U.S. patent application number 17/174860 was filed with the patent office on 2021-06-17 for antigen detection using photocleavable labels.
This patent application is currently assigned to Agilent Technologies, Inc.. The applicant listed for this patent is Agilent Technologies, Inc.. Invention is credited to Xiyi CHEN, Stacie FRYE, Mimi HEALY, Sophia PETRICHENKO, Jinchun WANG, Weidong WU, Su ZHANG.
Application Number | 20210181191 17/174860 |
Document ID | / |
Family ID | 1000005419677 |
Filed Date | 2021-06-17 |
United States Patent
Application |
20210181191 |
Kind Code |
A1 |
HEALY; Mimi ; et
al. |
June 17, 2021 |
ANTIGEN DETECTION USING PHOTOCLEAVABLE LABELS
Abstract
Provided herein are methods of using photocleavable labels for
multiplex and serial antigen detection. The methods comprise
detecting the presence of photocleavable labels, which are
conjugated through functional linkers to antigen-binding complexes,
which in turn non-covalently bind to antigens. The presence of a
photocleavable label is indicative of the presence of an antigen
specifically or selectively bound by an antigen-binding complex.
Also provided are apparatuses for using photocleavable labels for
multiplex and serial antigen detection.
Inventors: |
HEALY; Mimi; (Santa Clara,
CA) ; CHEN; Xiyi; (Santa Clara, CA) ; WANG;
Jinchun; (Santa Clara, CA) ; WU; Weidong;
(Santa Clara, CA) ; PETRICHENKO; Sophia; (Santa
Clara, CA) ; ZHANG; Su; (Santa Clara, CA) ;
FRYE; Stacie; (Santa Clara, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Agilent Technologies, Inc. |
Santa Clara |
CA |
US |
|
|
Assignee: |
Agilent Technologies, Inc.
Santa Clara
CA
|
Family ID: |
1000005419677 |
Appl. No.: |
17/174860 |
Filed: |
February 12, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15230876 |
Aug 8, 2016 |
10942176 |
|
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17174860 |
|
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|
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62201978 |
Aug 6, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2035/00138
20130101; G01N 33/53 20130101; B01L 2200/026 20130101; B01L
2300/0627 20130101; G01N 33/533 20130101; G01N 33/5308 20130101;
G01N 33/58 20130101; G01N 33/582 20130101; G01N 1/30 20130101; G01N
2458/10 20130101; G01N 1/312 20130101; B01L 3/502 20130101; G01N
2458/00 20130101; G01N 35/00029 20130101; B01L 2400/082 20130101;
G01N 2001/302 20130101; G01N 33/54306 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; B01L 3/00 20060101 B01L003/00; G01N 1/30 20060101
G01N001/30; G01N 1/31 20060101 G01N001/31; G01N 33/533 20060101
G01N033/533; G01N 33/53 20060101 G01N033/53; G01N 35/00 20060101
G01N035/00; G01N 33/58 20060101 G01N033/58 |
Claims
1. A method for detecting the presence of at least a first antigen
and a second antigen on or in a sample, the method comprising: (i)
contacting the sample with a first antigen-binding complex that is
conjugated to a first photocleavable label through a first
functional linker, wherein the first antigen-binding complex is
capable of non-covalently binding to the first antigen, thereby
forming an antigen-bound antigen-binding complex of formula (I):
(ii) detecting the presence of the first photocleavable label in
the sample, wherein the presence of the first photocleavable label
is indicative of the presence of the first antigen; (iii) exposing
the sample to ultraviolent light in order to cleave the first
photocleavable label; (iv) contacting the sample with a second
antigen-binding complex that is conjugated to a second
photocleavable label through a second functional linker, wherein
the second antigen-binding complex is capable of non-covalently
binding to the second antigen, thereby forming an antigen-bound
antigen-binding complex of formula (I-a): (v) detecting the
presence of the second photocleavable label in the sample, wherein
the presence of the second photocleavable label is indicative of
the presence of the second antigen, wherein the first functional
linker and the second functional linker are each a partially
double-stranded oligonucleotide, wherein each oligonucleotide of
the partially double-stranded oligonucleotide is 34 nucleotides in
length and wherein the partially double-stranded oligonucleotide
has a double-stranded section that is 28 nucleotides in length,
wherein each oligonucleotide in the partially double-stranded
oligonucleotide has a biotin on its 5' end.
2. The method of claim 1, wherein each oligonucleotide in the
partially double-stranded oligonucleotide has a photocleavable
label on its 3' end.
3. The method of claim 1, further comprising detecting the presence
of a third antigen in the sample by contacting the sample with a
third antigen-binding complex that is conjugated to a third
photocleavable label and detecting the presence of the third
photocleavable label in the sample, wherein the third
antigen-binding complex is capable of non-covalently binding to the
third antigen, wherein the presence of the third photocleavable
label is indicative of the presence of the third antigen.
4. The method of claim 3, wherein detecting the first
photocleavable label and detecting the third photocleavable label
is performed simultaneously.
5. The method of claim 4, wherein the first antigen-binding complex
and the third antigen-binding complex each comprise a unique
photocleavable label.
6. The method of claim 3, wherein detecting the second
photocleavable label and detecting the third photocleavable label
is performed simultaneously.
7. The method of claim 6, wherein the second antigen-binding
complex and the third antigen-binding complex each comprise a
unique photocleavable label.
8. The method of claim 3, wherein detecting the third
photocleavable label is performed sequentially after detecting the
second photocleavable label, wherein the second photocleavable
label is cleaved prior to contacting the sample with the third
antigen-binding complex.
9. The method of claim 1, wherein the first antigen-binding complex
and the second antigen-binding complex each comprise the same
photocleavable label.
10. The method of claim 1, wherein the first antigen-binding
complex and the second antigen-binding complex each comprise a
unique photocleavable label.
11. The method of claim 1, wherein the photocleavable label
comprises a reporter moiety covalently bound to a photocleavable
moiety.
12. The method of claim 11, wherein the photocleavable moiety is a
2-nitrobenzyl group.
13. The method of claim 12, wherein the 2-nitrobenzyl group
comprises a substitution on the .alpha.-carbon and/or a 5-methoxy
substitution on the benzene ring.
14. The method of claim 11, wherein the reporter moiety is a
colorimetric dye, a fluorescent dye, a radioactive label, a
chemiluminescent group, or a bioluminescent group.
15. The method of claim 1, wherein the first antigen-binding
complex and the second antigen-binding complex are each defined by
formula (VIII): wherein the first antigen-binding complex and the
second antigen-binding complex each comprise a primary antibody
bound by a secondary antibody, wherein the secondary antibody is
covalently bound to a first ligand, wherein the first ligand is
non-covalently bound to a first anti-ligand, wherein the
antigen-bound antigen-binding complex is further defined by formula
(IX):
16. The method of claim 1, wherein the sample is a tissue section,
biopsy sample, cell culture sample, cell smear, or protein lysate.
Description
[0001] The present application is a continuation of U.S. patent
application Ser. No. 15/230,876, filed Aug. 8, 2016, which claims
the priority benefit of U.S. provisional application No.
62/201,978, filed Aug. 6, 2015, the entire contents of each are
incorporated herein by reference.
[0002] Pursuant to 37 C.F.R. 1.821(c), a sequence listing is
submitted herewith as an ASCII compliant text file named
"LSGNP0011USC1_ST25.txt", created on Feb. 11, 2021 and having a
size of .about.5 kilobytes. The content of the aforementioned file
is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0003] The present invention relates generally to the fields of
histology, pathology, and molecular biology. More particularly, it
concerns the use of photocleavable labels (PCLs) for antigen
detection, including, for example, for multiplex and serial antigen
detection.
2. Description of Related Art
[0004] In typically used immunostaining protocols, an
antigen-specific antibody is added to an antigen-containing
specimen including, but not limited to, a tissue section, a cell,
or a protein blot, and allowed to bind to the antigen. This antigen
bound antibody is then detected using any of a variety of
techniques, including (a) a second antibody, with specific affinity
for the first antibody, conjugated to a fluorophore (e.g., Cy5) or
an enzyme used for colorimetric detection (e.g., HRP); (b) a second
antibody, with specific affinity for the first antibody, conjugated
to biotin which is subsequently detected using avidin or
streptavidin conjugated to a fluorophore (e.g., Cy5) or an enzyme
used for colorimetric detection (e.g., HRP); and (c) a fluorophore
(e.g., Cy5) or an enzyme used for colorimetric detection (e.g.,
HRP) or biotin that is directly conjugated to the first antibody.
In instance (a) and (b), such technologies allow for about three
antibodies, each uniquely labeled, to be used on the same sample.
In instance (c), such technologies are limited to about four
antibodies conjugated to distinct labels that can be separately
imaged. However, non-specific interactions between the secondary
antibodies and the limited ability to detect individual labels
prevent higher order multiplexing. New multiplex technologies are
emerging, but many suffer drawbacks, e.g., time consuming,
expensive, and/or require extensively optimized primary antibodies.
Improved methods and instruments for automating antigen detection
are desirable.
SUMMARY OF THE INVENTION
[0005] Provided herein are methods of using photocleavable labels
for multiplex and serial antigen detection. The methods comprise
detecting the presence of photocleavable labels, which are
conjugated through functional linkers to antigen-binding complexes,
which are in turn non-covalently bound to antigens. Thus, the
presence of a photocleavable label is indicative of the presence of
an antigen specifically or selectively bound by an antigen-binding
complex.
[0006] In one aspect, methods are provided for detecting the
presence of at least a first antigen on or in a sample comprising a
first antigen-binding complex capable of specifically binding the
first antigen, the method comprising detecting the presence of a
photocleavable label in the sample, wherein the photocleavable
label is conjugated to the first antigen-binding complex through a
functional linker, wherein the first antigen-binding complex is
non-covalently bound to the first antigen forming an antigen-bound
antigen-binding complex of formula (I):
wherein the presence of the photocleavable label is indicative of
the presence of the first antigen.
[0007] In some embodiments, the methods further comprise
photocleaving the photocleavable label. Photocleaving may comprise
exposing the sample to ultraviolet light.
[0008] In some embodiments, the photocleavable label comprises a
reporter moiety covalently conjugated to a photocleavable moiety.
The photocleavable moiety may be a 2-nitrobenzyl group, a benzoin
group, a coumarinyl group, or a p-hydroxyphenacyl group. In various
embodiments, the photocleavable moiety is a 2-nitrobenzyl group. In
certain embodiments, the 2-nitrobenzyl group comprises a
substitution on the .alpha.-carbon and/or on the benzene ring.
[0009] In some embodiments, the functional linker is a
single-stranded oligonucleotide, an at least partially
double-stranded oligonucleotide, a peptide, or an
alkanediyl.sub.(C.ltoreq.16). In certain embodiments, the at least
partially double-stranded oligonucleotide is a hairpin
oligonucleotide. In certain embodiments, the at least partially
double-stranded oligonucleotide is a fully double-stranded
oligonucleotide.
[0010] In some embodiment, the first antigen-binding complex is
defined by formula (II):
wherein the first antigen-binding complex comprises a primary
antibody bound to a conjugation moiety, wherein the antigen-bound
antigen-binding complex is further defined by formula (III):
[0011] In some embodiments, the primary antibody is modified by at
least one reducing agent, such as, for example, TCEP or 2-MEA. In
some embodiments, the conjugation moiety is a heterobifunctional
linker, such as, for example, Sulfo-SMCC or SM(PEG)n. In some
embodiments, the conjugation moiety is covalently attached to the
primary antibody. In some embodiments, the conjugation moiety is
covalently attached to the functional linker. In some embodiments,
the functional linker is a partially double-stranded
oligonucleotide with a C6 amino modification on the 5' end and a
photocleavable label on the 3' end.
