involving
FRET. The detectable biomolecules, as disclosed herein, can also be
used in fluorescent immunohistochemistry (IHC), fluorescent
immunocytochemistry (ICC), and in vivo imaging applications.
[0206] In some embodiments, a method for determining the presence
of a desired target in a biological sample is encompassed, the
method comprising: a) contacting the biological sample with a
composition comprising one or more fluorescent label, and one or
more spacer molecules, wherein the spacer and fluorescent label are
conjugated to the biomolecule but not to each other, b) detecting
fluorescence emitted by the plurality of fluorescent labels; and c)
determining the presence of the desired target in the biological
sample when fluorescence emitted by the plurality of fluorescent
labels is detected. In some embodiments, the biological sample
comprises cell lysate. In some embodiments, the biological sample
comprises intact cells. In some embodiments, the biological sample
comprises a tissues sample. Further such tissue samples may be
fixed. Further, compositions of the invention may be used for
applications such as immunohistochemistry. In some embodiments, the
biological sample comprises isolated protein. In some embodiments,
the biological sample comprises recombinant protein. In some
embodiments, the biological sample is immobilized on a solid
support. In some embodiments, the biological sample comprises
intact cells in fluid. In some embodiments, the biological sample
is a live animal. In some embodiments, the live animal is a mammal.
In some instances, the sample comprises tissues such as liver,
lung, muscle and skin.
[0207] In some embodiments, disclosed herein is a method for
imaging a target antigen in a living body, wherein the method
comprises; a) providing an antibody conjugated to a plurality of
fluorescent labels and a spacer agent, as disclosed herein, that
binds to the target antigen; b) introducing the antibody into the
body to form a contacted body; c) illuminating the contacted body
with an appropriate wavelength to form an illuminated body; and d)
observing the illuminated body wherein the target antigen is
imaged. In some embodiments, these antibodies or other targeted
proteins or peptides specific for a target or antigen in a living
body that has been conjugated with a fluorescent dye(s) having an
excitation wavelength compatible with in vivo imaging, typically
about 580 nm to about 800 nm. The target specific dye conjugates
travel relatively freely within the circulating blood until their
preferential sequestration occurs at a target pathological or
non-pathological tissue sites such as a diseased or injury tissue
sites.
[0208] h. Kits
[0209] The compositions of the invention may be incorporated into
kits that facilitate the practice of various assays. The kits may
be packaged with the composition in a dry form or with the
composition in solution. The kits may optionally further include
one or more buffering agents, typically present as an aqueous
solution, sample preparation reagents, additional detection
reagents, organic solvent, other fluorescent detection probes,
standards, microspheres, specific cell lines, antibodies and/or
instructions for carrying out an assay. Additional optional agents
include components for testing of other cell functions in
conjunction with the compound.
[0210] In some embodiments, the kits comprise a biomolecule, spacer
agent, and a fluorescent label, as disclosed herein. In some
embodiments, the kit further comprises a buffer. In some
embodiments, the biomolecule is already conjugated to the spacer
agent and the fluorescent label. In some embodiments, the kit may
comprise a polymer conjugated to fluorescent labels and a reactive
group as a bioconjugation kit.
[0211] Kits of the invention may further contain reagents used to
prepare fluorescently labeled biomolecules (e.g., antibodies).
Exemplary reagents include one or more of the following:
fluorescent dyes, spacers that are either prelabled of labels
according to instructions provided in the kits, and compounds that
may be used to conjugate (1) spacers to biomolecules and/or (2)
fluorescent labels to biomolecules.
[0212] This description and exemplary embodiments should not be
taken as limiting. For the purposes of this specification and
appended claims, unless otherwise indicated, all numbers expressing
quantities, percentages, or proportions, and other numerical values
used in the specification and claims, are to be understood as being
modified in all instances by the term "about," to the extent they
are not already so modified. Accordingly, unless indicated to the
contrary, the numerical parameters set forth in the following
specification and attached claims are approximations that may vary
depending upon the desired properties sought to be obtained. At the
very least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of the claims, each numerical
parameter should at least be construed in light of the number of
reported significant digits and by applying ordinary rounding
techniques.
EXAMPLES
[0213] The following examples are provided to illustrate certain
disclosed embodiments and are not to be construed as limiting the
scope of this disclosure in any way.
Example 1: Fluorescent Western Blotting Methods
[0214] a.) Antibody Labeling Using NHS Activated Fluorescent Dyes
and Sulfo NHS-Acetate/NHS-Acetate and NHS-MS(PEG).sub.4,
NHS-MS(PEG).sub.8 and NHS-MS(PEG).sub.12
[0215] NHS activated fluorescent dyes, such as DYLIGHT.TM.
650-4.times.PEG, were reconstituted in dimethylformamide (DMF) at
10 mg/ml. NHS-Acetate was prepared fresh in dimethyl formamide
(DMF) at 1 mg/ml. NHS-MS(PEG)4 (Cat. no. 22341 (Thermo Fisher
Scientific), NHS-MS(PEG).sub.8 (cat. no. 22509 (Thermo Fisher
Scientific), NHS-MS(PEG).sub.12 (cat. no. 22686 (Thermo Fisher
Scientific), were reconstituted in DMF at 100 mg/ml. The PEG
Reagents were further diluted to 1 mg/ml in DMF just prior to use.
1 mg of Goat Anti-Mouse (GAM) and Goat Anti-Rabbit (GAR) antibody
at 7-10 mg/ml in Borate Buffer 50 mM (cat. no. 28384 (Thermo Fisher
Scientific), pH 8.5 were labeled with each fluorescent dye, or a
mixture of each fluorescent dye and a spacer selected from
NHS-Acetate, NHS-MS(PEG).sub.4, NHS-MS(PEG).sub.8 or
NHS-MS(PEG).sub.12 at various molar excesses. The labeling
reactions were incubated for approximately 1 hour at room
temperature (RT). The NHS activated dye and the NHS activated
spacer agents were combined before addition to the antibody so that
both reactions were concurrent, allowing for a random spacing of
the dye substitution and of the spacers. 100 mM MES Buffer at pH
4.7 was added to each sample to lower the pH from 8.5 to
approximately 7.2. At this point the concentration of the
conjugates was adjusted down to approximately 6 mg/ml to
accommodate the final dilution in the storage buffer. The free dye
was removed using the Dye Removal Resin (Thermo Fisher Scientific,
cat. no. 22858) and 5 .mu.m Harvard columns (Harvard Apparatus,
cat. no. 74-3820). 0.2 ml of the 50% purification resin slurry was
used per mg of protein. The conjugates were diluted 1:50 with 0.1M
Sodium Phosphate Buffer, pH 7.2 (PBS) and scanned using the UV Cary
Spectrophotometer. OD scans (252 nm to 900 nm) were used to
determine concentrations of conjugates and calculate mole dye/mole
protein ratios (D/P). Finally, the conjugates were diluted to 1
mg/ml in STABILZYME.RTM. NOBLE Storage Buffer (Surmodics, cat. no.
SZ04) for long term storage.
[0216] Serially diluted cell lysates (500 ng to 2 ng) were combined
with SDS-PAGE sample buffer. The samples were heated for five
minutes at 95.degree. C., and loaded onto Thermo Fisher Scientific
Tris Glycine SDS-PAGE gels (Novex Gels, 4-20%, 10-well, cat. no.
WT4202BX10). Gels were electrophoresed according to manufacturer's
instructions and then transferred to nitrocellulose membranes using
a semi-dry transfer method. The membranes were blocked for thirty
minutes with SEA BLOCK Blocking Buffer (Thermo Fisher Scientific,
cat. no. 0037527). The primary antibodies were prepared to a final
concentration of 0.1 to 2.5 .mu.g/ml in Sea Block Blocking Buffer.
The blots were incubated with the antibody prepared in the Sea
Block Blocking Buffer for one hour with shaking at room temperature
(RT). The antibody solution was decanted and the membranes were
washed two times for ten minutes in 10 mM Tris, 150 mM NaCl, 0.05%
Tween-20, pH 7.2 (TBST). The fluorescently labeled secondary
antibody conjugates were diluted to a final concentration of 20 to
1000 ng/mL in SEA BLOCK Blocking Buffer. The washed membrane was
incubated with the relevant secondary antibody conjugate with
agitation for 30 to 60 minutes. The buffer was decanted and the
membranes were washed six times for five minutes with TB ST. The
membranes were imaged using a compatible fluorescent imager.
