U.S. patent application number 16/838623 was filed with the patent office on 2020-09-17 for method for evaluation of target in histological sample.
The applicant listed for this patent is Agilent Technologies, Inc.. Invention is credited to Kirsten Damgaard Hoff, Lars Christian Jacobsen, Kristian Jensen, Rikke Malene Krusenst Jerna-Hafstrom, Jesper Lohse.
Application Number | 20200292535 16/838623 |
Document ID | / |
Family ID | 1000004856499 |
Filed Date | 2020-09-17 |
View All Diagrams
United States Patent
Application |
20200292535 |
Kind Code |
A1 |
Lohse; Jesper ; et
al. |
September 17, 2020 |
METHOD FOR EVALUATION OF TARGET IN HISTOLOGICAL SAMPLE
Abstract
The present invention lies in the field of visualization and
quantification of immobilized targets in samples using
immunochemical means. In particular, the invention relates to a
method and reagents for detection, visualization and quantification
of a molecular target in immunosiained histological samples using
particular compositions of the target specific binding agent.
Methods and compositions of the invention are suitable for any
assay that uses a target visualization system in histological
samples based on detection of the target by the target specific
binding agent. The methods and compositions tire useful for
evaluation of targets that are biomarkers of diseases in medical
diagnostic.
Inventors: |
Lohse; Jesper; (Herlev,
DK) ; Jensen; Kristian; (Ringsted, DK) ;
Krusenst Jerna-Hafstrom; Rikke Malene; (Bikerod, DK)
; Hoff; Kirsten Damgaard; (Malov, DK) ; Jacobsen;
Lars Christian; (Copenhangen NV, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Agilent Technologies, Inc. |
Santa Clara |
CA |
US |
|
|
Family ID: |
1000004856499 |
Appl. No.: |
16/838623 |
Filed: |
April 2, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14356735 |
May 7, 2014 |
10648970 |
|
|
PCT/DK2012/000119 |
Nov 8, 2012 |
|
|
|
16838623 |
|
|
|
|
61556916 |
Nov 8, 2011 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5308 20130101;
G01N 33/6854 20130101; G01N 33/58 20130101 |
International
Class: |
G01N 33/53 20060101
G01N033/53; G01N 33/68 20060101 G01N033/68; G01N 33/58 20060101
G01N033/58 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 8, 2011 |
DK |
PCT/DK2011/000131 |
Claims
1-16. (canceled)
17. A kit-of-parts comprising a composition comprising a binding
agent which is capable of specifically binding to the binding
partner comprised in said one or more target sites, wherein the
amount of the binding agent is sufficient to bind to substantially
all units of the binding partner present in the sample, and wherein
the binding agent comprises first binding molecules and second
binding molecules, wherein the first binding molecules comprise a
binding part and a detectable part, and the second binding
molecules comprise a binding part, wherein the binding part of both
first and second binding molecules is capable of specifically
binding to the binding partner and competing for said binding, and
wherein the second binding molecules do not comprise a part that is
substantially identical to the detectable part of the first binding
molecules.
18. The kit-of-parts of claim 17, further comprising one or more of
the following (i) reagents for visualizations of the detectable
part of the first binding molecule; (ii) protocols for
visualizations of the detectable part of the first binding
molecule; (iii) guidance for quantification of a target in a test
sample; (iv) reference materials (v) instrument(s) for visual
analysis of the sample and/or image capture, or a reference to such
instruments; (vi) software for controlling the instruments; (vii)
software for image analysis; (viii) locked image analysis
algorithms; (ix) standards for evaluating the samples, e.g. scoring
standards and scoring guidelines; (x) instructions for use.
19. The kit-of-part of claim 17, for evaluation of a target in a
histological sample.
20. The kit-of-parts of claim 17, wherein the binding agent
comprises an antibody, or a fragment or a derivative thereof, or a
nucleic acid or a nucleic acid analog sequence.
21. The kit-of-parts of claim 17, wherein the first binding
molecules and the second binding molecules are antibodies that are
(i) specific for the same antigen; (ii) capable of inhibiting each
other binding to said antigen, and (iii) have different Fc
regions.
22. The kit-of-parts of claim, wherein the first binding molecule
is an antibody and the second binding molecule is the F(ab)2
fragment of the same antibody.
23. The kit-of-parts of claim 19, wherein the target is a
biological or chemical target molecule, particle, molecular or
cellular complex, molecular or cellular structure, virus or
microorganism, or a fragment of said target molecule, particle,
complex, structure, virus or microorganism.
24. The kit-of-parts of claim 17, comprising the reference material
which is one or more histological samples comprising cells with
predetermined amounts of the target.
25. The kit-of-parts of claim 17, for the quantitative evaluation
of the target.
26-30. (canceled)
31. A kit-of-parts comprising a composition comprising a binding
agent which is capable of specifically binding to the binding
partner comprised in said one or more target sites, wherein the
amount of the binding agent in the incubation medium is sufficient
to bind to at least 51% of the binding partners present in the
sample, and wherein the binding agent comprises a population of
first binding molecules and a population of second binding
molecules, wherein the first binding molecule comprises a binding
part and a detectable part, and the second binding molecule
comprises a binding part, wherein the binding part of the first
binding molecule and the binding part of the second binding
molecule are both capable of specifically binding to the binding
partner and competing against each other for said binding, wherein
the first binding molecule comprises an antibody molecule, or a
derivative thereof, comprising an Fc region, and wherein the second
binding molecule does not comprise a part that can be detected by a
detector of the detectable part of the first binding molecule and
the second binding molecule does not comprise the Fc region of the
first binding molecule.
32. The kit-of-parts of claim 31, wherein the binding partner is
the target or a substance associated with the target.
33. The kit-of-parts of claim 31, wherein the binding agent and the
binding partner are members of a specific binding pair.
34. The kit-of-parts of claim 31, wherein the first and second
binding molecules each comprise an antibody or an antigen-binding
portion of an antibody, and the target comprises an antigen.
35. The kit-of-parts of claim 31, wherein the antibody molecule, or
a derivative thereof, is specific for an epitope comprised in the
binding partner, and the second binding molecule comprises an
antigen binding portion that is specific for the same epitope as
said antibody or is capable of inhibiting the first binding
molecule binding to said epitope.
36. The kit-of-parts of claim 34, wherein the binding part of the
first binding molecule and the binding part of the second binding
molecule comprise an antigen binding part of the same antibody or
antigen binding parts of two different antibodies that are specific
for the same binding partner.
37. The kit-of-parts of claim 1, wherein the concentration of the
binding agent in the incubation medium is greater than the Kd
(dissociation constant) value of the binding agent-binding partner
complex.
38. A kit-of-parts comprising a composition comprising: a first
binding molecule comprising an antibody molecule, or a derivative
thereof, comprising an Fc region, wherein said antibody or
derivative thereof is capable of specifically binding said target,
a second binding molecule capable of specifically binding said
target, wherein said second binding molecule does not comprise the
Fc region of the first binding molecule, and a detectable agent
specific for said Fc region of said first binding molecule.
39. The kit-of-parts of claim 38, wherein said second binding
molecule is an F(ab)2 fragment.
40. The method of claim 39, wherein the amounts of the first
binding molecule and the second binding molecule together are
sufficient to bind to at least 51% of the target present in the
sample.
41. The method of claim 40, wherein said target is the Her2
protein.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/356,735, filed on May 7, 2014, which is a national
stage entry pursuant to 35 U.S.C. .sctn. 371 of International
Application No PCT/DK2012/000119, filed Nov. 8, 2012, which claims
the benefit of U.S. Provisional Application No. 61/556,916, filed
Nov. 8, 2011, and International Application No PCT/DK2011/000131,
filed Nov. 8, 2011, the contents of all of which are fully
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention lies in the field of visualization and
quantification of immobilized targets in samples using
immunochemical means in particular, the invention relates to a
method and reagents for detection, visualization and quantification
of a molecular target in immunostained histological samples using
particular compositions of the target specific binding agent.
Methods and compositions of the invention are suitable for any
assay that uses a target visualization system in histological
samples based on detection of the target by the target specific
binding agent The methods and compositions are useful for
evaluation of targets that are biomarkers of diseases in medical
diagnostic.
BACKGROUND OF THE INVENTION
[0003] In the field of immunohistochemistry, IHC, biological
targets of interest are typically stained with enzymatically
generated dyes. However, most of today's IHC enzymatic systems have
a limited usability of for target visualization due to restricted
sensitivity, if a target is of very low abundance, the amount of
deposited dye remains undetectable. Likewise, there is an upper
detection limit above which a further dye deposition does not lead
to detectably more intense stains. Using lower concentration of
reagents, the upper detection limit may be compromised to allow
differentiation between high and very high abundance targets,
however this also leads to an increase of the lower detection
limit, i.e. the loss in sensitivity of detection. Thus, most of the
today's systems have a limited dynamic range of detection. Further,
differences in sensitivity between different visualization systems
from same or different vendors makes comparison the staining
results difficult.
[0004] A further challenge is quantification of immunochemically
stained targets due to the dye deposition is not a linear function
of target concentration. Around the baseline of detection limit die
intensity increases rapidly as a direct function of target
concentration (as the going from no detectable signal to a signal,
even of a low strength, represents an infinite increase.
Conversely, close to the upper detection limit, even a large
increase in target concentration will lead to virtually no
perceptible increase in the already intense signal.
[0005] A further complication arises from the fact that no
internationally recognized standards exist, and invariable
reference samples are difficult to prepare. Even serial sections of
the same tissue sample usually exhibit biological variation.
Immortal cell lines might in principle provide the infinite
reference material, however differences in cultivation conditions,
cell cycle circles and biological variation wilt also in this case
lead to some batch to batch variation in target expression. Glass
slides chemically modified with peptides or proteins may be used as
surrogate targets, however comparison to tissue samples is not
straight forwards.
[0006] Thus there is a need for standardized quantitative detection
of immobilized targets in biological samples.
[0007] Recently described methods of immunochemical staining of
immobilized targets in biological samples, including histological
samples. (WO2010094284. WO2010094283, WO201047680. WO2012143010)
provide a visualization system characterized by an extreme
sensitivity, such as single units of the target can be visualized
and detected, which also allows a precise quantification of the
target (as described in WO2012075028 WO2012062318), as the amount
of deposited dye is in a linear correlation with the target
expression. However, the latter visualization methods due to their
extreme sensitivity, may in some cases have disadvantages, e.g. the
methods are best applicable for visualization of a sub-fraction of
a target in the sample, but not the whole expression range. This
disadvantage may be an obstacle for broad use of the methods for
visualization of targets in histological samples, especially when
robustness of the procedure and reproducibility of the results are
concerned. Further, the precise quantification of a target
according to the methods may be laborious. The present invention
solves the latter mentioned problems.
SUMMARY OF INVENTION
[0008] Use of lately developed reagents and methods allowing the
detection of all or substantially all target units in histological
samples, e.g. such as described in WO2010094284, WO2010094283,
WO201047680 or WO2012143010, while having advantages with
immunochemical staining of targets present in the samples at low to
medium expressions levels, may also be associated with such
drawbacks as overstaining of samples with targets present at
abundant levels. It may be difficult to evaluate quantify and
distribution of abundant targets in the samples and compare
different histological samples comprising the same target. To
reduce the target-specific signal in such samples, these methods
utilize very low amounts of target-specific detecting agents, such
target-specific binding agents, e.g. antibody reagents. This
approach allows reducing the target-specific signal, but it also
makes the detection of the target vulnerable to different
conditions and results of the evaluation of target not fully
reliable, especially, when the precision of the evaluation is
crucial, e.g. in medical diagnostics.
[0009] The present invention relates to a method for detection,
visualization and quantification of targets in samples, in
particular, molecular targets, such as biological markers of
diseases, where the target is immobilized on or within a solid
support. In particular, the invention relates to detection and
evaluation of the target expression in histological samples. The
present invention solves technical problems of the mentioned
extremely sensitive and powerful detection systems employing
immunostaining of targets in histological samples. In particular it
solves the problem of generation of excessive target-specific
signal produced due to super powerful amplification of the signal,
and the problem of insufficient robustness of the systems due to
necessity of using very tow amounts of target-specific detection
agents, by providing a method and reagents that make these
detection systems robust without compromising their advantages.
[0010] Thus, in one aspect the invention relates to a method for
detecting a target in a target site in a histological sample,
wherein the target site comprises a binding partner for a binding
agent, comprising [0011] a) Incubating the sample presumably
comprising the target in one or more target sites in an incubation
medium comprising a binding agent which is capable of specifically
binding to the binding partner comprised in said one or more target
sites, wherein the amount of the binding agent in the incubation
medium is sufficient to bind to substantially all units of the
binding partner present in the sample, and wherein the binding
agent is characterized in that it comprises first binding molecules
and second binding molecules, wherein the first binding molecules
comprise a binding part and a detectable part, and the second
binding molecules comprise a binding part, wherein the binding part
of both first and second binding molecules is capable of
specifically binding to the binding partner and competing for said
binding, and wherein the second binding molecules do not comprise a
part that is substantially identical to the detectable part of the
first binding molecules; [0012] (b) Detecting the detectable part
of the first binding molecules in the sample, thereby detecting the
target in the target sites.
[0013] In another aspect the invention relates to a kit-of-parts
comprising a composition comprising a binding agent which is
capable of specifically binding to the binding partner comprised in
said one or more target sites, wherein the amount of the binding
agent is sufficient to bind to substantially all units of the
binding partner present in the sample, and wherein the binding
agent is characterized in that it comprises first binding molecules
and second binding molecules, wherein the first binding molecules
comprise a binding part and a detectable part, and the second
binding molecules comprise a binding part, wherein the binding part
of both first and second binding molecules is capable of
specifically binding to the binding partner and competing for said
binding, and wherein the second binding molecules do not comprise a
part that is substantially identical to the detectable part of the
first binding molecules.
[0014] In another aspect the invention relates to an assay for
evaluation of a target in a histological sample, comprising a step
of detection of the target in a histological sample according to
the method of invention.
[0015] One advantage of the invention is that the methods and kits
may be implemented to any method for visualization targets in
histological samples based on use of target-specific binding agents
for detection and visualization of the target.
[0016] Another advantage is that the methods of the invention allow
identifying and counting single units of a target in a histological
sample and, allow determining both the absolute or relative
quantity of the target repeatedly precisely and independently of
the level of target expression. This of particular advantage for
quantification of diagnostic or therapeutic targets such as growth
factor receptors, e.g. Her2 or the like, and thus, utility of the
present methods in diagnostic and therapeutic applications cannot
be overrated.
[0017] The invention allows obtaining the target-specific signal of
any desirable intensity which makes its use in combination with new
powerful target visualization methods important for successful
implementing these visualization methods in medical diagnostics,
which standards and scoring systems have been developed based on
evaluation of target-specific signals produced in histological
samples by much less sensitive and capable visualization
systems.
[0018] Suitability of the invention for both manual and automatic
evaluation of the quantity of a target in samples is an additional
valuable feature.
[0019] The methods are also applicable to any sample comprising a
target immobilized on/within a solid support that is detectable by
a binding agent that has affinity to that target. Thus, virtually
any immobilized target which has an affinity binding partner, such
as e.g. a chemical and biological molecules particle,
microorganism, etc, can be detected and precisely quantified by the
methods of the invention.
DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 demonstrates selected non-limiting embodiments of the
invention.
[0021] FIG. 2 shows representative images of Her2 1+ (A and C) and
2+ (B and D) positive cells immunostained for Her2 using HRP-DAB
visualization system (Envision.TM.) and scored according to
HercepTest.TM., where the samples were incubated with a binding
agent that consisted of only first binding molecules of the
invention (A and B) and a binding agent that contained both the
first binding molecules and second binding molecules (C and D). The
corresponding target detection and visualization procedures are
described in Example 3 Experiment 4
DETAILED DESCRIPTION OF INVENTION
[0022] 1. Method
[0023] One aspect of the invention relates to a method for
detecting a target in a target site in a histological sample,
wherein the target site comprises a binding partner for a binding
agent, comprising [0024] a) Incubating the sample presumably
comprising the target in one or more target sites in an incubation
medium comprising a binding agent which is capable of specifically
binding to the binding partner comprised in said one or more target
sites, wherein the amount of the binding agent is sufficient to
bind to substantially all units of the binding partner present in
the sample, and wherein the binding agent is characterized in that
it comprises first binding molecules and second binding molecules,
wherein the first binding molecules comprise a binding part and a
detectable pail, and the second binding molecules comprise a
binding part, wherein the binding part of both first and second
binding molecules is capable of specifically binding to the binding
partner and competing for said binding, and wherein the second
binding molecules do not comprise a part that is substantially
identical to the detectable part of the first binding molecules;
[0025] (b) Detecting the detectable part of the first binding
molecules in the sample, thereby detecting the target in the target
sites;
[0026] Another aspect of the invention relates to an assay for
evaluation of a target in a sample, in particular, a histological
sample, comprising a step of detection of the target in a target
site of the sample according to the method of the invention.
[0027] Different embodiments of the invention as stated above are
described below and illustrated by non-limiting working examples
described in the section EXAMPLES.
Sample
[0028] The term "sample" means a representative part or a single
item from a larger whole of group, an amount or portion of a matter
or object that supposedly contains a target of interest, e.g. a
portion or amount f biological material comprising a target
molecule, particle, structure to be analyzed, e.g. a body tissue
sample, such as a biopsy sample, a blood sample, etc. A typical
sample shows what the rest of the matter or object is or should be
like. In one preferred embodiment a sample of the invention is a
histological sample.
[0029] Non-limiting examples of a histological sample in the
context of the present invention may be the following; [0030] 1. a
sample comprising suspended cells and/or cells debris, e.g. blood
sample, suspension of cloned cells, body tissue homogenate, etc;
[0031] 2. a sample comprising of intact or damaged cells of an
animal body, a body tissue, smear or fluid or a sample of a tumor,
e.g. a biopsy sample; It may be a fresh tissue sample or preserved
tissue sample, e.g. a formalin fixed paraffin embedded tissue
sample; [0032] 3. a sample comprising a living organism, e.g. a
sample of a medium comprising an animal, plant, bacterium, fungi,
etc; [0033] 4. a sample comprising viral particles, debris thereof,
or viral products, e.g., a body smear comprising viral nucleic
adds, proteins, peptides, etc; [0034] 5, a sample comprising a cell
organelle(s); [0035] 6. a sample comprising natural or recombinant
biological molecules, blood plasma sample, conditioned cell culture
media, etc. [0036] 7. a sample comprising plant cells or derbies
thereof.