[0012] In some embodiments, the first antigen-binding complex is
defined by the formula (IV):
wherein the first antigen-binding complex comprises a primary
antibody covalently conjugated to a first ligand, wherein the first
ligand is non-covalently bound to a first anti-ligand, wherein the
antigen-bound antigen-binding complex is further defined by formula
(V):
[0013] In some embodiments, the methods further comprise
sequentially contacting the sample with the primary antibody and
the first anti-ligand. In other embodiments, the methods further
comprise simultaneously contacting the sample with the primary
antibody and the first anti-ligand. The first ligand may be biotin,
avidin or streptavidin, or a first single-stranded oligonucleotide.
The first anti-ligand may be avidin or streptavidin, biotin, or a
second single-stranded oligonucleotide at least partially
complementary to the first single-stranded oligonucleotide.
[0014] In some embodiments, the photocleavable label is covalently
bound to the first anti-ligand through a functional linker. In some
embodiments, two occurrences of the same photocleavable label are
covalently bound to the first anti-ligand through a functional
linker. In other embodiments, two different photocleavable labels
are covalently bound to the first anti-ligand through a functional
linker. The functional linker may be a single-stranded
oligonucleotide, an at least partially double-stranded
oligonucleotide (such as, but not limited to, a hairpin
oligonucleotide), a peptide, or an alkanediyl.sub.(C.ltoreq.16). In
certain embodiments, the at least partially double-stranded
oligonucleotide is a fully double-stranded oligonucleotide.
[0015] In some embodiments, the first antigen-binding complex is
defined by formula (VI):
wherein the first antigen-binding complex comprises a primary
antibody covalently bound to a first ligand, wherein the first
ligand is non-covalently bound to a first anti-ligand, wherein the
second ligand is non-covalently bound to the first anti-ligand,
wherein the antigen-bound antigen-binding complex is further
defined by formula (VII):
[0016] In some embodiments, the methods further comprise
sequentially contacting the sample with the primary antibody, the
first anti-ligand, and/or the second ligand. In other embodiments,
the methods further comprise simultaneously contacting the sample
with the primary antibody, the first anti-ligand, and/or the second
ligand. The first ligand may be biotin, avidin, or an anti-avidin
antibody. The first anti-ligand may be avidin or streptavidin or
biotin. The second ligand may be biotin, avidin, or an anti-avidin
antibody. In some aspects, the first anti-ligand comprises a
single-stranded oligonucleotide and the second ligand comprises a
second single-stranded oligonucleotide at least partially
complementary to the first single-stranded oligonucleotide.
[0017] In some embodiments, the photocleavable label is covalently
bound to the second ligand through a functional linker. In some
embodiments, two occurrences of the same photocleavable label are
covalently bound to the second ligand through a functional linker.
In other embodiments, two different photocleavable labels are
covalently bound to the second ligand through a functional linker.
The functional linker may be a single-stranded oligonucleotide, an
at least partially double-stranded oligonucleotide (such as, but
not limited to, a hairpin oligonucleotide), a peptide, or an
alkanediyl.sub.(C.ltoreq.16). In certain embodiments, the at least
partially double-stranded oligonucleotide is a fully
double-stranded oligonucleotide.
[0018] In some embodiments, the first antigen-binding complex is
defined by formula (VIII):
wherein the first antigen-binding complex comprises a primary
antibody bound by a secondary antibody, wherein the secondary
antibody is covalently bound to a first ligand, wherein the first
ligand is non-covalently bound to a first anti-ligand, wherein the
antigen-bound antigen-binding complex is further defined by formula
(IX):
[0019] In some embodiments, the methods further comprise
sequentially contacting the sample with the primary antibody, the
secondary antibody, and/or the first anti-ligand. In other
embodiments, the methods further comprise simultaneously contacting
the sample with the primary antibody, the secondary antibody,
and/or the first anti-ligand. The first ligand may be biotin,
avidin or streptavidin, or a first single-stranded oligonucleotide.
The first anti-ligand may be avidin, streptavidin, biotin,
anti-avidin, or a second single-stranded oligonucleotide at least
partially complementary to the first single-stranded
oligonucleotide.
[0020] In some embodiments, the photocleavable label is covalently
bound to the first anti-ligand through a functional linker. In some
embodiments, two occurrences of the same photocleavable label are
covalently bound to the first anti-ligand through a functional
linker. In other embodiments, two different photocleavable labels
are covalently bound to the first anti-ligand through a functional
linker. The functional linker may be a single-stranded
oligonucleotide, an at least partially double-stranded
oligonucleotide (such as, but not limited to, a hairpin
oligonucleotide), a peptide, or an alkanediyl.sub.(C.ltoreq.16). In
certain embodiments, the at least partially double-stranded
oligonucleotide is a fully double-stranded oligonucleotide.
[0021] In some embodiments, the first antigen-binding complex is
defined by formula (X):
wherein the first antigen-binding complex comprises a primary
antibody bound by a secondary antibody, wherein the secondary
antibody is covalently bound to a first ligand, wherein the first
ligand is non-covalently bound to a first anti-ligand, wherein the
second ligand is non-covalently bound to the first anti-ligand,
wherein the antigen-bound antigen-binding complex is further
defined by formula (XI):
[0022] In some embodiments, the methods further comprise
sequentially contacting the sample with the primary antibody, the
secondary antibody, the first anti-ligand, and/or the second
ligand. In other embodiments, the methods further comprise
simultaneously contacting the sample with the primary antibody, the
secondary antibody, the first anti-ligand, and/or the second
ligand. The first ligand may be biotin or avidin or streptavidin.
The first anti-ligand may be avidin or streptavidin or biotin. The
second ligand may be biotin or avidin or streptavidin. In some
aspects, the first anti-ligand comprises a single-stranded
oligonucleotide and the second ligand comprises a second
single-stranded oligonucleotide at least partially complementary to
the first single-stranded oligonucleotide.
[0023] In some embodiments, the photocleavable label is covalently
bound to the second ligand through a functional linker. In some
embodiments, two occurrences of the same photocleavable label are
covalently bound to the second ligand through a functional linker.
In other embodiments, two different photocleavable labels are
covalently bound to the second ligand through a functional linker.
The functional linker may be a single-stranded oligonucleotide, an
at least partially double-stranded oligonucleotide (such as, but
not limited to, a hairpin oligonucleotide), a peptide, or an
alkanediyl.sub.(C.ltoreq.16). In certain embodiments, the at least
partially double-stranded oligonucleotide is a fully
double-stranded oligonucleotide.
[0024] In some embodiments, the first antigen-binding complex is
defined by formula (XII):
wherein the first antigen-binding complex comprises a primary
antibody bound by a secondary antibody, wherein the secondary
antibody is covalently bound to the photocleavable label through a
functional linker, wherein the antigen-bound antigen-binding
complex is further defined by formula (XIII):
[0025] In some embodiments, the methods further comprise
sequentially contacting the sample with the primary antibody and
the secondary antibody. In other embodiments, the methods further
comprise simultaneously contacting the sample with the primary
antibody and the secondary antibody.
[0026] In some embodiments, the photocleavable label is covalently
bound to the secondary antibody through a functional linker. In
some embodiments, two occurrences of the same photocleavable label
are covalently bound to the secondary antibody through a functional
linker. In other embodiments, two different photocleavable labels
are covalently bound to the secondary antibody through a functional
linker. The functional linker may be a single-stranded
oligonucleotide, an at least partially double-stranded
oligonucleotide (such as, but not limited to, a hairpin
oligonucleotide), a peptide, or an alkanediyl.sub.(C.ltoreq.16). In
certain embodiments, the at least partially double-stranded
oligonucleotide is a fully double-stranded oligonucleotide.
[0027] In some embodiments, the first antigen-binding complex
comprises an aptamer, wherein the aptamer is covalently bound to
the photocleavable label through a functional linker, wherein the
antigen-bound antigen-binding complex is further defined by formula
(XIV):
[0028] In some embodiments, the methods further comprise contacting
the sample with the aptamer.
[0029] In some embodiments, the methods are defined as methods of
detecting the presence of at least two antigens in the sample, the
methods further comprising detecting the presence of at least a
second photocleavable label in the sample, wherein the second
photocleavable label is conjugated to a second antigen-binding
complex through a functional linker, wherein the second
antigen-binding complex is bound to the second antigen, wherein the
presence of the second photocleavable label is indicative of the
presence of the second antigen. Likewise, the methods may be
defined as methods of detecting the presence of at least three
antigens in the sample, the methods further comprising detecting
the presence of at least a third photocleavable label in the
sample, wherein the third photocleavable label is conjugated to a
third antigen-binding complex through a functional linker, wherein
the third antigen-binding complex is bound to the third antigen,
wherein the presence of the third photocleavable label is
indicative of the presence of the third antigen.
[0030] In some embodiments, detecting the first photocleavable
label and detecting the second, and optionally the third,
photocleavable label are performed simultaneously. In some
embodiments, the first antigen-binding complex and the second
antigen-binding complex each comprise a unique photocleavable
label. In other embodiments, the first antigen-binding complex and
the second antigen-binding complex each comprise the same
photocleavable label. In some embodiments, detecting the first
photocleavable label and detecting the second photocleavable label
are performed sequentially.
[0031] In some embodiments, the methods further comprise
photocleaving the first photocleavable label prior to detecting the
second photocleavable label. In some embodiments, the methods
further comprise photocleaving the second photocleavable label
following detection of the second photocleavable label. In some
embodiments, the methods further comprise photocleaving the first
and second photocleavable labels simultaneously. Photocleaving may
comprise exposing the sample to ultraviolet light.
[0032] In some embodiments, the methods further comprise detecting
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, or 100 antigens in the sample. Some antigens may be detected
simultaneously while other antigens are detected sequentially
within the same sample. For simultaneous detection, each
antigen-binding complex comprises a unique photocleavable label. At
least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 different antigens may be detected simultaneously. In
methods of simultaneous antigen detection, each antigen-binding
complex may be added to the sample sequentially followed by a
single detection step. Alternatively, some of the antigen-binding
complexes may be added simultaneously. In various embodiments, at
least a portion of the steps of the method may be automated. In
some embodiments, all of the steps of the method may be
automated.
[0033] In some embodiments, the photocleavable label is a
colorimetric dye, a fluorescent dye, a radioactive label, a
chemiluminescent group, or a bioluminescent group. In some
embodiments, the sample is a tissue section, biopsy sample, cell
culture sample, cell smear, or protein lysate.
[0034] Certain embodiments include an apparatus configured to
perform any of the methods disclosed herein. Particular embodiments
include: an imaging system configured to image a sample; a sample
chamber comprising a fluid inlet and a fluid outlet; and a fluid
control system comprising a plurality of reservoirs, where the
fluid control system is in fluid communication with the fluid inlet
and the fluid outlet of the sample chamber.
[0035] In some embodiments, the fluid control system is configured
to provide automated serial staining of the sample. In specific
embodiments, the fluid control system further comprises a valve in
fluid communication with the sample chamber and with the plurality
of reservoirs. In certain embodiments, the valve is a rotary valve.