[0217] Results are shown in FIG. 8 for an experiment testing the
effect of addition of NHS Acetate (2.5.times., 5.times. and
10.times.) and MS(PEG).sub.4 (3.75.times.) spacer to a GAM
secondary antibody conjugated to DYLIGHT.TM. 488 at
5.times.-20.times. molar excess in a western blotting assay. A431
cell lysate was diluted 3-fold from 1 .mu.g/well. The primary
antibody rabbit used was anti-Hsp90 diluted 1/5000 from 1 mg/ml.
All DYLIGHT.TM. secondary antibodies were diluted 1/5000 from 1
mg/ml stock. In a Western blot application, there is noticeable
increase of fluorescent intensity over the base conjugate (made
without the spacer) at each dye molar excess from 7.5.times. to
20.times. for DYLIGHT.TM. 488-GAR conjugated with the addition of
NHS Acetate or MS(PEG).sub.4.
[0218] Results demonstrating the effect of addition of NHS Acetate
(5.times.) or MS(PEG).sub.4 (5.times.) spacer to a GAM secondary
antibody conjugated to DYLIGHT.TM. 650-4.times.PEG (at 7.5.times.
Dye) in a Western blot assay are shown in FIG. 9. HeLa cell lysate
was diluted 4-fold from 0.5 .mu.g/well. Primary antibody mouse
anti-PDI was diluted to 1/5000 of 1 mg/ml. All DYLIGHT.TM.
secondary antibodies were diluted to 1/5000 of 1 mg/ml stock. NHS
acetate added at 5.times. molar excess to GAM-DYLIGHT.TM.
650-4.times.PEG-7.5.times. conjugation improved intensity by
1.5-fold. NHS acetate added at 3.75.times. molar excess to
GAM-DYLIGHT.TM. 650-4.times.PEG-7.5.times. conjugation improved
intensity by 1.4-fold.
[0219] Results demonstrating the effect of addition of NHS Acetate
(2.5.times., 5.times.) and MS(PEG).sub.4 spacer to a
GAR-DYLIGHT.TM. 800-4.times.PEG secondary antibody in a Western
blot assay are shown in FIG. 10 and Table 10. A431 cell lysate was
serially diluted 1:1. Primary antibodies rabbit anti-Hsp90 and
anti-Cyclophilin B were diluted 1/5000. All DYLIGHT.TM. secondary
antibodies were diluted to 1/20,000 of 1 mg/ml stock. The addition
of MS(PEG).sub.4 (3.75.times. and 5.times.) and NHS Acetate
(2.5-5.times.) significantly enhanced the fluorescence intensity
and sensitivity of base DYLIGHT.TM. 800-4.times.PEG conjugate by
20-100% at different molar excesses of the dye in this Western
blotting application.
TABLE-US-00010 TABLE 10 Effect of .+-. NHS Acetate (2.5X, 5X) or
MS(PEG).sub.4 (5X) on GAR-DYLIGHT .TM. 800-4xPEG in Western Blot
Assays. +2.5X NHS +5X NHS +5X Fold Improvement (WB) NA Acetate
Acetate MS(PEG).sub.4 GAR-DYLIGHT .TM. 800- 1.0 1.4 1.2 1.8
4xPEG_5x GAR-DYLIGHT .TM. 800- 1.0 1.5 1.2 1.7 4xPEG_7.5x
GAR-DYLIGHT .TM. 800- 1.0 1.7 1.5 1.9 4xPEG_10x GAR-DYLIGHT .TM.
800- 1.0 1.9 1.5 2.0 4xPEG_15x NA = No Addition
[0220] Results demonstrating the effect of addition of NHS Acetate
(2.5.times., 5.times. and 10.times.) and MS(PEG).sub.4 (5.times.)
and MS(PEG).sub.8 (5.times.) spacer to a GAM-DYLIGHT.TM.
550-2.times.PEG conjugate (at 12.5.times. molar excess of dye) in a
western blotting assay are shown in FIG. 11. HeLa cell lysate was
diluted 4-fold from 0.5 .mu.g/well and was stained with anti-PDI
primary antibody diluted to 1/5000 of 1 mg/ml. All DYLIGHT.TM.
secondary antibodies were diluted to 1/5000 of 1 mg/ml stock. This
experiment showed that the addition of MS(PEG).sub.4 (5.times.) and
NHS acetate (2.5.times. and 5.times.) significantly enhanced the
fluorescence intensity and sensitivity of base DYLIGHT.TM.
550-2.times.PEG conjugates by at least by 2-fold in a Western Blot
assay. Conjugates prepared with longer chain MS(PEG).sub.8 did not
show significant improvement over the base conjugate in this
experiment.
[0221] Results demonstrating the effect of addition of NHS Acetate
(2.5.times., 5.times.) and MS(PEG).sub.4 (5.times.) spacer to
GAM-DYLIGHT.TM. 680-4.times.PEG-GAR (at 10.times. molar excess of
dye). are shown in FIG. 12. HeLa cell lysate was diluted 4-fold
from 0.5 .mu.g/well and anti-PDI primary antibody was diluted to
1/5000 of 1 mg/ml. All DYLIGHT.TM. 680-4.times.PEG-GAR secondary
antibodies were diluted to 1/20000 of 1 mg/ml stock. This
experiment shows that that the addition of MS(PEG).sub.4 (5.times.)
and NHS acetate (2.5.times. and 5.times.) significantly enhanced
the fluorescence intensity and sensitivity of base DYLIGHT.TM.
680-4.times.PEG conjugate by 3 to 4-fold in a Western blotting
assay.
Example 2: Dot Blot Assays
[0222] Serial dilutions (1:1) Mouse or Rabbit IgG were made from
chosen stock concentration. The dilutions were placed in a 96-well
plate from highest to lowest using a 20 .mu.L 12-channel multi
pipette. 1 or 2 .mu.L of the 11 serial dilutions was carefully
spotted onto Nitrocellulose membranes. The membranes were allowed
to dry overnight and then blocked with 2% BSA blocking buffer in
TBST. The membranes were incubated with agitation for one hour at
room temperature. The blocking solution was decanted from the
container. The secondary antibody conjugates were diluted in TBS or
blocking buffer. Secondary antibody conjugate dilutions varied
depending on the conjugated label: 1:5,000 (DYLIGHT.TM. 488 and
550-2.times.PEG conjugates); 1:10,000 (DYLIGHT.TM. 650-4.times.PEG
conjugates); and 1:20,000 (DYLIGHT.TM. 680-4.times.PEG and
DYLIGHT.TM. 800-4.times.PEG conjugates). The membranes were
incubated with the relevant secondary antibody conjugates with
agitation for 30 to 60 minutes. The membranes were washed five
times for five minutes with TBST buffer. The membranes were imaged
with a suitable imager, for example, ChemiDoc MP (488, 550, 650,
680 nm) and LiCOR Odyssey CLx (650, 680, 800 nm).
[0223] Results depicted in FIG. 3 and Table 11 show the effect of
NHS acetate (2.5.times., 5.times. and 10.times.) and MS(PEG).sub.4
(3.75.times.) spacer to GAM-DYLIGHT.TM. 488 (at 5.times.-20.times.
molar excess of dye) in a dot blot assay. In dot blot applications,
the DYLIGHT.TM. 488-GAM conjugates made with the addition of NHS
Acetate and MS(PEG).sub.4 resulted in definite improvement in
fluorescent intensity ranging from 1.2 to 1.8-fold over the base
conjugate (made without the spacer) at the various dye molar
excesses from 7.5.times. to 20.times..