[0037] The invention relates to samples comprising an immobilized
target, i.e. to samples, where the target is prevented from freedom
of movement during a detection procedure of the present invention,
e.g. samples, where the target motion is substantially reduced or
eliminate by mechanical or chemical means, as e.g. in case of
samples or targets attached to or within a certain support or
medium. Thus, a sample comprising single individual units of a
target of interest may in one embodiment be immobilized onto a
solid support before the detection procedure, e.g. a solid body
tissue sample immobilized on a glass slide. Examples of samples
comprising immobilized targets of the invention include but not
limited to body tissue samples immobilized on glass or plastic
slides; or samples comprising biological or chemical molecules
immobilized onto membranes, etc. A target of a sample in these
embodiments may be immobilized either within the sample, e.g. a
protein fixed within a tissue sample, or is immobilized on the
surface or within certain material, such as e.g. a portion of a
solid material or a gel such as a nitrocellulose membrane, etc. In
one embodiment the solid support may be a three-dimensional
structure, e.g. a collagen or agar block or the like. In this
embodiment a target, e.g. molecule or particle may be immobilized
within the structure. The term "solid support" means a piece of any
material that is insoluble under conditions of the procedures
according to the invention, e.g. it may be a nitrocellulose
membrane, glass slide etc. Examples of supports suitable for
immobilizing samples and/or targets include but not limited to
synthetic polymer supports, such as polystyrene, polypropylene,
substituted polystyrene, e.g. aminated or carboxylated polystyrene;
polyacrylamides; polyamides; polyvinylchloride; glass; agarose;
nitrocellulose; nylon; polyvinylidenedifluoride; surface-modified
nylon, etc. The invention relates to a solid support that is
chemically inert under conditions described herein, i.e. the chosen
support may not have any major influence on the results of
detection by the method. Accordingly, any such inert support
suitable for immobilizing a sample or target fitting the chosen
assay format, e.g. for INC, ELISA, blotting etc, may be
selected.
[0038] The invention is also applicable to environmental samples,
e.g. a sample of a soil or a sample of a spillage; food samples;
samples of a library of organic molecules; samples of warfare.
[0039] In one embodiment the invention relate to a sample (as arty
of the above) that does not comprise the target, i.e. a negative
control sample. In another embodiment, the invention relate to a
sample that supposedly comprise the target, i.e. a test sample. In
one embodiment, the invention relates to a sample comprising a
predetermined amount of the target of interest, i.e. a reference
sample. In one embodiment, the reference sample is a histological
sample, e.g. a sample comprising cells expressing certain levels of
the target of interest, e.g. a target protein or target nucleic
acid. It may be preferred that the reference sample is treated or
pretreated in the same way as the test sample and the amount of
target in the reference sample is determined according to the
method of the invention.
Target
[0040] The term "target" means in the present content an object of
interest supposedly present in a sample that can be characterized
by particular physical and/or functional features. In the context
of the invention the term "target" relates to the whole pool of
substantially identical entities of that object, not to a single
entity of that object in a sample; in samples where a target is
represented by the only single unit, this only single target unit
is understood as target (i.e. the whole target pool). The term
"substantially identical" in the present context means that all or
substantially all single entities of the target in a sample possess
one or more features that make them recognizable as the target. For
example, the target may be a particular protein including all
molecules of that particular protein in a sample; a target of the
invention may be a particular molecular complex or structure
including substantially aft objects of the sample that comprise
that particular molecular complex or molecular structure; another
example of a target of the invention may be a viral particle or a
bacterium, wherein total population of that viral particles or that
bacteria of the sample is the target.
[0041] Biological objects such as molecules, molecular complexes,
molecular structures, particles or organisms which are associated
with features that are characteristic for a particular cell type,
tissue, cellular structure, physiological condition, etc., are
termed "biological markers" of that particular cell type, tissue,
cellular structure, or physiological condition. Non-limited
examples of such biological markers, that may be targets of the
invention, include but not-limited to particular nucleotide
sequences, proteins or other biological molecules, e.g.
carbohydrates or lipids, chromosomal or membrane structures,
viruses, bacteria, microorganisms etc. In some embodiments of the
invention, the term "target" is used interchangeable with the term
"biological marker" and relates to a molecule, molecular complex,
structure or particle that is characteristic for a particular cell
type, tissue, physiologic condition, etc, wherein the total
population of any of the latter biological markers in the test
sample is considered to be the target.
[0042] In one embodiment, the target may be a protein, e.g. a
cellular membrane receptor or a cytoplasmic protein, in another
embodiment the target may be a nucleic acid. e.g. a cytoplasmic
nucleic acid. Derivatives of any latter mentioned targets, e.g.
fragments, precursors, mutants of target proteins or nucleic acids,
etc. may also be targets in some embodiments of the invention.
[0043] Among targets contained in chemical and environmental
samples may be different pollutants, toxins, warfare substances,
members of molecular libraries, industrial noxious waste compounds,
etc.
[0044] In one embodiment the invention relates to single units of
targets that in different embodiments may be represented by whole
single molecules of targets or fragments of said single molecules
of targets, or single molecular structures, particles, etc.
[0045] By the term "single unit of target" is meant a single
quantity of target that can be regarded as the whole in calculation
and that may, in some embodiments, possess a particular function
(which is also the function of the target as whole). The term
"single" in the present content means one target unit as opposed to
or in contrast with many, e.g. one protein molecule of the target
protein, i.e. one molecule of plurality molecules of the same
kind.
[0046] In some embodiments the invention relates to a single unit
of target, wherein said single unit is a part of a target molecule,
e.g. an epitope, a structural or functional domain of a protein
molecule, or the like.
[0047] In one preferred embodiment, the target is a biological
marker related to cancer, e.g. nucleic acids and polypeptides of
hormones and growth factors and their receptors, cell adhesion
molecules signal transduction molecules, cell cycle regulation
molecules, etc. e.g. genes, RNAs and proteins of the group
including growth factors PDGF, VEGF, TGF, HGF or EGF, their
receptors and the pathway related molecules, genes and their
products relating to signal transduction pathways, e.g. the
JAK/STAT pathway or Akt1/PKB cell survival pathway, or 5-FU
pathway, estrogen receptor ER and its gene (ERS1), etc. In one
preferred embodiment, the invention relates to nucleic acid
sequences, such as the genes. RNA molecules, and protein molecules
of the ErbB family of receptors. Protein molecules, aggregates of
said molecules, genes, RNAs, fragments of genes, proteins and RNAs,
structural and functional domains thereof are contemplated as
targets of the invention.
[0048] The invention allows detecting, visualizing and quantifying
single individual units of a target present in a histological
sample in a broad dynamic range, including quantifying single
target units. Two or more different targets may be visualized in
one or the same sample, e.g. a protein target and nucleic acid
target, or two or more different protein targets, or two or more
different nucleic acid targets, etc.
[0049] According to the invention immobilized target units are
located in discrete target sites of a sample. The target site may
comprise a single target unit or it may comprise a single target
unit which is directly or indirectly associated with one or more
substances, e.g. a target unit directly or indirectly bound to one
or more substances, e.g. a unit of the target bound to a primary
antibody molecule, or primary antibody bound to a hapten conjugated
with a unit of the target, or a secondary antibody bound to either
of these primary antibodies, or the like. Targets units located in
target sites of the sample or substances associated with the target
units, that can be detected by the target unit or the
substance-specific binding agents of the invention are termed
herein "binding partners" of corresponding binding agents. In one
embodiment, the invention relates targets, and/or substances
associated with targets that are the first members of specific
binding pairs, wherein the second members are the target or
substance (correspondingly) specific binding agents, i.e. the
targets and/or substances are specific binding partners for the
corresponding binding agents.
Binding Agent
[0050] The term "binding agent" in context of the present invention
relates to substances that can specifically bind to another
substance present in a test sample such as its binding partner
present in a target site of a sample; preferably, the binding
partner makes a specific binding pair with its binding partner in
the sample, wherein said specific binding pair can be characterized
by a particular value of the dissociation constant (Kd). A number
of different specific binding pairs are known in the art, these are
the pairs of two different molecules which are capable of specific
binding to each other. Non-limiting examples of specific binding
pairs suitable for the invention are discussed below.
[0051] In one embodiment the binding agent may be a member of a
specific binding pair with the target in a sample. In another
embodiment, the binding agent may be a member of a specific binding
pair with a substance which is directly or indirectly associated
with, the target in a sample.
[0052] In particular, binding agents of the invention are capable
of directly and specifically binding to their binding partners
present in target sites of the sample. The term "specifically
binding" means, in one embodiment, that the binding agent-binding
partner binding has affinity defined by the corresponding Kd value.
Some examples of affinity binding illustrating the latter may be
the primary antibody antigen binding; in another embodiment,
"specifically binding" may relate to binding of two complementary
nucleotide sequences under stringency conditions discussed
below.
[0053] The binding agent of the invention is represented by two
populations of binding molecules termed herein as first binding
molecules and second binding molecules. According to the invention,
both first and second binding molecules are capable of specific and
direct binding to the same binding partner present in a binding
site of the sample, wherein the binding partner is a target or a
substance associated with the target in a target site of the
sample. According to the invention, the first and the second
binding molecules are capable of inhibiting each other specific
binding to the binding partner. This means that the first binding
molecules and the second binding molecules comprised in one binding
agent has either affinity to the same single unit of the binding
partner in the sample (e.g. the same epitope or hapten, or the
like) and competitively inhibit each other binding to said binding
partner, or they have affinity to different single units of the
binding partner (e.g. different epitopes, or haptens or the like)
which different single units ere located within the binding partner
molecule so, that binding of one binding molecule to the first of
the different single units is capable of preventing binding of the
other binding molecule to the second of the different single units,
e.g. for the reasons of sterical hindrance. The term "inhibiting
binding" means in the present content one binding molecule of the
binding agent has a capability of reducing another binding molecule
of the binding agent to interact with the single unit of the
binding partner (or single unit of a substance that is associated
with the target) and form a specific binding pair with it.
Accordingly, the first binding molecule and the second binding
molecule in one embodiment have affinity to the same single unit
(e.g. same epitope) of same binding partner, in another embodiment
the first binding molecule has affinity to a first single unit of a
binding partner (e.g. first epotope) and the second binding
molecule has affinity to another single unit of the same binding
partner (e.g. a second epitope), wherein the first single unit and
the second single unit are located within the binding partner so
that binding to the first single unit prevents binding to the
second binding unit and vice versa. The latter is also valid in
embodiments when the single unit of a binding partner is a whole
single molecule, structure, particle etc.; then, in one embodiment,
the first and second binding molecules can prevent each other
binding to said molecule, structure, particle etc.; in another
embodiment the binding molecules can prevent each other binding to
two individual single units of said partner.
[0054] According to the invention, first and second binding
molecules comprised in the binding agent are similar in that they
both comprise a part ("binding part") that has is capable of
specific binding an individual single unit of the binding partner
present in a target site of the invention. This part may be
structurally identical in both molecules. In other embodiments, the
binding parts of the first and second binding molecules may be
structurally different. Accordingly, affinity of binding of the
first binding molecule to the binding partner may have first Kd
value and binding of the second binding molecules to the same
binding partner may have second Kd value, wherein the first and
second Kd values differs from each other. However, for both binding
molecules the binding part defines the function of the binding
molecule as the member of a specific binding pair with the same
binding partner.
[0055] Both the first and the second binding molecules comprised in
the same binding agent may further comprise a part that may have a
function as detectable label. i.e. be detectable by application of
the appropriate detection means. However, in any embodiment the
second binding molecules may not comprise a part that is
substantially identical to the detectable part of the first binding
molecules. The term "substantially identical" means that the
detectable part of the second binding molecules may not be detected
by the same detection means as the detectable part of the first
binding molecules.
[0056] The detectable part of the first binding molecule may be any
detectable substance available in the art, e.g. a chromogenic
fluorescent or luminescent label (collectively termed "optically
detectable" labels), radioactive, magnetic label, a hapten, an
enzyme, an enzyme substrate, etc. Some non-limiting particular
examples of suitable labels are discussed below.
[0057] The detectable part, in one embodiment of the invention, may
be a part of the same binding molecule, e.g. the Fc fragment of an
antibody; accordingly, in such embodiment the second binding
molecule comprised in the same binding agent may not comprise the
Fc fragment of the same antibody, while it may comprise the antigen
binding part (such as e.g the F(ab) fragment) of this antibody. The
latter means that in one embodiment where the binding agent
comprises two different antibodies specific for the same antigen or
epitope (e.g. a first and second antibody derived from different
host species), the first antibody is the first binding molecule and
the second antibody is the second binding molecule; the Fc region
of the first antibodies is the detectable part of the binding agent
of the invention. The Fc region of the second antibody is not the
detectable part in the context of the present invention.
[0058] Another example illustrating the above may be the binding
agent comprising two nucleic acid probes (first and second binding
molecules), each comprising a nucleotide sequence capable of
hybridizing under similar stringency conditions with the same
target nucleic acid (i.e. the binding partner) located in the
target sites of a sample. Only one of the probes, the first binding
molecule, is to comprise the detectable part, which may be a
detectable substance linked to the binding part, such any of the
described herein, or it may be a nucleotide sequence that is not
capable of hybridizing with the target sequence under the same
condition. Another probe, the second binding agent, may not
comprise the same detectable substance or the same nucleic acid
sequence, while it may comprise another nucleotide substance which
is not "detectable" in the context of the present invention.
[0059] Members of specific binding pairs suitable for use in
practicing the invention ra ay be of the immune or non-immune
type.
[0060] Non-immune specific binding pairs include systems wherein
the two components share a natural affinity for each other but are
not antibodies. Exemplary non-immune binding pairs are
biotin-avidin or biotin-streptavidin, folic acid-folate binding
protein, complementary nucleic acids, receptor-ligand, etc. The
invention also includes non-immune binding pairs which form a
covalent bond with each other. Exemplary covalent binding pairs
include sulfhydryl reactive groups such as maleimides and
haloacetyl derivatives and amine reactive groups such as
isothiocyanates, succinimidyl esters, sulfonyl halides, and coupler
dyes such as 3-methyl-2-benzothiazolinone hydrazone (METH) and
3-(dimethyl-amino)benzoic acid (DMAB), etc.
[0061] Immune specific binding pairs may be exemplified by
antibody-antibody systems or hapten-anti-hapten systems. In one
embodiment the immune specific binding pair of the invention may be
an antibody-antibody binding pair comprising two, or more antibody
molecules having affinity to each other, for example a primary
antibody and secondary antibody pair, wherein the primary antibody
represents the binding partner and the secondary antibody
represents the binding agent: Antibody systems comprising 3 or 4,
or more antibody members may also be used. In other embodiments of
the invention the immune binding pair may be represented by a
hapten-anti-hapten system. In such embodiments the binding agent
may comprise the first binging molecule which a conjugate
comprising a molecule having affinity to a binding partner in a
target site (e.g. a target) and linked to a hapten, e.g., a primary
antibody-hapten conjugate or nucleic acid sequence-hapten
conjugate, and the second binding agent which is the same antibody
molecule or the same anucleic acid sequence, both without the
hapten. The hapten (i.e. the detectable part of the first binding
molecule) may be then detected by a detection agent specific for
the hapten (e.g. indirectly with use of the anti-hapten antibody,
or directly via microscopic observation, if the hapten is a
visually detectable substance, e.g. a fluorescent label).
[0062] The term "hapten" designates a small molecule which can be
considered as an isolated epitope to which an antibody can be made,
although the hapten alone will not induce an immune response if
injected into an animal, it must be conjugated to a carrier
(usually a protein). As haptens are small molecules, multiple
copies of a hapten may be attached to a large molecule, e.g. a
polymer molecule, such as protein, nucleotide sequence, dextran,
etc. Haptens may serve as convenient label molecules for assay
formats where it is necessary or advantageous to amplify a signal.
Thus, the bound multiple copies of a hapten provide for enhanced
sensitivity, e.g. increased signal strength. Non-limited examples
of suitable haptens include Fluorescein (FITC), 2,4-Dinitrophenol
(DNP), myc Digoxigenin (DIG), tyrosine, nitrotyrosine biotin and
dyes. e.g. tetramethylrhodamine, Texas Red, dansyl, Alexa Fluor
488, BODIPY FL, lucifer yellow and Alexa Fluor 405/Cascade Blue
fluorophores, Haptens are described in US20080305497 may also be
used for the purposes of the invention.
[0063] The term `antibody`, as used herein, designates an
immunoglobulin or a part thereof, and includes any polypeptide
comprising an antigen binding site regardless of the source, method
of production, and other characteristics. The term includes for
example, polyclonal, monoclonal, monospecific, polyspecific,
humanized, single chain, chimeric, synthetic, recombinant, hybrid,
mutated, and CDR-grafted antibodies. The antigen binding part of an
antibody includes any antibody fragment which can still bind
antigen, for example, an Fab, F(ab').sub.2, Fv, scFv, or fragments
or derivatives thereof, e.g. recombinant molecules, etc. The origin
of the antibody is defined by the genomic sequence irrespective of
the method of production.
[0064] Primary antibody, in context of the present invention,
refers to an antigen binding agent, e.g. a whole primary antibody
molecule, a fragment or a derivative of said molecule, e.g. a
conjugate comprising a primary antibody or a polymerized primary
antibody, that specifically binds to its antigen, e.g. target
molecule or another single unit of a target, hapten, etc. In some
embodiments, a primary antibody may be a bivalent antibody which is
capable of binding to two (or more) single individual units of
different targets, e.g. an antibody that is capable of binding to a
receptor dimer, e.g. Her2/Her3 dimer. In this embodiment the single
unit of a target according to the invention is a single Her2/Her3
dimer, and the target is a population of Her2/her3 dimers in a
sample including all said dimers of the sample.
[0065] Secondary antibody, in context of the present invention,
refers to a binding agent capable of specifically binding to the
corresponding primary antibody. It may be a whole antibody
molecule, a fragment or a derivative of said molecule, e.g. a
conjugate comprising an antibody or a polymerized antibody, that
have an antigen binding domain that specifically binds to the
primary antibody.
[0066] Tertiary antibody, in context of the present invention,
refers to an antibody capable of binding to the corresponding
secondary antibody. It may be a whole antibody molecule, a fragment
or a derivative of said molecule, e.g. a conjugate comprising an
antibody or a polymerized antibody that comprise an antigen binding
domain that specifically binds to a secondary antibody or a hapten
linked to a secondary antibody.