Particular embodiments further comprise a light source configured
to photocleave the first antigen-binding complex from the first
antigen. In some embodiments, the plurality of reservoirs comprises
a first reservoir containing an imaging solution, a second
reservoir containing a wash solution, a third reservoir containing
a cleavage solution, a fourth reservoir containing a buffer
solution, a fifth reservoir containing a hybridization wash
solution, and/or a sixth reservoir containing a hybridization
solution.
[0036] In specific embodiments, the fluid control system further
comprises a valve in fluid communication with the sample chamber
and with the first, second, third, fourth, fifth, and sixth
reservoirs. In certain embodiments, the sample chamber comprises a
microscope slide, a coverslip, and a gasket disposed between the
microscope slide and coverslip.
[0037] In particular embodiments, the fluid inlet and the fluid
outlet of the sample chamber are coupled to the coverslip. In some
embodiments, the sample chamber is coupled to a platform configured
to move in an X-Y plane. In specific embodiments, the fluid outlet
of the sample chamber is coupled to a fluid transport device. In
certain embodiments, the fluid transport device is a syringe pump.
In particular embodiments, the imaging system comprises: a
microscope; a light source; a light guide adapter; and a
camera.
[0038] Particular embodiments include an apparatus for detecting
the presence of at least a first antigen on or in a sample, the
apparatus comprising: a sample chamber comprising a fluid inlet and
a fluid outlet; an imaging system configured to image a sample in
the sample chamber; a light source configured to photocleave a
first antigen-binding complex from a first antigen on or in the
sample; and a fluid control system in fluid communication with the
fluid inlet and the fluid outlet of the sample chamber, where the
fluid control system comprises a plurality of reservoirs in fluid
communication with the sample chamber.
[0039] In specific embodiments, the fluid control system is
configured to provide automated serial staining of the sample. In
particular embodiments, the imaging system is configured to provide
automated imaging of the sample. In some embodiments, the plurality
of reservoirs comprises a first reservoir containing an imaging
solution, a second reservoir containing a wash solution, a third
reservoir containing a cleavage solution, a fourth reservoir
containing a buffer solution, a fifth reservoir containing a
hybridization wash solution, and a sixth reservoir containing a
hybridization solution. In certain embodiments, the fluid control
system further comprises a valve in fluid communication with the
sample chamber and with the first, second, third, fourth, fifth,
and sixth reservoirs. In particular embodiments, the valve is a
rotary valve. In some embodiments, the sample chamber comprises a
microscope slide, a coverslip, and a gasket disposed between the
microscope slide and coverslip.
[0040] In specific embodiments, the sample chamber is coupled to a
platform configured to move in an X-Y plane. In certain
embodiments, the fluid inlet and the fluid outlet of the sample
chamber are coupled to the coverslip. In particular embodiments,
the fluid outlet of the sample chamber is coupled to a fluid
transport device. In some embodiments, the fluid transport device
is a syringe pump. In specific embodiments, imaging system
comprises a microscope, a light source, a light guide adapter, and
a camera. In certain embodiments, the apparatus is configured to
stain and image a plurality of sample chambers simultaneously or
via random access.
[0041] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0043] FIG. 1. Detection of an antigen in a tissue section using a
single-stranded biotin-oligonucleotide-photocleavable Cy5
fluorescent dye. Included as controls are biotinylated and
fluorescein-labeled probes.
[0044] FIG. 2. Exemplary partially double-stranded
biotin-oligonucleotide-photocleavable fluorescent dye. The sequence
of the top strand is provided as SEQ ID NO: 1; the sequence of the
bottom strand is provided as SEQ ID NO: 2.
[0045] FIG. 3. Detection of an antigen in a tissue section using a
single-stranded and a partially double-stranded
biotin-oligonucleotide-photocleavable Cy5 fluorescent dye.
[0046] FIG. 4. Exemplary single-stranded
biotin-oligonucleotide-photocleavable label complexed with a single
photocleavable fluorescent dye. The sequence of each example is
provided as SEQ ID NO: 3.
[0047] FIG. 5. Exemplary double-stranded
biotin-oligonucleotide-photocleavable label complexed with two
photocleavable fluorescent dyes of the same color. The sequence of
the top strand of the first (Cy5) example is provided as SEQ ID NO:
3; the sequence of the bottom strand of the first (Cy5) example is
provided as SEQ ID NO: 4. The sequence of the top strand of the
second (AF594) example is provided as SEQ ID NO: 5; the sequence of
the bottom strand of the second (AF594) example is provided as SEQ
ID NO: 6. The sequence of the top strand of the third (AF532)
example is provided as SEQ ID NO: 7; the sequence of the bottom
strand of the third (AF532) example is provided as SEQ ID NO: 8.
The sequence of the top strand of the fourth (AF488) example is
provided as SEQ ID NO: 9; the sequence of the bottom strand of the
fourth example (AF488) is provided as SEQ ID NO: 10.
[0048] FIG. 6. Exemplary hairpin
biotin-oligonucleotide-photocleavable label with a single
photocleavable fluorescent dye. The sequence of the first (Cy5)
example is provided as SEQ ID NO: 11; the sequence of the second
(AF594) example is provided as SEQ ID NO: 12; the sequence of the
third (AF532) example is provided as SEQ ID NO: 13; the sequence of
the fourth (AF488) example is provided as SEQ ID NO: 14.
[0049] FIG. 7. Exemplary photocleavable label covalently attached
to a biotin molecule through a functional linker. The functional
linker can be a nucleic acid, peptide, or fatty acid chain.
[0050] FIG. 8. Exemplary nucleotide-photocleavable
moiety-fluorescent dye structures, such as those illustrated in
FIGS. 2 and 4-6.
[0051] FIGS. 9A-E. Efficiency of photocleavage. FIG. 9A shows an
image of the stained tissue section before cleavage. FIG. 9B shows
an image of the stained section following 2 min of cleavage by UV
365 nm exposure. FIG. 9C shows an image of the stained section
following a buffer refresh. FIG. 9D is an image of a different FOV
showing the cleavage boundary (the upper left corner was cleaved).
FIG. 9E is an image pixel intensity profile along the line across
FIGS. 9A-C showing the image brightness change after cleavage and
after wash.
[0052] FIGS. 10A-H. Time course of photocleavage using various
PCLs. FIG. 10A shows the trace of maximum signal intensity. FIG.
10B shows the trace of absolute difference in signal (signal to
background). FIG. 10C shows that trace of relative difference in
signal (signal to background) as a percentage of the maximal
signal. FIG. 10D shows the images of the Standard Duplex PCL before
and after photocleavage in the center of the field. FIG. 10E shows
the images of the Long Duplex PCL before and after photocleavage.
FIG. 10F shows the images of the Short PCL before and after
photocleavage. FIG. 10G shows the images of the Single Biotin
Duplex PCL before and after photocleavage. FIG. 10H shows the
images of the oligonucleotide having a photocleavable biotin (IDT
Duplex PCL) both before and after cleavage.
[0053] FIG. 11. Serial tissue staining. FFPE tissue staining of two
target proteins with two photocleavable labels of different colors.
The same field of view was imaged in the first color and overlaid
on the previous color with ImageJ software.
[0054] FIG. 12. Illustration of the photocleavable labels used in
Example 5. The Standard Duplex PCL consisted of two 34-nucleotide
oligos having a biotin on their 5' ends and a photocleavable
fluorescent label on their 3' ends, the oligos were annealed to
form a 28-nucleotide double-stranded section with four nucleotide
single-stranded overhangs on each end. The sequence of the top
strand of the Standard Duplex PCL is provided as SEQ ID NO: 3; the
sequence of the bottom strand of the Standard Duplex PCL is
provided as SEQ ID NO: 4. The Long Duplex PCL consisted of two
69-nucleotide oligos having a biotin on their 5' ends and a
photocleavable fluorescent label on their 3' ends, the oligos were
annealed to form a 67-nucleotide double-stranded section with two
nucleotide single-stranded overhangs on each end. The sequence of
the top strand of the Long Duplex PCL is provided as SEQ ID NO: 15;
the sequence of the bottom strand of the Long Duplex PCL is
provided as SEQ ID NO: 16. The Short PCL consisted of a single
12-nucleotide oligo (SEQ ID NO: 17) having a biotin on its 5' end
and a photocleavable fluorescent label on its 3' end. The Single
Biotin Duplex PCL was the same as the Standard Duplex PCL except
that only one of the two oligos in each hybrid had a biotin on its
5' end. The sequence of the top strand of the Single Biotin Duplex
PCL is provided as SEQ ID NO: 3; the sequence of the bottom strand
of the Single Biotin Duplex PCL is provided as SEQ ID NO: 4. The
IDT Duplex PCL consisted of two 34-nucleotide oligos having a
photocleavable biotin on their 5' ends and a fluorescent label on
their 3' ends, the oligos were annealed to form a 28-nucleotide
double-stranded section with four nucleotide single-stranded
overhangs on each end. The sequence of the top strand of the IDT
Duplex PCL is provided as SEQ ID NO: 3; the sequence of the bottom
strand of the IDT Duplex PCL is provided as SEQ ID NO: 4.
[0055] FIG. 13. Schematic of an apparatus according to exemplary
embodiments disclosed herein.
[0056] FIG. 14. Exploded view of a sample chamber according to
exemplary embodiments disclosed herein.
[0057] FIG. 15. Assembly view of the sample chamber of FIG. 14.
[0058] FIG. 16. Schematic of a portion of the apparatus of FIG. 13
including a fluid control system and sample chamber.
[0059] FIG. 17. Schematic of a portion of the apparatus of FIG. 13
including a power and control system.
[0060] FIG. 18. Exemplary modified oligo with an SMCC
(Succinimidyl-4-[-maleimidomethyl]cyclohexane-1-carboxylate)
bifunctional crosslinker.
[0061] FIGS. 19A-E. FIG. 19A. Schematic of three consecutive
staining and imaging cycles. FIG. 19B. A stitched image of 25
pictures taken with a 20.times. objective (5.times.5) showing
Collagen, ALCAM and Hoechst. The dotted square outlines an area
magnified and split into channels in FIGS. 19C-E. FIG. 19C. ALCAM,
CD44, Hoechst. FIG. 19D. SMA, cytokeratin, Hoechst. FIG. 19E.
Fibronectin, Histone H3, Hoechst.
[0062] FIG. 20. Image of an FFPE tissue section with multiplex
staining using three color photocleavable labels simultaneously.
Images captured signal at the three appropriate wavelengths for
Cy3, Cy5, and Cy7, then image merging was performed using ImageJ
software to co-visualize the three distinct regions.
[0063] FIGS. 21A -C. FIG. 21A. Post-cleavage image of multiplex
stained tissue in FIG. 20. All signal was released after one
photocleaving event. FIG. 21B. shows trace of relative percent of
total signal over total cleavage time. FIG. 21C. shows the trace of
maximum signal intensity.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0064] Immunofluorescent detection of antigens in fixed tissue
specimens is used routinely in clinical practice and research
laboratories. Current methods are limited to the detection of one
to four antigens per tissue section. Consequently, the detection of
additional antigens requires multiple independent stains on
separate sections and limits the co-localization of antigens on a
single section. Additionally, current methods are exceedingly
time-consuming because staining and imaging are disconnected
processes.