TABLE-US-00011 TABLE 11 Effect of NHS Acetate (2.5X, 5X and 10X) or
MS(PEG).sub.4 (3.75x to 10X) addition in the conjugation of
GAM-DYLIGHT .TM. 488 at 5X-20X molar excess in a Dot Blot. +2.5X
+5X +10X FOLD OF NHS NHS NHS +3.75X +5X +10X IMPROVEMENT NA Acetate
Acetate Acetate MS(PEG).sub.4 MS(PEG).sub.4 MS(PEG).sub.4 DYLIGHT
.TM. 1.0 1.5 1.1 0.3 1.0 0.6 488_5x DYLIGHT .TM. 1.0 1.2 1.6 1.8
1.0 1.3 488_7.5x DYLIGHT .TM. 1.0 1.6 1.3 1.3 1.3 0.8 488_10x
DYLIGHT .TM. 1.0 1.4 1.0 0.8 1.6 1.2 1.1 488_15x DYLIGHT .TM. 1.0
1.0 0.7 06 1.5 1.3 1.0 488_20x NA = No Addition
[0224] Results shown in FIG. 4 and Table 12 demonstrate the effect
of addition of NHS acetate (2.5.times. and 5.times.) and
MS(PEG).sub.4 (3.75.times.) to GAM-DYLIGHT.TM. 488 (at
7.5.times.-20.times. molar excess of dye) in a dot blot assay. In
this experiment, a different secondary antibody source was used.
NHS acetate and MS(PEG).sub.4 added to the conjugation mixture
provided a significant improvement of signal intensity as compared
to the base conjugates at 5.times., 15.times. and 20.times. dye
molar excesses; ranging from 1.2 to 2.6-fold.
TABLE-US-00012 TABLE 12 Effect of NHS Acetate (2.5X, 5X and 10X) or
MS(PEG).sub.4 (3.75x) addition in the conjugation of GAM-DYLIGHT
.TM. 488 at 5X-20X molar excess in a Dot Blot Assay. FOLD OF +2.5X
NHS +5X NHS +3.75X IMPROVEMENT No addition Acetate Acetate
MS(PEG).sub.4 DYLIGHT .TM. 1.0 1.8 2.1 2.1 488_7.5x DYLIGHT .TM.
1.0 1.2 1.0 1.2 488_10x DYLIGHT .TM. 1.0 1.5 1.9 2.0 488_15x
DYLIGHT .TM. 1.0 2.0 2.6 1.3 488_20x
[0225] Results shown in FIG. 5 and Table 13 demonstrate the effect
of addition of NHS Acetate (2.5.times., 5.times. and 10.times.) and
MS(PEG).sub.4 (3.75.times.) to GAM-DYLIGHT.TM. 550-2.times.PEG-GAR
(at 10.times.-20.times. molar excess of dye) in a dot blot assay.
Mouse IgG was serially diluted 1:1 from 1000 ng/dot. All
DYLIGHT.TM. 550-2.times.PEG-GAR secondary antibodies were diluted
to 1/5000 of 1 mg/ml stock. NHS acetate and MS(PEG).sub.4 added to
the conjugation mix provided an improvement of signal intensity as
compared to the base conjugates at each respective dye molar
excesses. Improvement ranged from 1.2 to 1.6-fold.
TABLE-US-00013 TABLE 13 Effect of the addition of NHS Acetate
(2.5X, 5X and 10X) or MS(PEG)4 (3.75X) in the conjugation of
GAM-DYLIGHT .TM. 550-2xPEG-GAR at 10X-20X molar excess in a Dot
Blot Assay FOLD OF No +2.5X NHS +5X NHS +3.75X IMPROVEMENT addition
Acetate Acetate MS(PEG).sub.4 GAR-DYLIGHT .TM. 1.0 1.23 1.35 1.32
550-2xPEG_10x GAR-DYLIGHT .TM. 1.0 0.77 0.98 0.93 550-2xPEG_12.5x
GAR-DYLIGHT .TM. 1.0 1.12 1.1 1.52 550-2xPEG_15x GAR-DYLIGHT .TM.
1.0 0.9 1.51 1.63 550-2xPEG_20x
[0226] Results shown in FIG. 6 and Table 14 [Surbhi and Marie: The
data in FIG. 6 and Table 14 do't fully match. I'm not sure which is
correct. Please sort this out. This isn't a major problem but we
should get it right.] demonstrate the effect of the addition NHS
Acetate (2.5.times., 5.times. and 10.times.) and MS(PEG)4
(3.75.times.) to GAM-DYLIGHT.TM. 650-4.times.PEG-GAM (at
10.times.-20.times. molar excess) in a dot blot assay. Mouse IgG
was serially diluted 1:1 from 1000 ng/dot. All DYLIGHT.TM.
650-4.times.PEG-GAR secondary antibodies were diluted to 1/10000 of
1 mg/ml stock. Both NHS Acetate and (MS)PEG.sub.4 brought
significant improvement in sensitivity and signal/background over
the initial base conjugates. NHS acetate added at 2.5.times. molar
excess to GAM-DYLIGHT.TM. 650-4.times.PEG-15.times. improved
intensity by 1.7-fold. The improvement provided by NHS Acetate
showed 1.3-fold better performance than with the conjugate prepared
with the highest molar excess of dye (20.times.). All MS(PEG).sub.4
added to GAM-DYLIGHT.TM. 650-4.times.PEG-15.times. conjugation
improved fluorescence intensity by 1.8 to 2.2-fold and performed
better than the corresponding highest base conjugate
GAM-DYLIGHT.TM. 650-4.times.PEG-20.times..
TABLE-US-00014 TABLE 14 Effect of NHS Acetate (2.5X, 5X and 10X) or
MS(PEG).sub.4 (3.75X) addition in the conjugation of GAM-DYLIGHT
.TM. 650-4xPEG-GAM at (10X-20X) in a Dot Blot Assay. +2.5X +5X +10X
FOLD OF NHS NHS NHS +3.75X +5X +10X IMPROVEMENT NA Acetate Acetate
Acetate MS(PEG).sub.4 MS(PEG).sub.4 MS(PEG).sub.4 GAM-DYLIGHT .TM.
1.0 1.0 1.2 1.1 1.9 1.1 650-4xPEG_5x GAM-DYLIGHT .TM. 1.0 1.7 1.5
1.8 1.2 1.6 650-4xPEG_7.5x GAM-DYLIGHT .TM. 1.0 1.2 2.9 1.8 1.3 1.5
650-4xPEG_10x GAM-DYLIGHT .TM. 1.0 1.6 1.5 1.0 2.2 1.0 2.2
650-4xPEG_15x GAM-DYLIGHT .TM. 1.0 1.0 0.8 0.8 1.6 0.9 1.3
650-4xPEG_20x NA = No Addition
[0227] Results shown in FIG. 7 and Table 15 demonstrate the effect
of the addition of NHS Acetate (2.5.times., 5.times. and 10.times.)
and MS(PEG).sub.4 (3.75.times., 5.times., 10.times.) spacers to
GAM-DYLIGHT.TM. 800-4.times.PEG- in a dot blot assay. Mouse IgG was
serially diluted 1:2 from 1000 ng/dot. All DYLIGHT.TM.
800-4.times.PEG-GAR secondary antibodies were diluted to 1/20000 of
1 mg/ml stock. This experiments shows that the addition of
MS(PEG).sub.4 (3.75.times. and 5.times.) and NHS Acetate (5.times.)
significantly enhanced the fluorescence intensity and sensitivity
of base DYLIGHT.TM. 800-4.times.PEG conjugate by 1.5 to 6-fold in a
dot blot application.
