U.S. patent application number 16/856619 was filed with the patent office on 2020-10-08 for signaling conjugates and methods of use.
The applicant listed for this patent is VENTANA MEDICAL SYSTEMS, INC.. Invention is credited to Nelson Alexander, William Day, Jerome W. Kosmeder, Mark Lefever, Larry Morrison, Anne M. Pedata, Stacey Stanislaw.
Application Number | 20200319193 16/856619 |
Document ID | / |
Family ID | 1000004914931 |
Filed Date | 2020-10-08 |
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United States Patent
Application |
20200319193 |
Kind Code |
A1 |
Alexander; Nelson ; et
al. |
October 8, 2020 |
SIGNALING CONJUGATES AND METHODS OF USE
Abstract
Disclosed herein are embodiments of a signaling conjugate,
embodiments of a method of using the signaling conjugates, and
embodiments of a kit comprising the signaling conjugate. The
disclosed signaling conjugate comprises a latent reactive moiety
and a chromogenic moiety that may further comprise a linker
suitable for coupling the latent reactive moiety to the chromogenic
moiety. The signaling conjugate may be used to detect one or more
targets in a biological sample and are capable of being covalently
deposited directly on or proximally to the target. Particular
disclosed embodiments of the method of using the signaling
conjugate comprise multiplexing methods.
Inventors: |
Alexander; Nelson; (Marana,
AZ) ; Day; William; (Tucson, AZ) ; Kosmeder;
Jerome W.; (Tucson, AZ) ; Lefever; Mark; (Oro
Valley, AZ) ; Morrison; Larry; (Oro Valley, AZ)
; Pedata; Anne M.; (Tucson, AZ) ; Stanislaw;
Stacey; (Tucson, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VENTANA MEDICAL SYSTEMS, INC. |
Tucson |
AZ |
US |
|
|
Family ID: |
1000004914931 |
Appl. No.: |
16/856619 |
Filed: |
April 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16038374 |
Jul 18, 2018 |
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16856619 |
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13849160 |
Mar 22, 2013 |
10041950 |
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16038374 |
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61778093 |
Mar 12, 2013 |
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61710607 |
Oct 5, 2012 |
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61616330 |
Mar 27, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/682 20130101;
G01N 33/53 20130101; C12Q 1/68 20130101; G01N 33/581 20130101; G01N
33/542 20130101 |
International
Class: |
G01N 33/58 20060101
G01N033/58; C12Q 1/682 20060101 C12Q001/682; C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53; G01N 33/542 20060101
G01N033/542 |
Claims
1-28. (canceled)
29. An immunohistochemistry (IHC) method, comprising: (a)
contacting a slide comprising a tissue sample that possibly
contains a target antigen with a detection probe comprising a
primary antibody specific for the target antigen, whereby the
primary antibody binds to the target antigen if the target antigen
is present in the tissue sample, thereby producing an
antibody-target complex comprising the primary antibody bound to
the target antigen; (b) after step (a), washing the tissue sample
to remove detection probe that is not bound to the target antigen;
(c) after step (b), contacting the tissue sample with a labeling
conjugate comprising a secondary antibody and an enzyme, wherein
the secondary antibody is specific for the primary antibody, and
the enzyme is a peroxidase, whereby, if the target antigen is
present in the tissue sample, the secondary antibody binds to the
primary antibody of the antibody-target complex, thereby producing
a labeled complex comprising the labeling conjugate bound to the
primary antibody of the antibody-target complex; (d) after step
(c), washing the tissue sample to remove labeling conjugate that is
not bound to the primary antibody of the antibody-target complex;
(e) after step (d), contacting the tissue sample with a signaling
conjugate comprising a phenolic moiety and a chromogenic moiety,
whereby, if the target antigen is present in the tissue sample, the
enzyme of the antibody-target complex catalyzes conversion of the
phenolic moiety into a reactive species comprising the chromogenic
moiety which then covalently binds to: (i) a location on the tissue
sample near the labeled complex; and/or (ii) a location on the
labeled complex; thereby producing deposited chromogen comprising
the chromogenic moiety covalently bound to the location (i) and/or
(ii); (f) after step (e), washing the tissue sample to remove
signaling conjugate that is not bound to the tissue sample or the
labeled complex; wherein, if the target antigen is present in the
biological sample, the deposited chromogen produces a colored
signal that provides for the detection of the target antigen when
exposed to light, wherein the light comprises one or more of
visible light, infrared light, or near infrared light.
30. The method of claim 29, further comprising: (g) after step (f),
analyzing the tissue sample by light microcopy such that, if the
target antigen is present in the biological sample, the deposited
chromogen produces a colored signal that provides for the detection
of the target antigen when exposed to light, wherein the light
comprises one or more of visible light, infrared light, or near
infrared light.
31. The method of claim 29, wherein the target antigen comprises a
polypeptide.
32. The method of claim 29, wherein the secondary antibody is an
anti-species antibody against the species of the primary antibody,
wherein the primary antibody is a rabbit, or mouse antibody and the
secondary antibody is a goat antibody.
33. The method of claim 29, wherein the secondary antibody is an
anti-hapten antibody.
34. A chromogenic in situ hybridization (CISH) method, comprising:
(a) contacting a slide comprising a tissue sample that possibly
contains a target nucleic acid with a detection probe comprising a
hapten linked to a nucleic acid probe having a sequence that
hybridizes to the sequence of the target nucleic acid, whereby the
nucleic acid probe hybridizes to a target nucleic acid if the
target nucleic acid is present in the tissue sample, thereby
producing a probe-target complex comprising the nucleic acid probe
hybridized to the target nucleic acid; (b) after step (a), then
washing the tissue sample to remove detection probe that is not
hybridized to the target nucleic acid; (c) after step (b),
contacting the tissue sample with a labeling conjugate comprising
an anti-hapten antibody and an enzyme, wherein the anti-hapten
antibody is specific for the hapten of the detection probe, and the
enzyme is a peroxidase, whereby, if the target nucleic acid is
present in the tissue sample, the anti-hapten antibody binds to the
hapten of the probe-target complex, thereby producing a labeled
complex comprising the labeling conjugate bound to the hapten of
the probe-target complex; (d) after step (c), washing the tissue
sample to remove labeling conjugate that is not bound to the hapten
of the probe-target complex; (e) after step (d), contacting the
tissue sample with a signaling conjugate comprising a phenolic
moiety and a chromogenic moiety, whereby, if the target nucleic
acid is present in the tissue sample, the enzyme of the
probe-target complex catalyzes conversion of the phenolic moiety
into a reactive species comprising the chromogenic moiety which
then covalently binds to: (i) a location on the tissue sample near
the labeled complex; and/or (ii) a location on the labeled complex;
thereby producing deposited chromogen comprising the chromogenic
moiety covalently bound to the location (i) and/or (ii); (f) after
step (e), washing the tissue sample to remove signaling conjugate
that is not bound to the tissue sample or the labeled complex;
wherein, if the target nucleic acid is present in the biological
sample, the deposited chromogen produces a colored signal that
provides for the detection of the target nucleic acid when exposed
to light, wherein the light comprises one or more of visible light,
infrared light, or near infrared light.
35. The method of claim 34, further comprising: (g) after step (f),
analyzing the tissue sample by light microcopy such that, if the
target nucleic acid is present in the biological sample, the
deposited chromogen produces a colored signal that provides for the
detection of the target nucleic acid when exposed to light, wherein
the light comprises one or more of visible light, infrared light,
or near infrared light.
36. The method of claim 34, wherein the probe nucleic acid
comprises RNA, DNA, LNA, PNA, or a combination thereof.
37. The method of claim 34, wherein the hapten is selected from an
oxazole hapten, pyrazole hapten, thiazole hapten, nitroaryl hapten,
benzofuran hapten, triterpene hapten, urea hapten, thiourea hapten,
rotenoid hapten, coumarin hapten, cyclolignan hapten,
di-nitrophenyl hapten, biotin hapten, digoxigenin hapten,
fluorescein hapten, or rhodamine hapten
38. The method of claim 29, further comprising, prior to step (a),
inactivating endogenous tissue peroxidase activity with a
peroxidase inhibitor.
39. The method of claim 35, further comprising, prior to step (a),
inactivating endogenous tissue peroxidase activity with a
peroxidase inhibitor.
40. The method according to claim 29, wherein the signaling
conjugate further comprises a linker joining the chromogenic moiety
and the phenolic moiety.
41. The method according to claim 35, wherein the signaling
conjugate further comprises a linker joining the chromogenic moiety
and the phenolic moiety.
42. The method according to claim 40, wherein the linker comprises
polyethylene glycol.
43. The method according to claim 41, wherein the linker comprises
polyethylene glycol.
44. The method according to claim 29, wherein the signaling
conjugate comprises only a single phenolic moiety.
45. The method according to claim 35, wherein the signaling
conjugate comprises only a single phenolic moiety.
46. The method according to claim 29, wherein the chromogenic
moiety comprises rhodamine, a rhodamine derivative,
tetramethylrhodamine (TMR, TAMRA), diarylrhodamine derivatives, QSY
7, QSY 9, or QSY 21.
47. The method according to claim 35, wherein the chromogenic
moiety comprises rhodamine, a rhodamine derivative,
tetramethylrhodamine (TMR, TAMRA), diarylrhodamine derivatives, QSY
7, QSY 9, or QSY 21.
48. The method according to claim 29, wherein the chromogenic
moiety comprises tartrazine, 7-diethylaminocoumarin-3-carboxylic
acid, DABSYL, fluorescein isothiocyanate (FITC), Rhodamine Green
carboxylic acid succinimidyl ester (DY-505), eosin isothiocyanate
(EITC), 6-carboxy-2',4,7,7'-tetrachlorofluorescein succinimidyl
ester (TET), carboxyrhodamine 6G succinimidyl ester,
carboxytetramethylrhodamine succinimidyl ester (TMR, TAMRA)
(DY-554), QSY 9, sulforhodamine B sulfonyl chloride (DY-560), Texas
Red (sulforhodamine 101), Fast Green FCF, Malachite Green, QSY 21,
or Victoria Blue.
49. The method according to claim 35, wherein the chromogenic
moiety comprises tartrazine, 7-diethylaminocoumarin-3-carboxylic
acid, DABSYL, fluorescein isothiocyanate (FITC), Rhodamine Green
carboxylic acid succinimidyl ester (DY-505), eosin isothiocyanate
(EITC), 6-carboxy-2',4,7,7'-tetrachlorofluorescein succinimidyl
ester (TET), carboxyrhodamine 6G succinimidyl ester,
carboxytetramethylrhodamine succinimidyl ester (TMR, TAMRA)
(DY-554), QSY 9, sulforhodamine B sulfonyl chloride (DY-560), Texas
Red (sulforhodamine 101), Fast Green FCF, Malachite Green, QSY 21,
or Victoria Blue.
50. The method according to claim 29, wherein the peroxidase is
horseradish peroxidase.
51. The method according to claim 35, wherein the peroxidase is
horseradish peroxidase.
52. The method according to claim 29, wherein the light is visible
light.
53. The method according to claim 35, wherein the light is visible
light.
54. The method according to claim 29, further comprising, after
step (f), counterstaining the tissue sample.
55. The method according to claim 34, further comprising, after
step (f), counterstaining the tissue sample.
56. The method of claim 54, wherein the counterstaining comprises
hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcian
blue, or Nuclear Fast Red staining.
57. The method of claim 55, wherein the counterstaining comprises
hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcian
blue, or Nuclear Fast Red staining.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/616,330, filed on Mar. 27, 2012, U.S.
Provisional Patent Application No. 61/710,607, filed on Oct. 5,
2012, and U.S. Provisional Patent Application No. 61/778,093, filed
on Mar. 12, 2013, all of which are incorporated herein by
reference.
FIELD
[0002] The present disclosure concerns conjugates, compositions,
methods, and kits useful in performing assays for detecting one or
more targets within a biological sample.
BACKGROUND
[0003] Immunohistochemistry, or IHC, refers to the process of
detecting, localizing, and quantifying antigens, such as a protein,
in a biological sample, such as a tissue, and using specific
binding moieties, such as antibodies specific to the particular
antigens. This detection technique has the advantage of being able
to show exactly where a given protein is located within the tissue
sample. It is also an effective way to examine the tissues
themselves. In situ hybridization, or ISH, refers to the process of
detecting, localizing, and quantifying nucleic acids. Both IHC and
ISH can be performed on various biological samples, such as tissue
(e.g., fresh frozen, formalin fixed paraffin embedded) and
cytological samples. Upon recognition of the targets, whether the
targets be nucleic acids or antigens, the recognition event can be
detected through the use of various labels (e.g., chromogenic,
fluorescent, luminescent, radiometric).
[0004] In situ hybridization (ISH) on tissue includes detecting a
nucleic acid by applying a complementary strand of nucleic acid to
which a reporter molecule is coupled. Visualization of the reporter
molecule allows an observer to localize specific DNA or RNA
sequences in a heterogeneous cell population, such as a
histological, cytological, or environmental sample. Presently
available ISH techniques include silver in situ hybridization
(SISH), chromogenic in situ hybridization (CISH) and fluorescence
in situ hybridization (FISH).
[0005] Interrogation of gene expression in tissue sections using
PCR or microarrays has been successfully used to classify patients'
likelihood of tumor recurrence and identify those who may benefit
from specific therapies. However, tissue specificity and cellular
context, which improve the value of tissue-based assays, are lost
during the mRNA extraction for PCR or microarray analysis.
Moreover, false positive or negative results may be generated from
the presence of "contaminating" non-tumor cells in the section. As
such, there is a need for automated in situ hybridization assays
which target mRNA (mRNA-ISH) that enables robust and reproducible
evaluation of biomarker expression while preserving tissue context
and specificity, as well as cell-cell relationships.
[0006] Chromogenic substrates have been used widely for
immunohistochemistry for many years and for in situ hybridization
more recently. Chromogenic detection offers a simple and
cost-effective method of detection. Traditionally, chromogenic
substrates precipitate when activated by the appropriate enzyme.
That is, the traditional chromogenic substance is converted from a
soluble reagent into an insoluble, colored precipitate upon
contacting the enzyme. The resulting colored precipitate requires
no special equipment for processing or visualizing. There are
several qualities that successful IHC or ISH chromogenic substrates
share. First, the substance should precipitate to a colored
substance, preferably with a very high molar absorptivity. The
enzyme substrate should have high solubility and reagent stability,
but the precipitated chromogen products should be very insoluble,
preferably in both aqueous and alcohol solutions. Enzyme turnover
rates should be very high so as to highly amplify the signal from a
single enzyme in a short amount of time. Particular limitations of
current chromogenic techniques include the ability to multiplex,
incompatibility towards post-staining processing (e.g., solvent
washes, drying, subsequent staining), and limited color
options.
[0007] For in situ assays, such as ISH assays and IHC assays, of
tissue and cytological samples, especially multiplexed assays of
such samples, it is highly desirable to identify and develop
methods that provide desirable results without background
interference. Tyramide Signal Amplification (TSA) is a known method
based on catalyzed reporter deposition (CARD). U.S. Pat. No.
5,583,001 discloses a method for detection or quantitation of an
analyte using an analyte-dependent enzyme activation system relying
on catalyzed reporter deposition to amplify the reporter signal
enhancing the catalysis of an enzyme in a CARD or TSA method by
reacting a labeled phenol molecule with an enzyme. While tyramide
signal amplification is known to amplify the visibility of targets,
it is also associated with elevated background staining (e.g.,
amplification of non-specific recognition events).
SUMMARY
[0008] Disclosed herein are signaling conjugates, particularly
chromogen conjugates and methods of using the signaling conjugates
to detect targets within samples. The disclosed
chromogen-containing compositions and kits including the same, may
be used to detect targets in various analyses or assays. In
preferred embodiments, the targets are from a biological sample.
Illustrative targets include proteins and nucleic acids being
analyzed in the context of anatomical pathology or cytology. One
aspect of the disclosure is that the chromogen conjugates are fully
compatible with automated slide staining instruments and processes.
The chromogen conjugates enable previously unattainable detection
sensitivity and multiplexing capability, amongst various other
advantages, thus representing a significant advancement to the
state of the art.
[0009] In illustrative embodiments, a method of detecting a target
in a biological sample includes contacting the biological sample
with a detection probe, contacting the biological sample with a
labeling conjugate, and contacting the biological sample with a
signaling conjugate. The labeling conjugate includes an enzyme. The
signaling conjugate includes a latent reactive moiety and a
chromogenic moiety. The enzyme catalyzes conversion of the latent
reactive moiety into a reactive moiety which covalently binds to
the biological sample proximally to or directly on the target. The
method further includes illuminating the biological sample with
light and detecting the target through absorbance of the light by
the chromogenic moiety of the signaling conjugate. In one
embodiment, the reactive moiety reacts with a tyrosine residue of
the biological sample, the enzyme conjugate, the detection probe,
or combinations thereof.
[0010] In illustrative embodiments, the detection probe is an
oligonucleotide probe or an antibody probe. In further illustrative
embodiments, the labeling conjugate includes an antibody coupled to
the enzyme. Exemplary enzymes include oxidoreductases or
peroxidases. An exemplary antibody for the labeling conjugate would
be an anti-species or an anti-hapten antibody. The detection probe
may include a hapten selected from the group consisting an oxazole
hapten, pyrazole hapten, thiazole hapten, nitroaryl hapten,
benzofuran hapten, triterpene hapten, urea hapten, thiourea hapten,
rotenoid hapten, coumarin hapten, cyclolignan hapten,
di-nitrophenyl hapten, biotin hapten, digoxigenin hapten,
fluorescein hapten, and rhodamine hapten. In other examples, the
detection probe is monoclonal antibody derived from a second
species such as goat, rabbit, mouse, or the like. The labeling
conjugate is configured, through its inclusion of an anti-species
or an anti-hapten antibody to bind selectively to the detection
probe.
[0011] One aspect of the present disclosure is that the signaling
conjugates disclosed herein may be configured to absorb light more
selectively than traditionally available components, such as
traditional chromogens. Detection is realized by absorbance of the
light by the signaling conjugate; for example, absorbance of at
least about 5% of incident light would facilitate detection of the
target. In other darker stains, at least about 20% of incident
light would be absorbed. Non-uniform absorbance of light within the
visible spectrum results in the chromophore moiety appearing
colored. The signaling conjugates disclosed herein may appear
colored due to their absorbance; the signaling conjugates may
appear to provide any color when used in the assay, with certain
particular colors including red, orange, yellow, green, indigo, or
violet depending on the spectral absorbance associated with the
chromophore moiety contained therein. According to another aspect,
the chromophore moieties may have narrower spectral absorbances
than those absorbances of traditionally used chromogens (e.g., DAB,
Fast Red, Fast Blue). In illustrative embodiments, the spectral
absorbance associated with the first chromophore moiety of the
first signaling conjugate has a full-width half-max (FWHM) of
between about 30 nm and about 250 nm, between about 30 nm and about
150 nm, between about 30 nm and about 100 nm, or between about 20
nm and about 60 nm.
[0012] Narrow spectral absorbances enable the signaling conjugate
chromophore moiety to be analyzed differently than traditional
chromogens. While having enhanced features compared to
traditionally chromogens, detecting the signaling conjugates
remains simple. In illustrative embodiments, detecting comprises
using a bright-field microscope or an equivalent digital scanner.
The narrow spectral absorbances enable chromogenic multiplexing at
level beyond the capability of traditional chromogens. For example,
traditional chromogens are somewhat routinely duplexed (e.g., Fast
Red and Fast Blue, Fast Red and Black (silver), Fast Red and DAB).
However, triplexed or three-color applications, or greater, are
atypical, as it becomes difficult to discern one chromophore from
another. In illustrative embodiments of the presently disclosed
technology, the method includes detecting from two to at least
about six different targets using different signaling conjugates or
combinations thereof. In one embodiment, illuminating the
biological sample with light comprises illuminating the biological
sample with a spectrally narrow light source, the spectrally narrow
light source having a spectral emission with a second full-width
half-max (FWHM) of between about 30 nm and about 250 nm, between
about 30 nm and about 150 nm, between about 30 nm and about 100 nm,
or between about 20 nm and about 60 nm. In another embodiment,
illuminating the biological sample with light includes illuminating
the biological sample with an LED light source. In another
embodiment, illuminating the biological sample with light includes
illuminating the biological sample with a filtered light
source.
[0013] In illustrative embodiments, detecting targets within the
sample includes contacting the biological sample with a first
amplifying conjugate that is covalently deposited proximally to or
directly on the first labeling conjugate. The first amplifying
conjugate may be followed by contacting the biological sample with
a secondary labeling conjugate. Illustratively, the amplification
of signal using amplifying conjugates enhances signaling conjugate
deposition. The enhanced signaling conjugate deposition enables
easier visual identification of the chromogenic signal, that is,
the amplification makes the color darker and easier to see. For low
expressing targets, this amplification may result in the signal
becoming sufficiently dark to be visible, whereas without
amplification, the target would not be apparent. In one embodiment,
the signaling conjugate is covalently deposited proximally to the
target at a concentration of greater than about 1.times.10.sup.11
molecules per cm.sup.2.mu.m to about 1.times.10.sup.16 molecules
per cm.sup.2.mu.m of the biological sample. In one embodiment, the
first target and the second target are genetic nucleic acids.
Detecting the first target through absorbance of the light by the
first signaling conjugate includes detecting, in an exemplary
embodiment, a first colored signal selected from red, orange,
yellow, green, indigo, or violet, the first colored signal
associated with spectral absorbance associated with the first
chromogenic moiety of the first signaling conjugate. Detecting the
second target through absorbance of the light by the second
signaling conjugate includes detecting, in an exemplary embodiment,
a second colored signal selected from red, orange, yellow, green,
indigo, or violet, the second colored signal associated with
spectral absorbance associated with the second chromogenic moiety
of the second signaling conjugate. Detecting also may comprise
viewing an overlap in proximity through absorbance of the light by
the first signaling conjugate overlapping in proximity with the
second signaling conjugate so that a third colored signal
associated with overlapping spectral absorbance of the first
spectral absorbance and the second spectral absorbance. According
to one example, this third colored signals a normal genetic
arrangement and the first and second colors signal a genetic
rearrangement or translocation.
[0014] Also disclosed herein are compositions comprising a
biological sample comprising one or more enzyme-labeled targets and
a plurality of signaling conjugates comprising a chromogenic
moiety. The signaling conjugates are configured to bind proximally
to or directly on the one or more targets in the biological sample
and are configured to provide a bright-field signal.
[0015] In particular disclosed embodiments of the composition,
"configured to provide a bright-field signal" comprises absorbing
5% or more of incident light. In another embodiment of the
composition, "configured to provide a bright-field signal"
comprises absorbing 20% or more of incident light. In particular
disclosed embodiments of the composition, "configured to provide a
bright-field signal" comprises having an absorbance peak with a
.lamda..sub.max of between about 350 nm and about 800 nm. In one
embodiment, "configured to provide a bright-field signal" comprises
having an absorbance peak with a .lamda..sub.max of between about
400 nm and about 750 nm. In another embodiment, "configured to
provide a bright-field signal" comprises having an absorbance peak
with a .lamda..sub.max of between about 400 nm and about 700 nm. In
yet another embodiment, "configured to provide a bright-field
signal" comprises having a first absorbance peak with a first
.lamda..sub.max of between about 350 nm and about 500 nm, and a
second absorbance peak with a second .lamda..sub.max of between
about 500 nm and about 800 nm. In another embodiment, "configured
to provide a bright-field signal" comprises having a first
absorbance peak with a first .lamda..sub.max of between about 400
nm and about 500 nm, and a second absorbance peak with a second
.lamda..sub.max of between about 500 nm and about 750 nm. In yet
another embodiment, "configured to provide a bright-field signal"
comprises having a first absorbance peak with a first
.lamda..sub.max of between about 350 nm and about 450 nm, and a
second absorbance peak with a second .lamda..sub.max of between
about 450 nm and about 600 nm. In another embodiment, "configured
to provide a bright-field signal" comprises having a first
absorbance peak with a first .lamda..sub.max of between about 350
nm and about 450 nm, and second absorbance peak with
.lamda..sub.max of between about 600 nm and about 800 nm.