[0065] An automated Next Generation Histology (NGH) platform for
formalin-fixed paraffin embedded (FFPE) tissue sections was
developed to overcome these limitations. The platform was designed
to use a probe in which the traditional fluorescent tag is replaced
with a novel photocleavable fluorophore that is released by UV
irradiation. The photocleavable label enables rapid cycles of
repeated staining in the same tissue section without the need to
remove the antigen-detecting antibody. The NGH platform also
includes automated fluidics for reduced hands-on time and a
four-color epi-fluorescence microscopy imaging system for multiplex
PCL detection on a single platform.
I. METHODS OF ANTIGEN DETECTION
[0066] In various embodiments, the invention provides methods of
detecting the presence of at least a first antigen on, or in, a
sample comprising various incubating steps required to build an
"antigen-binding complex" to which a photocleavable label is
conjugated via a functional linker.
[0067] In some embodiments, the invention provides methods of
detecting the presence of at least a first antigen on or in a
sample comprising the following steps:
1. Prepare the sample for antigen detection as needed (e.g.,
deparafinization, rehydration, etc.), methods for which are well
known to one of skill in the art 2. Perform antigen retrieval
(e.g., heat+salt+pH) 3. Perform non-specific antigen blocking
(e.g., BSA, serum or a non-protein based blocking agent such as
Tween.RTM.-20) 4. Perform first ligand/first anti-ligand block
(e.g., avidin or streptavidin/biotin block) 5. Incubate with
primary antibody
6. Wash
[0068] 7. Incubate with first ligand-conjugated (e.g.,
biotinylated) secondary antibody
8. Wash
[0069] 9. Incubate with first anti-ligand (e.g., avidin or
streptavidin)
10. Wash
[0070] 11. Incubate with second ligand (e.g., biotin)-bound
photocleavable label
12. Wash
[0071] 13. Detect signal from photocleavable label
14. Photocleave
[0072] 15. Optionally, confirm successful photocleavage by
detecting an absence of signal from the photocleavable label
[0073] In some variations, steps 7 and 9 may be performed
simultaneously; in these variations, the first ligand-conjugated
(e.g., biotinylated) secondary antibody and the first anti-ligand
(e.g., avidin or streptavidin) are added at the same time and step
8 is not performed. In some embodiments, they are in the same
solution. In some variations, steps 5 through 15 may be repeated
for a second (or more) antigen. In some embodiments, steps of the
method may be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, or 100 times to detect a plurality of
antigens in the sample. For example, the method may be used to
detect the presence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, or 100 antigens in a single sample.
[0074] In some embodiments, the invention provides methods of
detecting the presence of at least a first antigen on or in a
sample comprising the following steps:
1. Prepare the sample for antigen detection as needed (e.g.,
deparafinization, rehydration, etc.), methods for which are well
known to one of skill in the art 2. Perform antigen retrieval
(e.g., heat+salt+pH) 3. Perform non-specific antigen blocking
(e.g., BSA, serum or a non-protein based blocking agent such as
Tween.RTM.-20) 4. Perform first ligand/first anti-ligand block
(e.g., avidin or streptavidin/biotin block) 5. Incubate with
primary antibody
6. Wash
[0075] 7. Incubate with first ligand-conjugated (e.g.,
biotinylated) secondary antibody
8. Wash
[0076] 9. Incubate with first anti-ligand (e.g., avidin or
streptavidin)-bound photocleavable label
10. Wash
[0077] 11. Detect signal from photocleavable label
12. Photocleave
[0078] 13. Optionally, confirm successful photocleavage by
detecting an absence of signal from the photocleavable label
[0079] In some variations, steps 7 and 9 may be performed
simultaneously; in these variations, the first ligand-conjugated
(e.g., biotinylated) secondary antibody and the first anti-ligand
(e.g., avidin or streptavidin) are added at the same time and step
8 is not performed. In some embodiments, they are in the same
solution. In some variations, steps 5 through 13 may be repeated
for a second (or more) antigen. In some embodiments, steps of the
method may be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, or 100 times to detect a plurality of
antigens in the sample. For example, the method may be used to
detect the presence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, or 100 antigens in a single sample.
[0080] In another aspect, the invention provides a method of
detecting the presence of at least a first antigen on or in a
sample comprising the following steps:
1. Prepare the sample for antigen detection as needed (e.g.,
deparafinization, rehydration), methods for which are well known to
one of skill in the art 2. Perform antigen retrieval (e.g.,
heat+salt+pH) 3. Perform non-specific antigen blocking (e.g., BSA,
serum or a non-protein based blocking agent such as Tween.RTM.-20)
4. Incubate with primary antibody
5. Wash
[0081] 6. Incubate with a secondary antibody conjugated to a
photocleavable label
7. Wash
[0082] 8. Detect signal from photocleavable label
9. Photocleave
[0083] 10. Optionally, confirm successful photocleavage by
detecting an absence of signal from the photocleavable label
[0084] In some variations, steps 4 through 10 may be repeated for a
second (or more) antigen. In some embodiments, steps of the method
may be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, or 100 times to detect a plurality of antigens
in the sample. For example, the method may be used to detect the
presence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, or 100 antigens in a single sample.
[0085] In some embodiments, the invention provides methods of
detecting the presence of at least a first antigen on or in a
sample comprising the following steps:
1. Prepare the sample for antigen detection as needed (e.g.,
deparafinization, rehydration, etc.), methods for which are well
known to one of skill in the art 2. Perform antigen retrieval
(e.g., heat+salt+pH) 3. Perform non-specific antigen blocking
(e.g., BSA, serum or a non-protein based blocking agent such as
Tween.RTM.-20) 4. Perform first ligand/first anti-ligand block
(e.g., avidin or streptavidin/biotin block) 5. Incubate with first
ligand-conjugated (e.g., biotinylated) primary antibody
6. Wash
[0086] 7. Incubate with first anti-ligand (e.g., avidin or
streptavidin)-bound photocleavable label
8. Wash
[0087] 9. Detect signal from photocleavable label
10. Photocleave
[0088] 11. Optionally, confirm successful photocleavage by
detecting an absence of signal from the photocleavable label
[0089] In some variations, steps 5 and 7 may be performed
simultaneously; in these variations, the first ligand-conjugated
(e.g., biotinylated) primary antibody and the first anti-ligand
(e.g., avidin or streptavidin) are added at the same time and step
6 is not performed. In some embodiments, they are in the same
solution. In some variations, steps 5 through 11 may be repeated
for a second (or more) antigen. In some embodiments, steps of the
method may be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, or 100 times to detect a plurality of
antigens in the sample. For example, the method may be used to
detect the presence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, or 100 antigens in a single sample.
[0090] In some embodiments, the invention provides methods of
detecting the presence of at least a first antigen on or in a
sample comprising the following steps:
1. Prepare the sample for antigen detection as needed (e.g.,
deparafinization, rehydration, etc.), methods for which are well
known to one of skill in the art 2. Perform antigen retrieval
(e.g., heat+salt+pH) 3. Perform non-specific antigen blocking
(e.g., BSA, serum or a non-protein based blocking agent such as
Tween.RTM.-20) 4. Perform first ligand/first anti-ligand block
(e.g., avidin or streptavidin/biotin block) 5. Incubate with first
ligand-conjugated (e.g., biotinylated) primary antibody
6. Wash
[0091] 7. Incubate with first anti-ligand (e.g., avidin or
streptavidin)
8. Wash
[0092] 9. Incubate with second ligand (e.g., biotin)-bound
photocleavable label
10. Wash
[0093] 11. Detect signal from photocleavable label
12. Photocleave
[0094] 13. Optionally, confirm successful photocleavage by
detecting an absence of signal from the photocleavable label
[0095] In some variations, steps 5 and 7 may be performed
simultaneously; in these variations, the first ligand-conjugated
(e.g., biotinylated) primary antibody and the first anti-ligand
(e.g., avidin or streptavidin) are added at the same time and step
6 is not performed. In some embodiments, they are in the same
solution. In some variations, steps 5 through 13 may be repeated
for a second (or more) antigen. In some embodiments, steps of the
method may be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, or 100 times to detect a plurality of
antigens in the sample. For example, the method may be used to
detect the presence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, or 100 antigens in a single sample.
[0096] In some embodiments, the invention provides methods of
detecting the presence of at least a first antigen on or in a
sample comprising the following steps:
1. Prepare the sample for antigen detection as needed (e.g.,
deparafinization, rehydration, etc.), methods for which are well
known to one of skill in the art 2. Perform antigen retrieval
(e.g., heat+salt+pH) 3. Perform non-specific antigen blocking
(e.g., BSA, serum or a non-protein based blocking agent such as
Tween.RTM.-20) 4. Incubate with a non-protein first ligand-bound
photocleavable label
5. Wash
[0097] 6. Detect signal from photocleavable label
7. Photocleave
[0098] 8. Optionally, confirm successful photocleavage by detecting
an absence of signal from the photocleavable label
[0099] In some variations, steps 4 through 8 may be repeated for a
second (or more) antigen. In some embodiments, steps of the method
may be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, or 100 times to detect a plurality of antigens
in the sample. For example, the method may be used to detect the
presence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, or 100 antigens in a single sample.
[0100] In some variations, the non-protein first ligand can be an
aptamer, a carbohydrate, a nucleic acid (e.g., DNA), a hormone, or
a small molecule.
[0101] In some embodiments, the invention provides methods of
detecting the presence of at least a first antigen on or in a
sample comprising the following steps:
1. Prepare the sample for antigen detection as needed (e.g.,
deparafinization, rehydration, etc.), methods for which are well
known to one of skill in the art 2. Perform antigen retrieval
(e.g., heat+salt+pH) 3. Perform non-specific antigen blocking
(e.g., BSA, serum or a non-protein based blocking agent such as
Tween.RTM.-20) 4. Perform first ligand/first anti-ligand block
(e.g., avidin or streptavidin /biotin block) 5. Incubate with first
ligand-conjugated (e.g., biotinylated) aptamer
6. Wash
[0102] 7. Incubate with first anti-ligand (e.g., avidin or
streptavidin)
8. Wash
[0103] 9. Incubate with second ligand (e.g., biotin)-bound
photocleavable label
10. Wash
[0104] 11. Detect signal from photocleavable label
12. Photocleave
[0105] 13. Optionally, confirm successful photocleavage by
detecting an absence of signal from the photocleavable label
[0106] In some variations, steps 5 and 7 may be performed
simultaneously; in these variations, the first ligand-conjugated
(e.g., biotinylated) aptamer and the first anti-ligand (e.g.,
avidin or streptavidin) are added at the same time and step 6 is
not performed. In some embodiments, they are in the same solution.
In some variations, steps 5 through 13 may be repeated for a second
(or more) antigen. In some embodiments, steps of the method may be
repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, or 100 times to detect a plurality of antigens in the
sample. For example, the method may be used to detect the presence
of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, or 100 antigens in a single sample.
[0107] In some embodiments, the methods provided herein may be used
in conjunction and/or combination with known techniques for antigen
detection, including, but not limited to, Western blotting,
microarray, enzyme-linked immunosorbent assay (ELISA), reverse
phase protein array (RPPA), and immunohistochemistry (based on
colorimetric or fluorescent read-outs).
[0108] In some embodiments, a sample will need to be prepared for
antigen detection prior to being incubated with the various
components of an antigen-binding complex. In some variations, a
target cell will need to be fixed, e.g., by adding chemical
fixatives, such as aldehydes or paraformaldehyde, to crosslink,
alcohols to precipitate, oxidizing agents, mercurials, and
picrates. In some variations, cell permeability will need to be
increased by, e.g., adding organic solvents, such as methanol and
acetone, or detergents, such as Triton.TM.-X 100, saponin, and
Tween.RTM.-20. In some variations, blocking steps will need to be
performed to reduce non-specific reactions by, e.g., adding bovine
serum albumin, goat serum, fish skin gelatin, horse serum, swine
serum, donkey serum, or rabbit serum.