TABLE-US-00015 TABLE 15 Effect of the addition of NHS Acetate
(2.5X, 5X and 10X) or MS(PEG).sub.4 (3.75X, 5X, 10X)) GAM-DYLIGHT
.TM. 800-4xPEG-in a Dot Blot Assay. +2.5X +5X +10X FOLD OF NHS NHS
NHS +3.75X +5X +10X IMPROVEMENT NA Acetate Acetate Acetate
MS(PEG).sub.4 MS(PEG).sub.4 MS(PEG).sub.4 GAM-DYLIGHT .TM. 1.0 1.7
0.2 3.1 5.5 1.8 800-4xPEG_5x GAM-DYLIGHT .TM. 1.0 0.6 1.4 1.8 2.9
1.3 800-4xPEG_7.5x GAM-DYLIGHT .TM. 1.0 1.7 1.3 2.1 0.7 1.0
800-4xPEG_10x GAM-DYLIGHT .TM. 1.0 1.3 1.6 1.2 3.1 1.3 1.9
800-4xPEG_15x GAM-DYLIGHT .TM. 1.0 1.3 1.8 1.5 3.2 1.3 1.6
800-4xPEG_20x NA = No Addition
[0228] Results shown in FIG. 11 demonstrate the effect of NHS
Acetate (2.5.times., 5.times. and 10.times.) and MS(PEG).sub.4
(5.times.) and MS(PEG).sub.8 (5.times.) spacers addition to
GAM-DYLIGHT.TM. 550-2.times.PEG-GAR (at 12.5.times. molar excess of
dye) in a dot blot assay. Mouse IgG was diluted 3 fold from 0.5
.mu.g/well. All DYLIGHT.TM. secondary antibodies were diluted to
1/5000 of 1 mg/ml stock. These dot blot assays showed that the
addition of MS(PEG).sub.4 (5.times.) and NHS acetate (2.5.times.
and 5.times.) significantly enhanced the fluorescence intensity and
sensitivity of base DYLIGHT.TM. 550-2.times.PEG conjugates by at
least by 2-fold. Conjugates prepared with longer chain
MS(PEG).sub.8 did not show significant improvement over the base
conjugate.
[0229] Results shown in FIG. 12 demonstrate the effect of addition
of NHS Acetate (2.5.times., 5.times.) and MS(PEG).sub.4 (5.times.)
spacers to GAM-DYLIGHT.TM. 680-4.times.PEG-GAR (at 10.times. molar
excess of dye) in a dot blot assay. Mouse IgG was serially diluted
1:2 from 1000 ng/dot. All DYLIGHT.TM. 680-4.times.PEG-GAR secondary
antibodies were diluted to 1/20000 of 1 mg/ml stock. These dot blot
assays show that the addition of MS(PEG).sub.4 (5.times.) and NHS
acetate (2.5.times. and 5.times.) significantly enhanced the
fluorescence intensity and sensitivity of base DYLIGHT.TM.
680-4.times.PEG conjugate.
Example 3: Plate Assay Methods
[0230] To prepare plates, eleven (1:1) serial dilutions of Mouse or
Rabbit IgG starting from 10 .mu.g/ml were prepared. 100 .mu.L of
each dilution was placed in 96-well plates in the corresponding
well from 1-11 from highest to lowest using a 300 .mu.L 12-channel
multi pipette; PBS was added to the last column (#12; negative
control). This was repeated from row A to H. The plates were
incubated overnight and then blocked and incubated with
SUPERBLOCK.TM. Blocking Buffer (Thermo Fisher, cat. not. 37515) as
follows: two times two hundred .mu.L for 5 minutes followed by one
times 200 .mu.L for ten minutes. The plates were allowed to dry and
then stored desiccated at 4.degree. C.
[0231] Mouse IgG- or Rabbit IgG-coated plates were washed two times
200 .mu.L with PBST 20 then one time with PBS. The secondary
antibody conjugates were diluted in TBS or PBS. Secondary antibody
conjugates were diluted 1:100 (DYLIGHT.TM. 488 and 550-2.times.PEG,
DYLIGHT.TM. 650-4.times.PEG DYLIGHT.TM. 680-4.times.PEG and
DYLIGHT.TM. 800-4.times.PEG conjugates). 100 .mu.L of the relevant
conjugates GAM in Mouse IgG-coated plate, GAR in Rabbit IgG coated
plate were added to the plate well. Each dilution was added to
different rows for each conjugate to be tested. All comparisons
were made on the same plate. The plates were incubated for sixty
minutes. Plates were washed three time 200 .mu.L with TBST or PBST
buffer. 100 .mu.L of PBS was added to each row in each well. The
fluorescence intensity was measured using the VariosKan instrument
or Image the fluorescent signal with a suitable imager, such as
ChemiDoc MP (488, 550, 650, 680 nm) and LiCOR Odyssey CLx (650,
680, 800 nm).
Example 4: Immunofluorescence (IFC) Methods (i.e., Cellular Imaging
Methods)
[0232] Method #1: Frozen U2OS cell plates stored at -80.degree. C.
were thawed for thirty minutes at 50.degree. C. Storage buffer
(PBS) was removed and the cells were permeabilized for fifteen
minutes (100 .mu.l/well) with 0.1% Triton-X100 in 1.times.PBS
buffer. Plates were blocked for thirty minutes in 2% BSA/PBS-0.1%
Triton-X100 blockers. Primary antibody Mouse anti-PDI or Rabbit
anti-HDAC2 (10 .mu.g/ml) (cat. no. PA1-861, Life Technologies
Corp., Carlsbad, Calif.), diluted in 2% BSA/PBS-0.1% Triton-X100
was added to the plate and incubated for one hour at RT. Negative
controls contain only 2% BSA/PBS-0.1% Triton-X100 blocker. After
incubation, the primary antibody solution was removed from the
plate and the plate was washed three times 100 .mu.l/well PBST and
one time 100 .mu.l/well PBS. Next, GAM or GAR secondary antibodies
labeled with various molar excesses of dyes were diluted to 4
.mu.g/ml in PBS and incubated for one hour at room temperature. The
plates were washed three times 100 .mu.l/well PBST and 1.times.100
.mu.l/well PBS and Hoechst 33342 (cat. no. 62249, Thermo
Scientific, Waltham, Mass.), (diluted to 0.1 .mu.g/ml in PBS) was
added to each well (100 .mu.l/well). The plates were scanned on
ARRAYSCAN.TM. Plate Reader VTI3, 20.times. objective.
[0233] Method #2: Certain experiments were done in A549 cells in
384-well plates. Primary antibody was used at the same
concentration (1 .mu.g/ml) with varying dilutions of secondary.
pH2AX measurements in etoposide (50 .mu.M for 3 hours, Tocris
Bioscience, cat no. 12-261-00) treated cells were used to measure B
Signal/Noise (S/N, also referred to as Signal to Background), and
brightness was used to compare different antibodies. Secondary
antibody conjugates were tested at 4 different dilutions (0.5, 1, 2
and 4 mg/ml). Standard procedures were used for antibody staining:
4% formaldehyde fixation for fifteen minutes. Permeabilization was
performed in 0.5% Triton x-100 for ten minutes. Blocking was
performed with 3% BSA for thirty minutes. Primary antibody
incubation was carried out at RT for one hour. This was followed by
three washes with PBS. Secondary antibody conjugates were incubated
at RT for one hour followed by three washes with PBS. The cells
were analyzed on ARRAYSCAN.TM. VTI (Thermo Fisher).
[0234] Results shown in FIG. 13 and Tables 16 and 17 demonstrate
the effect of NHS acetate (2.5.times. and 5.times.) and
MS(PEG).sub.4 (3.75.times.) spacer addition to GAM-DYLIGHT.TM. 488
(13A) and GAR-DYLIGHT.TM. 488 ((13B) at 7.5.times. to 20.times.
molar excess of dye) in a cellular imaging application. DYLIGHT.TM.
488-GAM and DYLIGHT.TM. 488-GAR. A549 cells were stained with a
pH2Ax primary antibody diluted to 1/1000 of the 1 mg/ml stock. All
DYLIGHT.TM. 488 secondary antibodies were diluted to 1/250 of the 1
mg/ml stock. NHS acetate added to the conjugation mix provided an
improvement of signal/background as compared to the base conjugates
at 15.times. dye molar excesses; ranging from 1.4 to 1.5-fold (GAM)
and 1.1 to 1.6-fold (GAR). For GAM conjugates the most significant
improvement was observed with NHS Acetate at 5.times. and with
MS(PEG).sub.4 at 3.75.times., and for GAR conjugates the more
noticeable improvement was observed with NHS Acetate at 2.5.times.
and with MS(PEG).sub.4 at 3.75.times..
