U.S. patent application number 11/338681 was filed with the patent office on 2006-06-15 for quality control for cytochemical assays.
Invention is credited to Steven A. Bogen, Halasya Ramanathan, Seshi R. Sompuram.
Application Number | 20060127956 11/338681 |
Document ID | / |
Family ID | 27807663 |
Filed Date | 2006-06-15 |
United States Patent
Application |
20060127956 |
Kind Code |
A1 |
Sompuram; Seshi R. ; et
al. |
June 15, 2006 |
Quality control for cytochemical assays
Abstract
A method and device is described for a quality control device
for immunohistochemical testing of surgical biopsies.
Immunohistochemistry testing involves the use of antibodies to
detect the presence of proteins that have diagnostic or prognostic
importance. In this invention, peptides are covalently attached to
glass microscope slides. The peptides represent antibody contact
sites to the native protein. Namely, the peptide contains the same
amino acid sequence as the epitope in the native protein to where
the antibody binds. Alternatively, peptides may not necessarily
share the exact sequence as found in the native protein but they
still bind to the antibody. To use this invention, tissue sections
derived from surgical biopsies may be mounted on glass slides
bearing the peptides. As the immunohistochemical stain is performed
on the tissue section, a similar reaction sequence occurs at the
site of the glass slide where the peptide is located. At the
conclusion of the immunohistochemical stain, a colored reaction
product deposits at the site of the peptide. Since the
immunohistochemical reaction at the peptide is largely the same as
that which occurs in the tissue section, the peptide serves a
quality control function.
Inventors: |
Sompuram; Seshi R.;
(Brighton, MA) ; Ramanathan; Halasya; (Worcester,
MA) ; Bogen; Steven A.; (Sharon, MA) |
Correspondence
Address: |
STEPTOE & JOHNSON LLP
1330 CONNECTICUT AVENUE, N.W.
WASHINGTON
DC
20036
US
|
Family ID: |
27807663 |
Appl. No.: |
11/338681 |
Filed: |
January 25, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09549855 |
Apr 14, 2000 |
7011940 |
|
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11338681 |
Jan 25, 2006 |
|
|
|
09291351 |
Apr 14, 1999 |
6281004 |
|
|
09549855 |
Apr 14, 2000 |
|
|
|
Current U.S.
Class: |
435/7.2 ;
435/287.2 |
Current CPC
Class: |
Y10T 436/105831
20150115; Y10T 436/106664 20150115; Y10T 436/2525 20150115; Y10T
436/25 20150115; G01N 1/312 20130101; G01N 21/93 20130101; Y10T
436/107497 20150115; G01N 33/54386 20130101; G02B 21/34 20130101;
G01N 2333/723 20130101; Y10T 436/25375 20150115 |
Class at
Publication: |
435/007.2 ;
435/287.2 |
International
Class: |
G01N 33/567 20060101
G01N033/567; C12M 1/34 20060101 C12M001/34; G01N 33/53 20060101
G01N033/53 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by a grant
1R43CA81950-01 from The National Institute of Health. The
Government has certain rights in the invention.
Claims
1. A method for verifying the proper performance of an assay of a
tissue section or cell containing an analyte that is detected in
the assay comprising: covalently attaching a soluble analyte mimic
to a microscope slide to form a covalent bond between the analyte
mimic and the slide, said analyte mimic being a smaller molecule
than the analyte and capable of producing a reaction in the assay;
performing the assay on both the tissue section or cell and on the
analyte mimic; and assessing assay performance by examining the
result of the assay on the tissue section or cell and the analyte
mimic.
2. The method of claim 1 wherein the tissue section or cells are
mounted on the same slide as the analyte mimic.
3. The method of claim 1 wherein the assay is an
immunohistochemical stain.
4. The method of claim 1 wherein the analyte mimic is covalently
attached to the microscope slide by first attaching chemically
reactive moieties to the slide and then reacting the chemically
reactive moieties with the analyte mimic.
5. The method of claim 4 wherein the microscope slide includes a
glass surface.
6. The method of claim 5 wherein the chemically reactive moieties
are attached to the slide through a silane linker.
7. The method of claim 4 wherein the chemically reactive moiety
comprises an isocyanate.