[0109] In some embodiments, various incubating steps are performed
to build the antigen-bound antigen-binding complex in the sample.
In some cases, more than one component of an antigen-binding
complex may be added to the sample simultaneously while others are
added sequentially. In some cases, each component of an
antigen-binding complex is added to the sample sequentially. In
some cases, every component of an antigen-binding complex may be
added to the sample simultaneously. The various incubating steps
may be performed under conditions that favor an interaction between
the primary antibody, aptamer, etc. and the target antigen that may
be present in the sample, or alternatively/additionally, between a
primary antibody and a secondary antibody, between a first ligand
and a first anti-ligand, or between a first anti-ligand and a
second ligand. Exemplary conditions that may be modulated to affect
a desired interaction include temperature and pH. Exemplary
temperatures for incubation steps include any temperature between
15.degree. C. and 30.degree. C., 18.degree. C. and 27.degree. C.,
or 20.degree. C. and 25.degree. C., or any range derivable therein.
Exemplary temperature for incubation steps include, for example,
15.degree. C., 16.degree. C., 17.degree. C., 18.degree. C.,
19.degree. C., 20.degree. C., 21.degree. C., 22.degree. C.,
23.degree. C., 24.degree. C., 25.degree. C., 26.degree. C.,
27.degree. C., 28.degree. C., 29.degree. C., and 30.degree. C.
Exemplary pH values for incubation steps include any pH between 6
and 8, 6.2 and 7.8, or 6.5 and 7.5, or any range derivable therein.
Exemplary pH values for incubation steps include 6.0, 6.1, 6.2,
6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,
7.6, 7.7, 7.8, 7.9, and 8.0.
[0110] In some embodiments, washing steps are performed following
various incubating steps to remove any components that are not
specifically bound to the antigen-bound antigen-binding complex in
the sample. Washing may be performed using a washing solution
comprising, for example, water, a buffer solution (e.g., PBS),
physiological saline, or a combination thereof.
[0111] In some embodiments, various components of an
antigen-binding complex may be biotinylated. In biochemistry,
biotinylation is the process of covalently attaching biotin to a
protein, nucleic acid, or other molecule. Biotinylation is rapid,
specific and is unlikely to perturb the natural function of the
molecule due to the small size of biotin (MW=244.31 g/mol). Various
methods are known in the art for biotinylating biological
macromolecules, such as, for example, proteins and nucleic acids.
See, e.g., Moritz and Wahle (2014); Hermanson (2013). Also,
multiple biotin molecules can be conjugated to a molecule of
interest. Proteins can be biotinylated chemically or enzymatically.
Chemical biotinylation utilizes various conjugation chemistries to
yield nonspecific biotinylation of amines, carboxylates,
sulfhydryls and carbohydrates (e.g., NHS-coupling gives
biotinylation of any primary amines in the protein). Enzymatic
biotinylation results in biotinylation of a specific lysine within
a certain sequence by a bacterial biotin ligase. Most chemical
biotinylation reagents consist of a reactive group attached via a
linker to the valeric acid side chain of biotin. As the biotin
binding pocket in avidin/streptavidin is buried beneath the protein
surface, biotinylation reagents possessing a longer linker are
desirable, as they enable the biotin molecule to be more accessible
to binding avidin/streptavidin/Neutravidin protein. This linker can
also mediate the solubility of biotinylation reagents; linkers that
incorporate poly(ethylene) glycol (PEG) can make water-insoluble
reagents soluble or increase the solubility of biotinylation
reagents that are already soluble to some extent. Oligonucleotides
are readily biotinylated in the course of oligonucleotide synthesis
by the phosphoramidite method using commercial biotin
phosphoramidite. Upon the standard deprotection, the conjugates
obtained can be purified using reverse-phase or anion-exchange
HPLC.
[0112] In some embodiments, detecting a signal from a
photocleavable label comprises measuring the signal generated from
the antigen-bound antigen-binding complex present in the sample.
Detecting may, for example, comprise measuring a signal generated
by a fluorescent dye of the antigen-bound antigen-binding complex
using a fluorescence microscope. The presence of a signal generated
by, e.g., a fluorescent dye may indicate that the antigen is
present in the sample. Thus, detecting may comprise determining the
presence of the antigen in the sample. In some variations,
detecting a signal may be defined as measuring a signal from a
photocleavable label, where said measuring provides either a
relative quantitative, absolute quantitative, or qualitative
measure of the amount of the antigen present in the same.
[0113] In some embodiments described herein, the photocleavable
label comprises a 2-nitrobenzyl or substituted 2-nitrobenzyl group,
which may be efficiently photochemically cleaved, for example, with
365 nm UV light. See U.S. Patent Appl. Publ. 2010/0041041, which is
incorporated herein by reference. It is generally understood that
wavelengths >300 nm are used to minimize damage to DNA and
proteins (Corrie, 2005) with several specific exemplary wavelengths
other than 365 nm being 340 nm and 355 nm (Seo, 2005). As such, the
terms "photocleaving" or "photocleave," as used herein, are meant
to refer generally to the act of exposing a sample to a wavelength
of light >300 nm so as to effect the cleavage of the
photocleavable bond.
[0114] In some embodiments, after the photocleaving step, the
method may further comprise repeating the incubating steps to build
an antigen-binding complex on or in a sample and detecting a signal
from the photocleavable label conjugated to an antigen-bound
antigen-binding complex to detect the presence of a second antigen
in the sample. For any given method of building an antigen-binding
complex, after photocleavage of the label from the previously built
antigen-binding complex, the steps of incubation to build the new
antigen-binding complex and detecting a signal from the
photocleavable label may be sequentially repeated at least once. In
some variations, the steps may be sequentially repeated at least
twice, three times, four times, five times, 10 times, 20 times, 30
times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times,
or 100 times. In some variations, the photocleavable label may be
subjected to conditions to effect photocleavage between each
repetition of the steps. In some variations, the steps may be
repeated once, twice, three times, four times, five times, six
times, seven times, eight times, nine times, or 10 times
consecutively before a photocleavage step is performed. In some
variations, the photocleavable label conjugated to an
antigen-binding complex in any given repetition may be the same or
different from the label used in any previous repetition.
[0115] In some variations, the incubating steps to build the
antigen-binding complex may be repeated once, twice, three times,
four times, five times, six times, seven times, eight times, nine
times, or 10 times consecutively before a detecting step is
performed. In these variations, the photocleavable labels
conjugated to each antigen-binding complex are different so that
they can be distinguished during one detecting step, i.e., the
labels are selected so that the overlap of the emission spectra of
the various labels is minimized.
[0116] In some variations, the components of an antigen-binding
complex used in each repetition may be the same or different from
the components used in an antigen-binding complex from a previous
cycle. In some variations, only the component that binds directly
to the antigen is different, while the other components are the
same.
II. PHOTOCLEAVABLE LABELS
[0117] Some aspects of the present invention are directed towards
providing photocleavable labels with improved UV-cleavage rates for
use in detection of multiple proteins in a single sample
sequentially and/or simultaneously. Cleavage of the 2-nitrobenzyl
or substituted 2-nitrobenzyl group with, e.g., 365 nm UV light
allows for the next cycle of antigen detection to be performed
without interference from the label(s) used in the previous
cycle(s). Without being bound by theory, at least two factors have
been found to influence UV-cleavage rates of substituted
2-nitrobenzyl groups: a) stereo-chemistry of the .alpha.-carbon
substitution of the 2-nitrobenzyl group, and b) substitution on the
benzyl ring.
[0118] The photocleavable labels are optionally conjugated to
ribonucleoside triphosphates (NTPs) and deoxyribonucleoside
triphosphates (dNTP). For example, a nucleotide or nucleoside
compound may include a chemically or enzymatically cleavable group
labeled with a reporter group, such as a fluorescent dye. The
nucleotide and nucleoside compounds may include chemically or
enzymatically removable protecting groups that are designed to
terminate DNA synthesis. The presence of such cleavable groups
labeled with fluorescent dyes on the nucleotide and nucleoside
compounds can enhance the speed and accuracy of indirect detection
of multiple proteins in a single sample. Examples of such
nucleotide and nucleoside compounds include those disclosed in PCT
Publn. Nos. WO 2003/006625, WO 2005/084367, WO 2008/070749, WO
2009/152353, WO 2013/040257, which are each incorporated herein by
reference in their entirety. In some cases, an oligonucleotide may
be produced that comprises a photocleavable linker (Gene Link Cat.
No. 26-6888) or photocleavable spacer (Gene Link Cat. No. 26-6889),
such as are commercially available. In addition, photocleavable
nucleotide-fluorophore conjugates are available from Ambergen
Technology. Also, the amino modifier C6-dT (Integrated DNA
Technologies) can be used to make oligonucleotides with
photocleavable fluorescent groups attached to a nucleoside
base.
[0119] As used herein, the term "reporter" or "label" refers to a
chemical moiety that is able to produce a detectable signal
directly or indirectly. Examples of reporters include fluorescent
dye groups, colorimetric dye groups, radioactive labels, or groups
affecting a signal through chemiluminescent or bioluminescent
means. Examples of fluorescent dye groups include xanthene derivate
dyes (e.g., fluorescein and its derivatives, fluorescein
isothiocyanate [FITC], carboxyfluorescein succinimidyl ester [CF
SE], carboxyfluorescein diacetate succinimidyl ester [CFDA-SE],
eosin Y, eosin B, rhodamine B, rhodamine 6G, rhodamine 123,
rhodamine red-X [RRX], carboxytetramethylrhodamine [TAMRA],
tetramethylrhodamine [TMR], i sothiocyanate-derivative of rhodamine
[TRITC], sulforhodamine 101, sulfonyl chloride derivative of
sulforhodamine 101 [Texas Red], Oregon Green), BODIPY derivative
dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581/591,
BODIPY TR, BODIPY 630/650, BODIPY 650/665), coumarin derivative
dyes (e.g., aminomethylcoumarin [AMCA]), allophycocyanin [APC],
pyrene derivative dyes (e.g., Cascade Blue),
4',6-diaminidino-2-phenylindole [DAPI], DyLight dyes (e.g.,
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, DyLight.TM. 800), phycoerythrin [PE], PI,
peridinin-chlorophyll-protein [PerCP], cyanine derivative dyes
(e.g., Cy.RTM.5.5, indodicarbocyanine (Cy.RTM.5), cyanine
(Cy.RTM.2), indocarbocyanine (Cy.RTM.3), Cy.RTM.3B, Cy.RTM.3.5,
Cy.RTM.7, Cy.RTM.7Q, oxacarbocyanine, thiacarbocyanine,
merocyanine, phthalocyanine), anthracene derivative dyes (e.g.,
Draq-5, Draq-7, CyTRAK Orange, IRIS 2, IRIS 3, IRIS 3.5, IRIS 5,
IRIS 5.5, IRIS 7G), eFluor dyes (e.g., eFluor.RTM. 450,
PE-eFluor.RTM. 615, eFluor.RTM. 660, eFluor.RTM. 710,
PE-eFluor.RTM. 610, PerCP-eFluor.RTM. 710, APC-eFluor.RTM. 780),
FluoProbes dyes (FluoProbes 390, FluoProbes 488, FluoProbes 532,
FluoProbes 547H, FluoProbes 594, FluoProbes 647H, FluoProbes 682,
FluoProbes 752, FluoProbes 782), GFP, IRDye 800, Pacific Blue,
Pacific Green, Pacific Orange, pyrene, phycobiliprotein,
Quasar.RTM. dyes (e.g., Quasar.RTM. 570, Quasar.RTM. 670,
Quasar.RTM. 705), SNAFL, sulfocyanine derivative dyes (e.g.,
sulfo-Cy3, sulfo-Cy5, sulfo-Cy7), Tokyo Green, Alexa fluor.RTM.