TABLE-US-00016 TABLE 16 Effect of NHS Acetate (2.5X, 5X and 10X) or
MS(PEG).sub.4 (3.75x) addition in the conjugation of GAM-DYLIGHT
.TM. 488 at 5X-20X molar excess in a Cellular Imaging
application-DYLIGHT .TM. 488-GAM +2.5X NHS +5X NHS +3.75X FOLD OF
IMPROVEMENT NA Acetate Acetate MS(PEG).sub.4 DYLIGHT .TM. 488_7.5x
1.00 1.07 1.14 1.23 DYLIGHT .TM. 488_10x 1.00 1.04 1.13 1.17
DYLIGHT .TM. 488_15x 1.00 1.11 1.48 1.53 DYLIGHT .TM. 488_20x 1.00
0.72 1.09 1.07 NA = No Addition
TABLE-US-00017 TABLE 17 Effect of NHS Acetate (2.5X, 5X and 10X) or
MS(PEG).sub.4 (3.75x) addition in the conjugation of GAR-DYLIGHT
.TM. 488 at 5X-20X molar excess in a Cellular Imaging
application-DYLIGHT .TM. 488-GAR +2.5X NHS +5X NHS +3.75X FOLD OF
IMPROVEMENT NA Acetate Acetate MS(PEG).sub.4 DYLIGHT .TM. 488_7.5x
1.00 1.13 1.07 1.13 DYLIGHT .TM. 488_10x 1.00 1.20 1.21 1.02
DYLIGHT .TM. 488_15x 1.00 1.60 1.31 1.67 DYLIGHT .TM. 488_20x 1.00
1.23 1.14 1.11 NA--No Addition
[0235] Results shown in FIG. 14 and Table 18 demonstrate the effect
of NHS Acetate (2.5.times., 5.times. and 10.times.) and
MS(PEG).sub.4 (3.75.times., 5.times. and 10.times.) on
GAM-DYLIGHT.TM. 550-2.times.PEG-GAM (at 7.5.times. to 20.times.
molar excess of dye) in a cellular fluorescence imaging
application. U2OS cells were stained with an anti-PDI primary
antibody diluted to 1/100 of the 1 mg/ml stock. All DYLIGHT.TM.
550-2.times.PEG-GAM secondary antibodies were diluted to 1/250 of
the 1 mg/ml stock. In this cellular imaging application, the
addition of 5.times.NHS Acetate generated about 50% improvement as
compared to the base conjugate (made without the additives) for
DYLIGHT.TM. 550-2.times.PEG GAM conjugate at 12.5.times. dye molar
excess, and addition of 3.75.times. MS(PEG).sub.4 resulted in about
50% improvement over the base conjugates at 20.times. dye molar
excess.
TABLE-US-00018 TABLE 18 Effect of 2.5X to 10X NHS Acetate and
MS(PEG)4 3.75X to 10X on fluorescence intensity of GAM-DYLIGHT .TM.
550-2xPEG) in a Cellular Imaging application +2.5X +5X +10X FOLD OF
NHS NHS NHS +3.75X +5X +10X IMPROVEMENT NA Acetate Acetate Acetate
MS(PEG).sub.4 MS(PEG).sub.4 MS(PEG).sub.4 GAM-DYLIGHT .TM. 1.0 0.5
0.7 0.5 0.9 0.7 550-2xPEG_7.5x GAM-DYLIGHT .TM. 1.0 1.7 1.7 0.9 1.4
1.5 550-2xPEG_10x -GAM-DYLIGHT .TM. 1.0 1.8 2.0 0.9 1.1 1.2
550-2xPEG_12.5x GAM-DYLIGHT .TM. 1.0 2.2 2.2 1.9 1.3 1.9 1.1
550-2xPEG_15x GAM-DYLIGHT .TM. 1.0 1.7 1.3 1.0 1.2 1.2 1.0
550-2xPEG_20x NA = No Addition
[0236] Results are shown in FIG. 15 and Table 19 for an experiment
testing the effect of NHS Acetate (2.5.times., 5.times. and
10.times.) and MS(PEG)4 (3.75.times., 5.times., 10.times.) spacer
to GAM-DYLIGHT.TM. 650-4.times.PEG) in a cellular imaging
application. U2OS cells were stained with anti-PDI primary antibody
diluted to 1/100 of 1 mg/ml stock. All DYLIGHT.TM.
650-4.times.PEG-GAM secondary antibodies were diluted to 1/250 of 1
mg/ml stock. In this cellular imaging application, the addition of
NHS Acetate-5.times. generated about 70% improvement as compared to
the base conjugate (made without the additives) for DYLIGHT.TM.
650-4.times.PEG-GAM conjugate at 20.times. molar excess, and
MS(PEG).sub.4-3.75.times. showed about 90% improvement over the
base conjugates at 20.times. molar excess.
TABLE-US-00019 TABLE 19 Effect of the addition of NHS Acetate
(2.5X, 5X and 10X) or MS(PEG)4 (3.75X, 5X, 10X) GAM-DYLIGHT .TM.
650-4xPEG) - Cellular Imaging application. +2.5X +5X +10X FOLD OF
NHS NHS NHS +3.75X +5X +10X IMPROVEMENT NA Acetate Acetate Acetate
MS(PEG).sub.4 MS(PEG).sub.4 MS(PEG).sub.4 GAM-DYLIGHT .TM. 1.0 1.0
1.0 0.9 1.2 1.1 650-4xPEG_5x GAM-DYLIGHT .TM. 1.0 0.5 0.5 0.5 0.4
1.6 650-4xPEG_7.5x GAM-DYLIGHT .TM. 1.0 1.3 1.7 0.9 1.0 1.5
650-4xPEG_10x GAM-DYLIGHT .TM. 1.0 0.8 0.8 0.8 0.8 0.7 2.2
650-4xPEG_15x GAM-DYLIGHT .TM. 1.0 1.0 0.9 1.1 1.9 1.1 1.3
650-4xPEG_20x NA = No Addition
[0237] Results are shown in FIG. 16 and Table 20 for an experiment
testing the effect of the addition of NHS Acetate (2.5.times.,
5.times. and 10.times.) and MS(PEG).sub.4 (3.3.75.times., 5.times.,
10.times.) spacer to the detectable fluorescence level of
GAM-DYLIGHT.TM. 680-4.times.PEG) in a cellular imaging application.
U2OS cells were stained with mouse anti-PDI primary antibody
diluted to 1/100 of 1 mg/ml stock. All DYLIGHT.TM.
680-4.times.PEG-GAM secondary antibodies were diluted to 1/250 of 1
mg/ml stock: In this cellular imaging application, the addition of
NHS Acetate-5.times. generated about 70% improvement for dyes
conjugates at both 7.5.times. and 10.times. as compared to the base
conjugate (made without the additives) for DYLIGHT.TM.
680-4.times.PEG. GAM conjugate at molar excesses of 15.times. molar
excess and MS(PEG).sub.4-3.75.times. showed about 80% improvement
over the base conjugates at 15.times. molar excesses.
TABLE-US-00020 TABLE 20 Effect of the addition of NHS Acetate
(2.5X, 5X and 10X) or MS(PEG)4 (3.3.75X, 5X, 10X) GAM-DYLIGHT .TM.
680-4xPEG) - Cellular Imaging application +2.5X +5X +10X FOLD OF
NHS NHS NHS +3.75X +5X +10X IMPROVEMENT NA Acetate Acetate Acetate
MS(PEG).sub.4 MS(PEG).sub.4 MS(PEG).sub.4 GAM-DYLIGHT .TM. 1.0 0.8
0.7 0.7 1.0 0.8 680-4xPEG_5x GAM-DYLIGHT .TM. 1.0 1.2 1.7 1.1 1.1
1.1 680-4xPEG_7.5x GAM-DYLIGHT .TM. 1.0 1.7 1.7 1.1 1.5 1.5
680-4xPEG_10x GAM-DYLIGHT .TM. 1.0 1.4 1.2 1.1 1.8 1.3 1.4
680-4xPEG_15x GAM-DYLIGHT .TM. 1.0 0.8 0.9 1.1 1.0 1.1 1.1
680-4xPEG_20x NA = No Addition
Results
[0238] The use of spacer agents such as NHS-Acetate, NHS-MS(PEG)
and NHS-Betaine increased fluorescent signal sensitivity and
intensity above optimal D/P levels that typically result in
quenching. This was demonstrated by labeling goat anti-mouse (GAM)
and goat anti-rabbit (GAR) secondary antibodies with
NHS-DYLIGHT.TM. 488, NHS-DYLIGHT.TM. 550 2.times.PEG,
NHS-DYLIGHT.TM. 650 4.times.PEG, NHS-DYLIGHT.TM. 680 4.times.PEG,
NHS-DYLIGHT.TM. 800 4.times.PEG in combination with spacer agents
which include, but are not limited to, NHS-Acetate and
NHS-MS(PEG).sub.4, NHS-MS(PEG).sub.8 and NHS-MS(PEG).sub.12. Our
calculations of D/P value following dye conjugation and
purification demonstrated that the addition of the spacer agents
did not make significant differences to the D/P ratios indicating
that these reagents and the fluorophores labeled different primary
amines on the antibody (i.e., they don't compete for the same
primary amines).