[0067] Antibodies used in the invention, including primary
antibodies, secondary antibodies and tertiary antibodies, may be
derived from any mammal species, e.g., a rat, a mouse, a goat, a
guinea pig, a donkey, a rabbit, horse, lama, camel, or any avian
species e.g., chicken, duck. Derived from any mammal or avian
species, as used herein, means that at least a part of the nucleic
acid sequence encoding a particular antibody originated from the
genomic sequence of a specific mammal, e.g., a rat, a mouse, a
goat, or a rabbit or a specific bird e.g., chicken, duck. The
antibody may be of any isotype, e.g., IgG, IgM, IgA, IgD, IgE or
any subclass, e.g., IgG1 IgG2, IgG3, IgG4.
[0068] In certain embodiments, any antibody (primary, secondary or
teriery) may be conjugated to a polymer. In some embodiments, 1-20
antibody molecules their fragments or derivatives, such as e.g.
5-15 molecules, 1-10, etc, may be conjugated with a polymer, e.g.
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 molecules per polymer.
[0069] Engineered antibodies including chimeric, CDR-grafted and
artificially selected antibodies produced using phage display or
alternative techniques mat be use as binding molecules comprised in
the binding agent of the invention.
[0070] Antibodies comprised in binding agents or as detection
agents of the invention may be produced by any of numerous methods
well-known in the art e.g., according to Harlow and Lane,
Antibodies: a Laboratory Manual, (1988) (Cold Spring Harbor Press,
Cold Spring Harbor, N.Y.). Techniques for the preparation of
recombinant antibody molecules are described in the above reference
and a number of other references, e.g., EP 0623679; EP 0368684; and
EP 0436597. Nucleic acids encoding antibodies may be isolated from
a cDNA library. Nucleic acids encoding antibodies may be isolated
from a phage library (see e.g. McCafferty et al. 1990, Nature
348:552, Kang et al. 1991, Proc. Natl. Acad. Sci. USA 88:4363; EP 0
589 877 B1). Nucleic adds encoding antibodies can be obtained by
gene shuffling of known sequences (Mark et al. 1992, Bio/Technol
10:779). Nucleic acids encoding antibodies can be isolated by in
viva recombination (Waterhouse et al. 1993, Nucl. Acid Res.
21:2265). The antibodies used in the methods of the invention
include humanized immunoglobulins (see U.S. Pat. No. 5,585,089,
Jones et al. 1986, Nature 332:323). Antibodies of the invention may
be altered any possible way, presuming that they retain their
binding affinity, e. g., they may fused with an effector protein,
toxin, label, etc. Methods of conjugation of antibody with
different agents are well known in the art.
[0071] In one preferred embodiment, both binding molecules of the
binding agent are or comprise an antibody or an antigen-binding
portion of an antibody, and the binding partner is a molecule
comprising the antigen, e.g. an antigenic entity, such as hapten or
amino acid sequence or the like.
[0072] In one preferred embodiment of the invention, the detectable
part of the first binding molecule is the Fc region of an antibody,
a fragment or a derivative thereof; one embodiments, is of the same
antibody as the binding part of the first binding molecule; in
other embodiments, is of another antibody, i.e, of an antibody that
has the antigen binding part different from the binding part of the
first binding agent.
[0073] In one preferred embodiment, the first binding molecule is
or comprises a primary antibody molecule, or a derivative thereof,
specific for an epitope comprised in the binding partner, and the
second binding molecule is selected from a molecule that comprises
an antigen binding portion of said primary antibody, or a molecule
that comprises an antigen binding portion of another antibody,
wherein said another antibody is specific for the same epitope as
said primary antibody, or is capable of inhibiting the first
binding molecule binding to said epitope
[0074] In another preferred embodiment, the binding agent comprises
labeled and unlabeled molecules comprising an antigen binding
portion of the same antibody, such as labeled and unlabeled normal
or derivatized molecules of a primary antibody, labeled and
unlabeled normal or derivatized antibody molecules of a secondary
antibody, or labeled and unlabeled conjugate molecules comprising
normal or derivatized molecules of a primary or a or labeled and
unlabeled conjugate molecules comprising normal or derivatized
molecules of secondary antibody.
[0075] In one embodiment of the invention, the binding agent is
represented by a first and a second binding molecules that comprise
an antigen binding region of an antibody, such as a Fab region,
e.g. F(ab)1 or F(ab)2 fragments of an antibody.
[0076] In one preferred embodiment, the binding part of the first
binding molecule and the second binding molecule are or comprise
the Rab)2 fragment of the same primary antibody specific for an
epitope comprised in the binding partner, or the F(ab)2 fragments
of two different primary antibodies that are specific for the same
epitope, or capable of inhibiting binding of the first binding
molecules to the epitope.
[0077] In another preferred embodiment, the binding part of the
first binding molecule and the binding part of the second binding
molecule are or comprise the F(ab)2 fragments of the same secondary
antibody or F(ab)2 fragments two different secondary antibodies
that are specific for the same primary antibody.
[0078] In some embodiments the binding agent may be a composition
comprising more than two different antibody molecules, e.g. 3, 4,
5, etc different antibody molecules. In this embodiment, one or
more antibodies may be the first binding molecules and all the
other antibody molecules comprised in the binding agent are
representatives the population of the second binding molecules.
[0079] As mentioned, in some embodiments, the invention relates to
binding agents that comprise first and second binding molecules
that comprise the binding part which is a member of a non-immune
specific binding pair with the binding partner in the target site,
e.g. nucleotide sequences, or nucleic acid analog molecules. One
preferred embodiment of the invention related to a binding agent
that comprises the first and second binding molecules that are or
omprise a nucleic acid or a nucleic acid analog, which binding
partner is a nucleic acid in the sample.
[0080] A binding agent comprising a nucleic acid or nucleic acid
analog molecule, e.g., a DNA molecule, an RNA molecule, a PNA
molecule, etc., may be useful for the detection of nucleic acid
targets. A binding agent that comprises the first and second
binding molecules that are or comprise a nucleic acid sequence or a
nucleic acid analog sequence, and the binding partner is a
nucleotide sequence in the sample may be a preferred embodiment.
Further, it may be preferred that the first and the second binding
molecules comprising a nucleic acid sequence or a nucleic acid
analog sequence, or the like, comprised in the binding part are
capable of hybridizing with the same binding partner under same
stringent conditions, such as low to medium stringent conditions.
In some embodiment, the binding molecules may preferably hybridize
with the partner nucleic acid in the sample under high stringency
conditions.
[0081] Nucleic acid sequences used as binding agents fiar the
purposes of the invention may be synthesized chemically or produced
in recombinant cells. Both modes of production are well known in ht
eart (see e.g. Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual, 2nd ed. Cold Spring Harbor Press). In some
embodiments, a nucleic acid binding agent may comprise a peptide
nucleic acid (PNA). A peptide nucleic acid is a nucleic acid
molecule in which the deoxyribose or ribose sugar backbone, usually
present in DNA and RNA is replaced with a peptide backbone. Methods
of making PNAs are known in the art (see e.g. Nielson, 2001,
Current Opinion in Biotechnology 12:16) (hereby incorporated by
reference). In other embodiments, the binding agent may comprise a
locked nucleic acid (LNA) (Sorenson et al, 2003, Chem. Commun.
7(17):2130).
[0082] A nucleic acid binding agent, in some embodiments, may
comprise at least one oligo- or at least one polynucleotide
sequence that specifically hybridizes to a single unit of a target
sequence in a biological sample, e.g. a single mRNA sequence, under
specific conditions of stringency. The term "hybridization under
stringent conditions," is used herein to describe conditions for
hybridization under which nucleotide sequences that are
significantly complementary to each other, such as at least 70%, at
least 80%, at least 85-90% complementary, remain bound to each
other. The percent complementary is determined as described in
Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402 (hereby
incorporated by reference).
[0083] Specified conditions of stringency are known in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, Inc. (Ausubel et al. 1995 eds.), sections 2, 4, and 6
(hereby incorporated by reference). Additionally, specified
stringent conditions are described in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor
Press, chapters 7, 9, and 11 (hereby incorporated by reference). In
some embodiments, the hybridization conditions are high stringency
conditions. An example of high stringency hybridization conditions
is hybridization in 4.times. sodium chloride/sodium citrate (SSC)
at 65-70.degree. C. or hybridization in 4.times.SSC plus 50%
formamide at 42-50.degree. C., followed by one or more washes in
1.times.SSC, at 65-70.degree. C. It will be understood that
additional reagents may be added to hybridization and/or wash
buffers, e.g., blocking agents (BSA or salmon sperm DNA),
detergents (SDS), chelating agents (EDTA), Ficoll, PVP, etc.
[0084] In some embodiments, the binding agents may hybridize to a
target sequence in a sample under moderately stringent conditions.
Moderate stringency, as used herein, include conditions that can be
readily determined by those having ordinary skill in the art based
on, for example, the length of the DNA. Exemplified conditions are
set forth by Sambrook et al. Molecular Cloning: A Laboratory
Manual, 2d ed. Vol, 1, pp. 1.101-104, Cold Spring. Harbor
Laboratory Press (1989) (hereby incorporated by reference), and
include use of a prewashing solution of 5.times.SSC, 0.5% SOS, 1.0
mM EDTA (pH 8.0), hybridization conditions of 50% formamide,
6.times.SSC at 42.degree. C. (or other similar hybridization
solution, such as Stark's solution, in 50% formamide at 42.degree.
C.), and washing conditions of 60.degree. C., 0.5.times.SSC, 0.1%
SOS.
[0085] In some embodiments, the binding agents hybridize to a
target sequence in a sample under low stringent conditions. Low
stringency conditions may include, as used herein, conditions that
can be readily determined by those having ordinary skill in the art
based on, for example, the length of the DNA. Low stringency may
include, for example, pretreating the DNA for 6 hours at 40.degree.
C. in a solution containing 35% formamide, 5.times.SSC, 50 mM
Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and
500 .mu.g/ml denatured salmon sperm DNA. Hybridizations are carried
out in the same solution with the following modifications: 0.02%
PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml salmon sperm DNA, 10%
(wt/vol) dextran sulfate, and 5-20.times.10.sup.6 CPM binding agent
is used. Samples are incubated in hybridization mixture for 18-20
hours at 40.degree. C., and then washed for 1.5 h at 55.degree. C.
in a solution containing 2.times.SSC, 25 mM Tris-HO (pH 7.4), 5 mM
EDTA, and 0.1% SDS. The wash solution is replaced with fresh
solution and incubated an additional 1.5 h at 60.degree. C.
[0086] In other embodiments the invention may relate to binding
agents comprising first and second binding molecules that comprise
peptide sequences that are derived from non-antibody proteins, e.g.
peptide sequences derived from nucleic acid binding domains of
different proteins, ligands of different cellular and nuclear
receptors and their derivatives. Some non-limiting examples of such
binding agents may be c1q protein of the classical pathway of the
complement cascade which can bind to an antibody constant region, a
MHC molecule, e.g., MHC class I and MHC class II and non
conventional MHC, a molecule having a specific binding partner,
such as molecules involved in cellular signaling pathways such as
molecules having leucine zipper domains, e.g., fos/jun, myc, GCN4,
molecules having SH1 or SH2 domains, such as Src or Grb-2; an
immunoglobulin receptor, e.g., an Fc receptor; a chimeric protein,
i.e., a protein engineered to combine the features of two or more
specific binding partners, e.g., a leucine zipper could be
engineered into a Fc region of an antibody, an SH2 domain could be
engineered to be expressed in a Fc region of an antibody. In other
embodiments, fusion proteins can be engineered comprising an Fc
portion of an antibody with a substituted variable domain.
[0087] The binding part of the binding molecules of a binding agent
of the invention may also be a small molecule which can bind
specifically to certain structural units of large biological
molecules.
[0088] As mentioned, the detectable part of the first binding agent
may be or may comprise a detectable substance, e.g. a fluorescent
label, hapten, enzyme, etc. Non-limiting hapten labels are
described above. Non-limiting examples of fluorescent, luminescent,
radioactive, chromogen or magnetic labels may be 5-(and
6)-carboxyfluorescein, 5- or 6-carboxyfluorescein,
6-(fluorescein)-5-(and 6-carboxamido hexanoic acid, fluorescein
isothiocyanate, rhodamine, tetramethylrhodamine, Cy2, Cy3, Cy5,
AMCA, PerCP, R-phycoerythrin (RPE) allophycoerythrin (APC), Texas
Red, Princeton Red, Green fluorescent protein (GFP) coated CdSe
nanocrystallites, DNP, digoxiginin, ruthenium derivatives, luminol,
isoluminol, acridinium esters, 1,2-dioxetanes and
pyridopyridazines, radioactive isotopes of hydrogen, carbon,
sulfur, iodide, cobalt, selenium, tritium, or phosphor; magnetic
particles or beads. Non-limiting examples of suitable enzyme labels
may be horseradish peroxidase (HRP), alkaline phosphatase (AP),
beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase,
beta-N-acetylglucosaminidase, .beta.-glucuronidase, invertase,
xanthine oxidase, firefly luciferase, glucose oxidase (GO). In one
preferred embodiment the first binding molecule may comprise HRP as
the detectable part.
[0089] The amount of the binding agent used in the method according
to the invention is predetermined so, that the first and second
binding molecules of the binding agent together bind to
substantially all units of their binding partner present in the
sample. The term "substantially all" means that at the binding
agent saturates at least 51% of the affinity binding sites of the
sample (i.e. the sites where the corresponding binding partner is
available for affinity binding), or more; preferably, more than 51%
of the binding sites, such as up to about 66% of the sites;
preferably, more than 60%, such as up to about 75% of the sites or
more, for example up to about 80% of the sites, or up to about 90%
of the sites or more. This means that the amount of the binding
agent should be at least equal or, preferably, above of the Kd
value of binding agent-binding partner complex in the sample,
preferably, above of the Kd value, such as 10-1000% above of the
value, or more. The Kd value of binding agent-binding partner
complex may be defined for a mixture of first and second binding
molecules or separately for each binding molecule following the
corresponding instructions of the art, e.g. as exemplified herein
in Example 1 or 2 for antibody binding agents.
[0090] The invention relates to a great variety of sample species,
binding partner species, binding molecule species, various values
of binding affinity between binding molecules of the binding agent
ant their binding partner, according the amounts of the binding
agent applied to a sample in different embodiments vary depending
on the embodiment. Using common general knowledge the skilled in
the art can select an appropriate binding agent molecules and
determine their affinity to the binding partner of interest in the
sample, and, thus, the amount of binding agent needed for every
particular embodiment.
[0091] In one embodiment of the invention, the binding agent may
contain an amount of the second binding molecules that is equal or,
preferably, higher than the Kd of the second binding
molecule-binding partner complex, such as 5-50% higher the Kd
value, such as 10-100% higher, 20-200% higher, 30-300% higher,
40-400% higher, etc.
[0092] According to the invention a high amount of the binding
agent comprising a mixture of first and second binding molecules
(as any of the described above), wherein a portion of the second
binding molecules is predetermined as above, has that advantage
that it allows detecting a fractional sub-population of the target
sites (comprising single target units) in the sample labeled with
the detectable part of first binding molecules that is
predetermined by the predetermined amount of the second binding
agent in the binding agent mixture. Thus, the quantity of the
corresponding binding target in the sample can be determined
precisely and repeatedly. Non-limiting examples of practical use of
the invention for evaluation of the amount of a target in a
histological sample are described herein in EXAMPLES.
Incubation Media
[0093] According to the invention a sample supposedly comprising
target sites of the invention is incubated in media comprising a
binding agent of the invention. Incubating of the sample means
maintaining the sample in incubation media for a period of time to
allow a certain event happening, e.g. a binding event, a chemical
reaction, etc. The term "incubation media" means in the present
context an aqueous solution comprising compounds that allows the
certain event happened, e.g. binding agent molecules.
[0094] Incubating time may vary, in different embodiments an
incubation may lasts from approximately 3 seconds to approximately
3 min, e.g. around 10 seconds, 20 seconds, 30 seconds, 1 minute, 2
minutes, 5 minutes, 10 minutes, or longer, e.g. one-two hours,
overnight. For example, incubating the sample in an aqueous
solution comprising a binding agent ("binding agent media") may
lasts 1-3 minutes.
[0095] Incubating may be performed at various temperatures,
depending on the type of target, binding agent, etc, e.g. it may be
performed at a temperature from around +4C.degree. to around +40
C.degree..
[0096] In one embodiment, the invention relates to a binding agent
comprising antibody or a derivative of an antibody. Accordingly,
the binding agent media may be an aqueous medium in which the
binding agent is soluble and stable and is capable of binding to
its binding partner in the target site. It is typically a buffered
aqueous solution that has pH in the range from 4 to 9, such as
between pH 3.5 and pH 9.5, e.g. between pH 5 and pH 7, between pH
5.5 and pH 6.5 or between pH 6.5 and 7.5, or between pH 7 and pH 8,
or between pH 7.5 and pH 8.5, or pH 8 and pH 9. Any buffer with a
suitable buffer capacity may be used, e.g. phosphate buffered
saline (PBS) and imidazole buffer. Other suitable buffers may be
found in Good, N E, et al (1966) Hydrogen ion buffers for
biological research. Biochem. 5(2), 467-477. The pH value of the
media may be essential for binding of the binding agent to the
binding partner; it may be optimized depending on the nature of the
binding agent and the target.
[0097] In some embodiments the binding agent medium may comprise an
organic or inorganic salt. The inorganic salt may be selected form
e.g. sodium chloride, magnesium chloride, potassium chloride,
calcium chloride, sodium phosphate, or ammonium sulfate. The
organic salt may be selected from e.g. sodium acetate, ammonium
acetate or imidazole salts, e.g. imidazole hydrochloride, etc. The
amount of salt in binding agent media may range from approximately
10.sup.-3 M to saturation, e.g. from approximately 20 mM to
approximately 200 mM, or from approximately 50 mM to approximately
500 mM. In one preferred embodiment, the media may comprise salt in
the amount from approximately 10 mM to 500 mM. In another preferred
embodiment the medium may be free of salt.
[0098] In some embodiments binding agent media may comprise an
organic modifier (by the term "organic modifier" is meant any non
water solvent), e.g. N-Methyl pyrolidone (NMP), dimethylsulphoxide
(DMSO), mono- and diethylene glycol, sulpholane,
N,N-dimethylformamide (DMF), polyethylene glycol (PEG), propylene
glycol, etc. The amount of the organic modifier may vary from
around 1% to around 20% (v/v or w/v), or, in some embodiments, be
higher than 20%.