[0016] The composition also may comprise a plurality of signaling
conjugates configured to have an absorbance peak with a full-width
half-max (FWHM) of between about 30 nm and about 250 nm. In one
embodiment, a plurality of signaling conjugates is configured to
have an absorbance peak with a full-width half-max (FWHM) of
between about 30 nm and about 150 nm. In another embodiment, a
plurality of signaling conjugates is configured to have an
absorbance peak with a full-width half-max (FWHM) of between about
30 nm and about 100 nm. In yet another embodiment, a plurality of
signaling conjugates is configured to have an absorbance peak with
a full-width half-max (FWHM) of between about 20 nm and about 60
nm.
[0017] The composition also may comprise signaling conjugates
wherein at least a portion of the plurality of signaling conjugates
has an average molar absorptivity of greater than about 5,000
M.sup.-1 cm.sup.-1 to about 90,000 M.sup.-1 cm.sup.-1. In one
embodiment, at least a portion of the plurality of signaling
conjugates has an average molar absorptivity of greater than about
10,000 M.sup.-1 cm.sup.-1 to greater than about 80,000 M.sup.-1
cm.sup.-1. In another embodiment, at least a portion of the
plurality of signaling conjugates has an average molar absorptivity
of greater than about 20,000 M.sup.-1 cm.sup.-1 to greater than
about 80,000 M.sup.-1 cm.sup.-1. In yet another embodiment, at
least a portion of the plurality of signaling conjugates has an
average molar absorptivity of greater than about greater than about
40,000 M.sup.-1 cm.sup.-1 to greater than about 80,000 M.sup.-1
cm.sup.-1.
[0018] In particular disclosed embodiments, the composition may
comprise a plurality of signaling conjugates wherein at least a
portion of the plurality of signaling conjugates has a solubility
in water of at least about 0.1 mM to about 1 M. In one embodiment,
at least a portion of the plurality of signaling conjugates has a
solubility in water of at least about 1 mM to about 1 M. In another
embodiment, at least a portion of the plurality of signaling
conjugates has a solubility in water of at least about 10 mM to
about 1 M. In yet another embodiment, at least a portion of the
plurality of signaling conjugates has a solubility in water of at
least about 100 mM to about 1M.
[0019] The disclosed composition also may comprise a plurality of
signaling conjugates that are stable against precipitation in an
aqueous buffered solution for greater than about 1 month to about
30 months. In one embodiment, a plurality of signaling conjugates
is stable against precipitation in an aqueous buffered solution for
greater than 12 months.
[0020] In particular disclosed embodiments, a plurality of
signaling conjugates are configured to provide an optically
apparent color under bright-field illumination. The optically
apparent color in exemplary embodiments is selected from red,
orange, yellow, green, indigo, violet, and mixtures thereof. In
particular disclosed embodiments, "configured to provide a
bright-field signal" comprises imparting a first optically distinct
color and a second optically distinct color. In one embodiment,
configured to provide a bright-field signal comprises imparting a
third color optically distinct from the first optically distinct
color and the second optically distinct color. In yet another
embodiment, configured to provide the bright-field signal comprises
imparting a fourth color optically distinct from the first
optically distinct color, the second optically distinct color, and
the third optically distinct color.
[0021] In particular disclosed embodiments of the composition, the
biological sample is a tissue or cytology sample. The tissue or
cytology sample, such as a formalin-fixed, paraffin embedded
sample, may be mounted on a glass microscope slide for use with an
automated slide staining instrument.
[0022] In certain embodiments, the biological sample comprises a
first target and the plurality of signaling conjugates are located
proximally to the first target. The biological sample also may
further comprise a second target and a second population of the
plurality of signaling conjugates that are located proximally to
the second target, wherein the first target and the second target
are different. In one embodiment, a first detection probe is used
to detect a first target and a second detection probe is used to
detect the second target.
[0023] Also disclosed herein are embodiments of a kit comprising a
signaling conjugate having a latent reactive moiety and a
chromogenic moiety as disclosed herein. In one embodiment, the kit
further comprises a peroxide solution. In another embodiment, the
kit further comprises an amplifying conjugate and an enzyme
conjugate.
[0024] The present disclosure contains information related to the
International Application entitled "Signaling Conjugates and
Methods of Use," filed on Mar. 22, 2013. The entirety of this
international application is incorporated herein by reference.
[0025] The foregoing and other objects, features, and advantages of
the presently disclosed technology will become more apparent from
the following detailed description, which proceeds with reference
to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0027] FIG. 1 is a flowchart providing the steps of one embodiment
of the method.
[0028] FIGS. 2(A-B) are schematic diagrams of embodiments of two
signaling conjugates. FIG. 2(A) illustrates a signaling conjugate
comprising a latent reactive moiety and a chromophore moiety. FIG.
2(B) illustrates an alternative signaling conjugate further
comprising a linker.
[0029] FIGS. 3(A-F) are schematic diagrams illustrating a manner in
which a target on a sample is detected. FIG. 3(A) shows a detection
probe binding to the target. FIG. 3(B) shows a labeling conjugate
binding to the detection probe. FIG. 3(C) shows a signaling
conjugate being enzymatically deposited onto the sample. FIG. 3(D)
shows an alternative embodiment in which an antibody-based
detection probe is used to detect a different target. FIG. 3(E)
shows an approach for detecting a target using an amplifying
conjugate. FIG. 3(F) shows that the amplifying conjugate was bound
to the sample and was labeled with a secondary labeling
conjugate.
[0030] FIGS. 4(A-B) are schematic diagrams illustrating (A) a
cross-sectional depiction of distribution of labeling conjugates
proximally to target (T); and (B) a graph depicting the
relationship between power of incident radiation (P.sub.0) across
the sample shown in (A) and power of transmitted radiation (P)
through the sample, the y-axis representing radiation power and the
x-axis representing linear distance across the sample.
[0031] FIGS. 5(A-B) are schematics showing the differences between
signals obtained with chromogens and signals obtained with
fluorophores. FIG. 5(A) illustrates detection of a chromogen
wherein the transmitted light is detected. FIG. 5(B) illustrates
the detection of a fluorophore wherein the emitted light is
detected.
[0032] FIGS. 6(A-B) are images illustrating the color
characteristics discussed herein. FIG. 6(A) is a color wheel
depicting the relationship between an observed color and FIG. 6(B)
is an image of absorbed radiation for the signaling conjugate.
[0033] FIGS. 7(A-B) are images illustrating results from a
particular embodiment of the disclosed method. FIG. 7(A) is a graph
illustrating the absorption spectrum of a 5-TAMRA-tyramide
conjugate, and FIG. 7(B) is a photomicrograph illustrating a
biological sample having targets detected by this particular
signaling conjugate.
[0034] FIGS. 8(A-B) are images illustrating results obtained from a
particular embodiment of the disclosed method. FIG. 8(A) is a
photomicrograph of a dual stain of two gene probes on a lung tissue
section testing for ALK rearrangements associated with non-small
cell lung cancer, and FIG. 8(B) is a UV-Vis spectra of fast red and
fast blue in ethyl acetate solutions as well as traces obtained
from tissue samples.
[0035] FIGS. 9(A-B) are graphs of absorbance versus wavelength and
illustrate the two sets of traces provided in FIG. 8(B). FIG. 9(A)
illustrates the traces obtained from tissue samples, whereas FIG.
9(B) illustrates traces obtained from ethyl acetate solutions of
Fast Red and Fast Blue.
[0036] FIGS. 10(A-B) are images and a schematic illustrating the
difference between a dual ISH chromogenic detection, where FIG.
10(A) shows a SISH/Red combined detection protocol, and FIG. 10(B)
shows a purple and yellow signaling conjugate as described herein.
The signal produced by combining these two chromogens is detected
as a third, unique color.
[0037] FIGS. 11(A-B) are photomicrographs showing two examples of
depositing two colors proximally to create a visually distinct
third color.
[0038] FIGS. 12(A-C) are photomicrographs showing the use of LED
illumination to separate the signal from a chromogenic dual stain,
where FIG. 12(A) shows white light illumination, FIG. 12 (B) shows
green light illumination and FIG. 12 (C) shows red light
illumination.
[0039] FIGS. 13(A-B) are photomicrographs, where FIG. 13(A) shows a
control slide to which no BSA-BF was added, and FIG. 13(B) shows a
slide to which the BSA-BF had been attached to the sample.
[0040] FIGS. 14(A-B) are photomicrographs showing a sample stained
with a signaling conjugate, where FIG. 14(A) is without tyrosine
enhancement and FIG. 14(B) is with tyrosine enhancement.
[0041] FIGS. 15(A-B) are photomicrographs showing a HER2 (4B5) IHC
in Calu-3 xenografts stained with two different signaling conjugate
having the absorption spectra shown in FIG. 16.
[0042] FIG. 16 illustrates absorbance spectra of two signaling
conjugates in solution and as used to stain the samples shown in
FIGS. 15(A-B).
[0043] FIGS. 17(A-E) show photomicrographs (FIG. 17(A-D)) of
tissues stained with signaling conjugates having different
chromogenic moieties, and FIG. 17(E) shows UV-Vis spectra with
traces corresponding to the absorbance of the signaling conjugates,
the traces corresponding to the associated photomicrograph.
[0044] FIGS. 18(A-E) show (A-D) photomicrographs of tissues stained
with signaling conjugates having different chromogenic moieties.
FIG. 18(E) shows UV-Vis spectra with traces corresponding to the
absorbance of the signaling conjugates, the traces corresponding to
the associated photomicrograph.
[0045] FIGS. 19(A-E) show (A-D) photomicrographs of tissues stained
with signaling conjugates having different chromogenic moieties.
FIG. 19(E) shows UV-Vis spectra with traces corresponding to the
absorbance of the signaling conjugates, the traces corresponding to
the associated photomicrograph.
[0046] FIGS. 20(A-E) show (A-D) photomicrographs of tissues stained
with signaling conjugates having different chromogenic moieties.
FIG. 20(E) shows UV-Vis spectra with traces corresponding to the
absorbance of the signaling conjugates, the traces corresponding to
the associated photomicrograph.
[0047] FIGS. 21(A-B) are photomicrographs of a tonsil tissue sample
comprised of normal non-cancerous B cells, where FIG. 21(A) is a
40.times. magnified view of a positive staining for KAPPA (brown)
and LAMBDA (purple) mRNA, and FIG. 21(B) is a 20.times. magnified
view of the same.
[0048] FIG. 22 is a schematic showing expected Kappa/Lambda copy
numbers associated with different types of non-Hodgkins B-cell
lymphomas.
[0049] FIGS. 23(A-B) are photomicrographs, where FIG. 23(A) is a
first lymphoma tissue sample showing a dual staining of KAPPA mRNA
(brown) and LAMBDA mRNA (purple, minimally observed), showing very
few cells expressing LAMBDA mRNA, and FIG. 23(B) a second lymphoma
tissue sample showing a dual staining for KAPPA mRNA (brown,
minimally observed) and LAMBDA mRNA (purple), showing very few
cells expressing KAPPA mRNA.
[0050] FIGS. 24(A-B) are photomicrographs which demonstrate dual
chromogenic mRNA ISH for a sample that would confound molecular
methods of diagnosis.
[0051] FIGS. 25(A-B) are photomicrographs of breast tissue, where
FIG. 25(A) is a negative staining for ACTB mRNA, and FIG. 25(B) is
positive staining for ACTB mRNA.
[0052] FIGS. 26(A-C) are photomicrographs of breast tissue samples
showing dual staining of ACTB, where FIG. 26(A) is a negative (0+)
staining for HER2 mRNA, FIG. 26(B) is a positive (1/2+) staining
for HER2 mRNA, and FIG. 26(C) is a positive (3+) staining for HER2
mRNA.
[0053] FIG. 27 is data from a number of tissue blocks comparing the
results of HER2 ISH analysis, HER2 IHC analysis, and HER2 mRNA
two-color ISH.
[0054] FIGS. 28(A-B) are photomicrographs illustrating direct
detection of the gene PTEN using a DNA ISH assay incorporating
direct deposition of a Rhod-tyramide conjugate. FIG. 28(A) is a
photomicrograph at 40.times. magnification and FIG. 28(B) is a
photomicrograph of a separate area at 63.times. magnification.
[0055] FIG. 29 is a photomicrograph illustrating direct detection
of an ERG5' target in MCF-7 human breast adenocarcinoma cells using
a DNA ISH assay with a Rhod-tyramide signaling conjugate.
[0056] FIG. 30 is a photomicrograph illustrating direct detection
of an ERG3' target in MCF-7 human breast adenocarcinoma cells using
a DNA ISH assay with a DABSYL-tyramide signaling conjugate.
[0057] FIG. 31 is photomicrograph illustrating amplified detection
of both ERG3' and ERG5' gene targets in MCF-7 human breast
adenocarcinoma cells using a DNA ISH assay with a Rhod-tyramide
signaling conjugate and a DABSYL-tyramide signaling conjugate.
[0058] FIG. 32 is a photomicrograph obtained using a multiplexed
DNA ISH assay showing rearrangement of the ERG gene in VCaP
prostate cancer epithelial cells.
[0059] FIG. 33 is a photomicrograph obtained using a multiplexed
DNA ISH assay illustrating rearrangement of the gene coding for
anaplastic lymphoma kinase in a CARPUS cell pellet.
[0060] FIG. 34 is a photomicrograph obtained using a multiplexed
DNA ISH assay illustrating rearrangement of the gene coding for
anaplastic lymphoma kinase in a section of lung adenocarcinoma.
[0061] FIGS. 35(A-C) are photomicrographs illustrating direct
detection of gene targets in Calu-3 cells using an mRNA ISH assay.
FIG. 35(A) shows detection of 18S RNA target using a Rhod-tyramide
conjugate. FIG. 35(B) shows detection of 18S RNA target using
direct deposition of a DABSYL-tyramide conjugate. FIG. 35(C)
illustrates a dual assay using the DABSYL-tyramide conjugate and
the Rhod-tyramide conjugate.
[0062] FIG. 36 is a photomicrograph illustrating detecting,
directly, HER2 and P53 proteins in Calu-3 cells using a multiplexed
IHC assay. HER2 is detected by direct deposition of DABSYL-tyramide
conjugate. P53 is detected by direct deposition of
Rhodamine-tyramide conjugate.
DETAILED DESCRIPTION
I. Definitions and Abbreviations
[0063] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes VII, published by
Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers
(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN
0471186341); and other similar references.
[0064] As used herein, the singular terms "a," "an," and "the"
include plural referents unless context clearly indicates
otherwise. Similarly, the word "or" is intended to include "and"
unless the context clearly indicates otherwise. The term "includes"
is defined inclusively, such that "includes A or B" means including
A, B, or A and B. It is further to be understood that all
nucleotide sizes or amino acid sizes, and all molecular weight or
molecular mass values, given for nucleic acids or polypeptides or
other compounds are approximate, and are provided for description.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present disclosure, suitable methods and materials are described
below.
[0065] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
explanations of terms, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
[0066] Disclosed herein are one or more generic chemical formulas.
For the general formulas provided herein, if no substituent is
indicated, a person of ordinary skill in the art will appreciate
that the substituent is hydrogen. A bond that is not connected to
an atom, but is shown, for example, extending to the interior of a
ring system, indicates that the position of such substituent is
variable. A curved line drawn through a bond indicates that some
additional structure is bonded to that position, typically a linker
or the functional group or moiety used to couple two moieties
together (e.g., a chromophore and a tyramide or tyramide
derivative). Moreover, if no stereochemistry is indicated for
compounds having one or more chiral centers, all enantiomers and
diasteromers are included. Similarly, for a recitation of aliphatic
or alkyl groups, all structural isomers thereof also are included.
Unless otherwise stated, R groups (e.g., R.sup.1-R.sup.24) in the
general formulas provided below independently are selected from:
hydrogen; acyl; aldehyde; alkoxy; aliphatic, particularly lower
aliphatic (e.g., C.sub.1-10alkyl, C.sub.1-10alkylene,
C.sub.1-10alkyne); substituted aliphatic; heteroaliphatic (e.g.,
organic chains having heteroatoms, such as oxygen, nitrogen,
sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms,
and even more typically lower alkyl having 10 or fewer atoms, such
as methyl, ethyl, propyl, isopropyl, and butyl); substituted alkyl,
such as alkyl halide (e.g., --CX.sub.3 where X is a halide, and
combinations thereof, either in the chain or bonded thereto);
oxime; oxime ether (e.g., methoxyimine, CH.sub.3--O--N.dbd.);
alcohols (i.e., aliphatic or alkyl hydroxyl, particularly lower
alkyl hydroxyl); amido; amino; amino acid; aryl; alkyl aryl, such
as benzyl; carbohydrates; monosaccharides, such as glucose and
fructose; disaccharides, such as sucrose and lactose;
oligosaccharides; polysaccharides; carbonyl; carboxyl; carboxylate
(including salts thereof, such as Group I metal or ammonium ion
carboxylates); cyclic; cyano (--CN); ester, such as alkyl ester;
ether; exomethylene; halogen; heteroaryl; heterocyclic; hydroxyl;
hydroxylamine; keto, such as aliphatic ketones; nitro; sulfhydryl;
sulfonyl; sulfoxide; exomethylene; and combinations thereof.
[0067] "Absorbance" or "Absorption" refers to the logarithmic ratio
of the radiation incident upon a material (P.sub.0), to the
radiation transmitted through a material (P). The absorbance A of a
material varies with the light path length through it (z) according
to Equation 1.
A = log P 0 P = - ( log T ) = lc Equation 1 ##EQU00001##
P.sub.0 and P are the incident and transmitted light intensities, T
is the optical transmission, and is the molar extinction
coefficient (M.sup.-1 cm.sup.-1), l is the length or depth of
illuminated area (cm), and c is the concentration of the absorbing
molecule.
[0068] "Amplification" refers to the act or result of making a
signal stronger.
[0069] "Amplifying conjugate" refers to a molecule comprising a
latent reactive species coupled to a hapten, such as, for example,
a hapten-tyramide conjugate. The amplifying conjugate may serve as
a member of a specific binding pair, such as, for example, an
anti-hapten antibody specifically binding to the hapten. The
amplification aspect relates to the latent reactive species being
enzymatically converted to a reactive species so that a single
enzyme can generate a multiplicity of reactive species. Reference
is made to U.S. Pat. No. 7,695,929, which is hereby incorporated by
reference, in its entirety.
[0070] "Antibody," occasionally abbreviated "Ab", refers to
immunoglobulins or immunoglobulin-like molecules (including by way
of example and without limitation, IgA, IgD, IgE, IgG and IgM,
combinations thereof, and similar molecules produced during an
immune response in any vertebrate, (e.g., in mammals such as
humans, goats, rabbits and mice) and antibody fragments that
specifically bind to a molecule of interest (or a group of highly
similar molecules of interest) to the substantial exclusion of
binding to other molecules (for example, antibodies and antibody
fragments that have a binding constant for the molecule of interest
that is at least 103 M-1 greater, at least 104 M-1 greater or at
least 105 M-1 greater than a binding constant for other molecules
in a biological sample. Antibody further refers to a polypeptide
ligand comprising at least a light chain or heavy chain
immunoglobulin variable region which specifically recognizes and
binds an epitope of an antigen. Antibodies may be composed of a
heavy and a light chain, each of which has a variable region,
termed the variable heavy (VH) region and the variable light (VL)
region. Together, the VH region and the VL region are responsible
for binding the antigen recognized by the antibody. The term
antibody also includes intact immunoglobulins and the variants and
portions of them well known in the art. Antibody fragments include
proteolytic antibody fragments [such as F(ab')2 fragments, Fab'
fragments, Fab'-SH fragments and Fab fragments as are known in the
art], recombinant antibody fragments (such as sFv fragments, dsFv
fragments, bispecific sFv fragments, bispecific dsFv fragments,
F(ab)'2 fragments, single chain Fv proteins ("scFv"), disulfide
stabilized Fv proteins ("dsFv"), diabodies, and triabodies (as are
known in the art), and camelid antibodies (see, for example, U.S.
Pat. Nos. 6,015,695; 6,005,079, 5,874,541; 5,840,526; 5,800,988;
and 5,759,808).
[0071] The term "antibody" includes monoclonal antibody which are
characterized by being produced by a single clone of B lymphocytes
or by a cell into which the light and heavy chain genes of a single
antibody have been transfected. Monoclonal antibodies are produced
by methods known to those of skill in the art. Monoclonal
antibodies include humanized monoclonal antibodies.
[0072] "Antigen" refers to a compound, composition, or substance
that may be specifically bound by the products of specific humoral
or cellular immunity, such as an antibody molecule or T-cell
receptor. Antigens can be any type of molecule including, for
example, haptens, simple intermediary metabolites, sugars (e.g.,
oligosaccharides), lipids, and hormones as well as macromolecules
such as complex carbohydrates (e.g., polysaccharides),
phospholipids, nucleic acids and proteins.
[0073] "Chromophore" refers to a molecule or a part of a molecule
responsible for its color. Color arises when a molecule absorbs
certain wavelengths of visible light and transmits or reflects
others. A molecule having an energy difference between two
different molecular orbitals falling within the range of the
visible spectrum may absorb visible light and thus be aptly
characterized as a chromophore. Visible light incident on a
chromophore may be absorbed thus exciting an electron from a ground
state molecular orbital into an excited state molecular
orbital.
[0074] "Conjugate" refers to two or more molecules that are
covalently linked into a larger construct. In some embodiments, a
conjugate includes one or more biomolecules (such as peptides,
nucleic acids, proteins, enzymes, sugars, polysaccharides, lipids,
glycoproteins, and lipoproteins) covalently linked to one or more
other molecules, such as one or more other biomolecules. In other
embodiments, a conjugate includes one or more specific-binding
molecules (such as antibodies and nucleic acid sequences)
covalently linked to one or more detectable labels (haptens,
enzymes and combinations thereof). In other embodiments, a
conjugate includes one or more latent reactive moieties covalently
linked to detectable labels (haptens, chromophore moieties,
fluorescent moieties).
[0075] "Conjugating," "joining," "bonding," "coupling" or "linking"
are used synonymously to mean joining a first atom or molecule to
another atom or molecule to make a larger molecule either directly
or indirectly.
[0076] "DABSYL" refers to
4-(dimethylamino)azobenzene-4'-sulfonamide, a yellow-orange
chromophore.
[0077] "Derivative" refers to a compound that is derived from a
similar compound by replacing one atom or group of atoms with
another atom or group of atoms.