8. The method of claim 1 wherein the analyte mimic is a
peptide.
9. The method of claim 1 wherein the assay further comprises an
antibody that binds to the analyte in tissue sections or cells.
10. The method of claim 9 wherein the antibody also binds to the
analyte mimic.
11. The method of claim 10 wherein the antibody binds to the
analyte mimic at an amino acid sequence that is similar to an amino
acid sequence in the analyte.
12. The method of claim 1 wherein the assay performance is assessed
by comparing the result of the assay with a previously determined
range for the assay result on the analyte mimic, the range
representing a proper performance of the assay.
13. The method of claim 1 further comprising a plurality of analyte
mimics on the slide.
14. A microscope slide on which an assay is performed, said assay
being capable of detecting an analyte in a tissue section or cell
and further comprising an antibody that binds to the analyte,
wherein the slide comprises a soluble analyte mimic covalently
attached to the slide such that the bonds between the analyte mimic
and the slide are covalent, said analyte mimic being a smaller
molecule than the analyte and wherein the antibody is capable of
binding to the analyte mimic.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 09/549,855, filed Apr. 14, 2000, which is a
continuation-in-part of U.S. application Ser. No. 09/291,351, filed
Apr. 14, 1999, which issued as U.S. Pat. No. 6,281,004, the entire
teachings of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] During the past decade, immunohistochemical (IHC) stains
have become an integral part of the diagnostic process in surgical
pathology. IHC stains are used with conventional histopathological
stains, as adjunctive assays. They are critical in correctly
diagnosing poorly differentiated malignancies, viral infections,
and tumor prognoses, such as in the use of estrogen receptor (ER)
analysis for breast carcinoma. Since IHC assays are relatively new
in medical practice, simple methods for accurately and reliably
monitoring quality control has not yet been developed.
[0004] Traditional microscopic analysis of biopsy samples
demonstrates overall cellular and tissue architecture. With the
commonly used hematoxylin and eosin stain, for example, nuclei are
colored purple (with hematoxylin) and the cytoplasm red/pink (with
eosin). Often, the type of information available from such stains
is insufficient for accurate diagnosis. IHC stains have the ability
to extend the level of analysis on a biopsy sample, beyond cellular
size and shape, to a molecular level. Tissue samples can be probed
for the presence of specific proteins using monoclonal and
polyclonal antibodies. The presence of such proteins can be
indicative of the cellular lineage of a tumor, facilitating a
diagnosis or prognosis with certain types of anti-tumor therapies.
Alternatively, the presence of microbiologic agents, such as
viruses or bacteria, can be detected using appropriate
antibodies.
[0005] Quality control is an important aspect of any clinical
assay. To assure that clinical test results are accurate, controls
should be run with all in vitro diagnostic tests. Quality control
in the clinical laboratory is mandated by the Clinical Laboratory
Improvement Act of 1988 (CLIA '88). This act outlines the
regulatory requirements that a clinical laboratory must meet in
order to obtain accreditation; controls and proficiency testing
comprise a significant portion of the act. Many tests in the
hematology and chemistry sections of the laboratory include
controls that are provided by the manufacturer. In contrast, most
histology laboratories generate their own controls from excess
tissue specimens. With laboratories generating their own control
tissues, there is little inter-laboratory standardization. The need
for better quality control of immunoreagents was recognized in the
late 1980's and led to workshops convened by the Biological Stain
Commission to address the issue.
[0006] The histology laboratory has lagged behind other sections of
the clinical laboratory in the implementation of optimal controls.
A negative control is easy to perform. It comprises a serial
section of the same tissue with an irrelevant, isotype-matched
primary antibody. Standardized positive controls have been harder
to achieve. Presently, each laboratory is left to fend for itself
in creating, storing, and validation positive tissues controls.
[0007] Most histopatholoy laboratories use tissue samples
previously documented to contain the particular antigen as positive
controls. The laboratory documents, sections, and archives a bank
of tissues that will serve as tissue controls for IHC. As the
tissue controls are depleted, new tissues/tumor samples are
procured to replace those expended. During each daily IHC assay
run, each antibody is tested on a positive tissue control. In this
manner, each antibody is validated at a specified dilution.