dyes (e.g., 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. 568, ALEXA
FLUOR.RTM. 594, ALEXA FLUOR.RTM. 610, ALEXA FLUOR.RTM. 633, ALEXA
FLUOR.RTM. 635, ALEXA FLUOR.RTM. 647, ALEXA FLUOR.RTM. 660, ALEXA
FLUOR.RTM. 680, ALEXA FLUOR.RTM. 700, ALEXA FLUOR.RTM. 750, ALEXA
FLUOR.RTM. 790), squaraine dyes (e.g., Seta.TM. dyes, SeTau dyes,
Square dyes), or combinations thereof. Additional examples of
fluorescent dye groups that may be used in some embodiments of the
present invention are disclosed throughout this Specification and
in Haugland, 2005 and U.S. Pat. Nos. 4,439,356 & 5,188,934,
which are incorporated by reference herein. Fluorescent dyes can be
attached to biological macromolecules (i.e., proteins, nucleic
acids, and fatty acid chains) via specific functional groups, such
as amino groups (e.g., via succinimide, isothiocyanate or
hydrazine), carboxyl groups (e.g., via carbodiimide), thiol (e.g.,
via maleimide or acetyl bromide), azide (e.g., via click
chemistry), or non-specifically (glutaraldehyde) or non-covalently
(e.g., via hydrophobicity, etc.). See, e.g., Proudnikov and
Mirzabekov, 1996; Riedel et al., 2012. Examples of radioactive
labels that may be used as reporters in some embodiments of the
present invention, which are well known in the art, include
.sup.35S, .sup.3H, .sup.32P, or .sup.33P. Examples of reporters
that function by chemiluminescent or bioluminescent means and that
may be used as reporters in some embodiments of the present
invention are described in Nieman, 1989; Givens & Schowen,
1989; Orosz et al., 1996; and Hastings, 1983, which are
incorporated by reference herein.
[0120] The photocleavable label of an antigen-binding complex used
in each sequential repetition of a method disclosed herein may be
the same or different than any photocleavable label used in an
antigen-binding complex for a previous repetition on the same
sample. In some cases, a photocleavable label may be comprised of
multiple reporters that in combination provide a unique signal
different from the signals of any of the reporter used
singularly.
[0121] The photocleavable labels of each of the antigen-binding
complexes used simultaneously in a multiplex repetition of a method
disclosed herein are preferably different to allow for each
reporter to be distinguishable from the others. In other words, in
the case of fluorescent reporters, each fluorescent reporter of a
plurality of antigen-binding complexes may be selected in such a
manner that an overlap among emission spectrum of each fluorescent
reporter is minimized. In some cases, two different dyes may be
used simultaneously but imaged independently to provide the
location. The number of different photocleavable labels that may be
used simultaneously in a multiplex repetition of a method disclosed
herein is two, three, four, five, six, seven, eight, nine, 10, 11,
12, 13, 14, or 15.
III. APPARATUS
[0122] As previously mentioned, exemplary methods disclosed herein
may be performed via an automated Next Generation Histology (NGH)
apparatus. Referring initially to FIG. 13, a schematic is provided
of one exemplary embodiment of such an apparatus 100. Apparatus 100
can enable multiple, rapid cycles of staining in the same tissue
section of a sample without the need to remove an antigen-detecting
antibody. As discussed further below, apparatus 100 also includes
automated fluidics for reduced hands-on time and a microscopy
imaging system for multiplex PCL detection. In the embodiment
shown, apparatus 100 comprises an imaging system 110, a sample
chamber 120, and a fluid control system 130.
[0123] In the embodiment shown, fluid control system 130 comprises
a valve 131 in fluid communication with sample chamber 120 and with
a plurality of reservoirs 132-137. Reservoirs 132-137 can contain
various fluids used in an automated staining process for a sample
under analysis. In particular embodiments, reservoir 132 may
comprise a protein blocking buffer, reservoir 133 may comprise a
wash solution, reservoir 134 may comprise an imaging solution,
reservoir 135 may comprise a cleavage solution, reservoir 136 may
comprise biotin, and reservoir 137 may comprise an avidin blocking
buffer.
[0124] Imaging system 110 comprises a microscope 101, as well as an
imaging light source 140 configured to image a sample in sample
chamber 120. In the embodiment shown, light source 140 is coupled
to a light guide adapter 148, liquid light guide 147, and a TTL
shutter control 105. in particular embodiments, imaging light
source 140 is a solid state light engine. In the illustrated
embodiment, microscope 101 comprises a base 115, a motorized stage
111, a stage controller 112, a camera 113, an objective 107, a
motorized nosepiece 109. In particular embodiments, imaging system
110 may also comprise a motorized filter turret, filter block and
filter cube (not labeled in the figures for purposes of
clarity).
[0125] In the embodiment shown, apparatus 100 can be controlled via
a remote control unit 114. Apparatus 100 further includes a
multifunction data acquisition (DAQ) device 116, a frame grabber
118, a computer 117 and a serial USB hub 119 in this embodiment. In
the embodiment shown, apparatus 100 also comprises a cooling
element 145 for sample chamber 120. In particular embodiments,
cooling element 145 may comprise a temperature control block (e.g.,
a Peltier unit) and a liquid cooler.
[0126] Referring now to FIGS. 14 and 15, respectively, an exploded
view and assembled view of one embodiment of sample chamber 120 is
provided. In this embodiment, sample chamber comprises a microscope
slide 121, a coverslip 122, and a gasket 123 disposed between
microscope slide 121 and coverslip 122. The embodiment shown also
comprises a fluid inlet 124 and a fluid outlet 126 coupled to
coverslip 122. Gasket 123 comprises an open portion 125 positioned
in the interior of gasket 123, such that gasket 123 surrounds open
portion 125. In this embodiment, open portion 125 comprises a
central volume and extended portions in fluid communication with
fluid inlet 124 and fluid outlet 126. In the assembled view of FIG.
15, a sample 127 is located in open portion 125 and between
microscope slide 121 and coverslip 122. Accordingly, sample 127 is
contained within sample chamber 120, and fluid inlet 124 and fluid
outlet 126 can provide for fluid flow into sample chamber 120.
[0127] Referring back now to FIG. 13, fluid inlet 124 and fluid
outlet 126 are each coupled to an optical bubble sensor 142 and
146, respectively. Fluid inlet 124 is also coupled to and in fluid
communication with valves 141 and 143, as well as reservoirs 138
and 139 (which can contain an antibody solution and a PCL solution,
respectively). In certain embodiments, valves 141 and 142 may be
configured as solenoid valves. Fluid outlet 126 is also coupled to
valve 151 and a fluid transport device 150, which in certain
embodiments may be configured as a syringe pump. Valve 151 is also
coupled to waste reservoir 152. During operation, the flow of
fluids from reservoirs 132-137 can be controlled (e.g., via
operation of valves 131, 141 and 143) to fluid inlet 124 to provide
automated labeling and staining of sample 127 according to the
methods disclosed herein.
[0128] FIG. 16 provides a partial schematic diagram of fluid
control system 130 and sample chamber 120. As shown in this
embodiment, gasket 123 is located between microscope slide 121 and
coverslip 122. Fluid inlet 124 and fluid outlet 126 are in fluid
communication with components of fluid control system 130 that
allows for automated serial staining of sample 127. For example,
fluid control system 130 can provide for the transfer of fluids
from reservoirs 132-139 to sample chamber 120 in order to perform
the steps disclosed in the methods described herein.
[0129] FIG. 17 provides a schematic diagram of one exemplary
embodiment of a power and control system 160 for a light source 106
configured to photocleave an antigen-binding complex from an
antigen on or in a sample in sample chamber 120. In particular
embodiments, light source 106 can be configured as an LED head
unit. It is understood that the component model numbers indicated
in FIG. 16 are merely exemplary of one embodiment, and that other
embodiments of exemplary systems may comprise different components
and model numbers.
[0130] In the embodiment shown in FIG. 17, system 160 is an
ultra-violet (UV) reverse termination sub-system in which LED head
unit 106 is a Hamamatsu LC-L5 UV LED array configured to provide
excitation at 365 nm for cleavage of the PCL. In this embodiment,
system 160 is controlled by computer 117 via LabView software
through multifunction data acquisition (DAQ) device 116 providing a
digital output line to turn LED head unit 106 on and off as well as
an analog output line to set the UV intensity via a 0-10 V signal.
A solid state relay 164 coupled with an electromechanical relay 162
switch a 12 VDC signal from a 120 VAC-12 VDC transformer 163 to
turn the UV LED array in LED head unit 106 on and off. A 5 VDC
switching mode power supply 165 provides power to solid state relay
164. LED head unit 106 gets power from its own 36 VDC power supply
161.
IV. DEFINITIONS
[0131] The term "antigen" as used herein refers to any target
material present on or in a sample. For example, the antigen may be
a protein, a sugar, a lipid, a nucleic acid, a ligand, a
carbohydrate, a drug target, or a combination thereof. The antigen
may be present in or on a cell. The antigen may be present only in
or on a cancerous cell but not a normal cell. The antigen may be
present in a cell-free sample, such as serum or a cell lysate.
[0132] The term "sample" as used herein refers to any biological
sample in which an antigen may be present. For example, the sample
may be a biopsy sample, tissue sample, cell suspension, cell
culture, or a combination thereof. Further, the sample may be
isolated from an animal, such as a primate (e.g., human), mouse,
rat, guinea pig, hamster, rabbit, cat, dog, pig, cow, or horse. In
some cases, the sample may be a plant sample, a bacterial sample,
or a fungal sample. The sample may be an animal body fluid such as,
for example, blood, bone marrow fluid, lymph, saliva, lachrymal
fluid, urine, mucosal fluid, amniotic fluid, or a combination
thereof. The sample may be a cell mixture including different types
of cells mixed therein. The mixture may include cells having the
antigen and cells that do not. The sample may comprise circulating
tumor cells (CTCs), cancer stem cells, immune cells, fetal stem
cells, fetal cells, cancer cells, tumor cells, and/or normal
cells.
[0133] The term "antigen-binding complex" as used herein refers to
any complex that comprises a component that is capable of
selectively or specifically binding to an antigen. An
antigen-binding complex will also comprise a component to which a
photocleavable label is covalently bound, which component may be
the same as or different than the component that binds to the
antigen. The component that is capable of selectively or
specifically binding to an antigen may be, without limitation, a
primary antibody; an aptamer; a ligand for a receptor where the
receptor is the antigen; a receptor for a ligand where the ligand
is the antigen; a substrate, inhibitor, or cofactor for an enzyme
where the enzyme is the antigen; a drug molecule where the drug
target is the antigen; a lectin where a carbohydrate is the
antigen; an RNA molecule where an at least partially complementary
nucleic acid (e.g., DNA or RNA) is the antigen; a DNA molecule
where an at least partially complementary nucleic acid (e.g., DNA
or RNA) is the antigen; etc. Additional examples include use of
Annexin A5 to specifically bind to phosphatidylserine on a cell
surface, use of lectins to specifically bind to carbohydrate
moieties, use of a nuclear receptor that specifically binds to a
hormone ligand and the antigen is either the hormone ligand or a
DNA regulatory element bound by the nuclear receptor in the
presence of the hormone ligand.