[0099] In some embodiments binding agent media may comprise a
detergent, e.g. polyethylenglycol-p-isooctyphenyl ether (NP-40)) or
a surfactant (e.g. selected from the surfactants based on
polyoxyethylene sorbitanmonolaurate (TWEEN), or a surfactant based
on block copolymers (PLURONIC etc.), etc. The amount of the
detergent may vary from about 0.001% to about 5%/v/v or w/v).
[0100] In some embodiments binding agent media may comprise a
binding agent stabilizing agent, e.g. bovine serum albumin or
dextran. The amount of the stabilizing agent may vary from 0.01% to
20% (w/v).
[0101] In some embodiments binding agent media may comprise an ion
chelator (e.g. ethylene diamine tetra acetic acid (EDTA) or
ethylene diamine hydroxyl phenylacetic acid type chelator (EDHPA),
etc.). The amount of the chelator may vary from about 10.sup.-9 M
to about 10.sup.-8 M.
[0102] In some embodiments, binding agent media may comprise one or
more blocking agents for saturating non-specific binding sites,
i.e. sites of the solid support that do not comprise the target.
Some non-limiting examples of blocking agents suitable for
different embodiments may be the Denhard's solution, bovine serum
albumin, skimmed milk, etc.
[0103] As discussed above, the invention contemplates a great
variety of species of targets, binding agents and assay formats;
accordingly, the composition of binding agent medium may vary and
should be adjusted for every particular embodiment using the
knowledge of the art.
[0104] Amounts of the binding molecules in a binding agent may vary
depending on the species of said binding molecules, their affinity
to the binding partner in the sample (or in target sites of the
sample), species of the binding partner, sample species,
composition of the media, etc. As discussed above, the amount of
the binding agent in a media is predetermined to bind substantially
all units of the binding agent partner present the sample.
Depending on the affinity of the first and second binding molecules
to the binding partner, the quantity of each in the binding agent
may vary, however, according to the invention the amount of the
binding agent (including both first and second binding molecules)
should not be less, preferably higher than the value of Kd of
binding agent-binding partner complex, preferably above this value,
such as about 5-10% higher the Kd value, preferably more than 10%
than Kd value such as about 11-14%, 15-20%, 20-25%, 25-50%, or more
that 50% higher than the Kd value, such as 100% higher, 200%
higher, e.g. 300%-1000% higher or more. The amount of the second
binding molecule in the binding agent mixture may be above or below
the corresponding value of Kd (i.e. second binding molecule-binding
partner complex), however, in some embodiments it may be preferred
that the amount of the second binding molecules in the mixture is
higher of the Kd, at least 1% higher, such as 5% to 25% higher,
10-50% higher, 15-75% higher, etc. The amount of the first binding
molecule may according to the invention be defined individually for
different embodiments; in some embodiments it may be below of the
Kd value of first binding molecule-binding partner complex, on
other embodiments equal or above of the Kd.
Detection
[0105] A method of the invention may comprise one or more steps
preceding or following the step of incubating of the sample with
the binding agent of the invention in order to detect the
detectable part of the first binding molecules in the sample.
[0106] The detectable part of the first binding molecule may
comprises an optically detectable label, an enzyme, a member of a
specific binding pair, a particle, a radioactive substance, a
combination of any thereof. Accordingly, a label may be detected
directly, i.e. by observing the label using means allowing the
observation this detectable label, e.g. a microscope. For some
detectable parts of the invention there are no appropriate
detectable means exist, so these parts may be detected, so called,
"indirectly, by using detecting agents capable of specifically
detecting and making them "detectable", e.g. label the sites of the
presence of the detectable parts of the first binding molecules
with color or fluorescence or the like. In one embodiment, the
detectable part may comprise a member of a specific binding pair.
Detecting of such detectable parts may comprise one or more steps
of using binding agents which comprise the corresponding member of
this specific binding pair. This detecting procedure may further
comprise a step of visualization of the target site comprising the
first binding agent, e.g. use an enzyme-mediated deposition of a
reporter molecule at said target site. In one embodiment, the
enzyme-mediated deposition is a horse-radish peroxidase (HPR)
mediated deposition of a reporter. It may be any traditional
HRP-DAB (3,3'-diaminobenzidine) labeling of the target sites well
known in the art. A procedure recently described in WO2010094284,
WO2010094283, WO201047680, or WO02012143010 (all references are
incorporated herein by reference) may also be used. Some examples
of visualization of target sites according the later procedures are
described in Examples.
[0107] The HPR-mediated visualization of the detectable part of the
first binding molecules is, in some embodiments, preferred
visualization method, but not limiting, as any visualization
procedure that that is suitable for detecting the detectable part
of the first binding molecules in a sample of the invention may be
used. This flexibility of applicable visualization procedures is
one of the advantages of the method of the invention.
[0108] The detection procedure may also comprise one or more steps
of blocking, washing, mounting, or the like.
[0109] The method of the invention may be used in an assay for
evaluation of a target in a histological sample, wherein the assay
comprises a step of detection of the target in the sample using a
binding agent that have a specific affinity to the target. The
method may practically be implemented into any such assay, both to
those that already exist in the art or to those that will be
developed in the art of evaluation of a target in a histological
sample by use of target specific binding agents. Accordingly, as
assay for evaluation of a target in a histological sample,
comprising a step of detection of the target in the sample
according to the present method is another aspect of the
invention.
[0110] The assay comprising the target site detecting step of the
invention may be both for qualitative and quantitative evaluation
of the target in a sample. Both evaluations are embodiments of the
assay of the invention. The assay of the invention may be used for
a qualitative and/or quantitative evaluation of a biological
marker, e.g. a biomarker of a disease. In one embodiment, the
invention relates to a gene of the ErbB family of growth factor
receptors, or a product of said gene as a biomarker of a disease,
e.g. Her2, Her2, etc. In some embodiments, the biomarker may be a
combination of two or more biomarkers which are capable of
functioning as a single target unit in the context of the
invention, e.g. the ErbB receptor dimmers or the like.
[0111] The method of the invention is comparable with automatic,
semiautomatic or manual target detection and visualization methods
and assays.
[0112] 2. Kit-of-Part
[0113] Another aspect of the invention relates to a kit-of-parts
comprising a composition comprising a binding agent which is
capable of specifically binding to the binding partner comprised in
said one or more target sites, wherein the amount of the binding
agent is sufficient to bind to substantially all units of the
binding partner present in the sample, and wherein the binding
agent is characterized in that it comprises first binding molecules
and second binding molecules, wherein the first binding molecules
comprise a binding part and a detectable part, and the second
binding molecules comprise a binding part, wherein the binding part
of both first and second binding molecules is capable of
specifically binding to the binding partner and competing for said
binding, and wherein the second binding molecules do not comprise a
part that is substantially identical to the detectable part of the
first binding molecules.
[0114] In different embodiments a kit-of-parts of the invention may
further comprise one or more of the following [0115] (i) reference
materials; [0116] (ii) reagents for detection and/or visualization
of the detectable part of the first binding molecules; [0117] (iii)
protocols for detection and/or visualization of the detectable part
of the first binding molecules; [0118] (iv) protocols for target
quantification; [0119] (v) instrument(s) for visualization of a
target and/or image capture or a reference to such instruments;
[0120] (vi) software for controlling the instruments; [0121] (vii)
software for image analysis; [0122] (viii) locked image analysis
algorithms; [0123] (ix) standards for evaluating the samples, e.g.
scoring standards and scoring guidelines; [0124] (x) instructions
for use.
[0125] All embodiments of the method of the invention regarding the
binding agent, binding molecules, sample, media, target,
visualization means, etc., are also embodiments of the kit-of-parts
of the invention.
[0126] In particular, the binding agent of a kit-of-parts of the
invention may comprise binding molecules that are members of any of
specific binding pairs with the target in the sample or with a
substance associated with the target (embodiments of all the latter
are discussed above).
[0127] In one preferred embodiment the binding agent may comprise
an antibody, or a fragment or a derivative thereof, in another
embodiment, it may comprise a nucleic acid or a nucleic acid analog
sequence.
[0128] In one embodiment, the first binding molecules and the
second binding molecules of the binding agent may be antibodies
that are (i) specific for the same antigen; (ii) capable of
inhibiting each other binding to said antigen, and (iii) have
different Fc regions; in another embodiment, the first binding
molecule may be an antibody and the second binding molecule is the
F(ab)2 fragment of the same antibody.
[0129] The kit-of-parts of the invention is, in a preferred
embodiment, for evaluation of a target in a histological sample. In
one preferred embodiment for quantitative evaluation of the
target.
[0130] The target may be in different embodiments be a biological
or chemical target molecule, particle, molecular or cellular
complex, molecular or cellular structure, virus or microorganism,
or a fragment of said target molecule, particle, complex,
structure, virus or microorganism. In one preferred embodiment, the
target is a biomarker of a disease, such as one of the discussed
above.
[0131] In one embodiment, the kit-of-parts comprises a reference
materials, such as one or more histological samples comprising
cells with predetermined amounts of the target.
[0132] In one embodiment the kit-of-parts may comprise a binding
agent comprising a mixture of the first and the second binding
molecules, wherein the binding molecules are present in a
predetermined ratio, characterized in that the amount of the second
binding molecules is higher than the amount of the first binding
molecules.
[0133] The above mentioned embodiments are not-limiting.
EXAMPLES
[0134] The following is a description of non-limiting working
examples illustrating some embodiments of the disclosed invention.
The theoretical considerations are part of the description and not
bounding. The described embodiments are exemplary and not
limiting.
Abbreviations
[0135] MBHA 4-Methylbenzhydrylamine [0136] NMP N-Methyl Pyrolidon
[0137] HATU 2-(1h-7-azabenzotriazole-1-yl)-1,1,3,3 tetramethyl
uranium hexafluorophosphate; methenamminium [0138] DIPEA
Dilsopropyl EthylAmine [0139] DCM Dichloro Methane [0140] TFA
TriFluoroacetic Acid [0141] TFMSA TriFluor Methyl Sulphonic Acid
[0142] Flu Fluorescein [0143] Dex Dextran [0144] HPLC High
Performance Liquid Chromatography [0145] equi. Equivalent [0146]
L30
1,10,16,25-tetraaza-4,7,13,19,22,28-hexaoxa-11,15,26,30-tetraoxo-triacont-
ane [0147] L60, L90, L120, L150 different polymers of L30,
comprising 2, 3, 4 or 5 L30 reapeats [0148] CIZ 2-chloroZ=2chloro
Benzyloxycarbonyl [0149] FITC FlouresceinIsoThioCyanate [0150] HRP
Horse Radish Peroxidase [0151] GaM Goat anti-Mouse antibody [0152]
DNP 2,4 dinitro-fluorbenzene (DiNitroPhenyl) [0153] ACim
4-amino-Cinnamic acid [0154] LPR Liquid Permanent Red (Dako K0540)
[0155] Sin sinnapinic acid (4-hydroxy-3,5-dimethoxy cinnamic acid)
[0156] Caf caffeic acid (3,4-dihydroxy cinnamic acid) [0157]
Alpha-CHC apha-ciano-4-hydroxycinnamic acid [0158] PNA-X peptide
nucleic acid oligomer (N-(2-aminoethyl)-glycine) comprising
different substituents coupled to the central nitrogen [0159] A
adenine-9-acetic acid, [0160] C cytosine-1-acetic acid, [0161] D
2,6-diaminopurine-9-aceti acid, [0162] G guanuine-9-acetic acid,
[0163] Gs 6-thuioguanine-9-acetic acid, [0164] P 2-pyrimidinone-1
acetic acid, [0165] T thymine-1-acetic acid, [0166] Us
2-thiouracil-1-acetic acid. [0167] Dpr 2,3 diamino-propioninc acid,
[0168] Phe phenylalanine, [0169] Tyr tyrosine, [0170] Trp
tryptophane, [0171] Lys lysine, [0172] Cys cysteine, [0173] betaala
betaalanine, N,N diacetic acid [0174] FFPE formaldehyde fixed
paraffin embedded [0175] SMD single molecule detection [0176]
Cross-linker a first substrate of an enzyme with oxidoreductase
activity [0177] Reporter a detectable molecule [0178] RDM Reporter
Deposition Medium [0179] BAM Binding Agent Medium
Materials and Protocols for Visualization of a Protein Target in
Histological Sample According to the Methods Described in
WO2010094283. WO201047680, Both Incorporated by Reference).
[0180] 1. Reporter Molecules:
[0181] 1.1.
Sin-Lys(Sin)-Lvs(Sin-L150-Lys(Flu)(0328-018/D21047/D21067)
[0182] Synthesis is performed solution phase following solid phase
synthesis of intermediates carrying free N-terminal amino groups
and free lysine side chains amino groups.
Alpha-N-Boc-(epsilon-N-2-Cl-Z)-lysine was used to introduce lysine
residues giving free epsilon-N-amino groups following cleavage from
resin. The solution phase labeling is basically an extension of
solid phase techniques, utilizing that the relative high molecular
weight intermediates can be almost quantitatively precipitated with
diethyl ether from TFA or NMP solution.
[0183] Boc-(Lys(2-Cl-Z))3-L150-Lys(Fmoc) is prepared on solid
phase. The Fmoc group is removed, followed by fluorescein labeling
as described above. The intermediate NH2-((Lys(NH2))3-L150-Lys(Flu)
results from cleavage from resin. It is precipitated with diethyl
ether, dissolved in TFA, precipitated then dissolved in NMP and
made basic with DIPEA. This solution is mixed with an equal volume
of 0.2 M sinnapinic acid (4-hydroxy-3,5-dimethoxy cinnamic acid) in
NMP activated by HATU and DIPEA. After 10 min the labeling is
complete and the crude product is further "scrubbed" by addition of
ethylene diamine to a concentration of 10% for 5 minutes. Following
precipitation with diethyl ether, the product is further dissolved
in TFA and precipitated with diethyl ether three times to remove
low molecular weight debris. Prior to "scrubbing" with ethylene
diamine, mass spectroscopy shows two kinds of adducts (and
combinations thereof): +(176)n indicating extra ferulic acids
(phenolic esters on other ferulic acids and fluorescein) and +98
(N,N'-tetramethyl uronium adducts, likewise on unprotected phenolic
groups). These are completely removed by the ethylene diamine
treatment, and active esters and ferulic acid oligomers are
likewise decomposed.
[0184] 1.2.
Fer-Lys(Fer)-Lys(Fer)Lys(Fer)-L150-Lys(Flu)(D19185/D120068)
[0185] On 1 g of MBHA resin with standard solid phase chemistry
Boc-Lys(2CIZ)-Lys(2CIZ)-L.sub.150-Lys(Fmoc) was prepared. The Fmoc
protected Lysine side chain was deprotected with 20% piperidine in
NMP (2.times.5 min) and subjected to repeated carboxy fluorescein
labeling (3 mL 0.2 M in NMP, preactivated for 2 min with 0.9 equi
HATU, a equi DIPEA) 3.times.20 min. The resin was treated with 20%
piperidine in NMP then washed with NMP, DCM and TFA. The
intermediate product was cleaved of the resin with
TFA:TFMSA:thioanisol:m-cresol (6:2:1:1, 3 mL, for 1 h),
precipitated with diethyl ether, resuspended in TFA, precipitated
with diethyl ether, resuspended in NMP and again precipitated with
diethyl ether. It was made basic with 200 .mu.L DIPEA and dissolved
directly in 2 mL 0.3 M Ferulic acid preactivated with 0.9 equi HATU
and 2 equi DIPEA. After 10 min. the crude product was precipitated
with diethyl ether, redissolved in 1350 .mu.L NMP and 160 .mu.L
ethylendiamine was added. After 2 min. the product was precipitated
with diethyl ether. Resuspended in TFA, precipitated with diethyl
ether, dissolved in 25% acetonitril in water (24 mL) and subjected
to RP-HPLC purification.
[0186] Other suitable reporter molecules (second HRP substrate) are
described in PCT/DK2010/000137 and incorporated herein by reference
i.e. reporter conjugate molecules described on pages 86-100 of
WO2011047680 (PCT/DK2010/000137).
[0187] 2. Binding Agents:
[0188] 2.1. Goat anti-Rabbit antibody conjugated with Dex70
conjugated with HRP (L348.111, fractions 10-11)
[0189] 11 nmol 70 kDA MW dextran was reacted with 484 nmol HRP in
316 microliters of buffer A (100 mM NaCl, 25 mM NaHCO.sub.3, pH
9.5) for 3 h at 40 C. Thereafter 44 nmol Goat-anti-Rabbit 196
microL water was added to the dextran-HRP conjugate and allowed to
react for further 1 h at 40 C. The reaction mixture was quenched by
addition of 70 microL 0.165M cystein for 30 min and the product was
purified on Sephacryl 300 (GE Medical) in buffer B (100 mM NaCl, 10
mM HEPES pH 7.2). The eluded product was a dextran conjugate
comprising Goat-anti-Rabbit (GaR) and HRP. The product was divided
into 4 fractions based on conjugate size; The first two fraction
containing product (Frac. 8-9) eluded as a first peak, presumably
containing some cross linked conjugates, then followed by a broad
shoulder that was divided into fractions 10-11 (homogeneous large
conjugates) and fractions 12-21 (smaller variable conjugates) and
finally unconjugated enzymes and antibodies in fractions 22-42.
Measurements on individual product fractions, as well as fractions
containing non-conjugated antibody and HRP, showed a total
conjugate recovery of 87%; ratio dex:GaM:HRP=1:0.96:10.9.
[0190] 2.2. Goat-Anti-Mouse-Dex70-HRP (D18033/D18175)
[0191] 13.7 nmol divinylsulphone were activated 70 kDA MW dextran
and reacted with 602 nmol HRP were in 600 microL buffer (100 mM
NaCl, 25 mM NaHCO.sub.3, pH 9.5) for 3 h at 30 C. Then 41.1 nmol
Goat-anti-Mouse F(ab), antibody in 105 microL water was added, and
the reaction was continued for additional 16 h. The reaction
mixture was quenched by addition of 70 microL 0.165M cystein for 30
min and the product was purified on superdex 200 in 100 mM NaCl, 10
mM HEPES pH 7.2. The eluded product was a Dextran conjugate
comprising Goat-anti-Mouse (GaM) and HRP (Ratio
dex:GaM:HRP=1:1.1:7.5).