[0008] An improvement was described by Battifora (U.S. Pat. Nos.
4,820,504 and 5,610,022), whereby multiple positive control tissue
fragments, often-of tumors, are embedded together in a single
paraffin block. This "multi-tissue tumor block" simplifies the
sectioning process of positive tissue controls. Rather than
archiving and sectioning numerous blocks of tissue, the tissue
controls are embedded together in a single paraffin block.
Therefore, the group of archived tissues can be sectioned
simultaneously, with a single stroke of a microtome blade.
[0009] A slightly simpler method of preparing multi-tumor tissue
paraffin blocks was described by Furmanski et al. (U.S. Pat. No.
4,914,022). The improvement involved embedding tissue cores in a
paraffin block. The cores were cut from the tissue of origin with
the use of an ordinary plastic drinking straw.
[0010] The use of multi-tumor tissue blocks as positive controls
does not solve three important problems. One of the most important
aspects that a positive control should address is the early
detection of reagent failure. The ideal method of detecting early
failure is to determine the level of sensitivity at the working
concentration of antibody. Sensitivity is determined by titrating
the antigen concentration until the antigen is no longer detected.
In this manner, the assay can be stated as capable of detecting a
certain amount of antigen, e.g., nanomoles or picomoles of antigen.
The limit of sensitivity should ideally be checked daily so that
trends (towards increasing or decreasing sensitivity) can be
detected. It is impossible to perform this type of analysis using
tissue sections as controls since there is no practical method for
quantitation and titration of antigen in a tissue section.
[0011] In addition, tissue sections as controls do not control for
performance error. Cutting (with a microtome) and mounting tissue
sections on glass slides is labor intensive. Therefore, the
aforementioned types of tissue positive controls (such as
multi-tumor tissue blocks) are usually tested once per assay run.
Because of the associated labor costs, few laboratories place a
positive tissue control on each microscope slide. Thus, if there is
an error by placing an incorrect antibody (or no antibody) on the
sample, it may be impossible to detect. Importantly, the control
slide may be correctly treated (verifying the reagent quality) but
the sample slide can still be incorrectly treated. The sample would
therefore be interpreted as a negative result, although the cause
of the negative result is an error in the assay procedure. The
present system of positive tissue controls does not control for
errors in procedure.
[0012] A third problem with tissue sections as positive IHC assay
controls is that tumor tissues inherently have a varied,
non-standardized amount of antigen. Therefore, tissues do not
provide a ready means for calibrating the intensity of the
immunologic reaction to an external reference standard. For certain
IHC assays, the absence of external reference calibrators is a
serious problem. Notably, IHC assays for estrogen receptor and
progesterone receptor have become the gold standard for previously
quantitative assays that were performed in a test tube. In the
absence of such calibrators, staining is. typically quantified as
0-4+ staining intensity, an arbitrary standard that depends upon
the reagents, protocol, and time duration of calorimetric
development. Because of significant inter-laboratory variability in
IHC assay sensitivity, each hospital laboratory must develop its
own threshold for determining a positive result. This feature leads
to non-standard and sometimes incorrect results. These errors can
have therapeutic impact on patient care.
[0013] Therefore, a standardized, practical positive tissue control
for clinical IHC assays should have the following characteristics
to be clinically accepted and scientifically meaningful:
[0014] 1. Antigen specific. A positive reaction should indicate the
presence of only the antigen being assayed.
[0015] 2. Available in virtually unlimited quantities, so that the
positive control has constantly controlled characteristics with the
passage of years.
[0016] 3. Inexpensive. With cost pressures mounting on hospital
laboratories, an expensive positive control will most likely not be
broadly adopted into routine practice.
[0017] 4. Stable over a prolonged period of time, ideally without
the need for freezing.
[0018] 5. Standardized, so that each laboratory will have the exact
same positive control substrate.
[0019] Currently, there is not a quality control reagent or device
available for cytochemical procedures that has all of the above
characteristics.