[0134] The term "primary antibody" as used herein generally refers
to the component in an antigen-binding complex that selectively or
specifically binds to the antigen. A primary antibody may be, for
example, an antibody, an antibody mimetic, an aptamer, a receptor,
a ligand, an enzyme substrate, an enzyme inhibitor, an enzyme
cofactor, or an enzyme.
[0135] The term "antibody mimetic" as used herein refers to organic
compounds (including artificial peptides or proteins with a molar
mass of about 3 to 20 kDa, nucleic acids, and small molecules)
that, like antibodies, can specifically bind antigens, but that are
not structurally related to antibodies. Examples of antibody
mimetics include, without limitation, affibody molecules, affilins,
affimers, affitins, alphabodies, anticalins, avimers, DARPins,
fynomers, Kunitz domain peptides, and monobodies.
[0136] The term "aptamer" as used herein refers to oligonucleotide
or peptide molecules that bind to a specific target molecule, e.g.,
an antigen. Some aptamers are single-stranded nucleic acids that
fold into a well-defined three-dimensional structure. Aptamers show
a high affinity and specificity for their target molecules and in
some cases inhibit their biological functions. Aptamers can be
created by selecting them from a large random sequence pool.
Natural aptamers also exist in the form of riboswitches, which can
be used to selectively or specifically detect a ligand antigen. In
the case of oligonucleotide-based aptamers, the photocleavable
label may be covalently bound directly to the aptamer itself, thus
producing a single component antigen-binding complex.
[0137] The term "secondary antibody" as used herein refers to
antibodies of any immunoglobulin class that specifically bind to
the Fc portion of a primary antibody. The Fc portion of an
immunoglobulin (Ig) monomer corresponds to the stem of the Y-shaped
Ig molecule and consists of the C-terminal sections of the two
heavy chains linked by one or more disulfide bonds.
[0138] The term "first ligand" as used herein refers to any
molecule that can be covalently bound to a component (e.g., a
primary antibody, a secondary antibody, etc.) of an antigen-binding
complex. One example of a first ligand is biotin. Biotin is a
vitamin present in all living cells that binds with high affinity
to avidin or streptavidin. The binding between biotin and
avidin/streptavidin is the strongest (K.sub.a=10.sup.15 M.sup.-1)
known non-covalent interaction between a protein and ligand. Since
biotin is a relatively small molecule, it can be conjugated to
proteins (e.g., antibodies) without altering their biological
activity. Furthermore, a single protein (e.g., antibody) may be
conjugated to several occurrences of biotin that can each then bind
a molecule of avidin. Biotin occurs naturally; thus, surface
blocking of non-specific binding of biotin or avidin/streptavidin
is required when biotin-avidin/streptavidin systems are used. A
commonly used two-step blocking strategy includes: incubation with
an excess of avidin/streptavidin to block endogenous biotin-rich
enzymes and then incubation with an excess of biotin to block in
the introduced avidin/streptavidin. Another example of a first
ligand is an oligonucleotide. Such an oligonucleotide may be
covalently bound to a component (e.g., a primary antibody, a
secondary antibody, etc.) of an antigen-binding complex. Such an
oligonucleotide may be single stranded. Such an oligonucleotide may
be comprised of ribonucleic acids, deoxyribonucleic acids, or
derivatives thereof. Such an oligonucleotide may be at least 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides long.
[0139] The term "first anti-ligand" as used herein refers to any
molecule that can specifically and/or selectively bind to the first
ligand. For example, if the first ligand is biotin, then the first
anti-ligand may be avidin. The term "avidin" as used herein
generically refers to any biotin-binding protein, including both
natural proteins and recombinant and genetically engineered
proteins. The term includes the two common biotin-binding proteins
known as "egg white or avian avidin" and "streptavidin." Egg white
or avian avidin, commonly referred to simply as avidin, is a
protein that is a constituent of egg white and forms a non-covalent
complex with biotin. Streptavidin is a protein isolated from the
actino-bacterium Streptomyces avidinii and also forms a
non-covalent complex with biotin. Other bacterial sources of biotin
binding proteins are also known. Both egg white avidin and
streptavidin are tetrameric proteins in which the biotin binding
sites are arranged in pairs on opposite faces of the avidin
molecule. Accordingly, both of the above avidins have the ability
to bind to up to four molecules of biotin, either in the free form
or in a derivative form and, thereby, form a "complex." A
derivative form of biotin results from the conjugation of biotin to
another molecule. As another example, if the first ligand is an
oligonucleotide, then the first anti-ligand may be a second
oligonucleotide that is at least partially complementary to the
first ligand.
[0140] The term "second ligand" as used herein refers to any
molecule that can specifically and/or selectively bind to the first
anti-ligand. For example, if the first ligand is biotin and the
first anti-ligand is avidin, then the second ligand may be
biotin.
[0141] The term "functional linker" as used herein refers to any
molecule that can be covalently bound by a photocleavable label and
a component of an antigen-binding complex (e.g., a first ligand, a
first anti-ligand, a second ligand, a primary antibody, a secondary
antibody, or an aptamer). The functional linker may be a nucleic
acid. Such a nucleic acid may be at least two, three, four, five,
six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or
100 nucleotides long. Such a nucleic acid may be single stranded,
double stranded, or partially double stranded. The functional
linker may be a peptide. The functional linker may be a fatty acid
chain. The functional linker may be photo-stable.
[0142] As used herein, "essentially free," in the context of a
specified component, means that none of the specified component has
been purposefully formulated into a composition and/or is present
only as a contaminant or in trace amounts. The total amount of the
specified component resulting from any unintended contamination of
a composition is therefore well below 0.05%, preferably below
0.01%. Most preferred are compositions in which no amount of the
specified component can be detected with standard analytical
methods.
[0143] The use of the word "a" or "an," when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one."
[0144] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" may mean at least a second or
more.
[0145] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0146] The terms "comprise," "have" and "include" are open-ended
linking verbs. Any forms or tenses of one or more of these verbs,
such as "comprises," "comprising," "has," "having," "includes" and
"including," are also open-ended. For example, any method that
"comprises," "has" or "includes" one or more steps is not limited
to possessing only those one or more steps and also covers other
unlisted steps.
[0147] The above definitions supersede any conflicting definition
in any reference that is incorporated by reference herein. The fact
that certain terms are defined, however, should not be considered
as indicative that any term that is undefined is indefinite.
Rather, all terms used are believed to describe the invention in
terms such that one of ordinary skill can appreciate the scope and
practice the present invention.
V. EXAMPLES
[0148] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1--Detection of an Antigen in a Tissue Section Using
Single-Stranded Biotin-Nucleotide-Fluorophore Tags
[0149] This Example illustrates an antigen-bound antigen-binding
complex of formula (IX). First, formalin-fixed paraffin embedded
(FFPE) prostate cancer tissue sections were prepared and blocked
with BSA in PBS. Then, the sections were incubated with a mouse
anti-cytokeratin antibody, followed by washing. Next, the sections
were incubated with biotinylated goat anti-mouse IgG secondary
antibody, followed by washing. Then, the sections were incubated
with either avidin or streptavidin or buffer alone, followed by
washing. Then, the sections were incubated with either a
biotin-conjugated photocleavable Cy5 or avidin-fluorescein,
followed by washing. The biotin-conjugated photocleavable Cy5 had a
biotin modification at its 5' end and a dC-Cy5 (FIG. 8) at its 3'
end. Finally, the sections were imaged using a 20.times. water
immersion lens. FIG. 1 shows that the biotin-conjugated
photocleavable Cy5 produced a signal in a manner dependent on the
biotinylation of the secondary antibody.
[0150] In another experiment, sections of a formalin-fixed paraffin
embedded human prostate were incubated with biotinylated
anti-smooth muscle actin. Then, the sections were incubated
sequentially with avidin or streptavidin and a biotin-conjugated
probe containing photocleavable Cy5. In this instance, an automated
fluid exchange program was used in coordination with the probe,
which consisted of a double stranded nucleotide linking two
opposing biotins. The Cy5 was incorporated into the nucleotide
backbone. Unbound probe was washed and the tissue was imaged prior
to and after photocleavage. After the slide was imaged with a
fluorescent microscope with Cy5, a single location in the tissue
was photocleaved using a 2 min
[0151] UV exposure and re-imaged in a pre-programmed manner. FIG.
9A shows an image of the stained section prior to cleavage while
FIG. 9B shows the same area after photocleavage. The section was
imaged again following a buffer wash (FIG. 9C). FIG. 9E shows the
image pixel intensity profile along the line in FIGS. 9A-C,
illustrating the image brightness change after cleavage and wash.
FIG. 9D shows a different field-of-view following cleavage and
illustrates the cleavage boundary (the upper-left corner was
exposed to UV light).
Example 2--Automated Serial FFPE Tissue Staining of Two Target
Proteins with Two Photocleavable Labels of Different Colors
[0152] This Example illustrates an antigen-bound antigen-binding
complex of formula (VII). For serial FFPE, the tissue was first
incubated with biotinylated anti-smooth muscle actin for 2 h,
avidin for 30 min, and biotin-conjugated probe containing
photocleavable AF532 for 20 min before the green channel was
imaged. After UV exposure of 2 min at 150 mW/cm.sup.2, the tissue
underwent the same staining procedure with histone H3 antibody and
red photocleavable Cy5. The same field of view was imaged in the
red channel and overlaid on the previous green channel using ImageJ
software (FIG. 11).
[0153] Direct comparison of PCL with chemical inactivation and
photobleaching of Cy dyes demonstrates the release of dye from PCL
is 10-100 times more rapid. A similar comparison with other
photocleavable bonds demonstrated that the PCL resulted in faster
and more complete dye release than other photocleavable
reactions.
[0154] An automated NGH platform for FFPE tissue sections was
developed and designed to use a photocleavable fluorophore that is
released within seconds using UV irradiation. This unique
photocleavable label enables rapid cycles of repeated staining in
the same tissue section without the need to remove the
antigen-detecting antibody. The NGH platform also includes
automated fluidics for reduced hands-on time and a four-color
epi-fluorescence microscopy imaging system for multiplex reporter
detection.
[0155] FFPE tissue sections were mounted onto standard glass
slides, heat-fixed, dewaxed, hydrated, and placed in a specialized
flowcell. Using automated fluidics, the tissue was blocked, washed,
and exposed to biotinylated primary antibody using standard
reagents. Antibody binding was detected using avidin or
streptavidin-photocleavable label complexes, which were imaged
prior to the label being photocleaved in preparation for the next
staining cycle.
[0156] The NGH approach enabled serial staining of prostate tissue
sections. This staining strategy enabled rapid imaging of
antigens.
Example 3--Multiplex Staining of 8 Target Regions on a Single FFPE
Tissue Section with Two Color Photocleavable Labels
[0157] In another experiment, a 6 .mu.m section of human prostate
(normal) was prepared and stained in three consecutive cycles.