[0078] "Enhanc(e/er/ement/ing)" An enhancer or enhancing reagent is
any compound or any combination of compounds sufficient to increase
the catalytic activity of an enzyme, as compared to the enzyme
activity without such compound(s). Enhancer(s) or enhancing
reagent(s) can also be defined as a compound or combination of
compounds that increase or accelerate the rate of binding an
activated conjugate to a receptor site. Enhanc(e/ement/ing) is a
process by which the catalytic activity of an enzyme is increased
by an enhancer, as compared to a process that does not include such
an enhancer. Enhanc(e/ement/ing) can also be defined as increasing
or accelerating the rate of binding of an activated conjugate to a
receptor site. Enhanc(e/ement/ing) can be measured visually, such
as by scoring by a pathologist. In particular embodiments, scores
range from greater than 0 to greater than 4, with the higher number
indicating better visual detection. More typically, scores range
from greater than 0 to about 4++, such as 1, 1.5, 2, 2.5, 3, 3.5,
3.75, 4, 4+, and 4++. In addition, enhanc(e/ement/ing) can be
measured by determining the apparent V.sub.max of an enzyme. In
particular embodiments, the term encompasses apparent V.sub.max
values (measured as optical density/minute) ranging from greater
than 0 mOD/min to about 400 mOD/min, such as about 15 mOD/min, 18
mOD/min, about 20 mOD/min, about 40 mOD/min, about 60 mOD/min,
about 80 mOD/min, about 100 mOD/min, about 120 mOD/min, about 140
mOD/min, about 160 mOD/min, about 200 mOD/min, about 250 mOD/min,
about 300 mOD/min, about 350 mOD/min, and about 400 mOD/min. More
typically, the Vmax ranges from greater than 0 mOD/min to about 160
mOD/min, such as about 20 mOD/min, about 40 mOD/min, about 60
mOD/min, about 80 mOD/min, about 100 mOD/min, about 120 mOD/min,
about 140 mOD/min, and about 160 mOD/min. In addition, enhancement
can occur using any concentration of an enhancer greater than 0 mM.
Reference is made to US Pat. Publ. No. 2012/0171668, which is
hereby incorporated by reference in its entirety, for disclosure
related to enhancers useful within the present disclosure.
[0079] "Epitope" refers to an antigenic determinant. These are
particular chemical groups or contiguous or non-contiguous peptide
sequences on a molecule that are antigenic, that is, that elicit a
specific immune response. An antibody binds a particular antigenic
epitope.
[0080] "Functional group" refers to a specific group of atoms
within a molecule that is responsible for the characteristic
chemical reactions of the molecule. Exemplary functional groups
include, without limitation, alkane, alkene, alkyne, arene, halo
(fluoro, chloro, bromo, iodo), epoxide, hydroxyl, carbonyl
(ketone), aldehyde, carbonate ester, carboxylate, ether, ester,
peroxy, hydroperoxy, carboxamide, amine (primary, secondary,
tertiary), ammonium, imide, azide, cyanate, isocyanate,
thiocyanate, nitrate, nitrite, nitrile, nitroalkane, nitroso,
pyridyl, phosphate, sulfonyl, sulfide, thiol (sulfhydryl), and
disulfide.
[0081] "FWHM" refers to the full width of an absorbance peak at the
half maximum absorbance.
[0082] "Hapten" refers to a molecule, typically a small molecule,
which can combine specifically with an antibody, but typically is
substantially incapable of being immunogenic on its own.
[0083] "Linker" refers to a molecule or group of atoms positioned
between two moieties. For example, a signaling conjugate may
include a chemical linker between the chromophore moiety and a
latent reactive moiety. Typically, linkers are bifunctional, i.e.,
the linker includes a functional group at each end, wherein the
functional groups are used to couple the linker to the two
moieties. The two functional groups may be the same, i.e., a
homobifunctional linker, or different, i.e., a heterobifunctional
linker.
[0084] "MG" refers to Malachite green.
[0085] "Moiety" refers to a fragment of a molecule, or a portion of
a conjugate.
[0086] "Molecule of interest" or "Target" each refers to a molecule
for which the presence, location and/or concentration is to be
determined. Examples of molecules of interest include proteins and
nucleic acid sequences.
[0087] "Multiplex, -ed, -ing" refers to detecting multiple targets
in a sample concurrently, substantially simultaneously, or
sequentially. Multiplexing can include identifying and/or
quantifying multiple distinct nucleic acids (e.g., DNA, RNA, mRNA,
miRNA) and polypeptides (e.g., proteins) both individually and in
any and all combinations.
[0088] "Proximal" refers to being situated at or near the reference
point. As used herein, proximal means within about 5000 nm, within
about 2500 nm, within about 1000 nm, within about 500 nm, within
about 250 nm, within about 100 nm, within about 50 nm, within about
10 nm, or within about 5 nm of the reference point.
[0089] "Reactive groups" refers to a variety of groups suitable for
coupling a first unit to a second unit as described herein. For
example, the reactive group might be an amine-reactive group, such
as an isothiocyanate, an isocyanate, an acyl azide, an NHS ester,
an acid chloride, such as sulfonyl chloride, aldehydes and
glyoxals, epoxides and oxiranes, carbonates, arylating agents,
imidoesters, carbodiimides, anhydrides, and combinations thereof.
Suitable thiol-reactive functional groups include haloacetyl and
alkyl halides, maleimides, aziridines, acryloyl derivatives,
arylating agents, thiol-disulfide exchange reagents, such as
pyridyl disulfides, TNB-thiol, and disulfide reductants, and
combinations thereof. Suitable carboxylate-reactive functional
groups include diazoalkanes, diazoacetyl compounds,
carbonyldiimidazole compounds, and carbodiimides. Suitable
hydroxyl-reactive functional groups include epoxides and oxiranes,
carbonyldiimidazole, N,N'-disuccinimidyl carbonates or
N-hydroxysuccinimidyl chloroformates, periodate oxidizing
compounds, enzymatic oxidation, alkyl halogens, and isocyanates.
Aldehyde and ketone-reactive functional groups include hydrazines,
Schiff bases, reductive amination products, Mannich condensation
products, and combinations thereof. Active hydrogen-reactive
compounds include diazonium derivatives, Mannich condensation
products, iodination reaction products, and combinations thereof.
Photoreactive chemical functional groups include aryl azides,
halogenated aryl azides, benzophonones, diazo compounds, diazirine
derivatives, and combinations thereof.
[0090] "Rhod" refers to Rhodamine, a chromophore.
[0091] "Sample" refers to a biological specimen containing genomic
DNA, RNA (including mRNA), protein, or combinations thereof,
obtained from a subject. Examples include, but are not limited to,
peripheral blood, urine, saliva, tissue biopsy, surgical specimen,
amniocentesis samples and autopsy material.
[0092] "Specific binding moiety" refers to a member of a
specific-binding pair. Specific binding pairs are pairs of
molecules that are characterized in that they bind each other to
the substantial exclusion of binding to other molecules (for
example, specific binding pairs can have a binding constant that is
at least 103 M.sup.-1 greater, 104 M.sup.-1 greater or 105 M.sup.-1
greater than a binding constant for either of the two members of
the binding pair with other molecules in a biological sample).
Particular examples of specific binding moieties include specific
binding proteins (for example, antibodies, lectins, avidins such as
streptavidins, and protein A), nucleic acid sequences, and
protein-nucleic acids. Specific binding moieties can also include
the molecules (or portions thereof) that are specifically bound by
such specific binding proteins. Exemplary specific binding moieties
include, but are not limited to, antibodies, nucleotides,
oligonucleotides, proteins, peptides, or amino acids.
[0093] "TAMRA" refers to Carboxytetramethylrhodamine, a pink
rhodamine chromophore.
[0094] "TMR" refers to Tetramethylrhodamine, a red rhodamine
chromophore.
[0095] "TSA" refers to tyramide signal amplification.
[0096] "TYR" refers to tyramine, tyramide, tyramine and/or tyramide
derivatives.
II. Methods for Detecting a Target in a Sample
[0097] Disclosed herein are embodiments of a method for using
disclosed exemplary conjugates for detecting one or more targets in
a biological sample. In particular disclosed embodiments, one or
more of the conjugates are used in standard assays, such as in situ
hybridization (ISH), immunocytochemical, and immunohistochemical
(IHC) detection schemes. In particular disclosed embodiments, any
one of these assays may be combined with signal amplification,
and/or the assays may concern multiplexing wherein multiple
different targets may be detected. Particular disclosed embodiments
may also include one or more enhancers. Embodiments of the method
also may be combined. For example, a method using an IHC detection
scheme may be combined with an ISH detection scheme. Exemplary
embodiments of the disclosed method may be used for determining
cell clonality (e.g., a cell expresses either one of two
biomarkers, but not both), predicting response of cancer patients
to cancer therapy (e.g., detecting predictive biomarkers to
determine whether a particular patient will respond to treatment),
simultaneous analysis of biomarker expression and internal control
gene expression to monitor assay performance and sample integrity,
and combinations thereof.
[0098] Methods may be used on a biological sample having a solid
phase, such as protein components of cells or cellular structures
that are immobilized on a substrate (e.g., a microscope slide). In
illustrative embodiments, the sample is a tissue or cytology
sample, such as a formalin-fixed paraffin embedded sample, mounted
on a glass microscope slide. In one embodiment, the method is
particularly for an automated slide staining instrument.
[0099] A person of ordinary skill in the art will appreciate that
numerous types of targets may be detected using the disclosed
method. In certain disclosed embodiments, the target may be a
particular nucleic acid sequence, a protein, or combinations
thereof. For example, the target may be a particular sequence of
RNA (e.g., mRNA, microRNA, and siRNA), DNA, and combinations
thereof. The sample may be suspected of including one or more
target molecules of interest. Target molecules can be on the
surface of cells and the cells can be in a suspension, or in a
tissue section. Target molecules can also be intracellular and
detected upon cell lysis or penetration of the cell by a probe. One
of ordinary skill in the art will appreciate that the method of
detecting target molecules in a sample will vary depending upon the
type of sample and probe being used. Methods of collecting and
preparing samples are known in the art.
[0100] Samples for use in the embodiments of the method and with
the composition disclosed herein, such as a tissue or other
biological sample, can be prepared using any method known in the
art by of one of ordinary skill. The samples can be obtained from a
subject for routine screening or from a subject that is suspected
of having a disorder, such as a genetic abnormality, infection, or
a neoplasia. The described embodiments of the disclosed method can
also be applied to samples that do not have genetic abnormalities,
diseases, disorders, etc., referred to as "normal" samples. Such
normal samples are useful, among other things, as controls for
comparison to other samples. The samples can be analyzed for many
different purposes. For example, the samples can be used in a
scientific study or for the diagnosis of a suspected malady, or as
prognostic indicators for treatment success, survival, etc. Samples
can include multiple targets that can be specifically bound by one
or more detection probes. Throughout this disclosure when reference
is made to a target protein, it is understood that the nucleic acid
sequences associated with that protein can also be used as a
target. In some examples, the target is a protein or nucleic acid
molecule from a pathogen, such as a virus, bacteria, or
intracellular parasite, such as from a viral genome. For example, a
target protein may be produced from a target nucleic acid sequence
associated with (e.g., correlated with, causally implicated in,
etc.) a disease.
[0101] In some embodiments, the disclosed method may be used to
detect microRNA (miRNA or miR). MicroRNAs are small, non-coding
RNAs that negatively regulate gene expression, such as by
translation repression. For example, miR-205 regulates epithelial
to mesenchymal transition (EMT), a process that facilitates tissue
remodeling during embryonic development. However, EMT also is an
early step in tumor metastasis. Down-regulation of microRNAs, such
as miR-205, may be an important step in tumor progression. For
instance, expression of miR-205 is down-regulated or lost in some
breast cancers. MiR-205 also can be used to stratify squamous cell
and non-small cell lung carcinomas (J. Clin Oncol., 2009,
27(12):2030-7). Other microRNAs have been found to modulate
angiogenic signaling cascades. Down-regulation of miR-126, for
instance, may exacerbate cancer progression through angiogenesis
and increased inflammation. Thus, microRNA expression levels may be
indicative of a disease state. For microRNA within the scope of the
present disclosure, reference is made to PCT Application No.
PCT/EP2012/073984, which is hereby incorporated by reference in its
entirety.
[0102] In a particular disclosed embodiment, the disclosed method
may be used to analyze clinical breast cancer FFPE tissue blocks
that have been characterized for HER2 gene copy number and Her2
protein expression using INFORM HER2 Dual ISH and IHC assays
(Ventana Medical Systems, Inc., "VMSI"), respectively. HER2 mRNA
expression levels relative to ACTB (.beta.-actin) can be determined
using qPCR according to known methods. Results of the gene copy,
protein expression, and qPCR analyses can be compared to results
obtained through mRNA-ISH detection of HER2 and ACTB mRNA using the
method disclosed herein to analyze FFPE samples. Further results
from this method are discussed subsequently herein.
[0103] In another embodiment, the disclosed method may be used to
identify monoclonal proliferation of certain types of cells. Cancer
results from uncontrolled growth of a cell population; this
population may arise from a single mutant parent cell and,
therefore, comprise a clonal population. An example of cancer
derived from a clonal population is B-cell non-Hodgkin lymphomas
(B-NHL) which arise from monoclonal proliferation of B cells.
Clonal expansion of a specific B cell population can be detected by
sole expression of either KAPPA or LAMBDA light chain mRNA and
protein as part of their B cell receptor antibody. Accordingly, one
embodiment of the method disclosed herein concerns identifying
monoclonal proliferation of B cells using chromogenic dual staining
of KAPPA and LAMBDA mRNA.
[0104] Uniform expression of either light chain by malignant B
cells enables differentiation of monoclonal B cell lymphomas from
polyclonal KAPPA and LAMBDA light chain expressing B cell
populations that result during the normal immune response.
Determining light chain mRNA expression patterns is complicated by
the copy number range of light chain mRNA and antibody protein
expressed by B cell neoplasms derived from a variety of B cell
stages (naive and memory cells: 10-100 copies per cell; plasma
cells: .about.100 thousand copies per cell).
Methods
[0105] In illustrative embodiments, a method of detecting a target
in a biological sample includes contacting the biological sample
with a detection probe, contacting the biological sample with a
labeling conjugate, and contacting the biological sample with a
signaling conjugate FIG. 1 is a flowchart providing the steps of
one exemplary embodiment of a method according to the present
disclosure. In particular, the method includes a step 1 of
contacting the sample with a detection probe(s). The step can
include either a single detection probe or a plurality of detection
probes specific to a plurality of different targets. A subsequent
step 2 includes contacting the sample with a labeling conjugate. A
further subsequent step 7 includes contacting the sample with a
signaling conjugate. Dashed lines to step 3, contacting sample with
an amplifying conjugate, and step 5, contacting sample with a
secondary labeling conjugate, represent that these steps are
optional. Dashed lines to step 10 of contacting sample with an
enzyme inhibitor indicates that an optional loop can be used to
detect multiple targets according to a multiplexed approach. In
particular disclosed embodiments, one or more steps may be used
wherein an enzyme inhibitor is added to the biological sample. For
example, in embodiments wherein two or more signaling conjugates
are added to the sample, an enzyme inhibitor (e.g., a peroxidase
inhibitor) can be added in order to prevent any enzymatic activity
after one signaling conjugate has been covalently deposited and
before a second, different signaling conjugate is added.
[0106] In illustrative embodiments, detecting targets within the
sample includes contacting the biological sample with a first
amplifying conjugate that associates with the first labeling
conjugate. For example, the amplifying conjugate may be covalently
deposited proximally to or directly on the first labeling
conjugate. The first amplifying conjugate may be followed by
contacting the biological sample with a secondary labeling
conjugate. Illustratively, the amplification of signal using
amplifying conjugates enhances the deposition of signaling
conjugate. The enhanced deposition of signaling conjugate enables
easier visual identification of the chromogenic signal, that is,
the amplification makes the color darker and easier to see. For low
expressing targets, this amplification may result in the signal
becoming sufficiently dark to be visible, whereas without
amplification, the target would not be apparent. In embodiments
wherein an amplification step is used, the biological sample may
first be contacted with the detection probe and labeling conjugate
and then subsequently contacted with one or more amplifying
conjugates. In particular disclosed embodiments, the amplifying
conjugate comprises a latent reactive moiety coupled with a
detectable label. For example, a tyramine moiety (or a derivative
thereof) may be coupled with a hapten, directly or indirectly, such
as with a linker. The amplifying conjugate may be covalently
deposited by the enzyme of the enzyme conjugate, typically using
conditions described herein or are known to a person of ordinary
skill in the art that are suitable for allowing the enzyme to
perform its desired function. The amplifying conjugate is then
covalently deposited on or proximal to the target.
[0107] Conditions suitable for introducing the signaling conjugates
with the biological sample are used, and typically include
providing a reaction buffer or solution that comprises a peroxide
(e.g., hydrogen peroxide), and has a salt concentration and pH
suitable for allowing or facilitating the enzyme to perform its
desired function. In particular disclosed embodiments, this step of
the method is performed at temperatures ranging from about
35.degree. C. to about 40.degree. C. These conditions allow the
enzyme and peroxide to react and promote radical formation on the
latent reactive moiety of the signaling conjugate. The latent
reactive moiety, and therefore the signaling conjugate as a whole,
will deposit covalently on the biological sample, particularly at
one or more tyrosine residues proximal to the immobilized enzyme
conjugate, tyrosine residues of the enzyme portion of the enzyme
conjugate, and/or tyrosine residues of the antibody portion of the
enzyme conjugate. The biological sample is then illuminated with
light and the target may be detected through absorbance of the
light produced by the chromogenic moiety of the signaling
conjugate.
[0108] Depending on the level of multiplexing, the optional loop
can be repeated one, two, three, four, five, six, seven, eight, or
more times depending on the number of targets that are to be
detected in the sample. During subsequent detection steps, the
labeling conjugate can be the same or different depending on the
blocking reagents used. An example of different labeling conjugates
would be a first enzyme-anti-hapten antibody conjugate and a second
enzyme-anti-hapten antibody conjugate, wherein the first
anti-hapten antibody and the second anti-hapten antibody are
specific to different haptens. According to another example, the
difference could involve different anti-species antibodies, wherein
the targets were detected using primary antibodies derived from
different species. During subsequent detections, the signaling
conjugate used for each target would typically be different. For
example, the different targets could be detected as different
colors.
[0109] While step 1 of contacting the sample with detection
probe(s) is shown in FIG. 1 to be the simultaneous detection of
multiple targets during one step, multiplexing may also be
performed sequentially. A sequential method would include adding a
first detection probe followed by carrying out the various
subsequent method steps (i.e., steps 2, 7, optionally 3, and 5). A
second detection probe may then be added after the first signaling
conjugate has been covalently deposited on or proximal to the first
target, thereby providing the ability to detect a second target.
This process may then be iteratively repeated using a different
signaling conjugate comprising a chromophore moiety that differs
from the others deposited.
[0110] The method also comprises a step 9 of illuminating sample
with light and a detecting target(s) step 11. The signal produced
by the signaling conjugate is detected, thereby providing the
ability to detect a particular target. In particular disclosed
embodiments, the signal produced by the signaling conjugate may be
fluorescent, chromogenic, or combinations thereof. Exemplary
embodiments concern detecting a chromogenic signal. The signal may
be detected using suitable methods known to those of ordinary skill
in the art, such as chromogenic detection methods, fluorogenic
detection methods, and combinations thereof. For example, the
signal may be detected using bright-field detection techniques or
dark-field detection techniques.
[0111] FIGS. 2(A-B) are schematic diagrams of two embodiments of
signaling conjugates. FIG. 2(A) illustrates a signaling conjugate
12 comprising a latent reactive moiety 4 and a chromophore moiety
6. FIG. 2(B) illustrates an alternative signaling conjugate 14,
comprising chromophore moiety 6, latent reactive moiety 4, and
further comprising a linker 8.
[0112] FIGS. 3(A-F) are schematic diagrams illustrating an
embodiment of a method for detecting a target 17 on a sample 16.
FIG. 3(A) shows a detection probe 18, which is shown illustratively
to be a nucleic acid molecule with a hapten 19, binding to target
17, which, in this case, would be a nucleic acid target. FIG. 3(B)
shows a labeling conjugate 20 binding to detection probe 18.
Labeling conjugate 20 is depicted as an anti-hapten antibody
specific to hapten 19 conjugated to two enzymes, which are depicted
as circles containing an "E." While illustrated as being a
conjugate of one antibody and two enzyme molecules, the number of
enzymes per antibody can be altered and optimized for particular
applications by a person of ordinary skill in the art. In
particular, the number of enzymes could be modified from about 1 to
about 10, depending on various factors, including the size of the
antibody and the size of the enzymes. FIG. 3(C) shows signaling
conjugate 12 being enzymatically deposited onto sample 16. In
particular, enzymes "E," part of labeling conjugate 20, catalyze
conversion of the first latent reactive moiety of signaling
conjugate 12 into a first reactive species 13. This catalysis is
represented by a first large arrow 21 directing signaling conjugate
12 to enzymes "E" and a second large arrow 22 emanating from
enzymes "E" to reactive species 13, which is made of chromophore
moiety 6 and a reactive moiety, which is represented by the dot
replacing the arrow as shown on signaling conjugate 6. Reactive
species 13 covalently binds to the biological sample proximally to
or directly on the first target, to form a covalently bound
chromophore 15. FIG. 3(D) shows an alternative embodiment in which
an antibody-based detection probe 28 is used to detect a protein
target 27. FIG. 3(D) is included to show that the steps of
detecting either nucleic acid target 17 and/or protein target 27
are analogous except that detection probe 28 is represented as an
antibody as opposed a nucleic acid (e.g., detection probe 18).
Detection probe 28 is shown as not being haptenated, implying that
labeling conjugate 30 is an anti-species antibody conjugated to
enzymes "E." However, in alternative embodiments, detection probe
28 could be haptenated and labeling conjugate 30 could include an
anti-hapten antibody.
[0113] FIG. 3(E) shows an approach to detecting the target that
uses an amplifying conjugate 42. In particular, amplifying
conjugate 42 is enzymatically deposited onto a sample 36. In
particular, enzymes "E," part of labeling conjugate 40, catalyze
conversion of the first latent reactive moiety of amplifying
conjugate 42 into a first reactive species 43. This catalysis is
represented by a first large arrow 31 directing amplifying
conjugate 42 to enzymes "E" and a second large arrow 32 emanating
from enzymes "E" to reactive species 43, which is made of a hapten
(shown as a cross) and a reactive moiety, which is represented by
the dot replacing the arrow as shown on amplifying conjugate 42.
Reactive species 43 covalently binds to the biological sample
proximally to or directly on the first target, to form a covalently
bound hapten 45. The scheme depicted in FIG. 3(E) is shown here to
make apparent the similarities between the scheme of FIG. 3(E) and
the scheme of FIG. 3(C). In particular, the schemes are nearly
identical except for the substitution of the chromophore moiety of
signaling conjugate 12 for the hapten of amplifying conjugate 42.
FIG. 3(F) shows that the amplifying conjugate bound to the sample
(covalently bound hapten 45 as seen in FIG. 3(E)) can be labeled
with a secondary labeling conjugate 41. While not shown, the scheme
shown in FIG. 3(C) can then be used to form a covalently bound
chromophore, as deposition of amplifying conjugate 42 onto the
sample provides a larger number of enzyme molecules (i.e., enzymes
from labeling conjugate 40 and secondary labeling conjugate 41 are
shown proximally to the target in FIG. 3(F)).