[0192] 2.3. Anti-HER2-Antibody Conjugated with Dex70 Conjugated
with HRP (D21100, Fractions 9-10)
[0193] 4.6 nmol 70 kDA MW dextran was reacted with 202 nmol HRP in
125 microliters of buffer A (100 mM NaCl, 25 mM NaHCO.sub.3, pH
9.5) for 3 h at 30 C. Thereafter 18 nmol antiHer2 in 489 microL of
water was added to the dextran-HRP conjugate and the mixture was
allowed to react for further 21 h at 30 C. The reaction mixture was
quenched by addition of 70 microL 0.165M cystein for 30 min and the
product was purified on Sephacryl 300 (GE Medical) in buffer B (100
mM NaCl, 10 mM HEPES pH 7.2). The eluded product was a dextran
conjugate comprising antiHer2 and HRP. The product was divided into
4 fractions based on conjugate size: The first two fraction
containing product (Frac. 7-8) eluded as a first peak, presumably
containing some cross linked conjugates, then followed by a broad
shoulder that was divided into fractions 9-10 (homogeneous large
conjugates) and fractions 11-19 (smaller variable conjugates) and
finally unconjugated enzymes and antibodies in fractions 20-41.
Measurements on individual product fractions, as well as fractions
containing non-conjugated antibody and HRP, showed a total
conjugate recovery of 68%. Assuming direct proportionality between
incorporated HRP and Dextran showed that fractions 9-10 contained
9.1 HRPs and 0.6 antibodies per Dextran. Only these two fractions
were used for experiments.
[0194] 2.3. AntiFITC Antibody Conjugated with Dex70 Conjugated with
HRP (AMM 353-022 Fractions 8-11.)
[0195] 11 nmol 70 kDA MW dextran was reacted with 484 nmol HRP in
316 microliters of buffer A (100 mM NaCl, 25 mM NaHCO.sub.3, pH
9.5) for 3 h at 40 C. Thereafter 66 nmol antiFITC in 196 microL of
water was added to the dextran-HRP conjugate and allowed to react
for further 1 h at 40 C. The reaction mixture was quenched by
addition of 70 microL 0.165M cystein for 30 min and the product was
purified on Sephacryl 300 (GE Medical) in buffer 8 (100 mM NaCl, 10
mM HEPES pH 7.2). The eluded product was a dextran conjugate
comprising antiFITC and HRP. The product was divided into 3
fractions based on conjugate size: The first fractions (8-11)
containing product eluded as a first peak, then followed by a broad
shoulder (smaller variable conjugates, frac. 12-27) and finally
unconjugated enzymes and antibodies in fractions 28-45.
Measurements on individual product fractions, as well as fractions
containing non-conjugated antibody and HRP, showed a total
conjugate recovery of 90%. Assuming direct proportionality between
incorporated HRP and Dextran showed that fractions 10-11 contained
11.7 HRPs and 0.80 antibodies per Dextran. Only these two fractions
were used for experiments.
[0196] 2.4 Other binding agents suitable for the purposes of
present invention are described in PCT/DK2010/000137 and
incorporated herein by reference, i.e. binding agent molecules
described on pages 100-106 of WO2011047680 (PCT/DK2010/000137).
[0197] 3. First Substrate
[0198] DAB, ferulic acid and alpha-ciano-4-hydroxycinnamic acid
(alpha-CHC) were used as the first substrate at the following
conditions:
TABLE-US-00001 DAB Ferulic acid Alpha-CHC Optimal amount 0.14 mM
1.5 mM 5 mM (Range) (0.1 mM-less (0.5 mM to (1.5 mM and than 1 mM)
5 mM) 15 mM) Optimal H.sub.2O.sub.2 amount 1.5 mM 0.9 mM 0.6 mM
Optimal deposition 5-10 min 10-15 min 10-15 min time Optimal second
Contains Fer Contains Sin Contains Fer substrate or Sin Dot
diameter 3-4 microns 3-4 microns 2-3 microns
[0199] Compared to DAB, dots of a similar diameter with ferulic
acid were obtained when incubation time was doubled; with
alpha-ciano-4-hydroxycinnamic acid the incubation time was as for
DAB, however the dots were smaller (2-3 microns in diameter
compared to 3-4 microns for DAB).
[0200] 4. Incubation Media
[0201] Binding Agent Medium (BAM)
[0202] 0.1% 4-aminoantipurine, 0.2% Procline 2% BSA, 0.2% Casein,
2% PEG, 0.1% TWEEN20 (polyoxyethylene (20) sorbitan monolaurate),
0.1 M NaCL, 10 mM HEPES, pH 7.2. (ABCPT-buffer)
[0203] 5. Reporter Deposition Medium (RDM):
[0204] 50 mM imidazole HCl pH 7.5, 0.1% Nonidet P40, 0.1%,
benzalkonium chloride, 0.005% (1.5 mM) hydrogen peroxide.
[0205] 6. Other Reagents [0206] DAB chromogen solution (Dako K3465)
[0207] LPR chromogen solution (Dako K0640) [0208] Haematoxiin
counterstain (Dako S3301) [0209] Wash buffer (Dako S3306) [0210]
Target retrieval solution (Dako S1699) [0211] Mounting media Dako
Fairmount (S3025)
[0212] 7. Instruments.
[0213] Dako Autostainer Classic. This instrument is a totally open
and freely programmable automated IHC instrument where reagents and
incubation times can be used and set at will. The instrument
performs four basic actions [0214] 1. Aspirate reagent, [0215] 2.
Blow wash buffer off horizontally placed slide. [0216] 3. Dispense
reagent onto slide. (Known as sip and spit.) [0217] 4. Wash a slide
by flushing it with wash buffer.
[0218] A typical program for a single slide is described below in
protocol 1 (see Example 1). For all SMD experiments the initial
peroxidase block and the target visualization steps were kept
invariable.
Example 1. Quantification of a Target in a Histological Sample.
Determination of Kd1, Kd2 and Pr (Method I)
[0219] Theoretic Considerations:
[0220] In order to define a number of single entities of a target
in a sample and, in particularly, total number of said units, e.g.
single target protein molecules, several complex equilibrium
experiments may be performed, employing: [0221] 1. Several
Reference samples of a test material with identical, but unknown,
levels of an immobilized protein molecules, Pr. (e.g. serial
sections of a single block of homogeneous Her2 reference cells
lines); [0222] 2. A primary antibody, Ab1 (e.g. a high affinity
monoclonal Rabbit-anti-HER2) with unknown dissociation constant,
Kd1 that binds to said protein, [0223] 3. An Enzyme labeled
secondary antibody, Ab2 with unknown dissociation constant, Kd2,
that binds to said primary antibody. [0224] 4. Technologies for
visualizing almost every single molecule of said secondary
antibodies as discrete visually distinguishable dots (termed herein
"single molecule dots" or "SMD") (e.g. as described in
PCT/DK2010/000137 or herein).
[0225] According to the present invention the level of immobilized
target in a sample, e.g. a protein, can be expressed as counted SMD
per nucleus (e.g. in reference cell lines samples), or per area or
volume of a tissue sample, etc; the number of molecules can via
Avogadro's Number be translated into concentration of said
molecules in the sample.
[0226] It is generally accepted that theoretical framework for
antibody protein interaction is a complex equilibrium. The antibody
will reach equilibrium with the target protein:
Ab1+PrAb1:Pr F1
Governed by the dissociation constant, Kd1 of the antibody:
[ Ab 1 ] .times. [ Pr ] [ Ab 1 : Pr ] = Kd 1 F2 ##EQU00001##
[0227] Under such equilibrium conditions, total protein, PrTotal
and total antibody, Ab1 Total will be distributed between free
protein and complex and free antibody and complex
PrTotal=Pr+Ab1:Pr P3
Ab1Total=Ab1+Ab1:Pr F4
From F2 follows:
[ Pr ] = [ Ab 1 : Pr ] .times. Kd 1 [ Ab 1 ] F5 ##EQU00002##
[0228] Substituting F5 into F3 gives:
PrTotal = [ Ab 1 : Pr ] .times. Kd 1 [ Ab 1 ] + [ Ab 1 : Pr ] F6
##EQU00003##
[0229] P6 can then be arranged as the following:
PrTotal = [ Ab 1 : Pr ] .times. Kd 1 + [ Ab 1 ] [ Ab 1 ] F7
##EQU00004##
[0230] The first experimental challenge lies in determining when
this first equilibrium has been reached [Ab1:Pr] can be detected
and determined by a subsequent second equilibrium experiment with
enzyme labeled Ab2 followed by SMD visualization. The first series
of experiments, Exp1, can be used to establish that a sequential
application of a constant concentration of Ab1 to samples with a
constant amount of immobilized protein will eventually result in a
constant amount of Ab1:Pr being detected in a subsequent second
visualization step using enzyme labeled Ab2 and SMD detection.
[0231] The need to use multiple sequential additions of Ab1 arises
from the fact that a single addition of Ab1 to a sample with
immobilized protein will result in Ab1:Pr complex formation, and
thus in a decrease in both Ab1 and Pr concentration. The first
equilibrium may apparently be reached, but sequential additions of
Ab1 to identical reference samples until a constant level of Ab1:Pr
is detected must be used to access when a true equilibrium
reflecting the concentration of Ab1 has been reached, i.e. when
further additions of Ab1 will no longer result in an increase in
Ab1:Pr being detected. A single or a few additions of Ab1 will
result in equilibriums reflecting the total amount of protein in
the immobilized samples rather than the concentration of Ab1. Ab1
will be depleted due to complex formation and the effective
concentration in equilibrium will be significantly lower than the
concentration of Ab1 applied.
[0232] Formula 4 reflecting the effects of lowered concentration of
free antibody can be ignored, if multiple additions of antibody
confirm that depletion or slow kinetics is not a case.
[0233] Experimental set-up to confirm the above theory may be
designed as the following: A constant concentration of Ab1 is
sequentially applied to samples with constant concentration of
immobilized protein. The Ab1:Pr complexes formed are subsequently
detected using an enzyme labeled secondary antibody and SW)
visualization. Thus, a true equilibrium reflecting the
concentration of Ab1, not the amount of immobilized protein, can be
established. (The experiment confirming this theory is described
below in Experiment 3a, which shows that, after four to five
sequential 10 min-incubations reference samples with Ab1 no further
increase in Ab1:Pr complexes is detected).
[0234] The theory behind the second complex equilibrium step is
identical to the theory regarding the first (discussed above).
[0235] The second equilibrium is established between the enzyme
labeled secondary antibody and the immobilized primary antibody
protein complex:
Ab1:Pr+Ab2Ab2:Ab1:Pr F8
Governed by the dissociation constant, Kd2 of the labeled secondary
antibody:
[ Ab 2 ] .times. [ Ab 1 : Pr ] [ Ab 2 : Ab 1 : Pr ] = Kd 2 F9 Ab 1
: PrTotal = Ab 1 : Pr + Ab 2 : Ab 1 : Pr F10 Ab 2 Total = Ab 2 + Ab
2 : Ab 1 : Pr F11 ##EQU00005##
[0236] From F9 follows:
[ Ab 1 Pr ] = [ Ab 2 : Ab 1 : Pr ] .times. Kd 2 [ Ab 2 ] F12
##EQU00006##
[0237] Substituting F12 into F10 gives:
Ab 1 : PrTotal = [ Ab 2 : Ab 1 : Pr ] .times. Kd 2 [ Ab 2 ] + [ Ab
2 : Ab 1 : Pr ] F13 ##EQU00007##
[0238] F13 can then be rearranged into F14
Ab 1 : PrTotal = [ Ab 2 : Ab 1 : Pr ] .times. Kd 2 + [ Ab 2 ] [ Ab
2 ] F14 ##EQU00008##
[0239] This second equilibrium can only be established, if the
concentration of Ab1:Pr remains essentially constant during the
second equilibrium experiment, i.e. that no significant
dissociation between protein and primary antibody takes place
during washing steps and incubation with enzyme labeled secondary
antibody. If this condition is observed, it is possible to
substitute Ab1:PrTota of Formula 14 for [Ab1:Pr] of Formula 7.
[0240] This gives the next equation (Formula 15):
PrTotal = [ Ab 2 : Ab 1 : Pr ] .times. Kd 1 + [ Ab 1 ] [ Ab 1 ]
.times. Kd 2 + [ Ab 2 ] [ Ab 2 ] F15 ##EQU00009##
[0241] Formula 15 can be regarded as the theoretical foundation of
the absolute count experiments, i.e. experiments where the total
number of target molecules in a sample is determined, because it
describes a relationship between Kd1 and Kd2, which can be
determined in equilibrium experiments in connection with the
antibody titrations, and the total protein concentration and
complexes of the protein with the antibodies that are visualized as
dots.
[0242] These experiments may be performed as the following: A
constant concentration of Ab1 is sequentially applied to samples
with constant concentration of immobilized protein. The Ab1:Pr
complexes formed are subsequently detected using an enzyme labeled
secondary antibody and SMd visualization. The enzyme labeled
secondary antibody (a constant amount thereof) is likewise
sequentially applied multiple times. The experiment confirming this
theory (described in Experiment 3b) has shown that after four to
five sequential 10 min-incubations of enzyme labeled Ab2 with
reference samples previously equilibrated with primary antibody no
further increase in formation of Ab2:Ab1 Pr complexes was detected,
neither a decrease (potentially resulting from a significant
protein-Ab1 dissociation during washing steps and establishment of
the second equilibrium) was detected. Thus, a true equilibrium
reflecting the concentration of immobilized protein, (Ab1) and
enzyme labeled [Ab2] can be established confirming the equation of
Formula 15.
[0243] For the same reasons as discussed for Formula 4, now Formula
11 may be ignored. The effects of lowered concentration of free
secondary enzyme labeled antibody can be ignored if multiple
additions of this antibody confirm that depletion or slow kinetics
is not a problem.
[0244] Tissue samples with unknown protein concentration level may
be routinely incubated with primary antibodies in order to
determine said unknown protein concentration. This step may be
followed by steps of incubation with enzyme labeled secondary
antibody followed by, yet, extra steps of visualization.
[0245] As a rule, in routine INC staining procedures only single
incubations with primary and secondary antibodies are used, and a
physical agitation, either uncontrolled (due to gravity,
evaporation or wicking) or controlled by active stirring of
reagents on the slide, is an established practice. However, using
mixing and/or relative high concentrations of both primary and
secondary antibody, pseudo equilibrium conditions may be reached by
a single reagent application, resulting in reproducible results
(this is how the well-known histological staining systems work now,
e.g. Envision system). Consecutive additions of an antibody reagent
(primary or enzyme labeled secondary) results in relative stable
equilibriums, and thus can also act as a safeguard against antibody
depletion and allow, in contrary to the traditional IHC staining,
the precise evaluation of the amount of the target in an IHC
sample.
[0246] As described in Experiments below, the necessity of use of
tow amounts of high affinity primary antibody arises from the low
value of Kd1 of the Her2 clone tested in combination with the need
to use concentrations below Kd1 in order to measure Kd1. For
routine use concentration well above Kd1 may be used, reducing the
need for multiple additions. In case of the secondary antibody, it
is the need to reduce dot overlap that prevents use of higher
concentration. At higher concentrations the overlapping dots may
prevent an accurate dot count, at least when counting is done
manually.
[0247] When the staining conditions leading to forming
non-ovelapping SMOs are observed, the SMD can be counted as Pr,
and, if PrTotal can be kept constant (e.g. in case of use of
sequential sections of same reference material), experiments with
varying [Ab1] and constant [Ab2] will allow determining kd1;
PrTotal and Kd2 will still remain unknown, but constant. This
allows rearrangement of Formula 15 into Formula 16:
Dots = Constant .times. [ Ab 1 ] Kd 1 + [ Ab 1 ] F16
##EQU00010##
[0248] The Constant (C) reflects the value of PrTotal of the sample
and the fraction of Ab1:Pr complexes that are detected in the
second equilibrium reaction with constant [Ab2]. And it is the
absolute number of Dots that can be detected under those
conditions. The equation of F16 means that at high and increasing
[Ab1] the number of Dots will approach, but never reach a constant
level. At low and decreasing [Ab1] the number of Dots, which is a
hyperbolic function of [Ab1], will approach a linear function of
[Ab1].
[0249] The number of Dots as function of [Ab1] is a hyperbolic
function, and Formula 16 is used to determine Kd1 by fitting
experimental data correlating Dots with [Ab1] in experiments with
constant reference material and constant [Kd2]. However, using
sequential additions of Ab1 at concentrations close to Kd1
reproducibly allow accurate determination of Kd1 via an excellent
fit to Formula 16.
[0250] Experimental set-up that allows determination of Kd2 is
slightly more complex. The challenge is that concentrations of
enzyme labeled secondary antibody that are close to Kd2 invariably
will lead to formation of dots the number of which will be too high
to count due to overlap problems. Use of a very low concentration
of primary antibody and/or use of reference material with a low
protein concentration would not be a solution, as a background from
high concentrations of secondary antibody will give a very high
background noise due to unspecific bound secondary antibodies, thus
would not accurately reflect the protein concentration. This is
further compounded by difficulties of establishing the equilibrium
at very low primary antibody concentrations. An approach to
overcome these challenges is to use both primary and secondary
antibody in relative high concentrations, in case of the secondary
antibody with concentrations around Kd2, and visualizing the bound
secondary antibody by conventional IHC. By conventional IHC is
meant that the enzyme labeled secondary antibodies are used to
generate a brown deposit of 3,3'-diaminobenzidine (DAB), e.g. by
using the Envision system, rather than SMD visualization. The
intensity of such conventional DAB deposits is not linear and does
not correctly reflect the quantity of molecules of a target in the
sample, however the intensity of two deposits may be visually
compared and determined to be of approximately of the same
intensity. Indeed, this is how the IHC-staining results are at
present interpreted: they are evaluated by comparing the intensity
of the brown deposit in test samples and reference samples and
follow the graphic or descriptive guidelines for the
interpretation.
[0251] Using identical reference material, PrTotal (of F15) can be
kept constant. If [Ab1] and [Ab2] are also constant, and Ab2:Ab1:Pr
is visualized by conventional IHC as a brown deposit, the staining
will be of constant intensity. Evidently, the intensity has to be
within the dynamic range of conventional INC so that variations in
Ab2:Ab1:Pr are reflected in variable intensity of the brown
deposit. INC slides are normally scored on a scale: +0 (no color at
all). +1 (weak intensity), +2 (moderate intensity), and +3 (highest
intensity/brownish-black). In order to accurately reflects
[Ab1:Ab2:Pr], the score should be within the +0.5 to +2.5 range, so
that upwards or downwards variation is detected, and, preferably,
within the +1 to +2 range, where the intensity variation as
function of [Ab1:Ab2:Pr] is most pronounced and the background
noise is minimal.