[0239] Conjugates made with different dye and spacer agents were
tested in a variety of applications including IFC, Western
blotting, dot blotting or IgG Binding plate-based assays. In each
instance, with certain spacer agents at certain molar excess values
of the spacer agent to the dye, an increase in fluorescence
intensity was observed when spacer agents were used as compared to
controls lacking spacer agents.
[0240] In addition to the above described experiments, antibodies
labeled with NHS-Rhodamine and conjugated with a variety of
NHS-Betaine concentrations (Betaine 2.5, Betaine 5, Betaine 10
molar ratios) displayed an increase in total fluorescence when the
antibodies were conjugated with Betaine as the spacer modification
reagent. See FIG. 17 and Tables 21 and 22 below. Amongst the
different Betaine chain lengths, Betaine 10 had a positive effect
on TAMRA at all molar excesses of the dyes and on ALEXA FLUOR.RTM.
555 above a D/P ratio of 12 (data not shown).
TABLE-US-00021 TABLE 21 Effect of 2.5X to 10X Betaine on
fluorescence of TAMRA-GAM conjugates. NHS- NHS- Relative Betaine
TAMRA TAMRA/IgG Quantum Total MR MR DOL Yield Fluorescence 2.5 5 NA
NA NA 2.5 10 6.3 0.4 2.52 2.5 20 8.6 0.3 2.58 5 5 5.9 0.56 3.3 5 10
7.1 0.34 2.41 5 20 6.4 0.18 1.15 10 5 5.4 0.56 3.02 10 10 8.4 0.5
4.2 10 20 15.7 0.34 5.34 0 5 4.2 0.54 2.27 0 10 6.7 0.42 2.81 0 20
11.4 0.38 4.33 MR = Molar Ratio DOL = Degree of Labeling
Example 5: Reaction of Goat Anti-Mouse IgG (GAM) and 5-(and
-6)-carboxytetramethylrhodamine, succinimidyl ester (5(6)-TAMRA-SE)
with and without N,N,N-Trimethylglycine-N-hydroxysuccinimide ester
bromide (Betaine-SE)
[0241] TAMRA-SE was weighed out and made up as a stock solution at
10 mg/mL in anhydrous DMSO and Betaine-SE was weighed out and made
up at a stock of 4 mg/mL in anhydrous DMSO. The DMSO solutions were
then transferred into reaction vials with the TAMRA-SE+/-Betaine-SE
measured into the vials based on a 5, 10, or 20-fold molar ratio of
dye to IgG and the equivalent of molar ratio 0 or 10 Betaine-SE to
IgG also added to the vials. Separately, 0.417 mL (3.5 mg) of a 8.4
mg/mL solution of GAM in 10 mM potassium phosphate, 150 mM sodium
chloride buffer (PBS) was measured into a plastic tube and the pH
raised to >8.0 with 42 .mu.L of 1M sodium bicarbonate, pH 9.0.
0.5 mg of the GAM solution was added to the reaction vials
containing SE and reacted for 1 hour at RT. The dye-protein
conjugates were separated from free dye and Betaine by size
exclusion chromatography using 5-0.75.times.20 cm columns packed
with BioRAD.TM. BIO-GEL.RTM. P-30 fine in PBS and eluted with same.
The initial protein-containing band from each column was
collected.
[0242] Absorbance spectra were obtained on a Perkin-Elmer Lambda 35
UV/Vis spectrometer, and the degree of substitution (DOS) or moles
dye/mole GAM was determined for each sample. Fluorescence emission
spectra were obtained using a Perkin Elmer LS 55 Fluorescence
Spectrometer, using samples with matched optical density at 545 nm
and excited at 545 nm. Emission data collected from 550-750 nm.
Relative quantum yield (RQY) was measured as the area of the sample
spectrum/area of the dye standard spectrum. Total fluorescence was
then calculated as the product of the RQY*DOS.
TABLE-US-00022 TABLE 22 Total Fluorescent Output of Differing Molar
Ratios of TAMRA/GAM TAMRA/GAM (Molar Ratios) 5 10 20 TAMRA-GAM and
Betaine-SE 3.02 4.2 5.34 added at MR = 10 TAMRA-GAM (No Betaine-SE)
2.27 2.81 4.33
Example 6: Reaction of Goat Anti-Mouse IgG (GAM) and Alexa
Fluor.RTM. 488 Carboxylic Acid, Succinimidyl Ester, Dilithium Salt
(AF488-SE) with and without 1,3-propane sultone
(3-hydroxy-1-propanesulfonic acid .gamma.-sultone)
[0243] AF488-SE was weighed out and made up as a stock solution at
10 mg/mL in anhydrous DMSO and Propane sultone was weighed out and
made up at a stock of 1 mg/mL in E-Pure H.sub.2O.
[0244] 0.357 mL (4.0 mg) of a 11.2 mg/mL solution of GAM in 10 mM
potassium phosphate, 150 mM sodium chloride buffer (PBS) was
measured into a plastic tube and the pH raised to >8.0 with 36
.mu.L of 1M sodium bicarbonate, pH 9.0. 0.5 mg of the GAM solution
was transferred to reaction vials and reacted with a 0, 2, 5, or
10-fold molar excess of propane sultone for 2 minutes and then
AF488 stock was added to the mixtures in an 8 or 15-fold molar
excess over the GAM and let react for 1 hour at room temperature.
The dye-protein conjugates were separated from free dye and Propane
sultone by size exclusion chromatography using 5-0.75.times.20 cm
columns packed with BioRAD.TM. BIO-GEL.RTM. P-30 fine in PBS and
eluted with same. The initial protein-containing band from each
column was collected.
[0245] Absorbance spectra were obtained on a Perkin-Elmer Lambda 35
UV/Vis spectrometer, and the degree of substitution (DOS) or moles
dye/mole GAM was determined for each sample. Fluorescence emission
spectra were obtained using a Perkin Elmer LS 55 Fluorescence
Spectrometer, using samples with matched optical density at 475 nm
and excited at 475 nm. Emission data collected from 480-800 nm.
Relative quantum yield (RQY) was measured as the area of the sample
spectrum/area of the dye standard spectrum. Total fluorescence was
then calculated as the product of the RQY*DOS.
TABLE-US-00023 TABLE 23 Total Fluorescent Output of Differing Molar
Ratios of AF 488/GAM AF488/GAM (Molar Ratios) 8 15 AF488-GAM +
propanesultone/GAM MR = 2 2.52 3.67 AF488-GAM + propanesultone/GAM
MR = 5 2.62 3.73 AF488-GAM + propanesultone/GAM MR = 10 2.69 3.86
AF488-GAM control (no propanesultone) 3.56
Example 7: Labeling of SK3 Mouse Anti-Human CD4 Azide with 20 kDa
8-Arm PEG Amine (20K8 PEG) Modified with ALEXA FLUOR.RTM. 647 NHS
Ester, tris(triethylammonium Salt) (AF647-SE)
[0246] ALEXA FLUOR.RTM. 647 NHS/Succinimidyl Ester (Thermo Fisher
Scientific, cat. no. A37573), abbreviated as "AF647-SE", was
weighed out and made up as a stock solution at 32 mM in anhydrous
DMSO (Thermo Fisher Scientific, D12345). CLICK-IT.TM. SDP Ester
sDIBO Alkyne (sDIBO) (Thermo Fisher Scientific, cat. no. C20025)
was made up as a stock solution at 9 mg/mL in anhydrous DMSO. 8arm
PEG Amine (hexaglycerol), HCl salt (JenKem, Plano, Tex. 75024, cat.
no. 8ARM-NH2HCl), abbreviated as "20K8 PEG", having the following
structure:
R O CH.sub.2CH.sub.2O .sub.nCH.sub.2CH.sub.2--NH.sub.2HCl].sub.8
[0247] R=hexaglycerol core structure was weighed out and prepared
as a stock solution at 40 mg/mL in anhydrous DMSO.