[0114] In particular disclosed embodiments, the signaling conjugate
is detected using bright-field detection methods. An overview of
this process is illustrated in FIGS. 4(A-B). FIG. 4(A) is a
schematic of a cross-sectional view of sample 16 including an upper
surface 48 and a lower surface 49 in which a plurality of the
signaling conjugates 12 are located proximally to a target (T); the
sample is shown having a first arrow 46 representing incident
radiation directed towards upper surface 48 and a second arrow 47
representing transmitted radiation emanating from lower surface 49.
FIG. 4(B) is a graph depicting the relationship between power of
incident radiation (P.sub.0) across sample 16 shown in FIG. 4(A)
and power of transmitted radiation (P) through the sample, the
y-axis being radiation power and the x-axis being linear distance
across the sample. FIGS. 4(A-B) portray how a target could be
visualized using signaling conjugate 12. Equation 1 provides the
mathematical relationship between the power of the incident and
transmitted radiation.
[0115] The disclosed method steps may be carried out in any
suitable order, and are not limited to those described herein. In
particular disclosed embodiments, the method may comprise steps
wherein the labeling conjugates are added to the biological sample,
followed by the signaling conjugate. In other disclosed
embodiments, the method may comprise steps wherein the labeling
conjugates are added to the biological sample, followed by an
amplifying conjugate, an additional enzyme conjugate, and the
signaling conjugate. The conjugates disclosed herein may be added
simultaneously, or sequentially. The conjugates may be added in
separate solutions or as compositions comprising two or more
conjugates. Also, each class of conjugates used in the disclosed
method may comprise the same or different conjugate components. For
example, when multiple signaling conjugates are added to the
sample, the conjugates may comprise the same or different
chromogenic moieties and/or latent reactive moieties. Solely by way
of example, one signaling conjugate may comprise a coumarin
chromophore coupled to a tyramine moiety and another signaling
conjugate may comprise a rhodamine chromophore coupled to a
tyramine derivative moiety. The number of signaling conjugates
suitable for use in the disclosed multiplexing assay may range from
one to at least six, or more typically from two to five. In
particular disclosed embodiments, the method is used to detect from
three to five different targets using from three to five different
signaling conjugates. Multiple targets may be detected in a single
assay using the method disclosed herein. In another embodiment, any
one or more of the steps disclosed herein for the method are
performed by an automated slide staining instrument.
Chromogenic vs. Fluorescence
[0116] Historically, break-apart analysis has been done using FISH;
however, the present disclosure provides a three-color, break-apart
assay using chromogenic ISH. The differences between chromogenic
detection and fluorescence detection are pictorially illustrated in
FIGS. 5(A) and 5(B). FIG. 5(A) shows a red chromogen example 51, a
blue chromogen example 53, and a red and blue multiplexed chromogen
example 52. When chromogens are exposed to light (i.e., exposed to
light having an incident power of P.sub.0), which typically is
white light, the chromogens absorb various wavelengths. The
transmitted light will have a particular power (FIG. 5(A),
indicated as P.sub.1, P.sub.2, and P.sub.3) depending on the
absorbance of the chromogen and the amount of chromogen present.
The better detection event results in more chromogen being
deposited, which absorbs more light and makes the observed signal
smaller. Even for colored chromogens, a reduction of the
transmitted light will eventually cause the chromogen to appear
black as no light is transmitted. Multiplexing exacerbates this
effect, as shown in red and blue multiplexed chromogen example 52.
When a traditional red chromogen and a blue chromogen overlap in
space, the absorbance is broad and the detection event appears
blackish and dark, as illustrated by the P.sub.3 signal being
smaller than P.sub.1 and P.sub.2. Essentially, chromogenic
detection with overlapping signals will result in a subtractive
effect. This is in contrast to fluorescence which is illustrated in
FIG. 5(B). With reference to FIG. 5(B), a purple fluor example 61,
a green fluor example 63, and a purple and green multiplexed fluor
example 62 are shown. The excitation light (shown as .lamda..sub.ex
in the figure) can be the same across the three examples and
example 61 exhibits .lamda..sub.f1 (purple fluorescence), example
63 exhibits .lamda..sub.f2 (green fluorescence), and example 62
exhibits .lamda..sub.f1 (purple fluorescence) and .lamda..sub.f2
(green fluorescence). As more fluor is deposited on the sample a
stronger fluorescence signal is generated. Similarly, in a
multiplexed scenario, there is an additive affect for the
fluorophores, whereas a subtractive effect occurs with the
chromophores. This subtractive versus additive feature
significantly compounds the difficulty of multiplexing using
chromogens. As such, multiplexing with traditional chromogens has
not been broadly accepted. The current disclosure provides
signaling conjugates with narrow wavelength absorbance bands, which
enable combinations of colors heretofore not possible. As such, the
present disclosure provides unprecedented chromogenic multiplexing
despite the inherent disadvantages that chromogenic multiplexing
has when compared to fluorescent multiplexing.
Detecting & Illuminating
[0117] The signaling conjugate is configured to provide a variety
of characteristics that facilitate providing a detectable signal.
In particular disclosed embodiments, the signaling conjugate
comprises an appropriate chromophore moiety to provide a
bright-field signal. For example, the chromophore disclosed herein
may be selected to produce an optical signal suitable for detecting
the target disclosed herein. In particular disclosed embodiments,
the chromophore has optical properties, such as those discussed
below, that allow the signaling conjugate to be configured to
provide the desired signal.
[0118] When light (i.e., visible electromagnetic radiation) passes
through or is reflected by a colored substance, a characteristic
portion of the spectral wavelength distribution is absorbed. The
absorption of this characteristic portion imparts on the object a
complementary color corresponding to the remaining light. FIGS.
6(A) and 6(B) show a color wheel (FIG. 6(A)) that illustrates the
relationship between an observed color and absorbed radiation. The
color wheel includes a number of pie pieces representing colors (R)
Red, (O) Orange, (Y) Yellow, (G) Green, (B) Blue, (I) Indigo, and
(V) Violet. Each color is shown as a separate pie piece from the
next color with a series of lines terminating at numbers outside
the wheel. These numbers designate the wavelength of light in
nanometers (nm) of those wavelengths traditionally considered to be
the transition points between colors. FIG. 6(B) shows the same
distribution of colors on a linear graph having the wavelength of
light on the x-axis. That is, the region from 620 to 800 nm is
shown colored red as those wavelengths are "red" light wavelengths.
Typically, colors are perceived preferentially and some colors are
perceived only for a very narrow span of wavelengths. For example,
a laser having emission anywhere from 490 nm to 560 nm would be
perceived as green (a 70 nm span). To be perceived as orange, the
laser would have to emit light in the range of 580 nm and 620 nm
(40 nm). The graph is provided for representation only, and a
person of ordinary skill in the art appreciates that the
electromagnetic spectrum is continuous in nature and not discrete
as shown. However, the color classifications delineated herein
facilitate an understanding of the technology, as claimed
herein.
[0119] As described herein, when a substance absorbs a particular
wavelength, the substance appears to be the complementary color,
that color corresponding to the remaining light. The color wheel of
FIG. 6(A) shows complementary colors diametrically opposed to each
other. According to the color wheel, absorption of 420-430 nm light
imparts a yellow color to the substance (425 nm is opposite to that
portion of the wheel that is yellow). Similarly, absorption of
light in the range of 500-520 nm imparts a red color to the
substance since the red pie area is opposite the numerical range of
500-520 nm. Green is unique in that absorption close to 400 nm as
well as absorption near 800 nm can impart a green color to the
substance.
[0120] The concept that the absorption of light at wavelengths
between 420-430 nm results in the substance appearing yellow is an
over-simplification of many of the absorption phenomena described
herein. In particular, the absorption spectral profile has a strong
influence on the observed color. For example, a substance that is
black absorbs strongly throughout the range of 420-430 nm, yet the
black substance does not appear yellow. In this case, the black
absorber will absorb light across the entire visible spectrum,
including 420-430 nm. Thus, while absorption of light at a
particular wavelength is important, absorption characteristics
across the visible spectra (i.e., spectral absorption) also are
important.
[0121] Spectral absorption can be characterized according to
several measurable parameters. The wavelength at which the maximum
fraction of light is absorbed by a substance is referred to as
.lamda..sub.max. Because this wavelength is absorbed to the
greatest extent, it is typically referred to as the absorbance
wavelength. FIG. 7(A) is an absorption spectrum of a particular
signaling conjugate, and FIG. 7(B) illustrates a photomicrograph of
a protein stained using the signaling conjugate producing the
absorption spectrum of FIG. 7(A). FIG. 7(A) includes a first arrow
(70) illustrating the magnitude of the maximum absorbance. A second
arrow (71) shows the magnitude of half of the maximum. A third
arrow (72) shows the width of the peak at half of the maximum
absorbance. For this exemplary signaling conjugate, .lamda..sub.max
is 552 nm and the full width of the peak at the half maximum
absorbance (e.g., FWHM) is approximately 40 nm. While
.lamda..sub.max designates the wavelength of maximum absorption,
the FWHM designates the breadth of the spectral absorbance. Both
factors are important in describing the chromophore's color because
broad absorption spectra do not appear to have a color as would be
expected from their .lamda..sub.max. Rather, they appear to be
brown, black, or gray. Referring to FIG. 7(B), deposition of the
signaling conjugate is clearly evident in those locations that
would be expected for positive staining (HER2 (4B5) IHC in Calu-3
xenografts). Referring back to the color wheel (FIG. 6(A)), a
.lamda..sub.max of 552 nm should correspond to a complementary
color of red or red-violet. This matches the color observed in the
tissue sample shown in FIG. 7(B) (note that the sample further
includes hematoxylin nuclear counterstaining that is blue). Because
the counterstain is confined to the nucleus, it does not appear to
interfere or substantially affect the cell-membrane based HER2
staining.
[0122] Preferred chromophores have strong absorbance
characteristics. In some embodiments, the chromophores are
non-fluorescent or weakly fluorescent. By virtue of its absorbance
characteristics, a chromophore is a species capable of absorbing
visible light. A preferred chromophore is capable of absorbing a
sufficient quantity of visible light with sufficient wavelength
specificity so that the chromophore can be visualized using
bright-field illumination. In another embodiment, the chromophore
has an average molar absorptivity of greater than about 5,000
M.sup.-1 cm.sup.-1 to about 90,000 M.sup.-1 cm.sup.-1. For example,
the average molar absorptivity may be greater than about 5,000
M.sup.-1 cm.sup.-1, greater than about 10,000 M.sup.-1 cm.sup.-1,
greater than about 20,000 M.sup.-1 cm.sup.-1, greater than about
40,000 M.sup.-1 cm.sup.-1, or greater than about 80,000 M.sup.-1
cm.sup.-1. Strong absorbance characteristics are preferred to
increase the signal, or color, provided by the chromophore.
[0123] The deposition of signaling conjugates in the vicinity of
the target creates absorption of the incident light. Because the
absorption occurs non-uniformly across the sample, the location of
the target, within the sample, can be identified.
[0124] Certain aspects, or all, of the disclosed embodiments can be
automated, and facilitated by computer analysis and/or image
analysis system. In some applications, precise color ratios are
measured. In some embodiments, light microscopy is utilized for
image analysis. Certain disclosed embodiments involve acquiring
digital images, which can be done by coupling a digital camera to a
microscope. Digital images obtained of stained samples are analyzed
using image analysis software. Color can be measured in several
different ways. For example, color can be measured as red, blue,
and green values; hue, saturation, and intensity values; and/or by
measuring a specific wavelength or range of wavelengths using a
spectral imaging camera.
[0125] Illustrative embodiments involve using bright-field imaging
with the signaling conjugates. White light in the visible spectrum
is transmitted through the chromophore moiety. The chromophore
absorbs light of certain wavelengths and transmits other
wavelengths. This changes the light from white to colored depending
on the specific wavelengths of light transmitted.
[0126] The narrow spectral absorbances enable chromogenic
multiplexing at a level beyond the capability of traditional
chromogens. For example, traditional chromogens are somewhat
routinely duplexed (e.g., Fast Red and Fast Blue, Fast Red and
Black (silver), Fast Red and DAB). However, triplexed or
three-color applications are atypical. In illustrative embodiments,
the method includes detecting from two to about six different
targets, such as three to six, or three to five, using different
signaling conjugates or combinations thereof. In one embodiment,
illuminating the biological sample with light comprises
illuminating the biological sample with a spectrally narrow light
source, the spectrally narrow light source having a spectral
emission with a second full-width half-max (FWHM) of between about
30 nm and about 250 nm, between about 30 nm and about 150 nm,
between about 30 nm and about 100 nm, or between about 20 run and
about 60 nm. In another embodiment, illuminating the biological
sample with light includes illuminating the biological sample with
an LED light source. In another embodiment, illuminating the
biological sample with light includes illuminating the biological
sample with a filtered light source.
[0127] The samples also can be evaluated qualitatively and
semi-quantitatively. Qualitative assessment includes assessing the
staining intensity, identifying the positively-staining cells and
the intracellular compartments involved in staining, and evaluating
the overall sample or slide quality. Separate evaluations are
performed on the test samples and this analysis can include a
comparison to known average values to determine if the samples
represent an abnormal state.
[0128] In one embodiment, the signaling conjugate is covalently
deposited proximally to the target at a concentration suitable for
producing a detectable signal, such as at a concentration greater
than about 1.times.10.sup.11 molecules per cm.sup.2.mu.m to at
least about 1.times.10.sup.16 molecules per cm.sup.2.mu.m of the
biological sample. One of ordinary skill in the art could calculate
the number of molecules per cm.sup.2.mu.m of the biological sample
by using Equation 1 and absorbance measurements across the sample,
taking care to subtract the absorbance corresponding to the sample.
In one embodiment of the disclosed method, such as a multiplexing
method, detecting one signal includes detecting an absorbance of 5%
or more of incident light compared to a background, and detecting a
different, separate signal includes detecting an absorbance of 5%
or more of incident light compared to the background. In another
embodiment, detecting one signal includes detecting an absorbance
of 20% or more of incident light compared to a background, and
detecting a different, separate signal includes detecting an
absorbance of 20% or more of incident light compared to the
background.
[0129] In one embodiment, the first target and the second target
are genetic nucleic acids. Detecting the first target through
absorbance of the light by the first signaling conjugate includes
detecting a first colored signal selected from red, orange, yellow,
green, indigo, or violet. The first colored signal is associated
with spectral absorbance associated with the first chromogenic
moiety of the first signaling conjugate. Detecting the second
target through absorbance of the light by the second signaling
conjugate includes detecting a second colored signal selected from
red, orange, yellow, green, indigo, or violet. The second colored
signal is associated with spectral absorbance associated with the
second chromogenic moiety of the second signaling conjugate. An
overlap in proximity through absorbance of the light by the first
signaling conjugate overlapping in proximity with the second
signaling conjugate so that a third colored signal can be detected
that is associated with overlapping spectral absorbance of the
first spectral absorbance and the second spectral absorbance.
According to one example, this third colored signals a normal
genetic arrangement and the first and second colors signal a
genetic rearrangement or translocation.
ISH Three-Color Break Apart Probe
[0130] While providing a range of new colors for the recognition of
targets within biological samples is useful alone, the presently
disclosed signaling conjugates are particularly useful in
multiplexed assays, as well as assays using translocation probes.
FIG. 8(A) is a photomicrograph of a dual stain of two gene probes
on section of lung tissue testing for ALK rearrangements associated
with non-small cell lung cancer. FIG. 8(B) illustrates UV-Vis
spectra of fast red and fast blue in ethyl acetate solutions. The
3' probe was detected using fast red and the 5' probe was detected
using fast blue. FIGS. 9(A) and 9(B) illustrate the traces of FIG.
8(B) separately. FIG. 8(B) shows that fast red and fast blue have
broad and well-defined spectral absorption characteristics. Fast
red shows strong absorption between about 475 nm and about 560 nm.
Comparing this range to the color wheel, the expected color
corresponding to the spectral absorption characteristic would be
either red or orange. The range of absorption is so large it
essentially covers all of those wavelengths one would expect to
result in a red or an orange color. Fast blue exhibits strong
absorption between about 525 nm and about 625 nm, a range even
broader than fast red. Again, referring to the color wheel in FIG.
6(A), the absorption from 525-625 nm covers nearly half of the
color wheel with blue, indigo, and violet being complementary.
[0131] Referring now to FIG. 8(A), a fast red spot is highlighted
by the circle (R), a fast blue spot is highlighted by the circle
(B), a set of spots, one fast red spot and one fast blue spot, are
labeled as adjacent by the circle (A), and a fast red spot and a
fast blue spot overlapping each other is labeled by the circle (O).
As predicted, the fast red spot (A) is red, and the fast blue spot
(B) appears a dark bluish color one would expect from the mixture
of blue, indigo and violet. The adjacent spots within circle (A)
can be clearly distinguished from each other as separate red and
blue spots. However, the spot that includes an overlapping red and
blue spot results in an ambiguous color. It appears somewhat bluish
and has a red fringe on one side. The color of the spot is
difficult to distinguish and difficult to characterize. For an
overlapping spot, the absorption of the fast red and the fast blue
would be additive and the spectral absorption profile would span
from about 475 nm to about 625 and have .lamda..sub.max of around
550 nm. Referring again to the color wheel (FIG. 6(A)), this range
of wavelengths covers nearly the entire wheel. Broad based
absorption covering the entire spectra typically gives a black or
brown appearance with a tint of those colors absorbed least, in
this case indigo and violet. A pathologist considering the
photomicrograph in FIG. 8(A) may have difficulty distinguishing
between a blue to indigo spot (B) and the overlapping spot (O).
[0132] Accordingly, certain disclosed embodiments provide the
ability to choose different signaling conjugates that address this
issue. For example, different signaling conjugates can be
purposefully selected and made to comprise chromogenic moieties
that produce light at opposing ends of the UV-vis spectrum. FIGS.
10(A) and 10(B) illustrate how the disclosed signaling conjugates
and method can be used for resolving the issue associated with
probes comprising two different chromogenic moieties. With
reference to FIG. 10(A), a chromogenic moiety capable of producing
a black color ("B") is used in combination with a chromogenic
moiety that produces a red color ("R"). When the two signaling
conjugates overlap, it is unclear as two whether the observed black
color ("B") is produced by the black chromogenic moiety or if it is
produced by the overlap between the red and black chromogenic
moieties. However, referring to FIG. 10(B), this problem can be
solved by using two chromogenic moieties that, when combined,
produce a third unique color. For example, a purple chromogenic
moiety ("P") may be used in combination with a yellow chromogenic
moiety ("Y"). The overlap between the two is readily observed, as
an orange signal ("O") is produced. FIGS. 11(A-B) further show how
two colors can be deposited proximally to create a visually
distinct third color. In particular, FIG. 11(A) shows a yellow
signal, shown with a letter "y", combined with magenta signal,
shown with a letter "m", to create a vibrant cherry red color,
shown with a letter "r". FIG. 11(B) shows a magenta signal,
indicated by the letter "m," and a turquoise signal, indicated by
the letter "t," combine to create a dark blue signal, shown with a
letter "b".
Illumination
[0133] In particular disclosed embodiments, a traditional white
source and filter system may be used, such as those typically used
by persons of ordinary skill in the art. In other disclosed
embodiments, an LED light source may be used in the detection step
in order to generate narrower illumination light. Such light
sources may be used in embodiments wherein one or more different
signaling conjugates are used, particularly when three or more
different conjugates are used.
[0134] The method disclosed herein provides improved detection in
terms of the signal produced as well as the means by which the
signal is detected. Traditional detection techniques typically
comprise using narrow absorbing dyes with spectral filtering
wherein the dye absorbs only a narrow range of light having a
certain wavelength, and the filter passes only a small range of
wavelengths. Accordingly, combining the filter with such absorbance
produces a black spot in an otherwise bright-field, or other
chromogens may have absorbances that are within the spectral
absorbance ranges of the filter and therefore are not even apparent
under bright-field detection. This type of detection technique
typically is deconvulated into separate images or may further use
an overlaid image having false coloring. Using embodiments of the
method disclosed herein, bright-field detection may be used without
the problems typically associated with this particular technique in
analyzing chromogenic signals. The variety of signaling conjugates
contemplated by the present disclosure provides the ability to
analyze the biological sample in the bright-field and visually
detect the color signal(s) emitted without further manipulation.
Furthermore, the ability to use LED light sources with the
disclosed method provides flexibility in the range of wavelength
that can be absorbed by the disclosed signaling conjugate. In
particular disclosed embodiments, the signaling conjugates can be
visualized independently by illuminating the specimen with light of
a wavelength where the chromogen absorbs, thus causing the
chromogen to appear dark against a light background (light is
absorbed by the chromogen, reducing the light intensity at that
spot). In particular disclosed embodiments, illuminating the
specimen with light that is not absorbed by the chromogen causes
the chromogen to "disappear" because the intensity of the light is
not altered (absorbed) as it passes through the chromogen spot.
Solely by way of example, illuminating a biological sample slide
with green light causes the rhodamine chromogens to appear dark,
whereas the Cy5 chromogen disappears. Conversely, illuminating the
slide with red light causes the Cy5 chromogen to appear dark and
the rhodamine chromogens to disappear.
[0135] Slides stained using certain disclosed signaling conjugates
were illuminated using a multi-LED illuminator that was adapted to
Olympus BX-51 light microscope. Two LED illuminators were used: 1)
a homebuilt 3-LED illuminator comprising a Lamina RGB light engine
(EZ-43F0-0431) with 3 LEDdynamics BuckPlus current regulated
drivers with potentiometers and switches to permit on off control
and variation of the red, green, and blue LED intensities
independently; and 2) a TOFRA, Inc. RGBA Computer-Controlled LED
Illuminator for Upright Microscopes modified for manual LED
switching. To visualize only the tyramide chromogens, illuminating
the specimen with light of a wavelength where the chromogen absorbs
causes the chromogen to appear dark against a light background
(light is absorbed by the chromogen, reducing the light intensity
at that spot). Illuminating the specimen with light that is not
absorbed by the chromogen causes the chromogen to "disappear"
because the intensity of the light is not altered (absorbed) as it
passes through the chromogen spot.
[0136] FIGS. 12(A-B) are photomicrographs of a sample that has been
dual stained with a turquoise and magenta signaling conjugate under
(A) white light illumination, (B) green light illumination, and (C)
red light illumination. Illuminating the slide with green light
causes the turquoise signaling conjugates to appear dark, whereas
the magenta signaling conjugate disappears. Conversely,
illuminating the slide with red light causes the magenta signaling
conjugate to appear dark and the turquoise signaling conjugate to
disappear. Overlap between the magenta and the turquoise signaling
conjugates are dark in white light illumination, green light
illumination, and red light illumination. One of the perceived
benefits of fluorescence microscopy is the ability to use filters
to switch between the individual probe signals. Using the signaling
conjugates described herein, it is possible to enable switching
using chromogenic compounds. Matching the LED emission wavelength
with the absorbance wavelength of the tyramide dye causes the
matched chromogen signal to "disappear." LED power sources can be
easily added to a light microscope by replacing the condenser. The
emission wavelength of the LED can be switched between colors by
the user, with the push of a button.
Tyrosine Enhancement
[0137] Tyramide signal amplification and the signaling conjugates
described herein react with tyrosine residues available from the
sample and or the molecules/conjugates used to detect and label the
targets. The amount of protein surrounding the biomarker to be
detected is variable based on the natural variation between tissue
samples. When detecting biomarkers present at high levels, or when
detecting the co-localization of multiple biomarkers, the amount of
protein to which the tyramide molecules can attach may be a
limiting reactant in the deposition process. An insufficient amount
of protein in the tissue can result in the diffusion of tyramide
based detection, the potential to under-call the expression level
of biomarkers, and the inability to detect co-localized biomarkers.