[0252] Having established a reference system in the desired dynamic
range (i.e. within +1 to +2 and [Ab2] around [Kd2]) Experiment 3d
(described below) is carried out using a lower constant
concentration of Ab1 [Ab1].sub.2 with variable and increasing
concentration of Ab2 relative to the initial reference
experiment
[0253] By increasing [Ab2], the concentration of [Ab2:Ab1:Pr] will
at some point reach a level identical to the prior established
reference level, resulting in an identical intensity of brown
deposit. When the intensity of the brown DAB deposit is of
identical intensity to the deposit formed with [Ab1].sub.1 and
[Ab2].sub.1 it is to be concluded that:
(Ab2:Ab1:Pr).sub.1=[Ab2:Ab1:Pr].sub.2
Thus, the identical staining levels have been reached by two
different combinations of [Ab1] and [Ab2] and constant PrTotal. It
follows to the equation:
Kd 1 + [ Ab 1 ] 1 [ Ab 1 ] 1 .times. Kd 2 + [ Ab 2 ] 1 [ Ab 2 ] 1 =
Kd 1 + [ Ab 1 ] 2 [ Ab 1 ] 2 .times. Kd 2 + [ Ab 2 ] 2 [ Ab 2 ] 2
##EQU00011##
[0254] As Kd1 is known, as well as [Ab1].sub.1 and [Ab1].sub.2 from
experimental conditions, the equation may be reduced to Formula 17
(C1 and C2 are Constants):
C 1 .times. Kd 2 + [ Ab 2 ] 1 [ Ab 2 ] 1 = C 2 .times. Kd 2 + [ Ab
2 ] 2 [ Ab 2 ] 2 F17 ##EQU00012##
[0255] Dividing by C.sub.1 gives:
Kd 2 + [ Ab 2 ] 1 [ Ab 2 ] 1 = C 3 .times. Kd 2 + [ Ab 2 ] 2 [ Ab 2
] 2 F18 ##EQU00013##
[0256] Formula 18 may be rearranged to allow isolation of Kd2;
(Kd2.times.[Ab2].sub.2)+([Ab2].sub.1.times.[Ab2].sub.2)=(C.sub.3.times.K-
d2.times.[Ab2].sub.1)+(C.sub.3.times.[Ab2].sub.1.times.[Ab2].sub.2),
which can be reduced to:
Kd 2 = ( 1 - C 3 ) .times. ( [ Ab 2 ] 1 .times. [ Ab 2 ] 2 ) ( C 3
.times. [ Ab 2 ] 1 ) - [ Ab 2 ] 2 F19 ##EQU00014##
[0257] Where C.sub.3 (which is equal to C2/C1, see above) is
defined by:
C 3 = ( Kd 1 + [ Ab 1 ] 2 ) .times. [ Ab 1 ] 1 [ Ab 1 ] 2 .times. (
Kd 1 + [ Ab 1 ] 1 ) F20 ##EQU00015##
[0258] C.sub.3 relates to two hyperbolic functions on top of each
other reflects a constant level of the brown staining that is
derived from two different sets of experimental conditions: first,
a reference level is established by reaching a first equilibrium
reflecting [Ab1].sub.1 and [Ab2].sub.1; then, the same reference
level is reached by using [Ab1].sub.2 and [Ab2].sub.2. Kd1 is
known, Kd2 can thus be determined.
[0259] A reference level of the conventional staining intensity may
be produced using [Ab1].sub.1 and [Ab2].sub.1. Using a different
concentration of Ab1, [Ab1].sub.2 allows titration of [Ab2] until a
level of identical staining intensity is reached by [Ab2].sub.2.
This allows determination of Kd2 from Formula 19.
[0260] Returning to the original Formula 15, having determined Kd1
and Kd2, any SMO staining experiment fulfilling the proviso of
reaching equilibrium in both steps and allowing an accurate SMO dot
count, will allow determination of PrTotal in the reference
sample(s) used.
[0261] Any reference sample, wherein PrTotal has been determined in
this way, obtains a status of "absolute reference".
[0262] The absolute number of proteins (or any other immobilized
target compound) in the immobilized sample has been counted and may
be expressed in absolute terms such as molecules per
area/volume/cell etc. depending on the nature of the immobilized
sample.
Experimental Evidence
[0263] As a test material serial sections of pellets of formalin
fixed paraffin embedded cell lines sk45, df45, d123 expressing Her2
were used (these cell lines will further be referred to as the 0+,
the 1+ and the 3+ cell line, correspondingly). These cell lines are
the 0+, 1+ and 3+ control material for FDA approved Dako HercepTest
for breast cancer. Pellets of the cell lines were embedded in a
single block of paraffin to provide sections where the every cell
lines present. The choose of the test material reflects
availability of the material (e.g. each single block provides
hundreds of serial sections, the presence of three different cell
samples on each test slide allows inter correlation between the
results of one staining procedure of three different test
samples).
[0264] Slides with FFPE sections of blocks containing the three
cell lines (further referred as "slides") were deparaffinized by
emersion in xylene (2.times.5 min) followed by 96% ethanol
(2.times.2 min) and 70% ethanol (2.times.2 min). Then, the slides
were washed with deionized water and transferred to Target
retrieval solution, either the high pH solution (Dako S2375),
diluted 10.times. (examples 1 and 2 with anti cytokeration) or low
pH solution (Dako 51700) (see examples 10.3-10.8 below). The slides
were then heated to boiling in a microwave oven (approx 5 min) and
gently boiled for 10 min. Afterwards the slides were allowed to
cool for min 20 min and then were transferred to a wash buffer
(Dako S3006) diluted 10.times..
[0265] Pan specific anti-cytokeratin antibody (Dako M3515,
monoclonalmouse) was used both as concentrate and diluted solution.
Antibody dilutions were made based on total protein concentration
(indicated on each vial) and considering the molecular weight of
the antibody (150 kDa/mol). This antibody is further referred as
"anti-cytokeratin".
[0266] Anti-Her2 antibody was a monoclonal rabbit antibody Dako
clone 25-11-3). Dilutions were made based on calculated total
protein concentration in a concentrated solution and the molecular
weight of the antibody of (150 kDa/mol). The antibody is referred
herein as "anti-HER2".
Staining Protocol 1
[0267] Peroxidase block, 5 rain in Delco S2023 [0268] Wash [0269]
(a) Formation of target sites: [0270] Primary antibody, [0271] Wash
[0272] HRP-Labeled secondary antibody, [0273] Wash. [0274] (b)
Formation of reporter dot of deposits at target sites [0275]
incubation of samples (a) 10 minutes with 0.28 mM DAB and 5 .mu.M
reporter (D21047) in RDM. [0276] Wash [0277] c) Detection of dots
of the reporter deposits at single target sites [0278]
Anti-FITC-AP, 10 min, 20 nM D20038 in BAM [0279] Wash [0280] LPR,
10 min, Dako K0640 [0281] Wash [0282] d) Haemotoxylin counterstain
[0283] Haematoxylin, 5 min [0284] Wash with deionized neater [0285]
f) Mounting
[0286] Additional washes may be introduced into the automated
protocol. The automated scheduler will keep overall protocol time
at a minimum, by reducing duration of washing steps to a minimum;
however, duration of washing steps will depend on loading of the
instrument. If a single slide is programmed to be stained, a single
washing step might be reduced to 20 seconds, while a full load of
48 slides significantly increase washing time. To keep this time
variation minimal, 10 slides in average were stained in each run.
Accordingly, washing step duration was kept approximately 2 min per
step. Multiple washes following reporter deposition and incubation
of the deposits with anti-FITC-AP assures a minimal LPR background
staining. Despite of massive amplification (it is estimated that
each red Dots derived from a single antibody-dextran-HRP molecule
bound to the target comprise in average 100 billion molecules of
LPR) there can virtually no background be detected.
[0287] Extra washing might be recommended in order to reach the
highest level of amplification and lowest background staining,
while reporter and reporter binding agent are used in relative high
amounts.
Evaluation of Staining
[0288] Dot counting was initially performed manually, by visual
inspection of stained slides and their images. Automated image
analysis was performed using the freeware JMicrovision vs. 1.27. In
an exemplary embodiment, LPR red Dots produced as described and
haematoxylin stained nuclei were automatically counted. Automated
counts were verified by visual inspection and manual counts.
Segmentation and object abstraction could be based on hue alone in
Hue, Saturation, Intensity, (HSI) color space, i.e. both intensity
and saturation set to full 0-255 range. Dot hue was set to 188
(violet)-255 and 0-16 (orange), nuclear hue to 70 (green) to 163
(blue). Dot-nuclear contrast was enhanced by over exposing red
(1.2), neutral green (1.0) and under exposure of blue (0.56) during
image capture performed on an Olympus SX51 microscope fitted with a
DP50 5.5 Mpixel camera and CellD image capture software.
[0289] Experiment 1. Determination of Kd of Anti-Cytokeratin
Antibody
[0290] 8 slides with FFPE sections +0, +1 and +3 cell lines were
pretreated and stain as described above (see pretreatment and
protocol 1).
[0291] The primary antibody (anti-cytokeratin), was applied for 20
min in varying concentrations as described in the table:
TABLE-US-00002 Slide number Concentration of M3115 in BAM 1 40 nM 2
33 nM 3 25 nM 4 20 nM 5 13 nM 6 10 nM 7 5 nM 8 2.5 nM
[0292] The slides were then mounted with aqueous Faramount, 3
images of each cell line pellet on each slide were captured, red
colored dots were manually counted in each image and the number of
counted dots was compared to a theoretically calculated number of
dots in the samples.
[0293] Presuming that one molecule anti-cytokeratin (cAb) is
associated with one dot, the theoretical number of dots (Ndot) may
be calculated using the following formula
Nd = [ cAb c ] .times. Ndot max Kd + [ cAb ] . ( Formula 1 )
##EQU00016##
[0294] Wherein [cAb] is the concentration of anti-cytokeratin
antibody, and Kd is the dissociation constant of the
anti-cytokeratin antibody, i.e. cAb, and Ndot.sub.max is a
constant.
[0295] The constant named Ndot.sub.max means maximal number of dots
and in the present content means that the number of dots approaches
the maximum value when the used concentration of an antibody is
significantly above its Kd value, i.e. when the anti-cytokeratin
antibody are used in a concentration that is far beyond the Kd
value.
[0296] This formula is derived from the formula for the
dissociation constants for the primary and secondary antibodies
with the prerequisite that the absolute concentration of protein in
every test sample (i.e. samples of cells +0, +1 and +3, 8 slides of
each cells line with different concentrations of the antibody as
indicated in the table below) is constant and the concentration of
the secondary antibody is kept unvarying between slides.
[0297] The table (1) shows the number of experimentally obtained
and theoretically calculated dots for every sample 1-8 for all
three test cell lines:
TABLE-US-00003 Concentration of Dots counted and Dots counted and
Dots counted and primary calculated, total of 3 calculated, total
of 3 calculated, total of 3 antibody images in +0 cell line Images
in +1 cell line images in +3 cell line Slide nM counted calculated
counted calculated counted calculated 1 2.25 165 170 318 316 376
389 2 5 293 292 445 542 627 667 3 10 384 411 731 765 879 941 4 13.3
487 458 920 851 1043 1048 5 20 502 518 968 962 1140 1185 6 25 581
547 1026 1015 1333 1250 7 30 669 567 1159 1054 1546 1297 8 40 629
595 1269 1106 1663 1361
[0298] By fitting the curves generated from the formula to the
curves generated from the experimental data, approximate values of
Kd1 and Ndot.sub.max can be determined. Thus, Kd1 was set to 7 nM,
for all three calculated series, Ndot.sub.max to 700 (+0), 1300
(+1) and 1600 (+3).
[0299] A Kd value of 7 nM is in good agreement with experimental
count across all three cell lines. In case of the +1 and +3 cell
lines, calculated values are slightly below measured values for
high concentrations of antibody. Anti-cytokeratin antibody M 3515
has a broad specificity and it recognizes several different
cytokeratin subtypes. Theoretically, for each cytokeratin subtype
the antibody may have a slightly different Kd since the
surroundings the antigen may be different and it may influence the
antibody binding. This explains a "non-perfect fit" with the
hyperbolic curve. Furthermore, that some unspecific binding might
take place at concentrations well above the Kd value.
Conclusion
[0300] The performed quantification can be considered to be precise
because the results from experiments where different slides and
different cell lines were used can be directly compared, i.e. dot
staining pattern provides an easy and rapid digitalized
quantitative evaluation of samples, i.e. by counting the visually
distinct dots, e. g. 600 dots are easily distinguishable from 300
dots in another sample.
[0301] The Kd value of the used secondary antibody (D20168) is not
known, and it has not been shown that an equilibrium is reached in
this step of affinity binding, however control experiments did show
that further incubation with primary antibody (prolonged incubation
time and additional portions of antibodies) did not lead to
significant increase in signal. Thus, if a constant fraction of
primary antibodies is recognized by the secondary antibody during
the experiment, the latter has no influence on the Kd measurement.
Using multiple applications of secondary antibodies twice as many
dots can be produced. In these applications maximal number of dots
per slide (Ndot.sub.max) is also doubled, but these does not
influence measurement the Kd.
[0302] Experiment 2. Determination of Kd of a Second Binding Agent
(Goat-Anti-Mouse-Dextran-HRP Conjugate (D20168).)
[0303] This experiment was performed using conventional IHC stains
(Dako Envision system).
[0304] Slides were pretreated as described, and subjected to the
following staining protocol 2; [0305] 1. Peroxidase block, 5
min
[0306] Wash [0307] 2. Anti-Cytokeratin, 20 min in incubation media
1
[0308] Wash [0309] 3. HRP-labeled secondary antibody (D20168), 20
min in incubation media 1
[0310] Wash [0311] 4. DAB chromogen solution, 10 min
[0312] Wash [0313] 5. Haematoxilin stain, 5 min
[0314] Wash with water
[0315] Wash
[0316] Wash with de ionized water.
[0317] 12 samples of each of the three cell lines (+0, +1 and +3)
were divided in two series, wherein six slides of the first series
were inculcated with of 2.5 nM anti-cytokeratin antibody and
further incubated with 6 different concentrations of 020168 (100
nM, 50 nM, 25 nM, 15 nM, 10 nM and 5 nM), and six slides of the
second series were incubated with 10 nM anti-cytokeratin antibody
and further incubated with 6 different concentrations of 020168
(100 nM, 50 nM, 25 nM, 15 nM, 10 nM and 5 nM). The slides of both
series were than stained with DAB (as chromogen) and Haemotoxitin
according to the above protocol.
[0318] For all three cell lines staining intensity increased with
increasing concentration, but leveled off within the dynamic range
of the IHC staining (below a score of +2.5).
[0319] As expected, using a higher concentration of primary
antibody resulted in higher intensities of staining. The staining
of the slide treated with 2.5 nM anti-cytokeratin and 100 nM 020168
(further referred as slide A) (of each cell line) was compared to
the staining of slides with 10 nM anti-cytokeratin (within each
cell line). Two independent mock observers were used to estimate
the intensity of staining. They found that for all three cell lines
the intensity of staining of the slide A was identical to the
intensity of staining of the slide treated with 10 nM
anti-cytokeratin and 15 nM D20168 (slide B). Because of the
reference material was constant (same cell line control slides) and
approximately the same staining intensity was observed in slides
treated with different amounts of the primary and secondary
antibody. It was concluded that the number of
Cytokeratin-anti-Cytokeratin-D20168 complexes present in slides A
and B (within one cell line) was the same. Accordingly, the
following equation could be used to calculate Kd (i.e. Kd2) of the
secondary antibody of D20168:
Kd 2 = ( 1 - C 3 ) .times. ( [ Ab 2 ] 1 .times. [ Ab 2 ] 2 ) ( C 3
.times. [ Ab 2 ] 1 ) - [ Ab 2 ] 2 ##EQU00017##
[0320] Wherein C.sub.1, C.sub.2 and C.sub.3 [Ab1].sub.1=2.5 nM,
[Ab1].sub.2=10 nM, [Ab2].sub.1=100 nM, (Ab2).sub.2=15 nM, and
wherein C.sub.3 defined from the following equation:
C 3 = C 2 C 1 = ( Kd 1 + [ Ab 1 ] 2 ) .times. [ Ab 1 ] 1 [ Ab 1 ] 2
.times. ( Kd 1 + [ Ab 1 ] 1 ) ##EQU00018##
[0321] Thus, Kd2 of D20168 was calculated to be 25 nM.
[0322] Experiment 3a: Establishment of Equilibrium Conditions for
Primary HER2 Antibody.
[0323] Due to a low Kd (i.e. high affinity) value for the HER2
antibody clone tested, initial attempts to determine the Kd value
by means similar to example 1 might give results that would not fit
well with equilibrium conditions: a single application of a very
low concentrations (100 pM) of the primary antibody may lead to
formation of incomplete equilibrium. Therefore, in order to defined
and secure conditions of the equilibrium conditions for the HER2
antibodies, sequential additions of the primary antibody were
applied to the samples of all three lines. Slides treated with the
lowest concentration (100 pM) of the antibody, where antibody
depletion and incomplete equilibrium problems were expected to be
most severe, were as well treated with two sequential additions of
high concentrations of the secondary antibody, to compensate
depletion in of the primary antibody step.
[0324] The staining was done according to protocol 1 with the
specific concentrations, incubation times and number of sequential
additions for the primary and secondary antibodies, as the
following. [0325] 100 pM HER2 antibody, 1-6 sequential incubations,
10 minutes each:
TABLE-US-00004 [0325] Slide number Number of additions 1 1 2 2 3 3
4 4 5 5 6 6
[0326] One wash followed each addition (prior to the following
addition); [0327] 5 pM HRP-Labeled Goat-anti-Rabbit (L348-111 frac.
9-10), two sequential incubations, 10 min each.