Tissue section was probed for multiple target proteins at each
cycle, a single imaging event at each cycle at various wavelengths
captured signal from various target proteins. Following a cleaving
event through UV exposure, the tissue was probed again for the next
set of target proteins. Three such multiplex cycles resulted in
capture of the following 8 targets in a single tissue section:
Cycle I: Collagen (CNA35), Hoechst (nuclear DNA), anti-ALCAM and
anti-CD44; Cycle II: anti-cytokeratin and anti-SMA; Cycle III:
anti-Fibronectin and anti-Histone H3. Each cycle captured images
from four channels: Cy7 (750 nm), Cy5 (650 nm), Cy2 (488 nm) and
Hoechst (365 nm). At the completion of the experiment, the three
image data sets were co-registered using the Cy2 channel. Image
integration into a single 12-channel image was performed using
ImageJ software. FIG. 19 shows the cyclic multiplex staining and
imaging.
[0158] Cyclic multiplex staining successfully achieved
immunofluorescent detention with six distinct antibodies in
addition to two non-antibody stains (collagen and nuclear stains)
over three cycles of staining. There was no visible deterioration
or deformation of the tissue during the procedure and there was no
significant accumulation of non-specific signal across the cycles
when comparing the baseline images (capture before cycle I) and the
images after photo cleaving (captured after cycle I, II, and
III).
Example 4--Multiplex Staining of FFPE Tissue Section with Three
Color Photocleavable Labels
[0159] FFPE tissue section was prepared using procedures described
in Example 2. Multiplex staining was achieved on three tissue
regions on the same tissue section using three antibodies with
three unique biotin-photocleavable labels, each with a unique
fluorescent dye. The tissue section was blocked, and sequentially
incubated with biotin-labeled primary antibody, streptavidin,
biotin-labeled photocleavable label, and this cycle was repeated
for the subsequent two antibodies and photocleavable labels.
Anti-CD44 was detected by photocleavable Cy3, anti-29-7 by
photocleavable Cy5, and anti-Histone H3 by photocleavable Cy7. A
single image captured signal at the three appropriate wavelengths,
and image merging was performed using ImageJ software to
co-visualize the three distinct regions in three distinct colors
(FIG. 20). A single photocleaving event released signal from all
photocleavable labels, post-imaging (FIG. 21).
Example 5--Detection of an Antigen in a Tissue Section Using
Partially Double-Stranded Biotin-Nucleotide-Fluorophore Tags
[0160] To prepare partially double-stranded
biotin-oligonucleotide-photocleavable Cy5 fluorescent dye, dC-Cy5
(FIG. 8) was extended to 3' of the top oligo using DNA polymerase
as shown in FIG. 2. The duplex product was purified by RP-HPLC. The
volume of the collected duplex product solution was reduced. Then,
the product was diluted to 50 .mu.M final concentration in
1.times.PBS.
[0161] The partially double-stranded oligonucleotide-photocleavable
Cy5 fluorescent dye was used in an experiment as described in
Example 1 and compared to the results obtained using a single
stranded oligo. Both designs of the photocleavable label performed
well.
Example 6--Evaluation of Staining and Photocleavage with Various
PCLs
[0162] Individual tissue sections (prostate cancer, human) were
prepped in a single batch. Sections were blocked (avidin or
streptavidin, biotin, total protein) and stained with biotinylated
anti-SMA antibody followed by the addition of avidin, and then the
addition of one of a series of PCLs (Standard Duplex PCL, Long
Duplex PCL, Short Duplex PCL, and Single Biotin PCL; see FIG. 12)
at identical concentrations. The Standard Duplex PCL consisted of
two 34-nucleotide oligos having a biotin on their 5' end and a
photocleavable fluorescent label on their 3' end, the oligos were
annealed to form a 28-nucleotide double-stranded section with four
nucleotide single-stranded overhangs on each end. The Long Duplex
PCL consisted of two 69-nucleotide oligos having a biotin on their
5' end and a photocleavable fluorescent label on their 3' end, the
oligos hybridized to form a 67-nucleotide double-stranded section
with two nucleotide single-stranded overhangs on each end. The
Short Duplex PCL consisted of a single 12-nucleotide oligo having a
biotin on its 5' end and a photocleavable fluorescent label on its
3' end. The single biotin PCL was the same as the Standard PCL
except that only one of the two oligos in each hybrid had a biotin
on its 5' end. In addition, staining with an oligonucleotide having
a photocleavable biotin (IDT-PCL; from Integrated DNA Technologies,
Coralville, Iowa) was performed in a separate batch with an
identical concentration of PCL. The IDT-PCL consisted of two
34-nucleotide oligos having a photocleavable biotin on their 5' end
and a fluorescent label on their 3' end, the oligos hybridized to
form a 28-nucleotide double-stranded section with four nucleotide
single-stranded overhangs on each end (FIG. 12). The center of the
field of view for each section was photo cleaved (3.times.2 sec
with 2 min flush between each) during time-lapse imaging. Signal
intensity was measured in two areas (ROI) of equal size: one area
of specific stain and one area of background. The maximal intensity
of the stain was traced over time (FIG. 10A). The difference
between the signal and background was plotted both as an absolute
signal (FIG. 10B) and % of maximal signal (FIG. 10C). In addition
to the time-lapse capture, images of each stained section were
obtained both before and after photocleavage. FIGS. 11D-H show
images of each section that were produced by stitching together
about 25 images. These data show that all probe variations are
usable as PCLs.
Example 7--Detection of an Antigen in a Tissue Section Using PCLs
with SMCC Crosslinkers
[0163] This Example illustrates an antigen-bound antigen-binding
complex of formula (III). Two single stranded oligonucleotides were
obtained from IDT (Integrated DNA Technologies, Coralville, Iowa)
of which one oligonucleotide had a 5' C6 amino modification. The
oligonucleotides were annealed to form a partially double stranded
oligonucleotide and -dC-Cy5 or dU-Cy7PCLs were extended to the 3'
ends. PCL synthesis was followed by RP-HPLC purification.
Sulfo-SMCC (a bifunctional cross linker) was attached to the 5'
amino modified strand of the duplex and the reaction was purified
by RP-HPLC. Sulfo-SMCC was attached to the primary amine on the 5'
end of the oligonucleotide through an amide linkage. The SMCC
linker allowed direct linkage of the probe to the antibody,
eliminating the need for a separate ligand-receptor connection
between the probe and the antibody, such as the biotin-avidin or
streptavidin linkage, described previously. Eliminating additional
ligands reduced the risk of non-specific interactions and stearic
hindrance.
[0164] Direct conjugation of the probe to the antibody will
decrease hands-on time to complete the staining and allow for
multiplex antibody staining with minimal steps and reagents.
Multiple antibodies conjugated to uniquely labeled PCLs (at least
up to three colors) will be premixed and used in tissue staining
resulting in detection of three antigens in a single
staining/imaging cycle. Three such staining cycles with a cleavage
cycle at the end of each imaging event will thus result in
detection of nine antigens with 70% time reduction from the
previous ligand-based approach.
[0165] Other structural variations of the PCL with SMCC linker will
include adding an amino modification to the oligo on the base
instead of the phosphate, C12 linker in place of the C6 linker for
amino modification, linking the SMCC away from the end of the
oligo, and adding the SMCC linker on the same strand as
incorporation rather than the opposite strand.
[0166] Direct conjugation of PCL to the SMCC linker will also be
achieved through a SM(PEG)n heterobifunctional crosslinker in which
a polyethylene glycol (PEG) spacer replaces the hydrocarbon-based
spacer in the Sulfo-SMCC crosslinker. PEG spacer will offer more
flexibility between the two functional groups, improve solubility
of the reaction and final conjugate and reduce potential for
aggregation of the conjugates, thus preserving functionality. Based
on the size of the antibody, different lengths of the PEG linkers
will be chosen to minimize the stearic hindrance of the cross
linker with the PCL moiety and to maximize flexibility of the
spacer arm.
[0167] Direct conjugation of PCL molecules will be synthesized by
incubating heterobifunctional linkers with the amino modified
oligonucleotides under suitable pH (in PBS or PBS-EDTA at pH
7-7.2), followed by clean-up using RP-HPLC and/or desalting.
Subsequently, activated PCLs will be incubated with antibodies that
have exposed sulfhydryl groups (reduced using 5 mM TCEP). The
conjugation reaction will be further purified through a desalting
column to remove unbound oligos. Alternatively, conjugation of PCL
with antibodies will be achieved with the use of commercial
conjugation kits that provide optimized reagents and buffers
required to achieve conjugation (such as, Thunder-Link PLUS oligo
conjugation system, Cat #425-000, 425-0300 offered by Innova
Biosciences).
[0168] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
REFERENCES
[0169] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
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[0177] PCT Publn. No. WO 2013/040257 [0178] Corrie, in Dynamics
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Sequence CWU 1
1
17170DNAArtificial SequenceSynthetic polynucleotide 1cgtaccccgc
ttggtctttc tcccgtaccc cgcttggtct ttctccctgc cccgggttcc 60tcattctctc
70234DNAArtificial SequenceSynthetic polynucleotide 2tacggagcat
gagagaatga ggaacccggg gcag 34334DNAArtificial SequenceSynthetic
polynucleotide 3tacgctgccg gtgtcctcat tctctcactc gctc
34434DNAArtificial SequenceSynthetic polynucleotide 4tacggagcga
gtgagagaat gaggacaccg gcag 34535DNAArtificial SequenceSynthetic
polynucleotide 5tacgctgccg gtgtcctcat tctctcactc gctcc
35635DNAArtificial SequenceSynthetic polynucleotide 6tacggagcga
gtgagagaat gaggacaccg gcagc 35736DNAArtificial SequenceSynthetic
polynucleotide 7tacgctgccg gtgtcctcat tctctcactc gctccg
36836DNAArtificial SequenceSynthetic polynucleotide 8tacggagcga
gtgagagaat gaggacaccg gcagcg 36937DNAArtificial SequenceSynthetic
polynucleotide 9tacgctgccg gtgtcctcat tctctcactc gctccgt
371037DNAArtificial SequenceSynthetic polynucleotide 10tacggagcga
gtgagagaat gaggacaccg gcagcgt 371170DNAArtificial SequenceSynthetic
polynucleotide 11tacgctgccg gtgtcctcat tctctcactc gctctttttt
gagcgagtga gagaatgagg 60acaccggcag 701271DNAArtificial
SequenceSynthetic polynucleotide 12tacgctgccg gtgtcctcat tctctcactc
gctctttttt gagcgagtga gagaatgagg 60acaccggcag c 711372DNAArtificial
SequenceSynthetic polynucleotide 13tacgctgccg gtgtcctcat tctctcactc
gctctttttt gagcgagtga gagaatgagg 60acaccggcag cg
721473DNAArtificial SequenceSynthetic polynucleotide 14tacgctgccg
gtgtcctcat tctctcactc gctctttttt gagcgagtga gagaatgagg 60acaccggcag
cgt 731569DNAArtificial SequenceSynthetic polynucleotide
15agtaccccgc ttggtctttc tcccgtaccc cgcttggtct ttctccctgc cccgggttcc
60tcattctct 691669DNAArtificial SequenceSynthetic polynucleotide
16agagagaatg aggaacccgg ggcagggaga aagaccaagc ggggtacggg agaaagacca
60agcggggta 691712DNAArtificial SequenceSynthetic polynucleotide
17ggacaccggc ag 12
* * * * *