One solution to these problems is to provide more protein binding
sites (i.e., tyrosine) by coating the tissue with a proteinaceous
solution and permanently cross-linking the protein to the tissue
using formalin, or other fixatives.
[0138] The majority of work with TSA has been done in the context
of fluorescent detection. Fluorescent TSA detection is accomplished
by a single tyramide deposition of a fluorophore, and the
deposition times are typically quite short because the sensitivity
of the fluorescent detection is high, whereas the background
associated with traditional TSA becomes problematic with longer
deposition times. In contrast, chromogenic TSA detection may
include multiple depositions of tyramide conjugates with extended
deposition times. As such, the fluorescent TSA art does not suggest
solutions to chromogenic TSA problems because the nature of the
problem is so different. In particular, the saturation of a
sample's tyrosine binding sites by tyramide reactive species is
thought to be a unique problem particular to the detection
chemistries described herein. Enhancements to TSA originating from
the TSA fluorescence research typically addressed the diffusion of
the reactive tyramide moieties and the lack of TSA signal.
Solutions to these problems have been described in the art. For
example, an increase in the viscosity of the reaction solution
through the addition of soluble polymers was described for
decreasing diffusion and HRP activity was enhanced through the
addition of vanillin and/or iodophenol. These solutions were not
sufficient to address some of the problems observed for the
detection chemistries described herein.
[0139] Through various studies, it was discovered that the severity
of the identified problem varies depending on the sample used. For
example, it was found that breast cancer tissues and prostate
cancer tissues included different levels of available tyramide
binding sites. It is also known that there are differences in
protein content in the cellular compartments (nucleus, cell
membrane, cytoplasm, etc.) that are targeted in various IHC and/or
ISH tests. Hence, in addition to being necessary for TSA
co-localization, the proposed invention will normalize protein
content (e.g., tyramide binding sites) and reduce variation between
and across samples. In illustrative embodiments, the addition of a
tyrosine enhancement agent may increase inter- and intra-sample
reproducibility of assays described herein.
[0140] When using amplifying conjugates, as described herein,
especially in conjunction with the signaling conjugates described
herein, the amount of protein surrounding the target or targets may
be insufficient. When detecting biomarkers present at high levels,
or when detecting the co-localization of multiple biomarkers, the
amount of protein in the sample to which the tyramide-based
detection reagents can attach may be the limiting reagent. An
insufficiency in tyramide binding sites can cause a reduced
reaction rate, allow the tyramide reactive molecules to diffuse
away from the target, and generally results in a weaker response
due to lower quantities of the signaling conjugates reacting in the
vicinity of the target. It was discovered that providing more
binding sites to the sample enhanced the detection as described
herein. One approach to enhancing the available binding sites was
to introduce a protein solution to the sample. So that the protein
remains through various washes and so that the protein does not
diffuse during or after subsequent detection steps, the protein was
cross-linked to the sample using a fixative (e.g., formalin).
[0141] In illustrative embodiments, an additional amount of a
tyrosine-containing reagent, such as a protein, may be incubated
with and fixed to the biological sample in order to provide
additional binding sites for multiple signaling or amplifying
conjugates, such as in multiplexing or amplification. For example,
when a translocation probe is used, clearer three-color staining
may be obtained by adding an additional amount of protein to the
biological sample. Additionally, non-specific probe binding can be
decreased using this additional step. Exemplary embodiments concern
adding BSA to the biological sample, followed by fixing the protein
using a cross-linking agent, such as a fixative (e.g., 10%
NBF).
[0142] To demonstrate the efficacy of the solution, it was first
established that exogenous proteins can be fixed to a sample,
(e.g., a histologically prepared paraffin-embedded tissue sample).
To demonstrate that additional protein can be covalently attached
to paraffin tissue sections, bovine serum albumin (BSA) was
functionalized with a hapten (2,1,3-Benzoxadiaole-carbamide, "BF").
The BSA-BF was added to the tissue following a hybridization step
where no probe was added, and all experiments were completed on a
Benchmark XT automated slide stainer (Ventana Medical Systems,
Tucson Ariz.). 10 .mu.g of the BSA-BF conjugate was added to the
slide and incubated for 16 minutes. BF-labeled BSA protein was then
covalently fixed to the tissue by adding 100 .mu.l of 4%
paraformaldehyde, and incubating for 16 minutes. The presence of
covalently attached BSA-BF was detected by adding an anti-BF
monoclonal antibody that was functionalized with the horseradish
peroxidase (HRP) enzyme. FIGS. 13(A-B) show a photomicrograph (FIG.
13(A)) of a control slide to which no BSA-BF was added, and FIG.
13(B) is a photomicrograph of the slide to which the BSA-BF had
been used. The HRP enzyme catalyzed the deposition of
tyramide-TAMRA, which stains the slide with a pink chromogen where
the BSA-BF was attached to the tissue. Without the presence of the
BSA-BF, under the same experimental conditions, no pink chromogen
is deposited (FIG. 13(A)), suggesting that exogenously added BSA
protein can be permanently fixed into paraffin embedded tissue
sections.
[0143] It was discovered that applying a signaling conjugate, as
described herein, for certain embodiments is more efficient using a
tyrosine enhancement agent following non-staining tyramide
deposition cycles. To confirm this hypothesis, tissue samples were
subjected to multiple rounds of TSA with a tyramide-hapten
conjugate. FIGS. 14(A-B) are photomicrographs of a first sample
(FIG. 14(A)) to which a signaling conjugate, as described herein,
was deposited and FIG. 14(B) is a second sample in which a tyrosine
enhancement solution was used prior to detection with the signaling
conjugate. The difference between FIG. 14(A) and FIG. 14(B)
supports the hypothesis that the availability of protein within the
sample is diminished by TSA depositions and that the addition of
the tyrosine-containing enhancers can provide more robust staining.
In the absence of protein fixation (FIG. 14(A)) the subsequent
deposition of the signaling conjugate produced a low level of
chromogenic signal. When the exogenous protein was fixed into the
tissue section using paraformaldehyde (FIG. 14(B)), the signaling
conjugate produced signals significantly more intense and numerous.
The data suggests that fixation of exogenous protein to tissue
sections enhances tyramide signal amplification by providing
additional protein binding sites for the tyramide reagents to
covalently attach.
[0144] One disclosed embodiment of a method for detecting a target
in a sample comprises: contacting the sample with a detection probe
specific to the target; contacting the sample with a tyrosine
enhancer; contacting the sample with a cross-linking agent;
contacting the sample with a tyramide-based detection reagent; and
detecting the target in the sample; wherein the cross-linking
reagent covalently attaches the tyrosine enhancer to the sample. In
one embodiment, the method further comprises contacting the sample
with a labeling conjugate. In another embodiment, the method
further comprises contacting the sample with an amplifying
conjugate. In one embodiment, the method further comprises
detecting a second target, wherein contacting the sample with the
tyrosine enhancer occurs subsequent to contacting the sample with
the tyramide-based detection reagents for the first target and
prior to contacting the sample with tyramide-based detection
reagents for the second target. In one embodiment, the tyrosine
enhancer includes a protein. In another embodiment, the tyrosine
enhancer is a polymer containing tyrosine residues. In one
embodiment, the cross-linking agent is formalin or formaldehyde. In
another embodiment, the crosslinking agent is neutral buffered
formalin (NBF). In another embodiment the cross-linking agent is an
imidoester, a dimethyl suberimidate, or a
N-Hydroxysuccinimide-ester (NHS ester). In another embodiment, the
cross-linking agent is light radiation. In one embodiment, the
cross-linking agent is UV light or X-ray radiation. In one
embodiment, detecting the target in the sample includes imaging at
least one of the tyramide-based detection reagents. In another
embodiment, detecting the target includes fluorescently imaging at
least one of the tyramide-based detection reagents. In another
embodiment, detecting the target includes imaging at least one of
the tyramide-based detection reagents, the tyramide-based detection
reagents yielding a chromogenic signal detectable using
bright-field light microscopy. In another embodiment, detecting the
target includes imaging a signaling conjugate. In another
embodiment, detecting the target includes imaging a chromogen that
was deposited in the vicinity of at least one of the tyramide-based
detection reagents.
Counterstaining
[0145] Counterstaining is a method of post-treating the samples
after they have already been stained with agents to detect one or
more targets, such that their structures can be more readily
visualized under a microscope. For example, a counterstain is
optionally used prior to cover-slipping to render the
immunohistochemical stain more distinct. Counterstains differ in
color from a primary stain. Numerous counterstains are well known,
such as hematoxylin, eosin, methyl green, methylene blue, Giemsa,
Alcian blue, and Nuclear Fast Red. In some examples, more than one
stain can be mixed together to produce the counterstain. This
provides flexibility and the ability to choose stains. For example,
a first stain can be selected for the mixture that has a particular
attribute, but yet does not have a different desired attribute. A
second stain can be added to the mixture that displays the missing
desired attribute. For example, toluidine blue, DAPI, and pontamine
sky blue can be mixed together to form a counterstain. One aspect
of the present disclosure is that the counterstaining methods known
in the art are combinable with the disclosed methods and
compositions so that the stained sample is easily interpretable by
a reader.
III. Conjugates
[0146] Disclosed herein are various different conjugates suitable
for use in the disclosed method. The various classes of conjugates
contemplated by the present disclosure are described below.
[0147] A. Detection Probes
[0148] The present disclosure concerns particular detection probes
that may be used to detect a target in a sample, for example a
biological sample. The detection probes include a specific binding
moiety that is capable of specifically binding to the target.
Detection probes include one or more features that enable detection
through a labeling conjugate. Representative detection probes
include nucleic acid probes and primary antibody probes.
[0149] In illustrative embodiments, the detection probe is an
oligonucleotide probe or an antibody probe. As described herein,
detection probes may be indirect detection probes. Indirect
detection probes are not configured to be detected directly. In
particular, the probes are not configured for the purpose of direct
visualization. Instead, detection probes will generally be one of
two types, although these are not mutually exclusive types. The
first type of detection probe is haptenated and the second type of
detection probes are based on a particular species of antibody.
Other types of detection probes are known in the art and within the
scope of the current disclosure, but these are less commonly
implemented, for example aptamer-labeled probes or antibodies,
nucleic acid tagged probes or antibodies, antibodies that are
covalently bound to other antibodies so as to provide dual-binding
capabilities (e.g., through coupling techniques or through fusion
proteins). While not configured as such, some of the detection
probes may have properties that enable their direct detection. For
example, using haptens fluorophores is within the scope of the
present disclosure. According to one embodiment, the detection
probe includes a hapten label. Those of ordinary skill in the art
appreciate that a detection probe can be labeled with one or more
haptens using various approaches. The detection probe may include a
hapten selected from the group consisting an oxazole hapten,
pyrazole hapten, thiazole hapten, nitroaryl hapten, benzofuran
hapten, triterpene hapten, urea hapten, thiourea hapten, rotenoid
hapten, coumarin hapten, cyclolignan hapten, di-nitrophenyl hapten,
biotin hapten, digoxigenin hapten, fluorescein hapten, and
rhodamine hapten. In other examples, the detection probe is
monoclonal antibody derived from a second species such as goat,
rabbit, mouse, or the like. For labeling a hapten-labeled detection
probe, the labeling conjugate would include an anti-hapten
antibody. For labeling a species-based detection probe, the
labeling conjugate may be configured with an anti-species
antibody.
[0150] In illustrative embodiments, the present disclosure
describes nucleic acid probes which hybridize to one or more target
nucleic acid sequences. The nucleic acid probe preferably
hybridizes to a target nucleic acid sequence under conditions
suitable for hybridization, such as conditions suitable for in situ
hybridization, Southern blotting, or Northern blotting. Preferably,
the detection probe portion comprises any suitable nucleic acid,
such as RNA, DNA, LNA, PNA or combinations thereof, and can
comprise both standard nucleotides such as ribonucleotides and
deoxyribonucleotides, as well as nucleotide analogs. LNA and PNA
are two examples of nucleic acid analogs that form hybridization
complexes that are more stable (i.e., have an increased Tm) than
those formed between DNA and DNA or DNA and RNA. LNA and PNA
analogs can be combined with traditional DNA and RNA nucleosides
during chemical synthesis to provide hybrid nucleic acid molecules
than can be used as probes. Use of the LNA and PNA analogs allows
modification of hybridization parameters such as the Tm of the
hybridization complex. This allows the design of detection probes
that hybridize to the detection target sequences of the target
nucleic acid probes under conditions that are the same or similar
to the conditions required for hybridization of the target probe
portion to the target nucleic acid sequence.
[0151] Suitable nucleic acid probes can be selected manually, or
with the assistance of a computer implemented algorithm that
optimizes probe selection based on desired parameters, such as
temperature, length, GC content, etc. Numerous computer implemented
algorithms or programs for use via the internet or on a personal
computer are available. For example, to generate multiple binding
regions from a target nucleic acid sequence (e.g., genomic target
nucleic acid sequence), regions of sequence devoid of repetitive
(or other undesirable, e.g., background-producing) nucleic acid
sequence are identified, for example manually or by using a
computer algorithm, such as RepeatMasker. Methods of creating
repeat depleted and uniquely specific probes are found in, for
example, US Patent Publication No. 2012/0070862, which is hereby
incorporated by reference in its entirety. Within a target nucleic
acid sequence (e.g., genomic target nucleic acid sequence) that
spans several to several-hundred kilobases, typically numerous
binding regions that are substantially or preferably completely
free of repetitive (or other undesirable, e.g.,
background-producing) nucleic acid sequences are identified.
[0152] In some embodiments, a hapten is incorporated into the
nucleic acid probe, for example, by use of a haptenylated
nucleoside. Methods for conjugating haptens and other labels to
dNTPs (e.g., to facilitate incorporation into labeled probes) are
well known in the art. Indeed, numerous labeled dNTPs are available
commercially, for example from Invitrogen Detection Technologies
(Molecular Probes, Eugene, Oreg.). A label can be directly or
indirectly attached to a dNTP at any location on the dNTP, such as
a phosphate (e.g., .alpha., .beta. or .gamma. phosphate) or a
sugar. The probes can be synthesized by any suitable, known nucleic
acid synthesis method. In some embodiments, the detection probes
are chemically synthesized using phosphoramidite nucleosides and/or
phosphoramidite nucleoside analogs. For example, in some
embodiments, the probes are synthesized by using standard RNA or
DNA phosphoramidite nucleosides. In some embodiments, the probes
are synthesized using either LNA phosphoramidites or PNA
phosphoramidites, alone or in combination with standard
phosphoramidite nucleosides. In some embodiments, haptens are
introduced on a basic phosphoramidites containing the desired
detectable moieties. Other methods can also be used for detection
probe synthesis. For example, a primer made from LNA analogs or a
combination of LNA analogs and standard nucleotides can be used for
transcription of the remainder of the probe. As another example, a
primer comprising detectable moieties is utilized for transcription
of the rest of the probe. In still other embodiments, segments of
the probe produced, for example, by transcription or chemical
synthesis, may be joined by enzymatic or chemical ligation.
[0153] A variety of haptens may be used in the detectable moiety
portion of the detection probe. Such haptens include, but are not
limited to, pyrazoles, particularly nitropyrazoles; nitrophenyl
compounds; benzofurazans; triterpenes; ureas and thioureas,
particularly phenyl ureas, and even more particularly phenyl
thioureas; rotenone and rotenone derivatives, also referred to
herein as rotenoids; oxazole and thiazoles, particularly oxazole
and thiazole sulfonamides; coumarin and coumarin derivatives;
cyclolignans, exemplified by podophyllotoxin and podophyllotoxin
derivatives; and combinations thereof. Fluorescein derivatives
(FITC, TAMRA, Texas Red, etc.), Digoxygenin (DIG),
5-Nitro-3-pyrozolecarbamide (nitropyrazole, NP),
4,5,-Dimethoxy-2-nitrocinnamide (nitrocinnamide, NCA),
2-(3,4-Dimethoxyphenyl)-quinoline-4-carbamide (phenylquinolone,
DPQ), 2,1,3-Benzoxadiazole-5-carbamide (benzofurazan, BF),
3-Hydroxy-2-quinoxalinecarbamide (hydroxy quinoxaline, HQ),
4-(Dimethylamino)azobenzene-4'-sulfonamide (DABSYL), Rotenone
isoxazoline (Rot),
(E)-2-(2-(2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)phenozy)-
acetamide (benzodiazepine, BD),
7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid (coumarin 343,
CDO), 2-Acetamido-4-methyl-5-thiazolesulfonamide
(thiazolesulfonamide, TS), and p-Mehtoxyphenylpyrazopodophyllamide
(Podo). These haptens and their use in probes are described in more
detail in U.S. Pat. No. 7,695,929, which is hereby incorporated
herein by reference in its entirety.
[0154] B. Labeling Conjugates & Secondary Labeling
Conjugates
[0155] In illustrative embodiments, the labeling conjugate
specifically binds to the detection probe and is configured to
label the target with an enzyme. As described above, detection
probes configured from a second species or to include a hapten can
be detected by either an anti-species antibody or an anti-hapten
antibody. One approach to configuring a labeling conjugate has been
to directly couple an enzyme to the anti-species or anti-hapten
antibody. Conjugates of this kind, which may or may not include
various linkers, are also described in U.S. Pat. No. 7,695,929. The
labeling conjugate includes one or more enzymes. Exemplary enzymes
include oxidoreductases or peroxidases. The signaling conjugate
includes a latent reactive moiety and a chromogenic moiety. The
enzyme catalyzes conversion of the latent reactive moiety into a
reactive moiety which covalently binds to the biological sample
proximally to or directly on the target.
[0156] The secondary labeling conjugate is used in connection with
the amplifying conjugates, as described herein. Secondary labeling
conjugates are configured in the same manner as labeling conjugates
except that they are configured to label haptens deposited through
an amplification process instead of haptens conjugated to detection
conjugates. In illustrative embodiments, a secondary labeling
conjugate comprises an anti-hapten antibody conjugated to an
enzyme. In one embodiment, the enzyme is an oxidoreductase or a
peroxidase.
[0157] C. Signaling Conjugate
[0158] Another type of conjugate disclosed herein is a signaling
conjugate. The signaling conjugate provides the detectable signal
that is used to detect the target, according to the methods
disclosed herein. In particular disclosed embodiments, the
signaling conjugate comprises a latent reactive moiety and a
chromophore moiety.
[0159] One aspect of the present disclosure is that the signaling
conjugates may be configured to absorb light more selectively than
traditionally available chromogens. Detection is realized by
absorbance of the light by the signaling conjugate; for example,
absorbance of at least about 5% of incident light would facilitate
detection of the target. In other darker stains, at least about 20%
of incident light would be absorbed. Non-uniform absorbance of
light within the visible spectra results in the chromophore moiety
appearing colored. The chromogen conjugates disclosed herein may
appear colored due to their absorbance; the chromogen conjugates
may appear red, orange, yellow, green, indigo, or violet depending
on the spectral absorbance associated with the chomophore moiety.
According to another aspect, the chromophore moieties may have
narrower spectral absorbances than those absorbances of
traditionally used chromogens (e.g., DAB, Fast Red, Fast Blue). In
illustrative embodiments, the spectral absorbance associated with
the first chromophore moiety of the first signaling conjugate has a
full-width half-max (FWHM) of between about 30 nm and about 250 nm,
between about 30 nm and about 150 nm, between about 30 nm and about
100 nm, or between about 20 nm and about 60 nm.
[0160] Narrow spectral absorbances enable the signaling conjugate
chromophore moiety to be analyzed differently than traditional
chromogens. While having enhanced features compared to
traditionally chromogens, detecting the signaling conjugates
remains simple. In illustrative embodiments, detecting comprises
using a bright-field microscope or an equivalent digital
scanner.
[0161] An embodiment of the disclosed signaling conjugate is
illustrated in FIGS. 2(A) and 2(B). Referring to FIGS. 2(A-B), the
signaling conjugate 12 comprises a latent reactive moiety 4 and a
chromophore moiety 6; in another embodiment, an alternative
signaling conjugate 14 may include a linker 8 for conjugating
chromophore moiety 6 to latent reactive moiety 4. In particular
disclosed embodiments, the signaling conjugate has the following
general Formula 1:
##STR00001##
The disclosed signaling conjugate typically comprises a latent
reactive moiety as described herein. For example, the latent
reactive moiety may be the same or different from that of the
disclosed amplification conjugate; however, each latent reactive
moiety is capable of forming a reactive radical species and has the
general formula provided herein. As shown in Formula 1, the
signaling conjugate may comprise an optional linker. If a linker is
used, it may be selected from any of the linkers disclosed herein.
In particular disclosed embodiments, the linker is selected to
improve hydrophilic solution solubility of the signaling conjugate,
and/or to improve conjugate functionality on the biological sample.
In particular disclosed embodiment, the linker is an alkylene oxide
linker, such as a polyethylene glycol linker; however, any of the
linkers disclosed herein may be used for the signaling
conjugate.
[0162] 1. Chromophore Moiety
[0163] A chromophore moiety is generally described as the part of a
molecule responsible for its color. Colors arise when a molecule
absorbs certain wavelengths of visible light and transmits or
reflects others. The chromophore is a region in the molecule where
the energy difference between two different molecular orbitals
falls within the range of the visible spectrum, wherein visible
light interacting with that region can be absorbed. The absorbance
is usually associated with an electron transition from its ground
state to an excited state. Molecules having ground state to excited
state energy differences within the visible spectrum are often
conjugated carbon structures. In these compounds, electrons
transition between energy levels that are extended pi-orbitals,
created by a series of alternating single and double bonds, often
in aromatic systems. Common examples include various food
colorings, fabric dyes (azo compounds), pH indicators, lycopene,
.beta.-carotene, and anthocyanins. The structure of the molecule
imparts the characteristic of the pi-orbitals which result in the
energy level. Typically, lengthening or extending a conjugated
system with more unsaturated (multiple) bonds in a molecule will
tend to shift absorption to longer wavelengths. Woodward-Fieser
rules can be used to approximate ultraviolet-visible maximum
absorption wavelength in organic compounds with conjugated pi-bond
systems.
[0164] In illustrative embodiments, metal complexes can be
chromophores. For example, a metal in a coordination complex with
ligands will often absorb visible light. For example, chlorophyll
and hemoglobin (the oxygen transporter in the blood of vertebrate
animals) are chromophores that include metal complexes. In these
two examples, a metal is complexed at the center of a porphyrin
ring: the metal being iron in the heme group of hemoglobin, or
magnesium in the case of chlorophyll. The highly conjugated
pi-bonding system of the porphyrin ring absorbs visible light. The
nature of the central metal can also influence the absorption
spectrum of the metalloporphyrin complex or properties such as
excited state lifetime.
[0165] In illustrative embodiments, the chromophore moiety is a
coumarin or coumarin derivative. A general formula for coumarin and
coumarin derivatives is provided below.