[0328] Three images (10.times. magnification) of each +0 and +1
cell line samples were taken and the number of SMD dots per nucleus
was counted. The +3 cell line samples were disregarded due to a
very intensive staining which did not allow an accurate count the
dots. The results are presented in Table 2 below:
TABLE-US-00005 Additions of Dot/nuclei(0+) Dot/nuclei(1+) anti-HER2
(Series 1 of FIG. 5) (Series 2 of FIG. 5) 1 0.158 0.407 2 0.258
0.665 3 0.305 1.031 4 0.42 1.309 5 0.532 1.536 6 0.532 1.513
[0329] From the results of the experiment it was concluded that at
least 5 additions of the HER2 primary antibody solution, were the
amount of the antibody is 100 pM, is required to avoid depletion
and establish true equilibrium condition in the tested samples.
[0330] Experiment 3b: Establishment of Equilibrium Conditions for
Secondary Antibody.
[0331] To define the equilibrium conditions for the secondary
antibody, a high concentration of the HER2 primary antibody was
used in the first step of the procedure which would expected to
give a high level of bound primary antibody to the target, and a
series of applications of low concentration of the secondary
antibody (L348-111, fractions. 9-10), where depletion of the
antibody would be expected to be most sever, was performed in the
second step of the procedure.
[0332] The staining was done according to protocol 1 with the
specific concentrations, incubation times and number of additions
for the primary and secondary antibodies described below: [0333]
500 pM HER2 antibody, 2 sequential additions, 10 min each;
[0334] Wash [0335] 5 pM L348-111, 1-5 sequential additions, 10 min
each:
TABLE-US-00006 [0335] Slide number Number of additions 1 1 2 2 3 3
4 4 5 5
[0336] One wash was applied after each addition, prior to the
following addition.
[0337] Three images (10.times. magnification) of each +0 and +1
cell sample were taken and the number of SMD dots per nucleus was
counted. The +3 cell line samples were disregarded due to a very
intensive staining which did not allow an accurate count the
dots.
[0338] The results are presented in Table 3:
TABLE-US-00007 Additions of Dot/nucleus (0+) Dot/nucleus (1+)
Secondary antibody (Series 1 of FIG. 6) (Series 2 of FIG. 6) 1
0.077 0.327 2 0.083 0.609 3 0.195 0.889 4 0.318 1.216 5 0.364
1.31
[0339] From the results of the experiment, it was concluded that at
least 5 additions of 1.5 OA L348-111 frac. 9-10 was required to
reach the equilibrium.
[0340] Experiment 3c: Determination of the Kd Value of the
Anti-HER2.
[0341] From examples 3a and 3b it has been known that 6 sequential
additions of 100 pM HER2 antibody and subsequently 5 additions of 5
pM L348-111 were required in order to reach the equilibrium
conditions and measure the Kd values. Accordingly, SMD staining of
12 slides of samples of the tree cell lines was performed according
to protocol 1 with the specific concentrations, incubation times
and number of additions for the primary and secondary antibodies as
described below: [0342] 6 concentrations of the HER2 antibody, 8
sequential additions, 10 minutes each:
TABLE-US-00008 [0342] Slide number Concentration of HER2 1 and 2
100 pM 3 and 4 200 pM 5 and 6 300 pM 7 and 8 400 pM 9 and 10 500 pM
11 and 12 1 nM
[0343] One wash step was applied r each addition and prior to the
following; [0344] 5 pM L348111, 5 sequential additions, 10 min
each.
[0345] Three images (10.times. magnification) of samples of each +0
and +1 cell lines were taken and the number of SMD dots per nucleus
was counted. The +3 were disregarded due to very intensive
staining, likewise, the slides incubated with the highest
concentration of the primary antibody (1 nM).
[0346] The results of the experiment with samples of the +0 cell
line are presented in Table 4:
TABLE-US-00009 Theoretically calculated Dot/nucleus number of dots
experimentally Kd 280, max 0.7 counted Concentration dot/nucleus in
0+ cell line of Anti-HER2 (Series 1 of FIG. 7) (Series 2 of FIG. 7)
100 0.183246 0.186 200 0.290456 0.305 300 0.360825 0.358 400
0.410557 0.416 500 0.44757 0.451 1000 0.546022 0.69
[0347] Use of very low concentrations of both primary and secondary
antibodies (100-500 pM and 5 pM) correspondingly), combined with
multiple sequential additions is necessary to reach the equilibrium
conditions as demonstrated in experiments 3a and 3b. The 6 times
addition of primary antibody at a concentration well above Kd (1
nM) should led to some background, which is expected, however the
fit obtained from the 5 double determinations around Kd is very
good.
[0348] Experiment 3d: Determination of Kd of
Goat-Anti-Rabbit-Dextran-HRP Conjugate L348-111.
[0349] This experiment was performed using conventional IHC stains.
Slides were pretreated as described, and subjected to the following
staining protocol 3: [0350] 1. Peroxidase block, 5 min
[0351] Wash [0352] 2. Anti-HER2 in incubation media 1, 6 additions,
10 min each;
[0353] Wash [0354] 3. L348-111 in incubation media 1, 3 additions,
10 min each;
[0355] Wash [0356] 4. DAB stain, 10 min
[0357] Wash [0358] 5. Haematoxilin stain, 5 min
[0359] Wash with water
[0360] Wash
[0361] Wash with de ionized water.
[0362] For each of the three cell line, three slides were stained
(in triplicate) with 100 pM anti-HER2 and 50 nM L348-111. The other
six slides were stained with 500 pM anti-HER2 and with decreasing
concentrations of L348-111 (50 nM, 25 nM, 17 nM, 11 nM, 7.5 nM and
5 nM correspondingly). Two independent observes of the staining
results found that for all three cell lines the intensity of the
triplicate stain (100 pM anti-HER2 and 50 nM L348-111) was
identical to the slide treated with 500 pM anti-HER2 and 11 nM
L348-111. As the reference material was constant (same cell line
control slides) and a constant staining intensity was observed, it
could be concluded that the same number of HER2-antiHER2-L348-111
complexes were present. Accordingly, the following formula was used
to calculate the Kd of the secondary antibody:
Kd 2 = ( 1 - C 3 ) .times. ( [ Ab 2 ] 1 .times. [ Ab 2 ] 2 ) ( C 3
.times. [ Ab 2 ] 1 ) - [ Ab 2 ] 2 . ##EQU00019##
[0363] Wherein [Ab1].sub.1 and [Ab1].sub.2 are two different
concentrations of the primary antibody, and [Ab2].sub.1 and
[Ab2].sub.2 are different concentrations of the secondary
antibody.
[0364] Calculating C.sub.3 from the following equitation:
C 3 = C 2 C 1 = ( Kd 1 + [ Ab 1 ] 2 ) .times. [ Ab 1 ] 1 [ Ab 1 ] 2
.times. ( Kd 1 + [ Ab 1 ] 1 ) , , ##EQU00020##
[0365] And using the values of [Ab1].sub.1=100 pM, [Ab1].sub.2=500
pM, [Ab2].sub.1=50 nM, [Ab2].sub.2=11 nM, Kd2 of L348-111 was found
to be equal to 28 nM.
[0366] In the equilibrium titration of example 3c the results were
fitted to 0.70 dots per nucleus (at conditions of saturation with
primary antibody and use of L348-111 at 1.5 pM concentration).
Accordingly, using the following equation it is possible to
calculate the total amount of HER2 (PrTotal) present in +0
cells:
PrTotal = [ Ab 2 : Ab 1 : Pr ] .times. Kd 1 + [ Ab 1 ] [ Ab 1 ]
.times. Kd 2 + [ Ab 2 ] [ Ab 2 ] , ##EQU00021##
[0367] wherein [Ab2:Ab1:Pr] is the concentration of complexes
HER2-anti-HER2L348-111, Kd1 is the constant dissociation of
anti-HER2, and Kd2 is the constant dissociation of L348-111,
[Ab1].sub.1 and [Ab1].sub.2 two different concentrations of the
anti-HER2, and [Ab2].sub.1 and (Ab2).sub.2 are two different
concentrations of L348-111.
[0368] Setting [Ab2:Ab1:Pr] at 0.70 SMD dots/nucleus, the first
fraction to 1 and Kd2 to 28 nM and [Ab2] to 1.5 pM, the value of
PrTotal is calculated to be 13.000 molecules/nucleus.
[0369] This value is in a good agreement with the data of the field
(see, for example, David G. Hicks, D. G. and Schiffhauer, L.
Assessment of HER2 Status by Immunohistochemistry: Routine Use of
Controls for IHC Testing-Laboratory Medicine, 2011; 42(8):459-467)
that the 0+ cell line express 21,600.+-.6700 copies of the Her2
receptor on the surface of these cells.
Example 2. Quantification of a Target in a Histological Sample
(Method II)
[0370] 1. Theoretical Considerations
[0371] The method (II) for estimation of the total (absolute)
number of target molecules in cells has a number of similar
approaches compared to the method (I), however it has also some
differences.
[0372] One of the problems associated with the previously described
method is that equilibrium conditions should be established for
both primary antibody and labeled secondary antibody. In case of
high target concentrations this can be a problem as depletion of
binding agents during incubations will occur and it will thus
require multiple and prolonged incubations with the binding agents.
The present method utilizes that using very high concentration of
binding agents a "top" level of binding (which means that
essentially all binding sites in the sample will be saturated with
the corresponding binding agent) can be established without having
the depletion problems. Evidently never 100%, but 90-99% binding of
a protein target with a high affinity primary antibody, and 50-75%
binding of the primary antibody with labeled secondary antibody may
be reached. Within these ranges, experiments with a varying but
high concentration of reagents can be used to establish more
precise binding levels.
[0373] Further, using a mixture containing a high concentration of
unlabeled secondary antibody and low concentration of labeled (the
same) secondary antibody, equilibrium conditions can be reached,
while only a small fraction of the primary antibodies bound to the
target will be labeled.
[0374] The present method further utilizes the possibility provided
by the present visualization method that labeled secondary antibody
may be visualized in several ways, depending on degree of
amplification. In case of low amounts of the target bound primary
antibody, a labeled secondary (or a mixture of labeled and
unlabeled) antibody can be used to produce countable dots. In case
of high amounts of the target bound primary antibody, the same
reagent (or mixture) can be used to produce a conventional stain.
The experiment thus may comprise several steps:
[0375] 1. Incubations with high concentrations of binding agents
are used to establish equilibrium conditions leading to recognition
of a high and known fraction of targets. Such experiments are
carried out with both primary and labeled secondary antibody. Such
conditions will further be referred as "top level" conditions.
[0376] 2. Then, a mixture of labeled secondary and unlabeled
secondary antibody that recognizes an unknown fraction of primary
antibodies is prepared and used for incubation of a tissue sample
with a high target expression that has been treated with a primary
antibody at the top level conditions. The incubation is followed by
visualization of the bound labeled secondary antibody with a
conventional stain.
[0377] 3. Using conventional staining, titration of the target
bound primary antibody by the labeled secondary antibody at the top
level conditions is performed. The important point is that
equilibrium conditions need not be established between the target
and the primary antibody. It is sufficient that using constant test
material (the constant test material refers to a test material
wherein the amount of the target is constant), a reproducible
amount of the target is recognized. At some low concentration of
primary antibody, a staining intensity is obtained that is
identical to the level of staining that observed in step 2.
[0378] 4. Using a method for visualizing single molecules as dots
(as described in the present invention), a mixture of labeled and
unlabeled secondary antibody is used to access a fraction of the
target recognized by the same low concentration of the primary
antibody as in step 3, relative to the fraction of the target
recognized by the top level conditions of primary antibody.
[0379] 5. Using the low level of primary antibody as of step 3, and
the mixture of labeled and unlabeled secondary antibody as of step
2, single molecules are stained as dots and the number of dots per
nucleus is evaluated.
[0380] From these experiments, the absolute number of targets can
be determined. From experiments of steps 1 and 4 it is known which
fraction of the target is recognized by the low concentration of
the primary antibody. From experiments of steps 1 and 3, it is
possible to deduce which fraction of the primary antibodies is
recognized by the mixture of labeled and unlabeled secondary
antibody used in experiment 2. We use the fact that the identical
conventional staining levels are obtained in experiments of step 2
and 3 (which means that there is the identical number of the bound
labeled secondary antibodies in the samples). Thus, we now know
both the fraction of the target molecules recognized by the low
concentration of the primary antibody, and the fraction of the
primary antibodies recognized by the mixture of labeled and
unlabeled secondary antibody of experiment in step 5. Multiplying
these two factors gives the fraction of target molecules visualized
as dots (see description of Experiment 1c below). As we further
have counted the number of dots per nucleus, we know the number of
target molecules present per nucleus. Thus, an absolute count has
been performed.
[0381] 2. Experimental Evidence
[0382] Materials and methods used in the following experiments not
specifically disclosed, are as described above.
[0383] It is established that the Kd of the primary anti-Her2
antibody is 280 pM. (See experiment 3c) Using the antibody under
equilibrium conditions (multiple additions until no further
increase in signal is observed) at a concentration of 13.3 nM will
result in labeling of 13.3 nM/(13.3 nM+0.28 nM) which is equal to
approximately 97.9% of the primary target molecules.
[0384] Likewise, it is established that the Kd of the labeled
secondary antibody is 28 nM. (See experiment 3d). Using the labeled
secondary antibody under equilibrium conditions (multiple additions
until no further increase in signal is observed) at a concentration
of 25 nM will result in labeling of 25 nM/(25 nM+28 nM) which is
equal to approximately 47.1% of the bound primary antibodies.
[0385] Experiment 1a.
[0386] As constant test material was used serial sections of
pellets of formalin fixed paraffin embedded cell lines. The cell
lines used were 3+ control material from Dako HercepTest.
[0387] Slides with FFPE sections of blocks containing the cell
lines, from now on referred to as "slides" were de paraffinized by
emersion in xylene (2.times.5 min) followed by 96% ethanol
(2.times.2 min) and 70% ethanol (2.times.2 min). The slides were
washed with de ionized water and transferred to low pH target
retrieval solution (Dako 81700). The slides were then heated to
boiling in a microwave oven (approx 5 min) and then gently boiled
for 10 min. The slides were allowed to cool for min 20 min before
being transferred to wash buffer, Dako S 2343.
[0388] The slides were then stained on the Autostainer using the
following protocol: Peroxidase block, Dako S2023, 5 min
[0389] Wash
[0390] Several sequential 10 minute additions of 13.3 nM antiHER2
primary antibody
[0391] Wash
[0392] Several sequential 10 minute additions of 100 pM
Goat-anti-Rabbit-Dextran-HRP (L348.111) mixed with 5 nM unlabelled
Goat-anti-Rabbit
[0393] Wash
[0394] DAB (Dako K5007), 10 min
[0395] Wash
[0396] Haematoxylin (Dako S3301), 5 min
[0397] Wash with water
[0398] Wash
[0399] Results:
[0400] Three 10 minute additions of 13.3 nM antiHER2 were
sufficient to reach equilibrium conditions. A fourth addition did
not lead to increased staining level. Two 10 minute additions of
100 pM Goat-anti-Rabbit-Dextran-HRP (L348,111) mixed with 5 nM
unlabelled Goat-anti-Rabbit was sufficient to reach equilibrium
conditions. A third addition did not lead to increased staining
level. The maximum staining level reached corresponded to approx.
+1, (Although this cell line is referred to as +3, the use of low
concentration of labeled secondary antibody mixed with a high
concentration of unlabeled secondary antibody leads to labeling of
a small fraction of primary antibodies).
[0401] Experiment 1b.
[0402] Slides were pretreated as in Experiment 1a, and subjected to
the following protocol (conventional DAB staining):
[0403] Peroxidase block, Dako 32023, 5 min
[0404] Wash
[0405] 10 minutes anti-HER2 primary antibody in varying
concentration in the range 35 to 50 pM.
[0406] Wash
[0407] Two sequential 10 minute additions of 25 nM
Goat-anti-Rabbit-Dextran-HRP (L348.111). A control slide showed
that a third addition did not lead to increased signal.
[0408] Wash
[0409] DAB (Dako K5007), 10 min
[0410] Wash
[0411] Haematoxylin (Deka S33031), 5 min
[0412] Wash with water
[0413] Wash
[0414] Results:
[0415] An incubation with 40 pM anti-HER2 for 10 minutes resulted
in a staining intensity (+1) identical to the maximum staining
level reached in experiment 1a. The 43 pM incubation resulted in a
visibly higher staining intensity, whereas the 37 pM incubation
gave a visibly lower staining intensity.
[0416] Experiment 1c
[0417] The slides were pretreated as in experiment 1a and subjected
to the following protocol (SMO staining):
[0418] Peroxidase block, 5 min with Dako S2023
[0419] Wash
[0420] AntiHER2 primary antibody. Either 3 sequential 10 minute
additions of 13.3 nM (slide 1) or one 10 minute addition of 40 pM
(Slide 2-5)
[0421] Wash
[0422] Two sequential 10 minute additions of 500 femtoM
Goat-anti-Rabbit-Dextran-HRP (L348.111) mixed with 5 nM unlabelled
Goat-anti-Rabbit (slide 1-3) or two sequential 10 minute additions
of 100 pM Goat-anti-Rabbit-Dextran-HRP (L348.111) mixed with 5 nM
unlabelled Goat-anti-Rabbit (slides 4-5)
[0423] Wash
[0424] FITC-Reporter deposit: 10 min with incubation media 2 with
028 mM DAB and 10 microM 021087.
[0425] Three washes
[0426] Anti-FITC-AP: 10 min incubation, 20 nM 0036 in BAM
[0427] Three washes
[0428] LPR 10 min with Dako K0640
[0429] wash
[0430] Haematoxylin (Dako 53301), 5 min
[0431] Wash with water
[0432] Wash
[0433] The slides were subjected to image analysis. Images of the
entire cell pellets were captured at 20.times. (approx.
300.times.300 nm pixels) using a ScanScope (Aperio) slide scanner.
The images were analyzed using JMicrovision vs. 1.27 software. Red
dots were identified in Intensity, Hue, Saturation color space as
(1=0-234, H=187-37, S=52-255), blue nuclei were identified as
(I=0-201, H=146-221, S=0-190). A size threshold was further applied
to dots, objects bigger than 30 pixels were counted as two dots,
objects bigger than 45 pixels were counted as three dots. A lower
threshold of 100 pixels was applied to nuclei to filter away debris
and smaller fragments of nuclei.
[0434] Note that the partially overlapping color spaces allow
identifying individualpixels as both part of a red dot and as part
of a nucleus, consistent with the dark violet appearance of dots on
top of nuclei.