SUMMARY OF THE INVENTION
[0020] The present invention relates to the development of
accurate, reliable and easy-to-use quality control devices, and
methods of using the devices, to maintain quality control of assays
that measure analytes in cells or tissue sections, specifically
immunohistochemical analyses of biological samples. As used herein,
the term "biological sample" can be any cell-containing sample. For
example, the sample can be tissue, blood, urine, cerebral spinal
fluid (CSF), sputum, semen, cervicovaginal swab, or intestinal
wash. For example, the analyte assay can be an immunocytochemical
assay and the target molecule (analyte) to be detected is an
antigen. As used herein, the term "antigen" means a molecule
detected by an antibody. The term "immunocytochemical" is used
synonymously with "immunohistochemical", both referring to an
antibody test for in situ identification of analytes in cells or in
a tissue section.
[0021] The quality control device of the present invention is
useful for monitoring quality control of assays that measure
analytes in cells or tissue samples obtained from biological
samples. Specifically, the present invention relates to a quality
control device comprising one, or more, quality control moieties
that are affixed, or attached, to a reagent surface of a matrix.
The device comprises a matrix (a thin or single layer) having a
front (also referred to herein as top) surface and a back (also
referred to herein as bottom) surface. Typically, the quality
control device is used in an assay that is performed on a flat,
planar test platform, or surface (e.g., a glass microscope slide).
The matrix can be adhered to the test platform by an adhesive on
the bottom surface of the matrix. One example of a matrix is a
membrane, such as nitrocellulose. Each moiety is confined to a
discrete (and distinct) section, or "spot" on the matrix.
Typically, the moieties are covalently attached to the matrix. The
quality control moiety comprises one, or more, target molecules
(e.g., the molecule of interest that is being detected in the
biological sample) or a target molecule mimic (e.g., a synthetic
molecule that mimics the target molecule). Typically the target
molecule is a protein (e.g., a native protein, or an antibody such
as a goat anti-mouse immunoglobulin antibody or normal mouse
immunoglobulins) or polypeptide (e.g., synthetic protein, peptide,
or antibody fragment) that is detectable by antibody binding. Other
target molecules are proteins or polypeptides, carbohydrates,
lipids, or combinations thereof that are detectable by
histochemical stains. Representative histochemical stains include
the periodic acid-schiff stain, mucicarmine stain or reticulin
stain. Target molecules can also be nucleic acids (e.g., that are
detectable by in situ hybridization techniques). For example, in
this embodiment, the target is a nucleic acid detected by a nucleic
acid probe complementary to the nucleotide sequence of the target
molecule.
[0022] In one embodiment, the target molecule is a protein,
polypeptide, or fragment thereof, that is detectable by a specific
antibody (referred to herein as the primary antibody). The primary
antibodies of the present invention can be either polyclonal or
monoclonal, but are typically monoclonal. Such proteins,
polypeptides or fragments (e.g., small peptides) are referred to
herein as antigens. Antigens for use in the quality control device
described herein can be purified antigens, recombinantly produced
antigens or synthetic antigens. In order to simplify manufacture,
it is desirable to avoid the need for purification of the many
different antigens that are used in clinical diagnosis. Therefore,
a preferred form of the antigen of the quality control device
described herein is a synthetic, short peptide sequence that mimics
the antigen to be detected and specifically binds to the primary
antibody under substantially the same conditions as the antigen to
be detected (also referred to herein as a target molecule
mimic).
[0023] In this embodiment of the present invention, the antigen is
affixed to the quality control device, for example, by placing a
small spot of soluble antigen (i.e., antigen in a suitable
solution, such as a buffered saline solution) onto a spatially
discrete region, or section, of the matrix. That is, the antigen is
spotted onto the matrix in a discrete, limited area of the matrix,
so that the antigen spots to not overlap or touch, nor do they
overlap/touch the biological sample. The matrix can be any material
suitable for permanently affixing the antigen, including glass.
Preferably, a series of spots, each with soluble antigen in varying
concentrations, is placed onto the matrix. In this way, the
immunologic reaction on the matrix will cause some spots to be
intensely colored and others not at all. For example, to determine
the sensitivity of an immunocytochemical assay, the endpoint of
detection is determined as the spot of lowest antigen concentration
that still produces a 1+ clintensity (on a 0-4+ scale). In this
manner the sensitivity of the assay and initial stages of reagent
failure can be determined. In addition, an irrelevant antigen can
also applied to the matrix as a check for reaction specificity.