##STR00002##
[0166] With reference to Formula 2, R.sup.1-R.sup.6 are defined
herein. At least one of the R.sup.1-R.sup.6 substituents also
typically is bonded to a linker or the latent reactive moiety
(e.g., a tyramide or tyramide derivative). Certain working
embodiments have used the position indicated as having an R.sup.5
substituent for coupling to a linker or latent reactive moiety
(e.g., a tyramide or tyramide derivative). Substituents other than
hydrogen at the 4 position are believed to quench fluorescence, but
are useful within the scope of the present disclosure. Y is
selected from oxygen, nitrogen or sulfur. Two or more of the
R.sup.1-R.sup.6 substituents available for forming such compounds
also may be atoms, typically carbon atoms, in a ring system bonded
or fused to the compounds having the illustrated general formula.
Exemplary embodiments of these types of compounds include:
##STR00003##
A person of ordinary skill in the art will appreciate that the
rings also could be heterocyclic and/or heteroaryl.
[0167] Working embodiments typically comprise fused A-D ring
systems having at least one linker, tyramide, or tyramide
derivative coupling position, with one possible coupling position
being indicated below:
##STR00004##
[0168] With reference to Formula 3, the R and Y variable groups are
as stated herein. Most typically, R.sup.1-R.sup.14 independently
are hydrogen or lower alkyl. Particular embodiments of
coumarin-based chromophores include
2,3,6,7-tetrahydro-11-oxo-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizine-1-
0-carboxylic acid
##STR00005##
and 7-(diethylamino)coumarin-3-carboxylic acid
##STR00006##
[0169] Another class of chromogenic moieties suitable for use
herein include diazo-containing chromogens. These particular
chromophores may have a formula as illustrated below.
##STR00007##
With respect to this formula, ring E may be selected from phenyl,
imidazole, pyrazole, oxazole, and the like. Each R.sup.2
independently may be selected from those groups recited herein. In
particular disclosed embodiments, each R.sup.2 independently is
selected from amine, substituted amine, phenyl, hydroxyl, sulfonyl
chloride, sulfonate, carboxylate, and combinations thereof; and n
may range from zero to 5. Particular disclosed embodiments may be
selected from the following diazo chromophores: DABSYL, which has a
.lamda..sub.max of about 436 nm and has the following chemical
structure
##STR00008##
and Tartrazine, which has a .lamda..sub.max of about 427 nm and has
the following chemical structure
##STR00009##
[0170] In yet other embodiments, the chromophore may be a
triarylmethane compound. Triarylmethane compounds within the scope
of the present disclosure may have the following formula.
##STR00010##
With respect to Formula 4, each R.sup.a independently may be
selected from hydrogen, aliphatic, aryl, and alkyl aryl; and each
R.sup.24 may be selected from amine, substituted amine, hydroxyl,
alkoxy, and combinations thereof; each n independently may range
from zero to 5. Exemplary chromophores are provided below:
##STR00011##
[0171] In other disclosed embodiments, the chromophore moiety may
have the following formula
##STR00012##
wherein each R.sup.a independently may be selected from hydrogen,
aliphatic, aryl, and alkyl aryl; each R.sup.24 independently may be
selected from the groups provided herein, including substituted
aryl, which comprises an aryl group substituted with one or more
groups selected from any one of R.sup.1-R.sup.23, which are
disclosed herein; Y may be nitrogen or carbon; Z may be nitrogen or
oxygen; and n may range from zero to 4. In particular disclosed
embodiments, Z is nitrogen and each R.sup.a may be aliphatic and
fused with a carbon atom of the ring to which the amine comprising
R.sup.a is attached, or each Ra may join together to form a 4 or
6-membered aliphatic or aromatic ring, which may be further
substituted. Exemplary embodiments are provided as follows:
##STR00013## ##STR00014##
and other rhodamine derivatives, such as tetramethylrhodamines
(including TMR, TAMRA, and reactive isothiocyanate derivatives),
and diarylrhodamine derivatives, such as the QSY 7, QSY 9, and QSY
21 dyes.
[0172] Exemplary chromophores are selected from the group
consisting of DAB; AEC; CN; BCIP/NBT; fast red; fast blue; fuchsin;
NBT; ALK GOLD; Cascade Blue acetyl azide; Dapoxylsulfonic
acid/carboxylic acid succinimidyl ester; DY-405; Alexa Fluor 405
succinimidyl ester; Cascade Yellow succinimidyl ester;
pyridyloxazole succinimidyl ester (PyMPO); Pacific Blue
succinimidyl ester; DY-415; 7-hydroxycoumarin-3-carboxylic acid
succinimidyl ester; DYQ-425; 6-FAM phosphoramidite; Lucifer Yellow;
iodoacetamide; Alexa Fluor 430 succinimidyl ester; Dabcyl
succinimidyl ester; NBD chloride/fluoride; QSY 35 succinimidyl
ester; DY-485XL; Cy2 succinimidyl ester; DY-490; Oregon Green 488
carboxylic acid succinimidyl ester; Alexa Fluor 488 succinimidyl
ester; BODIPY 493/503 C3 succinimidyl ester; DY-480XL; BODIPY FL C3
succinimidyl ester; BODIPY FL C5 succinimidyl ester; BODIPY FL-X
succinimidyl ester; DYQ-505; Oregon Green 514 carboxylic acid
succinimidyl ester; DY-510XL; DY-481XL;
6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl
ester (JOE); DY-520XL; DY-521XL; BODIPY R6G C3 succinimidyl ester;
erythrosin isothiocyanate;
5-carboxy-2',4',5',7'-tetrabromosulfonefluorescein succinimidyl
ester; Alexa Fluor 532 succinimidyl ester;
6-carboxy-2',4,4',5'7,7'-hexachlorofluorescein succinimidyl ester
(HEX); BODIPY 530/550 C3 succinimidyl ester; DY-530; BODIPY TMR-X
succinimidyl ester; DY-555; DYQ-1; DY-556; Cy3 succinimidyl ester;
DY-547; DY-549; DY-550; Alexa Fluor 555 succinimidyl ester; Alexa
Fluor 546 succinimidyl ester; DY-548; BODIPY 558/568 C3
succinimidyl ester; Rhodamine red-X succinimidyl ester; QSY 7
succinimidyl ester; BODIPY 564/570 C3 succinimidyl ester; BODIPY
576/589 C3 succinimidyl ester; carboxy-X-rhodamine (ROX);
succinimidyl ester; Alexa Fluor 568 succinimidyl ester; DY-590;
BODIPY 581/591 C3 succinimidyl ester; DY-591; BODIPY TR-X
succinimidyl ester; Alexa Fluor 594 succinimidyl ester; DY-594;
carboxynaphthofluorescein succinimidyl ester; DY-605; DY-610; Alexa
Fluor 610 succinimidyl ester; DY-615; BODIPY 630/650-X succinimidyl
ester; erioglaucine; Alexa Fluor 633 succinimidyl ester; Alexa
Fluor 635 succinimidyl ester; DY-634; DY-630; DY-631; DY-632;
DY-633; DYQ-2; DY-636; BODIPY 650/665-X succinimidyl ester; DY-635;
Cy5 succinimidyl ester; Alexa Fluor 647 succinimidyl ester; DY-647;
DY-648; DY-650; DY-654; DY-652; DY-649; DY-651; DYQ-660; DYQ-661;
Alexa Fluor 660 succinimidyl ester; Cy5.5 succinimidyl ester;
DY-677; DY-675; DY-676; DY-678; Alexa Fluor 680 succinimidyl ester;
DY-679; DY-680; DY-682; DY-681; DYQ-3; DYQ-700; Alexa Fluor 700
succinimidyl ester; DY-703; DY-701; DY-704; DY-700; DY-730; DY-731;
DY-732; DY-734; DY-750; Cy7 succinimidyl ester; DY-749; DYQ-4; and
Cy7.5 succinimidyl ester.
[0173] In particular disclosed embodiments, the chromophore moiety
may be selected from tartrazine,
7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester,
Dabsyl sulfonyl chloride, fluorescein isothiocyanate (FITC) carboxy
succinimidyl ester (DY-495), Rhodamine Green carboxylic acid
succinimidyl ester (DY-505), eosin isothiocyanate (EITC),
6-carboxy-2',4,7,7'-tetrachlorofluorescein succinimidyl ester
(TET), carboxyrhodamine 6G succinimidyl ester,
carboxytetramethylrhodamine succinimidyl ester (TMR, TAMRA)
(DY-554), QSY 9 succinimidyl ester, sulforhodamine B sulfonyl
chloride (DY-560), Texas Red (sulforhodamine 101), gallocyanine,
Fast Green FCF, Malachite Green, isothiocyanate, and QSY 21
succinimidyl ester. In certain disclosed embodiments, the
chromophore moiety of the signaling conjugate is other than Dabsyl
sulfonyl chloride, FITC, 7-diethylaminocoumarin-3-carboxylic acid,
succinimidyl ester, Rhodamine Green carboxylic acid succinimidyl
ester (DY-505), eosin isothiocyanate (EITC),
6-carboxy-2',4,7,7'-tetrachlorofluorescein succinimidyl ester
(TET), carboxytetramethylrhodamine succinimidyl ester (TMR, TAMRA)
(DY-554), sulforhodamine B sulfonyl chloride (DY-560), Texas Red
(sulforhodamine 101), and gallocyanine.
[0174] Further exemplary chromogenic moieties that are used for the
signaling conjugate are provided below:
##STR00015## ##STR00016##
[0175] In illustrative embodiments of the present disclosure, the
signaling conjugate has absorption maxima and absorption breadths
particularly suited for bright-field imaging of targets in
biological samples. In one embodiment, a signaling conjugate is
configured to provide an absorbance peak having a .lamda..sub.max
of between about 350 nm and about 800 nm, between about 400 nm and
about 750 nm, or between about 400 nm and about 700 nm. These
wavelength ranges are of particular interest because they translate
into colors visible to humans. However, the approaches described
herein could also be applied to chromophore moieties useful for
near infrared (NIR), infrared (IR), or ultraviolet (UV) diagnostic
methodologies.
[0176] In one embodiment the signaling conjugate is configured to
produce a colored signal selected from the group consisting of red,
orange, yellow, green, indigo, violet, or mixtures thereof. In one
embodiment, a signaling conjugate has a .lamda..sub.max of between
about 400 nm and 430 nm. In another embodiment, the signaling
conjugate produces a yellow signal. In one embodiment, a signaling
conjugate has a .lamda..sub.max of between about 430 nm and 490 nm.
In another embodiment, the signaling conjugate produces an orange
signal. In one embodiment, a signaling conjugate has a
.lamda..sub.max of between about 490 nm and 560 nm. In another
embodiment, the signaling conjugate produces a red signal. In one
embodiment, a signaling conjugate has a .lamda..sub.max of between
about 560 nm and 570 nm. In another embodiment, the signaling
conjugate produces a violet signal. In one embodiment, a signaling
conjugate has a .lamda..sub.max of between about 570 nm and 580 nm.
In another embodiment, the signaling conjugate produces an indigo
signal. In one embodiment, a signaling conjugate has a
.lamda..sub.max of between about 580 nm and 620 nm. In another
embodiment, the signaling conjugate produces a blue signal. In one
embodiment, a signaling conjugate has a .lamda..sub.max of between
about 620 nm and about 800 nm. In another embodiment, the signaling
conjugate produces a green signal.
[0177] In one embodiment, the signaling conjugate is configured to
have a full-width half-max (FWHM) of between about 20 nm and about
60 nm, between about 30 and about 100 nm, between about 30 and
about 150 nm, or between about 30 and about 250 nm. In particular
disclosed embodiments, the FWHM is less than about 300 nm, less
than about 250 nm, less than about 200 nm, less than about 150 nm,
less than about 100 nm, less than about 50 nm. In illustrative
embodiments, a signaling conjugate having a FWHM of less than about
150 nm is described. In one embodiment, the FWHM is less than about
150 nm, less than about 120 nm, less than about 100 nm, less than
about 80 nm, less than about 60 nm, less than about 50 nm, less
than about 40 nm, less than about 30 nm, between about 10 nm and
150 nm, between about 10 nm and 120 nm, between about 10 nm and 100
nm, between about 10 nm and 80 nm, between about 10 nm and 60 nm,
between about 10 nm and 50 nm, or between about 10 nm and 40
nm.
[0178] In another embodiment, the signaling conjugate has an
average molar absorptivity of greater than about 5,000 M.sup.-1
cm.sup.-1 to about 90,000 M.sup.-1 cm.sup.-1. For example, an
average molar absorptivity of greater than about 5,000 M.sup.-1
cm.sup.-1, greater than about 10,000 M.sup.-1 cm.sup.-1, greater
than about 20,000 M.sup.-1 cm.sup.-1, greater than about 40,000
M.sup.-1 cm.sup.-1, or greater than about 80,000 M.sup.-1
cm.sup.-1. In yet another embodiment, the signaling conjugate has a
solubility in water of at least about 0.1 mM to about 1 M. For
example, the signaling conjugate has a solubility in water of at
least about 0.1 mM, at least about 1 mM, at least about 10 mM, at
least about 100 mM, or at least about 1 M. In one embodiment, the
signaling conjugate is stable against precipitation in an aqueous
buffered solution for greater than about 1 month to about 30
months. For example, the signaling conjugate is stable against
precipitation in an aqueous buffered solution for greater than
about 1 month, greater than about 3 months, greater than about 6
months, greater than about 12 months, greater than about 18 months,
or greater than about 24 months.
[0179] As described herein, the FWHM of the absorption peak
significantly contributes to the observed color of the signaling
conjugate. Referring to FIG. 6(A-B), several colors are observed
for light observed over a relatively small span of wavelengths. In
particular, yellow light is only apparent across a relatively
narrow span of 20 nm. To impart a yellow color on a substance, a
relatively narrow span of visible wavelengths should be absorbed
(400-430 nm). Referring to FIGS. 7(A) and 7(B), the signaling
conjugate shown therein has a FWHM of approximately 40 nm. FIG.
15(A) is a first photomicrograph and FIG. 15(B) is a second
photomicrograph of a protein stained (HER2 (4B5) IHC in Calu-3
xenografts) using the signaling conjugate having the absorption
spectra shown in FIG. 16. Trace A corresponds to the signaling
conjugate used for FIG. 15(A) and trace B corresponds to the
signaling conjugated used for FIG. 15(B); note that each signaling
conjugate was analyzed with spectrometry in solution prior to
staining and on the slide subsequent to having detected the HER2
(the dashed traces representing the spectra obtained on the
tissue). The signaling conjugate used to stain the tissue shown in
FIG. 15(A) has a .lamda..sub.max of about 456 nm and a FWHM of
about 111 nm. The signaling conjugate used to stain the tissue
shown in FIG. 15(B) has a .lamda..sub.max of about 628 nm and a
FWHM of about 70 nm.
[0180] Table 1 shows a classification system for the spectral
properties of various signaling conjugates according to
illustrative embodiments of the present disclosure. According to
the classification system, there are six different colors, which a
particular chromogen could be classified as, the series numbered
roman numerals one through six (i.e., I-VI). For each color
classification, there are five band-width classifications, those
band-width classifications being made according to broader FWHM
measurements. Accordingly, band-width classification (a) is the
narrowest and includes those signaling conjugates that have FWHM
widths of between about 10 and about 40 nm. Band-width
classification (e) is the broadest and includes those signaling
conjugates that have FWHM widths of between about 130-160 nm. A red
signaling conjugate having a .lamda..sub.max of about 530 nm and a
FWHM of about 115 nm could be classified as a series III(d)
signaling conjugate.
TABLE-US-00001 TABLE 1 Classification system for signaling
conjugates spectral properties. FWHM (nm) color .lamda..sub.max
(nm) 10-40 40-70 70-100 100-130 130-160 I. yellow 350-430 (a) (b)
(c) (d) (e) II. orange 430-490 (a) (b) (c) (d) (e) III. red 490-560
(a) (b) (c) (d) (e) IV. indigo/violet 560-580 (a) (b) (c) (d) (e)
V. blue 580-620 (a) (b) (c) (d) (e) VI. green 620-800 (a) (b) (c)
(d) (e)
[0181] FIGS. 17(A-D) are photomicrographs of tissues stained with
signaling conjugates having different chromogenic moieties. FIG.
17(E) shows UV-Vis spectra with traces corresponding to the
absorbance of the signaling conjugates, the traces corresponding to
the associated photomicrograph. As such, trace (A) of FIG. 17(E)
corresponds to the signaling conjugate shown in FIG. 17(A). The
other traces are similarly associated with the corresponding
photomicrographs. The blue color apparent in the slide is a
commercially available bluing solution. FIG. 17(A) and trace "A" of
FIG. 17(E) shows a malachite green signaling conjugate. It is
classifiable as a I(b) signaling conjugate according to Table 1.
FIG. 17(B) and trace "B" of FIG. 17(E) shows a tartrazine signaling
conjugate. It is classifiable as a I(c) signaling conjugate
according to Table 1. FIG. 17(C) and trace "C" of FIG. 17(E) shows
a sulforhodamine B signaling conjugate. It is classifiable as a
IV(b) signaling conjugate according to Table 1. FIG. 17(D) and
trace "D" of FIG. 17(E) shows a Victoria Blue signaling conjugate.
It is classifiable as a VI(c) signaling conjugate according to
Table 1.
[0182] FIG. 18(A-D) are photomicrographs of tissues stained with
signaling conjugates having different chromogenic moieties. FIG.
18(E) shows UV-Vis spectra with traces corresponding to the
absorbance of the signaling conjugates, the traces corresponding to
the associated photomicrograph. FIG. 18(A) and trace "A" of FIG.
18(E) shows a coumarin
(4-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid) signaling
conjugate. It is classifiable as a I(b) signaling conjugate
according to Table 1. FIG. 18(B) and trace "B" of FIG. 18(E) show a
Dabsyl (dimethylaminoazobenzenesulfonic acid) signaling conjugate.
It is classifiable as a II(b) signaling conjugate according to
Table 1. FIG. 18(C) and trace "C" of FIG. 18(E) shows a TAMRA
signaling conjugate. It is classifiable as a III(b) signaling
conjugate according to Table 1. FIG. 18(D) and trace "D" of FIG.
18(E) shows a 5-(and-6)-carboxyrhodamine 110 signaling conjugate.
It is classifiable as a V(a) signaling conjugate according to Table
1.
[0183] FIGS. 19(AD) are photomicrographs of tissues stained with
signaling conjugates having different chromogenic moieties. FIG.
19(E) shows UV-Vis spectra with traces corresponding to the
absorbance of the signaling conjugates, the traces corresponding to
the associated photomicrograph. FIG. 19(A) and trace "A" of FIG.
19(E) shows a FITC
(1-(3',6'-dihydroxy-3-oxospiro(isobenzofuran-1(3H),9'-(9H)xanthen-5-yl)
signaling conjugate. It is classifiable as a III(b) signaling
conjugate according to Table 1. FIG. 19(B) and trace "B" of FIG.
19(E) shows a Rhodamine 6G signaling conjugate. It is classifiable
as a III(c) signaling conjugate according to Table 1. FIG. 19(C)
and trace "C" of FIG. 19(E) shows a Texas Red (sulforhodamine 101)
signaling conjugate. It is classifiable as a IV(c) signaling
conjugate according to Table 1. FIG. 19(D) and trace "D" of FIG.
19(E) shows a cy5 signaling conjugate. It is classifiable as a
VI(c) signaling conjugate according to Table 1.
[0184] FIGS. 20(AD) are photomicrographs of tissues stained with
signaling conjugates having different chromogenic moieties. FIG.
20(E) shows UV-Vis spectra with traces corresponding to the
absorbance of the signaling conjugates, the traces corresponding to
the associated photomicrograph. FIG. 20(A) and trace "A" of FIG.
20(E) shows a Rhodamine 110 signaling conjugate. It is classifiable
as a III(b) signaling conjugate according to Table 1. FIG. 20(B)
and trace "B" of FIG. 20(E) shows a JOE
(6-Carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, succinimidyl
ester) signaling conjugate. It is classifiable as a III(c)
signaling conjugate according to Table 1. FIG. 20(C) and trace "C"
of FIG. 20(E) shows a gallocyanine signaling conjugate. It is
classifiable as a III(c) signaling conjugate according to Table 1.
FIG. 19(D) and trace "D" of FIG. 19(E) shows a carboxyrhodamine B
signaling conjugate. It is also classifiable as a III(c) signaling
conjugate according to Table 1.
[0185] In illustrative embodiments, a method is disclosed for
detecting multiple targets in a sample using spectrally distinct
signaling conjugates. In one embodiment, the method includes using
two or more signaling conjugates selected from those
classifications shown in Table 1. In another embodiment, the method
includes using three or more signaling conjugates selected from
those classifications shown in Table 1. In another embodiment, the
method includes using a first signaling conjugate from a first
classification I-VI and a second signaling conjugate selected from
a second classification I-VI, wherein the first and second
classifications are not the same. In another embodiment, the method
includes using a first signaling conjugate from a first
classification I-VI, a second signaling conjugate from a second
classification I-VI, and a third signaling conjugate from a third
classification I-VI, wherein the first, second, and third
classifications are not the same. In another embodiment, at least
one of the signaling conjugates has a FWHM classification of (e) or
narrower. In another embodiment, at least one of the signaling
conjugates has a FWHM classification of (d) or narrower. In another
embodiment, at least one of the signaling conjugates has a FWHM
classification of (c) or narrower. In another embodiment, at least
one of the signaling conjugates has a FWHM classification of (b) or
narrower. In another embodiment, at least two signaling conjugates
have FWHM classification of (e) or narrower. In another embodiment,
at least three signaling conjugates have FWHM classification of (e)
or narrower.
[0186] 2. Latent Reactive Moiety
[0187] The latent reactive moiety is configured to undergo
catalytic activation to form a reactive species that can covalently
bond with the sample or to other detection components. The
catalytic activation is driven by one or more enzymes (e.g.,
oxidoreductase enzymes and peroxidase enzymes, like horseradish
peroxidase). In the presence of peroxide, these enzymes can
catalyze the formation of reactive species. These reactive species,
e.g., free radicals, are capable of reacting with phenolic
compounds proximal to their generation, i.e., near the enzyme. The
phenolic compounds available in the sample are most often tyrosyl
residues within proteins. As such, the latent reactive moiety can
be added to a protein-containing sample in the presence of a
peroxidase enzyme and a peroxide (e.g., hydrogen peroxide), which
can catalyze radical formation and subsequently cause the reactive
moiety to form a covalent bond with the biological sample.
[0188] In particular disclosed embodiments, the latent reactive
moiety comprises at least one aromatic moiety. In exemplary
embodiments, the latent reactive moiety comprises a phenolic moiety
and binds to a phenol group of a tyrosine amino acid. It is
desirable, however, to specifically bind the labeling conjugate via
the latent reactive moiety at, or in close proximity to, a desired
target with the sample. This objective can be achieved by
immobilizing the enzyme on the target region, as described herein.
Only latent reactive moieties in close proximity to the immobilized
enzyme will react and form bonds with tyrosine residues in the
vicinity of, or proximal to, the immobilized enzyme, including
tyrosine residues in the enzyme itself, tyrosine residues in the
antibody to which the enzyme is conjugated, and/or tyrosine
residues in the sample that are proximal to the immobilized enzyme.
In particular disclosed embodiments, the labeling conjugate can be
bound proximally, such as within about 100 nm, within about 50 nm,
within about 10 nm, or within about 5 nm of the immobilized enzyme.