[0248] To a plastic tube, 300 .mu.L of 20K8 PEG stock solution, 176
.mu.L of sDIBO stock solution (2.4-fold molar excess/20K8 PEG) and
6 .mu.L of neat Triethylamine (TEA) was added and allowed to react
for 3 hours at 25.degree. C. After reaction, 300 .mu.L of the
AF647-SE stock solution (2-fold molar excess/PEG-amine) was added
to the tube and the reaction proceeded overnight at 25.degree. C.
The AF647-20K8 PEG-sDIBO constructs were purified from free dye and
sDIBO by size exclusion chromatography using a 1.times.30 cm column
packed with BioRad BIO-GEL.RTM. P-10F in PBS and eluted in the
same. The initial dye-containing fractions were collected and
concentrated using EMD Millipore AMICON.TM. Ultra-4 10 kDa
centrifugal filters.
[0249] Azido (PEO).sub.4 propionic acid, succinimidyl ester (Thermo
Fisher Scientific, cat. no. A10280), abbreviated as "Azide-SE", was
weighed out and made up as a stock solution at 10 mM in anhydrous
DMSO (Thermo Fisher Scientific, D12345). 266 .mu.L SK3 mouse
anti-human CD4 antibody (2.5 mg) and 134 .mu.L PBS were added to a
plastic tube and the pH was raised to >8.0 with 50 .mu.L 1M
sodium bicarbonate, pH 9.0. 8.3 .mu.L of Azide-SE stock solution (5
fold molar excess/antibody) and 42 .mu.L of DMSO were added to the
antibody solution. The reaction was allowed to proceed for 2 hours
at 25.degree. C. The azido-SK3 antibody was purified using 2 mL
BioRad BIO-GEL.RTM. P-30M spin columns. Azido-SK3 antibody was
prepared with 5-fold to 20-fold excess of Azide-SE to antibody.
[0250] 24.2 .mu.L of a 2 mM solution of AF647-20K8 PEG-sDIBO
(concentration of DIBO) and 116 .mu.L of a 4.3 mg/mL azido-SK3 were
combined in a plastic tube. 360 .mu.L of PBS were added to bring
the final concentration of the solution to 100 .mu.M AF647-20K8
PEG-sDIBO (concentration of DIBO) and 1 mg/mL azido-SK3 antibody.
The click reaction was allowed to proceed for 2 hours at 37.degree.
C. followed by quenching with 5 mM NaN.sub.3 for 1 hour at room
temperature. AF647-20K8 PEG-SK3 conjugates were diluted to 0.5
mg/mL in PBS. Conjugation reactions were carried out at final DIBO
concentrations between 100 and 600 .mu.M, with azido-SK3 antibody
at 1-3 mg/mL, at reaction temperatures between 25.degree. C. and
37.degree. C., for 2 hours to 20 hours. Specific conditions per
experimental run are set out in Table 24.
[0251] 24.2 .mu.L of a 2 mM solution of AF647-20K8 PEG-sDIBO
(concentration of DIBO) and 116 .mu.L of a 4.3 mg/mL azido-SK3 were
combined in a plastic tube. 360 .mu.L of PBS were added to bring
the final concentration of the solution to 100 .mu.M AF647-20K8
PEG-sDIBO (concentration of DIBO) and 1 mg/mL azido-SK3 antibody.
The click reaction was allowed to proceed for 2 hours at 37.degree.
C. followed by quenching with 5 mM NaN.sub.3 for 1 hour at room
temperature. Conjugates were purified and concentrated using EMD
Millipore AMICON.TM. Ultra-4 100 kDa centrifugal filters.
AF647-20K8 PEG-SK3 conjugates were diluted to 0.5 mg/mL in PBS.
Conjugation reactions were carried out at final DIBO concentrations
between 100 and 600 .mu.M, with azido-SK3 antibody at 1-3 mg/mL, at
reaction temperatures between 25.degree. C. and 37.degree. C., for
2 hours to 20 hours. Specific conditions per experimental run are
set out in Table 24.
TABLE-US-00024 TABLE 24 Conjugation Conditions Click Incubation
condition starPEG SK3-N.sub.3 SK3 DIBO Example MR.sup.1 DOS.sup.2
starPEG.sup.3 mg/mL.sup.4 .mu.M.sup.5 time/temp.sup.6 sa1 5 3 HG 20
kD 1 100 2 h/37.degree. C. sa2 10 6.1 HG 20 kD 1 100 2 h/37.degree.
C. sa3 20 12 HG 20 kD 1 100 2 h/37.degree. C. sa4 5 3 HG 20 kD 1
200 2 h/37.degree. C. sa5 10 6.1 HG 20 kD 1 200 2 h/37.degree. C.
sa6 20 12 HG 20 kD 1 200 2 h/37.degree. C. sa2* 10 6.1 HG 20 kD 1
100 20 h/25.degree. C. sa7 5 3 TP 20 kD 1 100 2 h/37.degree. C. sa8
10 6.1 TP 20 kD 1 100 2 h/37.degree. C. sa9 20 12 TP 20 kD 1 100 2
h/37.degree. C. sa10 5 3 TP 20 kD 1 200 2 h/37.degree. C. sa11 10
6.1 TP 20 kD 1 200 2 h/37.degree. C. sa12 20 12 TP 20 kD 1 200 2
h/37.degree. C. sa8* 10 6.1 TP 20 kD 1 100 20 h/25.degree. C. sa13
10 6.1 HG 20 kD 3 600 3 h/37.degree. C. sa14 20 12 HG 20 kD 3 600 3
h/37.degree. C. .sup.1MR: Molar Ratio; x-fold excess used to tag
SK3 antibody with Azide-SE: antibody (mole/mole). .sup.2DOS: degree
of substitution, number of azide groups incorporated per antibody
molecule. .sup.3starPEG: indicates MW of starPEG (20 kD) and core.
HG: hexaglycerol, TP: tripentaerythritol. .sup.4In the click
incubation SK3 was present at indicated concentrations in mg/mL
(specified for each example). .sup.5The starPEG DIBO during the
click conjugation was present at the indicated concentrations in
.mu.M. .sup.6The click reactions were carried out for the indicated
times at the indicated temperatures.
Example 8: Quantum Yield Characterization of Branched PEG AF647
Constructs
[0252] To prepare samples for quantum yield measurement, solutions
of AF647 Branched PEG constructs (AF647-2K4, AF647-10K4, AF647-10K8
and AF647-20K8) were diluted to a final dye concentration of 0.16
.mu.M in deionized water. Quantum yield (.PHI.) was measured using
a Hamamatsu Absolute PL Quantum Yield Spectrometer. Quantum yields
for the Branched PEG constructs were compared to that of the free
dye to determine the degree of quenching in the final constructs.
Additionally, the brightness was determined to determine the
fluorescent enhancement achieved using the branched PEG spacers.
The smallest construct, AF647-2K4 (2,000 molecular weight branched
PEG with four arms) showed the greatest quenching (20% QY of the
free dye, 0.2 Fluorescent Ratio) and had the lowest overall
improvement in the total fluorescence. The greatest fluorescence
enhancement was seen for higher molecular weight constructs, with
either 4 or 8 arms (AF647-10K4 and AF647-20K8) where up to 89% of
the fluorescence quantum yield was retained of the free dye
(Fluorescent Ratio of 0.9), and up to 5.8 fold improvement in
brightness was noted.