[0435] Results and Conclusions:
TABLE-US-00010 Slide Dots Nuclei Dots/nucleus 1 56918 12388 4.59 2
151 13817 0.0109 3 177 13925 0.0127 4 52011 13618 3.82 5 61040
12939 4.72
[0436] Comparison of slide 1 to the average of slides 2 and 3 shows
388 times less bound primary antibody. As slide 1 represents around
97.9% (the value is derived from Kd1 of anti-Her2) of bound target
molecules, application of 40 pM primary antibody for 10 minutes on
the same test material (slides 2 and 3) gives rise to 1 in 396
target molecules being bound to the primary antibody (or
0.252%).
[0437] This data can now be used to analyze the results of
Experiment 1a and 1b.
[0438] As mentioned, application of 40 pM primary antibody for 10
minutes results in labeling of 0.252% of the primary target.
Subsequently, binding 47.1% (the value is derived from Kd of the
secondary antibody) of the bound to the target primary antibodies
to the secondary antibody results in 0.119% of the target being
(indirectly) bound to the secondary antibody. This corresponds to
Experiment 1c, i.e. using 40 pM primary antibody for 10 min. This
must also be the case (as staining levels are identical) for
Experiment 1b, where the 13.3 nM primary antibody incubation (97.9%
of primary targets bound) was followed by the incubation with the
mixture of 100 pM labeled secondary antibody with 5 nM unlabeled
secondary antibody. Thus, it can be concluded that the use of this
mixture leads to 0.119%/0.979=0.121% of the primary antibodies
being bound to the labeled secondary; 0.121% of 0.252% of the
target is equal to 3.06 ppm (parts per million). Accordingly, the
4.27 dots (in average) per nucleus observed in slides 4 and 5 count
to 1.395.000 target molecule per nucleus (this follow from from the
following calculation; 4.27/0.00000306=1.395.000).
[0439] The precision of this evaluation can be made by comparing
slide 2 and 3 with slides 4-5. There were observed 362 times more
dots (in average) using the mixture with 100 pM labeled secondary
(slides 2-3) antibody than with 500 fM (slides 4-5). As the mixture
with 100 pM results in 0.121% primary antibodies being labeled, the
mixture with 500 fM must lead to 362 times lower labeling the
target with antibody, i.e. 0.121%/362=3.34 ppm. It can be
calculated that the level of labeling of target molecules in this
slide: 97.9% of 3.34 ppm gives 3.27 ppm, and the observed 4.59 dots
per nucleus corresponds to 1.402.000 target molecules per nucleus
(4.59/0.00000327=1.402.000).
Example 3: Use of a Binding Agent Comprising a Mixture of First
Binding Molecules and Second Binding Molecules
[0440] Experiment 1. Binding Agent Comprising a Mixture of a
Rabbit-Anti-Her2 with a Mouse-Anti-Her2 Specific for the Same
Epitope
[0441] 8 Slides with FFPE sections of mamma carcinoma with
HercepTest.TM. Intensity score between 1+ and 2+ were
de-paraffinized in xylene and ethanol and epitope retrieved in
PT-Link (Dako) at 97 degrees for 20 min in 10 mM HEPES pH 8.0 with
0.1% NP-40.
[0442] The slides were then stained according to the following
protocol (A) on the Autostainer (Dako): [0443] (a) Peroxidase
block, 5 min in Dako 52023
[0444] Wash [0445] (b) incubation with the binding agent: [0446] A
mixture of the first binding molecule Rabbit-anti-Her2 (Monoclonal
Rabbit anti-Human HER2, clone DAK-H.sub.cT-2:DG44) and the second
binding molecule Mouse-anti-Her2 (Monoclonal Mouse anti-human Her2
antibody clone DAK-H2-364, F11, G4, C9) [0447] Wash [0448] (c)
Detection of the first binding molecule; [0449] Incubation with
HRP-Labeled secondary antibody against Rabbit-anti-Her2 [0450]
Wash. [0451] Deposition of a reporter molecule (Reporter
Fer-Lys(Fer)-Lys(Fer)-Lys(Fer)-L150-Lys(Flu) (D19185/D120068)
[0452] (d) Detection of the reporter at target sites [0453] DAB,
2.times.5 min, Dako K0640 [0454] Wash [0455] (e) Haematoxylin
counterstain [0456] Haematoxylin, 3 min [0457] Wash with deionized
water [0458] (f) Mounting
[0459] By mixing Rabbit-anti-Her2 antibody with the Mouse-anti-Her2
antibody specific for the same epitope, the portion of targets
bound to the Rabbit-anti-Her:2 antibody was manipulated by changing
the ratio between the Rabbit-anti-Her2 and Mouse-anti-her2 while
retaining equilibrium conditions. Thus, the signal intensity was
also be manipulated since the anti-Rabbit HRP conjugated detection
layer detect only Rabbit-anti-Her2 antibodies bound to Her2, but
not Mouse-anti-Her2 antibodies bound to Her2
[0460] The slides were treated with mixtures of the
Rabbit-anti-Her2 and Mouse-anti-Her2 having the ratios shown in the
Table:
TABLE-US-00011 Rabbit-anti-Her2 Mouse-anti-her2 Slide number
(ng/ml) (ng/ml) 1 200 0 2 200 50 3 200 100 4 200 200 5 200 300 6
200 400 7 200 800 8 200 1600
[0461] The slides were analyzed and scored within the intensity
score range from 0 no intensity to 4 high intensity. The results
are presented in the table below.
TABLE-US-00012 Slide number Intensity results 1 1.8 2 1.6 3 1.6 4
1.4 5 1.4 6 1.4 7 1.2 8 1
[0462] From the results of the experiment it was concluded that a
mixture of Rabbit-anti Her2 and Mouse anti-Her2, can be used to
adjust the signal intensity while keeping the reaction at
equilibrium conditions.
[0463] Experiment 2. Binding Agent Comprising a Mixture of
Monoclonal Rabbit-Anti-Her2 with f(Ab).sub.2 Fragment of the
Antibody.
[0464] The staining of 4 slides of mamma carcinoma with a
HercepTest.TM. score of 2+ pretreated and immunostained as above
(Experiment 1, protocol A) using the binding agent comprising a
mixture of monoclonal Rabbit-anti-Her2 (see Experiment 1) with the
f(ab).sub.2 fragment of the antibody. Specific concentrations were
as indicated in the Table below,
TABLE-US-00013 Rabbit-anti-Her2-HRP with f(ab).sub.2 fragmentized
Slide number conjugated (ng/ml) Rabbit anti-Her2. 1 500 0 2 500
1000 3 500 1500 4 500 2000
[0465] Images of the slides were captured with Aperio Scanscope.
The images were analyzed with ISH-vs. 1.2 software from Indica
Labs. The slides where assessed and an intensity score was
evaluated (ranging from 0 no intensity to 4 high intensity). The
results are presented in the Table below:
TABLE-US-00014 Intensity results Slide number 0 = none, 4 = high 1
2.2 2 1.8 3 1.6 4 1.6
[0466] From the results of the experiment it is concluded that a
mixture of Rabbit-anti Her2 and the f(ab).sub.2 fragment of the
antibody can be used to adjust the signal intensity while keeping
the reaction at equilibrium conditions.
[0467] Experiment 3. Binding Agent Comprising a Mixture of Labeled
and Unlabeled Antibodies (First and Second Binding
Molecules)--Visualization of the First Binding Molecule in the
Target Site According to WO2012143010 (Incorporated by
Reference).
[0468] 18 Slides with FFPE sections of Her2+1 control cell lines
were de-paraffinized in xylene and ethanol and epitope retrieved in
PT-Link (Dako) at 97 degrees for 20 min in 10 mM HEPES pH 8.0 with
0.1% NP-40.
[0469] The slides were stained according to the following protocol
on the Autostainer (Dako): [0470] 1. Peroxidase block, 3% hydrogen
peroxide, 10 min. Wash Dako S3006. [0471] 2. Monoclonal
Rabbit-Anti-Her2 antibody, 6.6 nM in incubation media 1 (Tris:HCl
50 mM pH 7.6, NaCl 0.3 M, Bovine Serum Albumin 2%, Bronidox, 0.02%,
4-aminoantipyrine 2.44 mM, PolyEthyleneGlycol MW 3.000 3%, Caseine
0.05%, TWEEN 20 (polyoxyethylene (20) sorbitan monolaurate) 0.1%)
20 min. Wash Dako 53006. [0472] 3. Goat-Anti-Rabbit-Dextran70-HRP
(018033/018175), 200 fM+unlabelled Goat-Anti-Rabbit in varying
concentrations in incubation media 1), 20 min. Wash Dako 53006.
[0473] 4. 9 slides were again treated with the
Goat-Anti-Rabbit-Dextran70-HRP, 200 fM+unlabelled Goat-Anti-Rabbit
in varying concentrations in the incubation media, 20 min. Wash
Dako 53006, (identical to step 3) The other 9 slides were not
subjected to this step. [0474] 5. 2 microM reporter D19185/0120068,
5.3 mM alpha-CHC, 0.59 mM hydrogen peroxide in 50 mM in
imidazole:HCl pH 6.8, 10 min. Wash Dako 53006. [0475] 6.
Anti-FITC-Alkaline phosphatase, 40 nM in Media 1, 10 min. Wash Dako
S3006. [0476] 7. Liquid Permant Red, Dako K0640, 10 min. Wash Dako
S3006. [0477] 8. Haematoxilin counter stain, Dako 53301, 5 min.
Wash with water, wash 3' ako S3006.
[0478] The slides were dehydrated in 99.9% ethanol for 1 min and
cover slipped with Tissue-Tek Film cover slipper (Sakura).
[0479] Images of the slides were captured with Aperio Scanscope.
The images were analyzed with ISH-vs. 1.2 software from Indica
Labs. Settings were adjusted to detect blue nuclei (Red 0.650,
Green 0.704, Blue 0.291) and red dots (Red 0.072, Green 0.952, Blue
0.296). While these two types of objects show almost identical
transmission of blue light (Blue=0.291 vs. 0.296) the nuclei had
low transmission of both red and green (0.650 and 0.704) while the
red dots were had very high transmission of red and very low
transmission of green (0.072 and 0.952). This provided unequivocal
(as confirmed by visual inspection) identification of nuclei and
dots. To further enhance correct identification of the membrane
localized red dots, a logic filter was applied so that only dots
within 5 microns of a nucleus were counted. On each slide around
15.000 cells were counted to assure statistically valid data. The
results are summarized in the table below:
TABLE-US-00015 Concentration of unlabeled Goat- anti Rabbit in
steps 3 and 4 0 nM 3 nM 10 nM Average of three 0.134 dots/cell
0.073 dots/cell 0.033 dots/cell slides subjected to step 3 only
Average of three 0.242 dots/cell 0.087 dots/cell 0.036 dots/cell
slides subjected to step 3 and 4 Increase in Dot/cell 81% 19% 9% by
step 4
[0480] Results and Discussion:
[0481] As shown in other examples, when using low concentrations of
a binding agent alone, multiple additions are required to reach
equilibrium. In this example a 81% increase is observed by the
second addition of Goat-anti-Rabbit-Qex-HRP, showing that a single
addition is far from sufficient to reach a stable equilibrium. In
contrast, when using a mixture of labeled and unlabelled binding
molecules, significantly fewer dots marking the target sites are
detected by a single addition, yet a second addition leads only to
small increase in dot number. Comparing 2 binding agent comprising
3 nM and 10 nM unlabeled binding molecules, it may be drawn the
conclusion that the higher the concentration of the unlabeled
binding molecules, the fewer binding sites comprising the target
are visualized (i.e. fewer dots are detected) by single addition
and the smaller the increase in the visualized target sites by a
second addition. The fact that fewer dots are detected, and even
fewer are further detectable by a second addition, using mixtures
of labeled and unlabeled binding molecules directed to the same
target, shows that saturation of the target by the binding
molecules takes place. Thus, the assay has been transformed from an
equilibrium measurement (as of the Example 1 and Example 2
described above), that requires multiple additions of the binding
agent or prolonged time to reach stable equilibrium conditions, to
a competitive saturation assay where the target can be essentially
saturated by a single addition of mixture of labeled and unlabeled
molecules of one and the same binding agent. This is evidently
advantageous, as the procedure is significantly faster, less
laborious and much more robust with regard to minor fluctuations in
time, temperature, reagent qualify, instrumentation or human
operator variation. Such competitive saturation assays don't
preclude assays that measure immobilized targets in absolute terms,
i.e. a target concentration per cell or volume of immobilized
sample. As equilibrium experiments with primary antibody and
labeled secondary antibody on constant test material, i.e. a
specific cell line, can be used to determine absolute target
concentration in that cell line, other assays using a mixture of a
"detectable" and "non-detectable" binding agents can be calibrated
against the constant test material, i.e. if it has been determined
that a given cell line has 50.000 units of the target per cell, and
a robust saturation assay with labeled-unlabeled binding agent
produces 0.10 dots/cell (1 dot corresponding to one single unit
(see for explanation WO20110476680 or WO2012062318, incorporated
herein by reference), it can be concluded that that the assay
detects 1 target unit in a sample comprising 500.000 target
units.
[0482] Experiment 4: Binding Anent Comprising a Mixture of Labeled
and Unlabeled Antibodies First and Second Binding Molecules
Visualization of the First Binding Molecule in the Target Site by
Conventional HRP-DAB Stain (Envision.TM.)
[0483] 6 Slides with FFPE sections of Her2+0, +1, +2 and +3 control
cell lines were de-paraffinized in xylene and ethanol and epitope
retrieved in PT-Link (Dako) at 97 degrees for 20 min in 10 mM HEPES
pH 8.0 with 0.1% NP-40.
[0484] They were then stained according to the following protocol
on the Autostainer (Dako): [0485] 1. Peroxidase block, 3% hydrogen
peroxide, 10 min. Wash Dako 53006. [0486] 2. Monoclonal
Rabbit-Anti-Her2 antibody, 6.6 nM in Media 1 (see above), 20 min.
Wash Dako S3006. [0487] 3. Goat-Anti-Rabbit-Dextran70-HRP
(018033/D18175), 25 nM+unlabelled Goat-Anti-Rabbit in varying
concentrations (0, 8, 13, 21, 34 and 55 nM) in Media 1, 20 min.
Wash Dako S3006. [0488] 4. DAB, K5007 Dako, 5 min, ash Dako 53006.
[0489] 5. Haematoxilin counter stain, Dako S3301, 5 min. Wash with
water, wash Dako S3006.
[0490] After the staining the slides were dehydrated in 99.9%
ethanol for 1 min and cover slipped with Tissue-Tek Film cover
slipper, (Sakura).
[0491] Images of the slides were captured with Aperio Scanscope.
The images were analyzed using "Membrane vs. 9.0" from Aperio. The
original settings from Aperio were used, with the sole exception
that "membrane completeness" was reduced from 50% to 20%. This
feature adjusts the degree of membrane completeness required for a
cell to be scored as +3. This reduction gave a better separation
between +2 and +3 cells as virtually all intense cells scored +3
with 20% completeness requirement, whereas a 50% completeness
requirement only lead to around 50%+3 cells even in case of very
intense stains.
[0492] The algorithm has three basic outputs: [0493] 1. An
Intensity score based on average of the membrane intensity
staining. The dynamic range in case of the cell lines is from
around 35 (very intense membranes) to 170-180 (no membrane stain).
[0494] 2. A "Histoscore". Based on membrane intensity, cells are
binned as either +3, +2, +1 or +0. The score is calculated as (% of
+3.times.3)+(% of +2.times.2)+(% of +1.times.1)+(% of +0.times.0).
The dynamic range is thus from 300 (all cells +3) to 0 (all cells
+0). [0495] 3. A categorical score. Based on the Histoscore
distribution the entire sample (cell pellet) is classified as
either +3, +2 +1 or +0. In (rare) cases of extremely homogenous
material; i.e. all cells are of same category, the categorical
score is identical to the Histoscore. In most cases what decides
the categorical score is the "rule of 10%". This implies that if
more than 10% of the cells are +3, the score will be +3, if more
than 10% of the cells are +2 (but less than 10% are +3), the score
will be +2, if more than 10% of the cells are +1 (but less than 10%
are +2 or +3), the score will be +1, otherwise (less than 10% are
+1, +2 or +3) the scar will be +0.
[0496] The results of the evaluation of the 4 different cell lines
and the binding agent comprising 6 different concentrations of
unlabelled Goat-anti-Rabbit antibody are summarized in the Table
below: I=intensity, H=Histoscore and C=categorical score.
TABLE-US-00016 [GaR] +3 cell line +2 cell line +1 cell line +0 cell
line 0 nM 38(I), 297(H), +3(C) 67(I), 294(H), +3(C) 123(I), 157(H),
+3(C) 176(I), 47(H), +1(C) 8 nM 39(I), 298(H), +3(C) 77(I), 290(H),
+3(C) 139(I), 131(H), +3(C) 172(I), 27(H), +1(C) 13 nM 39(I),
298(H), +3(C) 79(I), 277(H), +3(C) 148(I), 124(H), +2(C) 176(I),
10(H), +0(C) 21 nM 44(I), 298(H), +3(C) 89(I), 274(H), +3(C)
164(I), 94(H), +2(C) 177(I), 4(H), +0(C) 34 nM 49(I), 298(H), +3(C)
101(I), 251(H), +3(C) 180(I), 62(H), +1(C) 174(I), 1(H), +0(C) 55
nM 51(I), 298(H), +3(C) 110(I), 230(H), +3(C) 182(I), 40(H), +1(C)
175(I), 0(H), +0(C)
[0497] Discussion of Results:
[0498] Each of the three scoring output represents different
mathematical "views" of the same image, neither one the complete
picture. It is, however, clear that without addition of unlabeled
Goat-anti-Rabbit, the staining level is excessively intense with 1
g little discrimination between (especially) the +3 and +2 cell
lines. Further the +1 is categorized as +3 and the +0 as +1.
[0499] The best results are obtained with either 34 nM or 55 nlul
unlabeled Goat-anti-Rabbit. All cell lines, except the +2, are
given the correct categorical score and discrimination between
these two cell lines is readily possible via the values of
intensity or histoscore. Indeed, the histoscore discriminates
readily between all four cell lines with 34 nM or 55 nM unlabeled
Goat-anti-Rabbit, whereas intensity score is of little value for
very weakly stained cells as cell morphology, and intensity of
haematoxylin counter stain subtly effects membrane intensity.
[0500] Illustrative examples of the signal intensity attention by
addition of unlabelled Goat-anti-Mouse are provided in FIG. 2.
* * * * *