For example, the tyrosine residue may be within a distance of about
10 angstroms to about 100 nm, about 10 angstroms to about 50 nm,
about 10 angstroms to about 10 nm, or about 10 angstroms to about 5
nm from the immobilized enzyme. Such proximal binding allows the
target to be detected with at least the same degree of specificity
as conventional staining methods used with the detection methods
disclosed herein. For example, embodiments of the disclosed method
allow sub cellular structures to be distinguished, e.g., nuclear
membrane versus the nuclear region, cellular membrane versus the
cytoplasmic region, etc.
[0189] In particular disclosed embodiments, the latent reactive
moiety has the general formula illustrated below.
##STR00017##
With reference to Formula 5, R.sup.25 is selected from the group
consisting of hydroxyl, ether, amine, and substituted amine;
R.sup.26 is selected from the group consisting of alkyl, alkenyl,
alkynyl, aryl, heteroaryl, --OR.sub.m, --NR.sub.m, and --SR.sub.m,
where m is 1-20; n is 1-20; Z is selected from the group consisting
of oxygen, sulfur, and NR.sup.a where R.sup.a is selected from the
group consisting of hydrogen, aliphatic, aryl, and alkyl aryl. An
exemplary embodiment of the latent reactive moiety is tyramine (or
tyramide, which is the name given to a tyramine molecule conjugated
with the detectable label and/or optional linker), or a derivative
thereof.
[0190] In particular disclosed embodiments, the signaling conjugate
has a minimum concentration, when covalently deposited on the
sample, of greater than about 1.times.10.sup.11 molecules per
cm.sup.2.mu.m or greater than about to about 1.times.10.sup.13
molecules per cm.sup.2.mu.m within the biological sample. In
particular disclosed embodiments, the concentration of signaling
conjugate deposited ranges from about to about 1.times.10.sup.11
molecules per cm.sup.2.mu.m to about to about 1.times.10.sup.16
molecules per cm.sup.2.mu.m.
[0191] Embodiments of the disclosed signaling conjugate can be made
using the general procedure illustrated in Scheme 1. In particular
disclosed embodiments, the conjugate is formed without an optional
linker. For example, a carboxylic acid moiety of the chromophore
may be coupled with a tyramine molecule or tyramine derivative by
first converting the carboxylic acid to an activated ester and then
forming an amide bond between the chromophore and the tyramine
molecule or tyramine derivative. An exemplary method for making a
signaling conjugate without a linker is illustrated below in Scheme
1.
##STR00018##
[0192] In embodiments wherein the linker is present, the carboxylic
acid moiety of the chromophore may be coupled with an
amine-terminated linker (e.g., an alkylene oxide) by first
converting the carboxylic acid to an activated ester and then
forming an amide bond between the chromophore and the
amine-terminated linker. The remaining terminus of the linker may
then be activated and subsequently coupled with a tyramine molecule
or tyramine derivative. An exemplary method for making the
signaling conjugate is provided below in Scheme 2.
##STR00019##
[0193] Exemplary signaling conjugates are provided below.
##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025## ##STR00026## ##STR00027## ##STR00028##
[0194] D. Amplifying Conjugates
[0195] Also disclosed herein are conjugates suitable for amplifying
a signal obtained from carrying out the method disclosed herein.
The amplifying conjugates typically comprise a latent reactive
moiety, a detectable label, and an optional linker.
[0196] The detectable label of the amplifying conjugate may be any
detectable label provided herein. In particular disclosed
embodiments, the detectable label is a hapten, such as any of the
haptens disclosed herein. U.S. Pat. No. 7,695,929 is hereby
incorporated by reference herein in its entirety for disclosure
related to the structures and synthetic approaches to making
amplifying conjugates and their corresponding specific antibodies.
In particular disclosed embodiments, a hapten having an
electrophilic functional group (or having a functional group
capable of being converted to an electrophilic functional group) is
conjugated to the latent reactive moiety or to a linker, (e.g., an
aliphatic or poly(alkylene oxide) linker). In certain embodiments,
the hapten includes a carboxylic acid functional group, which is
converted to an activated, electrophilic carbonyl-containing
functional group, such as, but not limited to, an acyl halide, an
ester (e.g., a N-hydroxysuccinimide ester), or an anhydride. The
latent reactive moiety includes a nucleophilic functional group
(e.g., amino, hydroxyl, thiol, or anions formed therefrom) capable
of reacting with the hapten's activated electrophilic functional
group. The hapten's electrophilic group can be coupled to the
latent reactive moiety's nucleophilic group using organic coupling
techniques known to a person of ordinary skill in the art of
organic chemistry synthesis. In embodiments where the conjugate
includes a linker, the linker typically has a nucleophilic
functional group at one end and an electrophilic functional group
at the other end. The linker's nucleophilic group can be coupled to
the hapten's electrophilic group, and the linker's electrophilic
group can be activated and coupled to the latent reactive moiety's
nucleophilic group using organic coupling techniques known to a
person of ordinary skill in the art of organic chemical
synthesis.
[0197] In further illustrative embodiments, the signaling conjugate
is used as an amplifying conjugate. The signaling conjugate can be
used as an amplifying conjugate where the chromophore moiety is an
effective labeling moiety. In illustrative embodiments, an antibody
specific to a chromophore moiety enables that chromophore moiety to
serve as a signaling and labeling conjugate. From another
perspective, a hapten which possesses physical attributes, as
disclosed herein, for effective chromophore moieties, may be used
as both a chromophore moiety and as a hapten. There are particular
benefits of using a signaling conjugate as an amplifying conjugate.
In particular, the amplifying step would result in the deposition
of significant, e.g., potentially detectable, amounts of the
chromophore moiety. As such, the subsequent chromogenic detection
could be stronger. Similarly, as described herein with respect to
mixing chromogens from different classifications, a unique color
could be generated using the overlap of absorbances from two or
more chromophore moieties.
IV. Compositions
[0198] An illustrative composition according to the present
disclosure comprises a biological sample and a plurality of
signaling conjugates. In particular disclosed embodiments, the
composition comprises a biological sample that comprises one or
more enzyme-labeled targets. The enzyme used to label the target
may originate from a labeling conjugate, such as an enzyme
conjugate. The composition also may further comprise one or more
detection probes. The plurality of signaling conjugates are as
disclosed herein and are configured to provide a bright-field
signal. The plurality of signaling conjugates are covalently bound
proximally to or directly on the one or more targets. In particular
disclosed embodiments, configured to provide a bright-field signal
comprises choosing a particular chromogenic moiety for the
signaling conjugate that is capable of absorbing about 5% or more
of incident light. In particular disclosed embodiments, about 20%
of the incident light may be absorbed.
[0199] In additional disclosed embodiments, the composition
comprises a signaling conjugate that has been configured to provide
the particular wavelength maxima disclosed herein for the
chromogenic moieties of the signaling conjugates. Solely by way of
example, the signaling conjugate is configured to provide a
bright-field signal such that an absorbance peak having a
.lamda..sub.max as is disclosed herein. Two different absorbance
peaks also may be obtained by configuring different signaling
conjugates to comprise different chromogenic moieties that have
absorbance peaks of differing values, as disclosed herein. The
composition also may comprise a plurality of signaling conjugates
configured to provide a bright-field signal by being selected as
having a particular FWHM value. Suitable FWHM values are disclosed
herein. In other disclosed embodiments, at least a portion of the
plurality of signaling conjugates has an average molar absorptivity
selected from the particular values provided herein.
[0200] Particular disclosed embodiments of the composition also
concern a plurality of signaling conjugates that have a particular
solubility in water, such as those values provided herein. Also,
the plurality of signaling conjugates also may be stable in an
aqueous buffer solution for the period of time provided herein.
[0201] In particular disclosed embodiments, the composition
comprises a plurality of signaling conjugates that are configured
to impart an optically apparent color under bright-field
illumination, such as red, orange, yellow, green, indigo, or
violet. The optically apparent color may also be a mixture, such as
that a first optically distinct color, a second optically distinct
color, a third optically distinct color, a fourth optically
distinct color, and even a fifth optically distinct color may be
obtained and visualized.
[0202] The biological sample present in the disclosed composition
can be a tissue or cytology sample as is disclosed herein. In
particular disclosed embodiments, the biological sample may
comprise two targets, a first target and a second target and the
composition may further comprise a first detection probe that is
specific for the first target and a second detection probe that is
specific for the second target.
V. Kits
[0203] Also disclosed herein are embodiments of a kit comprising
the signaling conjugate disclosed herein. In another embodiment,
the kit includes a detection probe. In another embodiment, the kit
includes a labeling conjugate. In another embodiment, the kit
includes a amplifying conjugate and a secondary labeling conjugate.
In another embodiment, the kit may further comprise a peroxide
solution. In illustrative embodiments, the kit includes a detection
probe. In illustrative embodiments, the reagents of the kit are
packaged in containers configured for use on an automated slide
staining platform. For example, the containers may be dispensers
configured for use and a BENCHMARK Series automated slide
stainer.
[0204] In illustrative embodiments, the kit includes a series of
reagents contained in different containers configured to work
together to perform a particular assay. In one embodiment, the kit
includes a labeling conjugate in a buffer solution in a first
container. The buffer solution is configured to maintain stability
and to maintain the specific binding capability of the labeling
conjugate while the reagent is stored in a refrigerated environment
and as placed on the instrument. In another embodiment, the kit
includes a signaling conjugate in an aqueous solution in a second
container. In another embodiment, the kit includes a hydrogen
peroxide solution in a third container for concomitant use on the
sample with the signaling conjugate. In the second or third
container, various enhancers (e.g., pyrimidine) may be found for
increasing the efficiency by which the enzyme activates the latent
reactive species into the reactive species. In a further
embodiment, the kit includes an amplifying conjugate.
VI. Working Embodiments
General Procedures and Preparation
[0205] All ISH detection was performed on a Ventana Benchmark XT.
DNP or DIG labeled (0.25 ng/ml final concentration) probes were
hybridized for one to three hours in a formamide containing buffer,
followed by stringency washing in 2.times.SSC. Probe detection was
mediated by an anti-DNP or anti-DIG monoclonal antibody (2.5 ng/ml
final concentration) that had been conjugated to horseradish
peroxidase. Deposition of the signaling conjugate (12.5 .mu.M final
concentration) was catalyzed by the addition of H.sub.2O.sub.2
(final percentage of 0.003%).
[0206] For assays utilizing an intermediate amplification step, the
HRP conjugated anti-DNP or anti-DIG monoclonal antibody bound to
the probe catalyzes the deposition of the amplifying conjugate
(6.25 .mu.M final concentration) by the addition of H.sub.2O.sub.2.
The covalently bound amplifying conjugates in the tissue served as
binding sites for monoclonal enzyme conjugates (2.5 ng/ml final
concentration), and deposition of the signaling conjugate was
catalyzed by the addition of the signaling conjugate (25 .mu.M
final concentration) and H.sub.2O.sub.2.
Signaling Conjugate Testing:
[0207] Each tyramide dye solution was tested for functionality at a
range of micromolar to millimolar concentrations using an
immunohistochemistry model against Her2 protein on formalin-fixed,
paraffin embedded Calu-3, ZR75-1 and MCF-7 xenograft tissues
mounted on Superfrost slides. Tissues were stained using a
Benchmark XT Ventana automated slide staining instrument. Reagents
necessary for the testing include VMSI Her2 (4B5) Primary Antibody
VMSI product #790-2991, UltraMap anti-Rb HRP #760-4315, AmpMap
Detection Kit with TSA #760-121, Hematoxylin II #790-2208 and
Bluing Reagent #760-2037. Slides were de-paraffinized then antigen
retrieved using cell conditioning 1 solution (#950-124), followed
by the addition of the primary antibody for 16 minutes at
37.degree. C., secondary antibody for 16 minutes at 37.degree. C.
and amplification using a single tyramide solution in TSA Diluent
(#60900) or phosphate buffered saline with the addition of
TSA-H.sub.2O.sub.2 (VMSI #760-4141) and incubating the reaction for
20 min. Each slide was counterstained with 4 minute incubation of
Hematoxylin followed by a 4 minute incubation of Bluing solution
and dehydrated using gradient alcohols and coverslipped.
Signaling Conjugate Evaluation:
[0208] Evaluation of the tyramide signal was visualized by use of a
bright-field white light microscope. Each slide comprised of a
positive control for Her2 protein of high expression (Calu-3
xenograft) an intermediate protein level control (ZR75-1 xenograft)
and negative control for Her2 protein expression (MCF7 xenograft).
Tyramide solutions that had specific staining were further tested
for optimal dye intensity in the above assay before tissue staining
was performed for nucleotide targets.
[0209] Signaling Conjugate Solubility and pH:
[0210] Solubility and pH proved to be variables unique to each
tyramide dye. For instance, malachite green tyramide proved to be
insoluble in the basic, pH 8.5, TSA Diluent (VMSI product #60900)
but using a neutral pH of 7.4, phosphate buffered saline showed
better solubility and no alteration of color properties. Any pH
range less than 6.0 for malachite green tyramide turned the
original green solution to a yellow color which was undesired. It
was also found that for the tyramide dyes to be visualized in a
bright-field white light manner, very high concentrations, on the
order of 10 to 20 fold higher than used for fluorescence, needed to
be achieved to generate enough colored material on the tissue
slide. Stock solutions were formulated at millimolar or greater
concentrations and the working solution was diluted in an aqueous
buffer at optimal pH and solubility for each unique tyramide
dye.
Example 1
[0211] Interrogation of gene expression in tissue sections using
PCR or microarrays has been successfully used to classify patients'
likelihood of tumor recurrence and identify those who may benefit
from specific therapies. However, tissue specificity and cellular
context, which improve the value of tissue based assays are lost
during mRNA extraction. Moreover, false positive or negative
results may be generated from the presence of "contaminating"
non-tumor cells in the section. As such, there is a need for
automated in situ hybridization assays which target mRNA (mRNA-ISH)
that enables robust and reproducible evaluation of biomarker
expression while preserving tissue context and specificity, as well
as cell-cell relationships. Preservation of context and the ability
to minimize cell-cell nucleic acid (RNA) contamination is desired
for tests that interrogate cell clonality in which a cell expresses
either one of two biomarkers but never both.
[0212] Methods for analyzing a sample for expression of an mRNA
target are described. In illustrative embodiments the methods
include contacting the sample with a labeled nucleic acid probe.
Detection of the labeled probe creates a signal that corresponds to
the expression of the mRNA target. This disclosure further
describes compositions, kits, and methods for determination of cell
clonality in human cancer samples. Specifically, B cell lymphomas
resulting from clonal expansion of a specific B cell population
expressing either KAPPA or LAMBDA mRNA are described.
[0213] In illustrative embodiments, a method for simultaneously
analyzing a sample for expression of two mRNA targets includes
contacting the sample with a mRNA target probe, wherein the mRNA
target probe is labeled with a first hapten, contacting the sample
with an internal mRNA standard probe, wherein the internal mRNA
standard probe is labeled with a second hapten, contacting the
sample with a first chromogenic detection reagent, contacting the
sample with a second chromogenic detection reagent, detecting a
second signal from the second chromogenic detection reagent, the
second signal providing the expression of the internal mRNA
standard, and detecting a first signal from the first chromogenic
detection reagent, the first signal providing the expression of the
mRNA target. In one embodiment, detecting the second signal below a
predetermined signal level indicates the sample lacks integrity for
analysis of the mRNA target.
[0214] Cancer results from uncontrolled growth of a cell
population; this population may arise from a single mutant parent
cell and, therefore, comprise a clonal population. An example of
cancer derived from a clonal population is B-cell non-Hodgkin
lymphomas (B-NHL) which arise from monoclonal proliferation of B
cells. Clonal expansion of a specific B cell population can be
detected by sole expression of either Kappa or Lambda light chain
mRNA and protein as part of their B cell receptor antibody. One
approach for the identification of monoclonal proliferation of B
cells is chromogenic dual staining of Kappa and Lambda mRNA.
Referring to FIG. 21(A-B), shown is an exemplary chromogenic dual
staining approach.
[0215] Uniform expression of either light chain by malignant B
cells enables differentiation of monoclonal B cell lymphomas from
polyclonal Kappa and Lambda light chain expressing B cell
populations that result during the normal immune response.
Determination of light chain mRNA expression patterns is
complicated by the copy number range of light chain mRNA and
antibody protein expressed by B cell neoplasms derived from a
variety of B cell stages (naive and memory cells:10-100 copies per
cell; plasma cells: .about.100 thousand copies per cell). FIG. 22
is a schematic showing expected Kappa/Lambda copy numbers
associated with different types of non-Hodgkins B-cell
lymphomas.
[0216] While the present disclosure describes, in particularity,
sensitive methods of analyzing a sample using KAPPA and LAMBDA mRNA
in tissue samples expressing a range of light chain mRNA copy
numbers, the approaches described herein are general and applicable
to various useful biomarkers expressed uniquely by specific cell
populations. The application of the disclosed technology to
additional target and standard mRNA probes is within the scope of
the present disclosure. By so applying the disclosed technology,
the present method enables the interrogation of additional disease
states and development of improved predictive and prognostic
analyses for cancer patients as well as novel companion
diagnostics. Furthermore, while the disclosure describes two-color
mRNA ISH analysis, the scope of the present disclosure includes
additional colors (e.g., three-color, four-color, etc.).
[0217] In illustrative embodiments, a method for determining cell
clonality by analyzing a sample for expression of mRNA targets
which are uniquely expressed by a specific cell population
comprises contacting the sample with a first mRNA target probe,
wherein the first mRNA target probe is labeled with a first hapten,
contacting the sample with a second mRNA target probe, wherein the
second mRNA target probe is labeled with a second hapten,
contacting the sample with a first chromogenic detection reagent,
contacting the sample with a second chromogenic detection reagent,
detecting a first signal from the first chromogenic detection
reagent, the first signal providing the expression of the first
mRNA target, detecting a second signal from the second chromogenic
detection reagent, the second signal providing the expression of
the second mRNA target. In one embodiment, the first and the second
signal indicate cell clonality for the sample. In another
embodiment, the sample is a specific B cell population and the
first and the second signal correspond to KAPPA or LAMBDA mRNA.
[0218] Probe Preparation and Formulation: Complementary (antisense)
and non-complementary (sense) KAPPA and LAMBDA riboprobes were in
vitro transcribed from PCR amplified dsDNA templates containing the
T7 promoter. The nucleic acids were chemically labeled with
different haptens (DIG, DNP) using linker arms prepared as directed
by the manufacturer (Label IT.RTM. Technology, Minis Bio LLC,
Madison, Wis.) and NHS-PEGS-haptens. Twenty-five nanograms of each
probe was suspended in one mL of a hybridization buffer
(Ribohybe.TM., VMSI #760-104) and placed into a dispenser (VMSI,
#760-205) compatible with an automated slide staining instrument
(VMSI, Discovery XT #F-DISXT-750000). mRNA in situ hybridizations
and detection: Samples were stained using mRNA
[0219] ISH reagents (RiboMap, VMSI #760-102). Formalin-fixed,
paraffin-embedded clinical tonsil and lymphoma tissue samples were
mounted on slides (SuperFrost Ultra Plus.RTM., Menzel-Glaser) were
de-paraffined and antigen retrieved using cell conditioning
reagents (Cell Conditioning 1, VMSI #950-124 and protease 3, VMSI
#760-2020). Following retrieval, one drop (100 .mu.L) of cocktailed
hapten-labeled HER2 and ACTB anti-sense strand probes were
dispensed onto the slide, denatured at 80.degree. C. for 8 min, and
hybridized at 65.degree. C. for 6 hrs. Following hybridization, the
slides were washed 3 times using a stringency buffer (0.1.times.SSC
VMSI #950-110) at 75.degree. C. for 8 minutes to remove
non-specifically hybridized probe.
[0220] A two-tiered amplification procedure was used to amplify the
signal for each of the binding events. Reagents included (1) an
HRP-conjugated anti-hapten antibody to catalyze deposition of (2) a
tyramide-hapten conjugate which was then bound by (3) a second
HRP-conjugated anti-hapten antibody. The HRP was used to catalyze
deposition of a chromophore and tyramide conjugate for LAMBDA and
DAB for KAPPA.
[0221] Endogenous tissue peroxidase activity was inactivated by
dispensing one drop an inhibitor (PO inhibitor, VMSI #760-4143) and
incubating the reaction for 12 min. Following several washes, one
drop of a second amplification blocking reagent (TSA block, VMSI
#760-4142) was dispensed onto the slide and incubated 4 min. Next,
a drop of HRP-conjugated anti-hapten monoclonal antibody solution
was dispensed (2.5 .mu.g/ml conjugate prepared in avidin diluent
plus B5 blocker, VMSI #90040); the mixture was incubated for 28
min. Tyramide-mediated hapten amplification was accomplished by
dispensing one drop of tyramide-hapten conjugate on the slide
followed by one drop of a hydrogen peroxide solution (TSA-H2O2,
VMSI #760-4141) and allowing the reaction to incubate for 20
min.
[0222] The procedure was repeated to direct tyramide-mediated
amplification of the second hapten in the probe cocktail. Control
studies demonstrated the use of three successive applications of
the peroxide inhibitor to inactivate the previous HRP-conjugated
anti-hapten antibody was preferred. Omission of the inactivation
step resulted in co-localization of signals and non-specific mRNA
signals. The LAMBDA amplified hapten was then sequentially detected
using a similar amplification strategy which included three
applications of the peroxide inhibitor, application of a cognate
anti-hapten monoclonal antibody and application of a
tyramide-chromophore conjugate and peroxide. The hapten designating
KAPPA was detecting using a DAB detection reagent (OptiView DAB,
VMSI #760-700).
[0223] Tissue nuclei were then stained using a hematoxylin solution
and bluing reagent (VMSI, Hematoxylin II, #790-2208 Bluing Reagent,
#760-2037). Slides were then dehydrated using gradient alcohols and
coverslipped.
[0224] Exemplary photomicrographs of tissue samples treated
according the above procedures are shown in FIGS. 23(A-B), which
are photomicrographs of (A) a first lymphoma tissue sample showing
a dual staining of KAPPA mRNA (brown) and LAMBDA mRNA (purple,
minimally observed), showing very few cells expressing LAMBDA mRNA
and (B) a second lymphoma tissue sample showing a dual staining for
KAPPA mRNA (brown, minimally observed) and LAMBDA mRNA (purple),
showing very few cells expressing KAPPA mRNA. The nearly monoclonal
populations observed are indicative of a cancer.
[0225] FIG. 24(A-B) are photomicrographs of a dual-color mRNA-ISH
KAPPA (brown) and LAMBDA (purple) assay for a tissue. In FIG.
24(A), the polyclonal B cell population is clearly stained with
either purple or brown indicating the cells are expressing either
LAMBDA or KAPPA mRNA. The sample exhibits high levels of expression
for both KAPPA and LAMBDA mRNA. FIG. 24(B) shows a portion of the
sample exhibiting a monoclonal cellular population indicative of
cancer. The high expressions of KAPPA and LAMBDA mRNA expression in
the sample, as a whole, would confound a molecular analysis of the
sample as the difference between the KAPPA and LAMBDA mRNA
expression is minimal. However, because the expression of KAPPA and
LAMBDA mRNA is visualized through a histopathological analysis, the
dual-staining approach described herein enables detection of the
monoclonal population.
[0226] Two-color mRNA-ISH is technically feasible for a large
majority of samples as a replacement or as a complement to existing
and yet undiscovered ISH and IHC analyses. Differentiation of
clonal lymphoma samples from non-clonal reactive processes was
empowered by the two-color detection system. Moreover, the assay's
utility for sensitive detection and discrimination of low copy mRNA
targets in various lymphoma cases was demonstrated. Collectively,
these observations indicate that the approach is useful for
determination of cell clonality using mRNA biomarkers expressed
uniquely by a specific population.