TABLE-US-00025 TABLE 25 Effect of Branched PEG spacers on the
percent quantum yield (QY) and brightness (B) of ALEXA FLUOR .RTM.
647 % QY of Dyes/ Quantum Free Dye Brightness Molecule Yield
(Fluorescent Brightness, B Ratio to Free Sample (N) (QY) Ratio) (QY
.times. .times. N) AU Dye AF647 1 0.404 100% (1.0) 1.09E+05 1.0
AF647-2K4 4 0.087 22% (0.2) 9.40E+04 0.9 AF647-10K4 4 0.359 89%
(0.9) 3.88E+05 3.6 AF647-10K8 8 0.145 36% (0.4) 3.13E+05 2.9
AF647-20K8 8 0.295 73% (0.7) 6.37E+05 5.8 An assumption is made
that effectively one fluorescent label is attached to the terminus
of each arm. In other words, an assumption is made that the PEG
molecules have been labeled to saturation, thus N = number of
arms/polymer. = 270,000 cm.sup.-1M.sup.-1 for ALEXA FLUOR .RTM.
647
Example 9: Flow Cytometry Evaluation of AF647-20K8 PEG-SK3
Constructs
[0253] Freshly collected anti-coagulated whole blood (human) was
lysed using ACK lysis buffer for 20 minutes at room temperature.
White blood cells were isolated by centrifugation (400.times.g, 5
mins.) and washes in 1% bovine serum albumin/PBS twice (1%
BSA/PBS). After isolation, the total number of white blood cells
was determined using the COUNTESS.RTM. Automated Cell Counter and
then diluted to 10 million cells per mL. One million cells/well in
a 96 well plate were stained with the AF647-20K8 PEG-SK3 conjugates
using a 7 point titration of 1 .mu.g to 0.015 .mu.g of antibody.
Stained cells were washed twice with 1% BSA/PBS. Analysis of the
stained cells was carried out using the ATTUNE.TM. NxT Flow
Cytometer and compared to APC (Thermo Fisher Scientific, cat. no.
MHCD0405), ALEXA FLUOR.RTM. 488 (Invitrogen, cat. no. MHCD0420),
FITC (Thermo Fisher Scientific, cat. no. MA1-81103) and BRILLIANT
VIOLET.TM. 605 (BioLegend, San Diego, Calif., cat. no. 300555) CD4
conjugates.
[0254] FIG. 22 shows a histogram plot of CD4 positive lymphocyte
cells as a function of fluorescence intensity in the RL1 channel of
the ATTUNE.TM. NxT Flow Cytometer ALEXAFLUOR.RTM. 647 conjugated to
CD4 is shown as a dashed line, and the AF647-20K8 PEG-SK3
conjugates are shown as dotted or solid lines. Compared to the
AF647 conjugate alone, the StarPEG conjugates show a greater that
0.5 log increase in brightness. FIG. 23 shows plotted signal to
noise (S/N) and percent positive (% Positive) as a function of
conjugate concentration in the flow cytometry experiment. It is
shown that the StarPEG constructs (here B1 and B2) have up to 2.5
fold increase in S/N versus the APC CD4 benchmark conjugate, and up
to 2 fold increase in S/N versus the AF647 CD4 benchmark conjugate
while retaining the ability to accurately assess the number of CD4
positive cells in the sample.
Example 10: Conjugation of ALEXA FLUOR.RTM. 488 to an Amino Dextran
Scaffold
[0255] Preparation of 70 kD amino dextran AF488 scaffold: 10 mg of
amino dextran (70,000 MW, 20 amino groups; Thermo Fisher
Scientific, Cat. No. D1862) was dissolved in 1.2 ml of dry DMSO
containing 1.0 .mu.l of DIEA. 0.9 mg of ALEXA FLUOR.RTM. 488
succinimidyl ester lithium salt (643 MF; Thermo Fisher Scientific,
Cat. No. A20000) was added to solution and the mixture was stirred
for 3.5 hours at ambient temperature. The solution was diluted with
12 mL of ethyl acetate and the resulting suspension was
centrifuged. The supernatant was discarded and the solid material
was shaken with 10 mL of fresh ethyl acetate and centrifuged. This
washing was repeated 3 more times with 10 mL of fresh ethyl acetate
and the resulting precipitate was dried in vacuum. The solid was
re-dissolved in 0.5 ml of water and solution put in 10 cm
Spectra/Por Dialysis membrane (Spectrum Labs, MWCO 12-14,000 flat
width 10 mm) clipped from both side. The dialysis membrane was
slowly stirred in 1 L of water for 1 week. The water was replaced
twice per day. The dialysis membrane was open from one end and
solution was lyophilized to give amino dextran ALEXA FLUOR.RTM.
scaffold. The measured DOL is 9.7 and relative QY is 0.6
(referenced to QY of ALEXA FLUOR.RTM. 488).
[0256] Attaching thiol linker to 70 kD amino dextran AF488
scaffold: Amino dextran AF488 scaffold (4.5 mg) was dissolved in
0.5 mL of DMSO containing 0.055 .mu.L of N,N-Diisopropylethylamine
(DIEA). Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (20
.mu.g) was added to solution and the mixture was kept at ambient
temperature overnight and then capped with acetic acid succimidyl
ester (1.0 mg, 3 hrs). The solution was diluted with 10 mL of ethyl
acetate. The resulting suspension was centrifuged and supernatant
discarded. The solid was shaken with 10 mL of fresh ethyl acetate
and centrifuged. The washing was repeated 5 more times. The
resulting solid was dried in vacuum. The measured DOL is 0.74. This
material was re-dissolved in 2 mL of water and 16 mg of DTT was
added to solution. The mixture was stirred for 5 min and loaded on
G15 SEPHADEX.RTM. column, the product was eluted with DE water as
green fluorescent solution which was used for conjugation to SMCC
modified streptavidin. The determined concentration was 48 .mu.M
(by dye adsorption).
[0257] Conjugation of amino dextran AF488 scaffold modified with
thiol linker to SMCC modified streptavidin: SMCC modified
streptavidin (35 .mu.L solution in water) was treated with 1, 2, 3
and 4 equivalents of thiol modified amino dextran AF488 scaffold
(48 .mu.M solution in water). The reaction was carried out at
ambient temperature for 3 hours and after that reaction mixture was
kept overnight at 4.degree. C. overnight. The conjugates are
purified on P100 size exclusion column with 10 nM PBS buffer.
TABLE-US-00026 TABLE 26 Streptavidin Labeled with 70 kD Amino
dextran AF488 Scaffold (average of 9.7 molecules of dye per
scaffold) DOL by Scaffold DOL by Dye (Avg.) (Avg.) QY Brightness
0.9 9.2 0.52 4.8 1.1 10.2 0.50 5.1 1.8 18 0.53 9.5 2.6 25.5 0.54
13.7
TABLE-US-00027 TABLE 27 Streptavidin labeled with AF488 dye DOL
(Avg.) QY Brightness 1.5 0.70 1.0 3.0 0.60 1.8 4.0 0.55 2.1 4.5
0.40 1.8 5.0 0.34 1.7
[0258] Results: As shown in Tables 26 and 27, conjugates made from
the scaffold are brighter as compared to conjugates made from
single AF488 dye. Also, QY of AF488 fluorophore drops from 0.70 to
0.34 for single dye conjugation in contrast to almost constant QY
for labeling with the amino dextran scaffold.
Sequence CWU 1
1
61104PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMOD_RES(3)..(102)Any amino
acidMISC_FEATURE(3)..(102)This region may encompass 2-100 residues
1Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1
5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa
Xaa Cys Cys 100 26PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 2Cys Cys Pro Gly Cys Cys 1 5
312PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Ala Gly Gly Cys Cys Pro Gly Cys Cys Gly Gly Gly
1 5 10 412PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Cys Cys Gly Gly Lys Gly Asn Gly Gly Cys Gly Cys
1 5 10 5106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMOD_RES(3)..(102)Any amino
acidMISC_FEATURE(3)..(102)This region may encompass 2-100
residuesMOD_RES(104)..(104)Any amino acidMOD_RES(106)..(106)Any
amino acid 5Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 100 105 66PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag 6His
His His His His His 1 5
* * * * *