Such antigen spots can serve as quantitative and qualitative
reference/standard controls for IHC assays.
[0024] Alternatively, different antigens can be affixed to the
matrix in order to detect multiple distinct primary antibodies. In
this alternative embodiment, the quality control invention would be
more versatile, in that it would provide an antigen-specific
binding site for multiple different primary antibodies.
[0025] In another embodiment of the present invention, the quality
control moiety is an antibody that recognizes the primary antibody
that binds to the target molecule. For example, the quality control
moiety can be goat anti-mouse immunoglobulin (IgG) if the primary
antibody is a mouse antibody, or rabbit anti-human antibody, if the
primary antibody is a human antibody. In this embodiment, the
quality control device monitors for the presence of primary
antibody and whether immunocytochemical reagents used during the
cytochemical assay (e.g., second antibody or enzyme) are applied in
the correct sequential order.
[0026] In another embodiment of the present invention, the quality
control moiety is normal mouse or rabbit immunoglobulins. These
immunoglobulins can serve as a target for the secondary (detecting)
reagent that is commonly used in immunohistochemical assays.
Namely, the normal mouse or rabbit immunoglobulins would be
recognized by anti-mouse or anti-rabbit IgG. The presence of normal
mouse or rabbit immunoglobulins would therefore test the proper
performance and sensitivity of the detection kit components in an
immunohistochemical assay.
[0027] In one embodiment of the present invention, the quality
control device is an adhesive device wherein the matrix of the
device has a front and back surface. The front surface is referred
to herein as the reagent surface and has one, or more, quality
control moieties affixed to the surface. The back surface of the
matrix is referred to herein as the adhesive surface, which permits
the quality control device to be affixed to a test platform. The
test platform can be a flat, optically transparent surface, for
example, a microscope slide. For simplicity, the quality control
device is often referred to herein as a "strip" of matrix material,
however, it is readily apparent that other shapes, or forms of the
matrix can be used in the device of the present invention, and
these alternative forms are also encompassed by the present
invention. For example, the quality control device comprising an
adhesive matrix strip can be peeled from a backing and applied to
the end of a microscope slide. The biological sample (e.g., tissue
section) to be tested is also affixed to the same microscope slide.
Therefore, the same reagents, temperature, and humidity conditions
that exist for the cytochemical reaction on the tissue section also
apply to the synthetic antigen control moieties affixed to the
device.
[0028] In another embodiment of the present invention, the antigens
can be attached, or affixed, directly onto a planar test platform,
e.g., a glass microscope slide. This alternative embodiment
eliminates the need for a separate matrix that adheres to the test
platform with an adhesive backing. One or more antigen spots can be
directly attached onto an area, or section, of the glass slide,
leaving an appropriately large area for placement of the biological
sample. In one embodiment of the present invention, the antigen is
covalently attached, or coupled, to the test platform. A variety of
covalent coupling chemistries are described herein, and are also
known to those of skill in the art. Typically, the coupling
utilizes a silane coupling chemistry to glass. These coupling
chemistries attach a protein, synthetic peptide, nucleic acid
(suitably derivatized for attachment), carbohydrate, or other
macromolecule to the glass. The use of this alternative embodiment,
with macromolecules attached directly to glass, is used analogously
to that with a matrix having an adhesive backing. The same
molecules that are detected in the tissue (or cell) sample are also
simultaneously detected in the spots containing the antigen (or
antigen mimic, such as a synthetic peptide).
[0029] Methods of using the devices described herein are also
encompassed by the present invention. For example, encompassed by
the present invention are methods for determining the sensitivity
of an assay for the detection of the presence or absence of one, or
more, target molecules in a biological sample. The method comprises
simultaneously processing the biological sample and a quality
control device described herein in the assay to detect the presence
or absence of one, or more target molecules. As used herein the
term "processing" means performing all the steps of an assay
required to detect the presence, or absence, of the target
molecule. For example, processing can mean performing the steps of
an immunocytochemical assay to detect the presence of a target
protein by contacting the protein with an antibody that
specifically binds to the protein, under appropriate conditions
wherein the antibody specifically binds to the target protein and
detecting the antibody bound to the target (e.g., by detecting a
colorimetric signal) wherein detection of the signal is indicative
of the presence of the target protein, and lack of signal detection
is indicative of the absence of the target protein. Such assay
steps are well-known to those of skill in the art. For example, the
device can comprise a matrix with quality control moieties with
different concentrations of a target molecule or target molecule
mimic, e.g., a synthetic antigen covalently attached to the matrix.