[0227] Furthermore, the use of chromophore and tyramide conjugates
enables a new class of two-color chromogenic analysis. The
conjugates are amenable to multiplexing due to their narrow
band-widths (e.g., FWHM). The conjugates are stable as reagents for
extended periods of time. The conjugates are covalently bound to
the tissue as opposed to traditional chromogen systems which
precipitate, thus the conjugates are not adversely affected by
post-staining processing or subsequent staining steps. The dramatic
amplification of the target enables bright-field detection and
significant concentrations of the chromophore localized proximally
to the target. These high concentrations overcome many concerns
associated with photo-bleaching, especially as compared to the
concentrations appropriate for fluorescent detection. Use of the
new chromophore and tyramide conjugates has enabled an important
new class of analytical methodologies--chromogenic mRNA ISH.
Example 2
[0228] Obstacles to mRNA-ISH assay utility in biological samples
(e.g., formalin-fixed paraffin embedded tissues, "FFPE tissues")
include variation in sample preparation (e.g., tissue fixation)
which influences sample mRNA integrity/accessibility and assay
performance. One aspect of the present disclosure is that automated
mRNA-ISH assays for FFPE samples have been developed which enable
simultaneous analysis of biomarker expression and an internal
control gene expression to monitor assay performance and sample
integrity. According to one specific example, clinical breast
cancer FFPE tissue blocks were characterized for HER2 gene copy
number and Her2 protein expression using INFORM HER2 Dual ISH and
IHC assays (Ventana Medical Systems, Inc.), respectively. HER2 mRNA
expression levels relative to ACTB (.beta.-actin) were determined
using qPCR according to known methods. Results of the gene copy,
protein expression, and qPCR analyses were compared to results
obtained through mRNA-ISH detection of HER2 and ACTB mRNA in FFPE
samples (FIG. 27). Varied tissue retrieval conditions were used to
test the utility of an internal mRNA standard to identify samples
for which mRNA integrity is compromised.
[0229] While the present disclosure describes, in particularity,
methods of analyzing a sample using HER2 and ACTB mRNA, the
approaches described herein are general and applicable to various
useful biomarkers. The application of the disclosed technology to
additional target and standard mRNA probes is within the scope of
the present disclosure.
[0230] In illustrative embodiments, a method for analyzing a sample
for expression of an mRNA target and an internal mRNA standard
includes contacting the sample with a mRNA target probe, wherein
the mRNA target probe is labeled with a first hapten, contacting
the sample with an internal mRNA standard probe, wherein the
internal mRNA standard probe is labeled with a second hapten,
contacting the sample with a first signaling conjugate, contacting
the sample with a second signaling conjugate, detecting a second
signal from the second signaling conjugate, the second signal
providing the expression of the internal mRNA standard, and
detecting a first signal from the first signaling conjugate, the
first signal providing the expression of the mRNA target. In one
embodiment, detecting the second signal below a predetermined
signal level indicates the sample lacks suitability for analysis of
the mRNA target. In another embodiment, detecting the first signal
includes determining the expression of the mRNA
semi-quantitatively.
[0231] In illustrative embodiments, contacting the sample with the
first signaling conjugate includes contacting the sample with a
first anti-hapten antibody and enzyme conjugate, the first
anti-hapten antibody and enzyme conjugate being specific to the
first hapten, contacting the sample with a third hapten and
tyramide derivative conjugate, contacting the sample with a third
anti-hapten antibody and enzyme conjugate, the third anti-hapten
antibody and enzyme conjugate being specific to the third hapten,
and contacting the sample with a first chromogen. In further
illustrative embodiments, contacting the sample with the second
signaling conjugate includes contacting the sample with a second
anti-hapten antibody and enzyme conjugate, the second anti-hapten
antibody and enzyme conjugate being specific to the second hapten,
contacting the sample with a fourth hapten and tyramide conjugate,
contacting the sample with a fourth anti-hapten antibody and enzyme
conjugate, the fourth anti-hapten antibody being specific to the
fourth hapten, and contacting the sample with a second chromogen.
In one embodiment, the first chromogen is selected from the group
consisting of DAB, AEC, CN, BCIP/NBT, fast red, fast blue, fuchsin,
NBT, and ALK GOLD. In another embodiment, the second chromogen
comprises a chromophore and tyramide conjugate. In one embodiment,
the second chromogen is selected from the group consisting of DAB,
AEC, CN, BCIP/NBT, fast red, fast blue, fuchsin, NBT, and ALK GOLD.
In yet another embodiment, the first chromogen comprises a
chromophore and tyramide conjugate.
[0232] Probe Preparation and Formulation: Complementary (antisense)
and non-complementary (sense) HER2 and ACTB riboprobes were in
vitro transcribed from PCR amplified dsDNA templates containing the
T7 promoter. The nucleic acids were chemically labeled with
different haptens (DIG, DNP) using linker arms prepared as directed
by the manufacturer (Label IT.RTM. Technology, Mirus Bio LLC,
Madison, Wis.) and NHS-PEGS-haptens. Twenty-five nanograms of each
probe was suspended in one mL of a hybridization buffer
(Ribohybe.TM., VMSI #760-104) and placed into a dispenser (VMSI,
#760-205) compatible with an automated slide staining instrument
(VMSI, Discovery XT #F-DISXT-750000).
[0233] mRNA in situ hybridizations and detection: Samples were
stained using mRNA ISH reagents (RiboMap, VMSI #760-102).
Formalin-fixed, paraffin-embedded clinical breast tissue samples
were mounted on slides (SuperFrost Ultra Plus.RTM., Menzel-Glaser)
were de-paraffined and antigen retrieved using cell conditioning
reagents (Cell Conditioning 1, VMSI #950-124 and protease 3, VMSI
#760-2020). Following retrieval, one drop (100 .mu.L) of cocktailed
hapten-labeled HER2 and ACTB anti-sense strand probes were
dispensed onto the slide, denatured at 80.degree. C. for 8 minutes,
and hybridized at 65.degree. C. for 6 hrs. Following hybridization,
the slides were washed 3 times using a stringency buffer
(0.1.times.SSC VMSI #950-110) at 75.degree. C. for 8 minutes to
remove non-specifically hybridized probe.
[0234] A two-tiered amplification procedure was used to amplify the
signal for each of the binding events. Reagents included (1) an
HRP-conjugated anti-hapten antibody to catalyze deposition of (2) a
tyramide-hapten conjugate which was then bound by (3) a second
HRP-conjugated anti-hapten antibody. The HRP was used to catalyze
deposition of a chromophore and tyramide conjugate for ACTB and DAB
for HER2.
[0235] Endogenous tissue peroxidase activity was inactivated by
dispensing one drop an inhibitor (PO inhibitor, VMSI #760-4143) and
incubating the reaction for 12 min. Following several washes, one
drop of a second amplification blocking reagent (TSA block, VMSI
#760-4142) was dispensed onto the slide and incubated 4 min. Next,
a drop of HRP-conjugated anti-hapten monoclonal antibody solution
was dispensed (2.5 .mu.g/ml conjugate prepared in avidin diluent
plus B5 blocker, VMSI #90040); the mixture was incubated for 28
min. Tyramide-mediated hapten amplification was accomplished by
dispensing one drop of tyramide-hapten conjugate on the slide
followed by one drop of a hydrogen peroxide solution (TSA-H2O2,
VMSI #760-4141) and allowing the reaction to incubate for 20
min.
[0236] The procedure was repeated to direct tyramide-mediated
amplification of the second hapten in the probe cocktail. Control
studies demonstrated the use of three successive applications of
the peroxide inhibitor to inactivate the previous HRP-conjugated
anti-hapten antibody was preferred. Omission of the inactivation
step resulted in co-localization of signals and non-specific mRNA
signals. The ACTB amplified hapten was then sequentially detected
using a similar amplification strategy which included three
applications of the peroxide inhibitor, application of a cognate
anti-hapten monoclonal antibody and application of a
tyramide-chromophore conjugate and peroxide. The hapten designating
HER2 was detecting using a DAB detection reagent (OptiView DAB,
VMSI #760-700).
[0237] Tissue nuclei were then stained using a hematoxylin solution
and bluing reagent (VMSI, Hematoxylin II, #790-2208 Bluing Reagent,
#760-2037). Slides were then dehydrated using gradient alcohols and
coverslipped.
[0238] Exemplary photomicrographs of tissue samples treated
according the above procedures are shown in FIGS. 25(A-B). FIG.
25(A) shows a photomicrograph of (A) an ACTB analysis performed on
a tissue sample fixed for 4 hours and (B) a tissue sample fixed for
24 hours. The first sample (FIG. 25(A)) includes weak ACTB staining
which was classified as lacking sample integrity due to the
improper fixing conditions. The second sample (FIG. 25(B)) includes
strong ACTB staining and was classified as suitable for HER2
evaluation (FIGS. 25(A-B) include only a single color). FIGS.
26(A-C) show examples of clinical tissue sample exhibiting
two-color mRNA ISH staining of ACTB mRNA and (A) negative (0+) HER2
mRNA ISH staining, (B) positive (1/2+) HER2 mRNA ISH staining, and
(C) positive (3+) HER2 mRNA ISH staining. FIG. 28 is data from 20
tissue blocks including the results of HER2 ISH analysis (VENTANA
INFORM HER2 Dual ISH assay, VMSI), HER2 IHC analysis (PATHWAY
HER-2/neu, OptiView DAB, VMSI), and HER2 mRNA two-color ISH.
[0239] It was discovered that mRNA ACTB signals were influenced by
assay pre-hybridization treatment and, therefore, useful for
evaluation of assay performance and determination of appropriate
assay conditions. HER2 mRNA-ISH signals predominantly correlated
with copy number and protein expression in samples with concordant
copy number and protein levels; in discordant samples (normal copy
number with increased protein expression or increased copy number
with little detectable protein expression) HER2 mRNA-ISH signals
were largely elevated. Collectively, these observations suggest
that the mRNA-ISH assay may serve as a companion assay to clarify
samples harboring discordant HER2 gene copy number and protein
levels. Moreover, these studies demonstrate utility of an
accessible bright-field assay platform for gene expression that
preserves cellular context in FFPE tissues.
[0240] From the above and the data included in FIG. 4, the
following conclusions were drawn. Two-color mRNA-ISH is technically
feasible for a large majority of samples as a replacement or as a
complement to existing and yet undiscovered ISH and IHC analyses.
The inclusion of an ACTB internal control, or a like internal
control, enables identification of tissues not suitable for
analysis and/or assay failures. Accordingly, the present disclosure
describes new approaches to diminishing false negative rates due to
unsuitability of the sample or from assay failure. HER2 mRNA-ISH
signals may be classified into three expression patterns largely
concordant with established conventional Her2 protein levels. Where
HER2 DNA-ISH and IHC are discordant in 10% and 5% of samples,
respectively. Gene expression analyses (qPCR and mRNA-ISH)
correlate with either DNA copy number or protein levels in
discordant samples. Two-color bright-field HER2/ACTB mRNA-ISH assay
may serve as a companion test to clarify discordant samples.
[0241] Furthermore, the use of chromophore and tyramide conjugates
enables a new class of two-color chromogenic analysis. The
conjugates are amenable to multiplexing due to their narrow
band-widths (e.g., FWHM). The conjugates are stable as reagents for
extended periods of time. The conjugates are covalently bound to
the tissue as opposed to traditional chromogen systems which
precipitate, thus the conjugates are not adversely affected by
post-staining processing or subsequent staining steps. The dramatic
amplification of the target enables bright-field detection and
significant concentrations of the chromophore localized proximally
to the target. These high concentrations overcome many concerns
associated with photo-bleaching, especially as compared to the
concentrations appropriate for fluorescent detection. Use of the
new chromophore and tyramide conjugates has enabled an important
new class of analytical methodologies--chromogenic mRNA ISH.
Example 3
[0242] DNP or DIG labeled (0.25 ng/ml final concentration) PTEN DNA
ISH probes were hybridized for one to three hours in a formamide
containing buffer, followed by stringency washing in 2.times.SSC.
Probe detection was mediated by an anti-DNP or anti-DIG monoclonal
antibody (2.5 ng/ml final concentration) that had been conjugated
to horseradish peroxidase. Deposition of Rhodamine-tyramide (12.5
.mu.M final concentration) was catalyzed by the addition of
H.sub.2O.sub.2 (final percentage of 0.003%). FIGS. 28(A-B) show
results obtained from using this embodiment to detect a PTEN DNA
ISH probe in VCAP xenograft tumor cells. FIG. 28(A) is an image
taken at 40.times. magnification, and FIG. 28(B) is an image of a
separate area of the tissue taken at 63.times. magnification.
Example 4
[0243] DNP or DIG labeled (0.25 ng/ml final concentration) ERG5'
DNA ISH probes were hybridized for one to three hours in a
formamide containing buffer, followed by stringency washing in
2.times.SSC. Probe detection was mediated by an anti-DNP or
anti-DIG monoclonal antibody (2.5 ng/ml final concentration) that
had been conjugated to horseradish peroxidase. Deposition of
Rhodamine-tyramide (12.5 .mu.M final concentration) was catalyzed
by the addition of H.sub.2O.sub.2 (final percentage of 0.003%).
[0244] Additionally, an HRP conjugated anti-DNP or anti-DIG
monoclonal antibody bound to the probe is used to catalyze
tyramide-BF deposition (6.25 .mu.M final concentration) by the
addition of H.sub.2O.sub.2. The covalently bound amplifying
conjugate in the tissue served as binding sites for monoclonal
anti-BF antibodies conjugated to HRP (2.5 ng/ml final
concentration), and deposition of the signaling conjugate was
catalyzed by the addition of the signaling conjugate (25 .mu.M
final concentration) and H.sub.2O.sub.2.
[0245] FIG. 29 shows results obtained from using this embodiment to
detect an ERG5' DNA ISH probe in MCF7 xenograft tumor cells.
Example 5
[0246] DNP or DIG labeled (0.25 ng/ml final concentration) ERG3'
DNA ISH probes were hybridized for one to three hours in a
formamide containing buffer, followed by stringency washing in
2.times.SSC. Probe detection was mediated by an anti-DNP or
anti-DIG monoclonal antibody (2.5 ng/ml final concentration) that
had been conjugated to horseradish peroxidase. Deposition of
Dabsyl-tyramide (12.5 .mu.M final concentration) was catalyzed by
the addition of H.sub.2O.sub.2 (final percentage of 0.003%).
[0247] Additionally, an HRP conjugated anti-DNP or anti-DIG
monoclonal antibody bound to the probe is used to catalyze
amplifying conjugate deposition (6.25 .mu.M final concentration) by
the addition of H.sub.2O.sub.2. The covalently bound amplifying
conjugate in the tissue served as binding sites for monoclonal
anti-NP antibodies conjugated to HRP (2.5 ng/ml final
concentration), and deposition of the signaling conjugate was
catalyzed by the addition of the signaling conjugate (25 .mu.M
final concentration) and H.sub.2O.sub.2.
[0248] FIG. 30 illustrates results obtained from using this
embodiment to detect an ERG3' DNA ISH probe in MCF7 xenograft tumor
cells.
Example 6
[0249] DNP or DIG labeled (0.25 ng/ml final concentration) ERG3'
and ERG5' DNA ISH probes were hybridized for one to three hours in
a formamide containing buffer, followed by stringency washing in
2.times.SSC. Probe detection was mediated by an anti-DNP or
anti-DIG monoclonal antibody (2.5 ng/ml final concentration) that
had been conjugated to horseradish peroxidase. Deposition of
Rhodamine-tyramide and Dabsyl-tyramide conjugates (12.5 .mu.M final
concentration) was catalyzed by the addition of H.sub.2O.sub.2
(final percentage of 0.003%).
[0250] Additionally, an HRP conjugated anti-DNP or anti-DIG
monoclonal antibody bound to the probe is used to catalyze
amplifying conjugate deposition (6.25 .mu.M final concentration) by
the addition of H.sub.2O.sub.2. The covalently bound amplifying
conjugate in the tissue served as binding sites for monoclonal
anti-BF and anti-NP antibodies conjugated to HRP (2.5 ng/ml final
concentration), and deposition of the signaling conjugates was
catalyzed by the addition of the signaling conjugate (25 .mu.M
final concentration) and H.sub.2O.sub.2.
[0251] FIG. 31 shows results obtained from using this embodiment to
detect both ERG3' and ERG5' DNA ISH probes in MCF7 xenograft tumor
cells. The red probe signals are generated from combined detection
of the ERG5'-rhodamine signal, and the ERG3' Dabsyl signal.
Example 7
[0252] This embodiment concerns detecting an ERG gene rearrangement
in prostate carcinoma cells using multiple signaling
conjugates.
[0253] DNP or DIG labeled (0.25 ng/ml final concentration) ERG3'
and ERG5' DNA ISH probes were hybridized for one to three hours in
a formamide containing buffer, followed by stringency washing in
2.times.SSC. Probe detection was mediated by an anti-DNP or
anti-DIG monoclonal antibody (2.5 ng/ml final concentration) that
had been conjugated to horseradish peroxidase. Deposition of
Rhodamine-tyramide and Dabsyl-tyramide conjugates (12.5 .mu.M final
concentration) was catalyzed by the addition of H.sub.2O.sub.2
(final percentage of 0.003%).
[0254] Additionally, an HRP conjugated anti-DNP or anti-DIG
monoclonal antibody bound to the probe is used to catalyze
amplifying conjugate deposition (6.25 .mu.M final concentration) by
the addition of H.sub.2O.sub.2. The covalently bound amplifying
conjugate in the tissue served as binding sites for monoclonal
anti-BF and anti-NP antibodies conjugated to HRP (2.5 ng/ml final
concentration), and deposition of the signaling conjugates was
catalyzed by the addition of the signaling conjugate (25 .mu.M
final concentration) and H.sub.2O.sub.2.
[0255] FIG. 32 illustrates results obtained from using this
embodiment to detect both ERG3' and ERG5' DNA ISH probes in VCAP
xenograft tumor cells. Individual and fused probe signals are
indicated with arrows: the fused ERG5'-Rhodamine and ERG3'-Dabsyl
signal (red signal at arrow) splitting into a separate purple
ERG5'-Rhodamine signal (at arrow head) and a separate yellow
ERG3'-Dabsyl signal (at thick, block arrow).
Example 8
[0256] This embodiment concerns detecting an ALK gene rearrangement
in the CARPUS carcinoma cells using multiple signaling
conjugates.
[0257] DNP or DIG labeled (0.25 ng/ml final concentration) Alk3'
and Alk5' DNA ISH probes were hybridized for one to three hours in
a formamide containing buffer, followed by stringency washing in
2.times.SSC. Probe detection was mediated by an anti-DNP or
anti-DIG monoclonal antibody (2.5 ng/ml final concentration) that
had been conjugated to horseradish peroxidase. Deposition of
Rhodamine-tyramide and Dabsyl-tyramide conjugates (12.5 .mu.M final
concentration) was catalyzed by the addition of H.sub.2O.sub.2
(final percentage of 0.003%).
[0258] Additionally, an HRP conjugated anti-DNP or anti-DIG
monoclonal antibody bound to the probe is used to catalyze
amplifying conjugate deposition (6.25 .mu.M final concentration) by
the addition of H.sub.2O.sub.2. The covalently bound amplifying
conjugate in the tissue served as binding sites for monoclonal
anti-BF and anti-NP antibodies conjugated to HRP (2.5 ng/ml final
concentration), and deposition of the signaling conjugates was
catalyzed by the addition of the signaling conjugate (25 .mu.NI
final concentration) and H.sub.2O.sub.2.
[0259] FIG. 33 illustrates results obtained from using this
embodiment to detect both Alk3' and Alk5' DNA ISH probes in a
CARPUS cell pellet. Probe signals in two cells with the ALK gene
rearrangement have been indicated with arrows; the fused
Alk5'-Rhodamine and Alk3'-Dabsyl signal (red signal at arrow)
splitting into a separate purple Alk5'-Rhodamine signal (at arrow
head) and a separate yellow Alk3'-Dabsyl signal (at thick, block
arrow).
Example 9
[0260] This embodiment concerns detecting an ALK gene rearrangement
in human lung cancer tissue using multiple signaling
conjugates.
[0261] DNP or DIG labeled (0.25 ng/ml final concentration) Alk3'
and Alk5' DNA ISH probes were hybridized for one to three hours in
a formamide containing buffer, followed by stringency washing in
2.times.SSC. Probe detection was mediated by an anti-DNP or
anti-DIG monoclonal antibody (2.5 ng/ml final concentration) that
had been conjugated to horseradish peroxidase. Deposition of
Rhodamine-tyramide and Dabsyl-tyramide conjugates (12.5 .mu.M final
concentration) was catalyzed by the addition of H.sub.2O.sub.2
(final percentage of 0.003%).
[0262] Additionally, an HRP conjugated anti-DNP or anti-DIG
monoclonal antibody bound to the probe is used to catalyze
amplifying conjugate deposition (6.25 .mu.M final concentration) by
the addition of H.sub.2O.sub.2. The covalently bound amplifying
conjugate in the tissue served as binding sites for monoclonal
anti-BF and anti-NP antibodies conjugated to HRP (2.5 ng/ml final
concentration), and deposition of the signaling conjugates was
catalyzed by the addition of the signaling conjugate (25 .mu.M
final concentration) and H.sub.2O.sub.2.
[0263] FIG. 34 illustrates results obtained from using this
embodiment to detect both Alk3' and Alk5' DNA ISH probes in a 4
micron section of lung adenocarcinoma. The area within the box
indicates a tumor cell where one copy of the ALK gene has
rearranged, splitting the combined Alk5'-Rhodamine and Alk3'-Dabsyl
signal (red signal at arrow) into a separate purple Alk5'-Rhodamine
signal (at arrow head) and a separate yellow Alk3'-Dabsyl signal
(at thick, block arrow).
Example 10
[0264] This embodiment concerns detecting 18S RNA targets using two
different colors of signaling conjugates simultaneously so as to
create a third color. FIGS. 35(A-C) are photomicrographs
illustrating direct detection of gene targets in Calu-3 cells using
an mRNA ISH assay. FIG. 35(A) shows detection of 18S RNA target
using a Rhodamine-tyramide conjugate. FIG. 35(B) shows detection of
18S RNA target using direct deposition of a DABSYL-tyramide
conjugate. FIG. 35(C) illustrates a detection with both the
DABSYL-tyramide conjugate and the Rhod-tyramide conjugate. The
signal observed in FIG. 35(A) appears purple, the signal in FIG.
35(B) appears orange, and the signal in FIG. 35(C) appears red.
FIG. 36 is a photomicrograph illustrating detecting, directly, HER2
and P53 proteins in Calu-3 cells using a multiplexed IHC assay.
HER2 is detected by direct deposition of DABSYL-tyramide conjugate.
P53 is detected by direct deposition of Rhodamine-tyramide
conjugate. While the two signaling conjugates shown in FIGS.
35(A-B) can be used together to generate a third, combination,
color, these two chromogens can also be used in a multiplexed
format in which each color is assignable to a particular
target.
[0265] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only preferred
examples of the invention and should not be taken as limiting the
scope of the invention. Rather, the scope of the invention is
defined by the following claims. We therefore claim as our
invention all that comes within the scope and spirit of these
claims.
* * * * *