The synthetic antigen mimics the antigenic site of the target
molecule and thus is also recognized by same antibody that
recognizes the target molecule. The processing results in the
detection of target molecule in the sample and target molecule or
target molecule mimic in the quality control moiety of the device
using one of the detection methods described herein. The moiety
that contains the lowest concentration of target molecule/target
molecule mimic is then determined, wherein determination of the
lowest concentration of detectable target molecule/target molecule
mimic is indicative of the sensitivity of the immunocytochemical
assay. Typically, the device is affixed to a flat, optically
transparent surface, e.g., a microscope slide.
[0030] Also encompassed by the present invention is an
immunocytochemical assay method for validating, or verifying, the
proper performance of the assay, for example, an assay that detects
the presence or absence of a target molecule in a biological
sample. The method comprises simultaneously processing the
biological sample and a quality control device in the assay,
wherein processing results in a detectable signal (e.g., a
calorimetric signal) produced by the target molecule and by the
quality control reagent moiety. As described above, the processing
results in the detection of the signal, therefore detection of the
target molecule in the biological sample and the target
molecule/target molecule mimic in the quality control reagent
moiety. As described above, the quality control moiety can be a
synthetic antigen/target molecule mimic. The fact that the quality
control reagent moiety develops a colorimetric signal provides
independent validation that the assay on the biological sample was
executed correctly. This is especially important in instances where
the biological sample yields a negative result, i.e., no color
development. The fact that the quality control strip yielded a
positive reaction establishes that the result is a true negative
and not due to errors in the procedure or problems with reagent
quality. Thus validation is established because the biological
sample and the quality control device are typically affixed to the
same microscope slide. Therefore, both the tissue sample and the
quality control device contacted the same series of reagents, for
the same time and temperature.
[0031] Another method of the present invention encompasses an assay
method (e.g., an immunocytochemical assay) for the determination of
the concentration of a target molecule in a biological sample. The
method comprises simultaneously processing the biological sample
and a quality control device in the immunocytochemical assay as
described above. The processing results in a detectable signal
generated by the presence of the target molecule in the biological
sample and target molecule/target molecule mimic in the quality
control moiety. The detectable signal generated from the target
molecule in the biological sample is compared with the detectable
signal generated from the target molecule (or synthetic antigen
mimic) in the quality control reagent moiety to determine the
concentration of target molecule in the biological sample.
[0032] As a result of the invention described herein, quality
control devices and methods are now available to serve as a quality
control tool to verify that a cytochemical, immunocytochemical, or
in situ hybridization procedure was executed correctly. Use of the
quality control device of the present invention verifies that each
and every tissue sample received the correct reagents, in the
proper sequence and timing, leading to proper staining. Moreover,
it verifies the integrity of the reagents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 is an en face view of a microscope slide with a
patient label and patient tissue sample. Additionally, the drawing
demonstrates where on the slide the adhesive antigen strip might be
positioned.
[0034] FIG. 2 is an en face view of the antigen strip showing the
positions of the antigen spots.
[0035] FIG. 3 is an en face view of the antigen strip showing the
degree of color development after a representative
immunohistochemical assay.
[0036] FIG. 4 is a graphic representation of an experiment to
determine linear assay range.
[0037] FIGS. 5A & 5B are graphic representations of the results
of an experiment for the detection of an early reagent failure.
FIG. 5A describes a failure test for a primary antibody. Control
strips with varying concentrations of ER peptide were subjected to
IHC staining with various dilutions ( 1/50, 1/450, and 1/900) of
anti-ER mouse monoclonal antibody 1D5.A 1/50 dilution is our
standard optimal 1D5. FIG. 5B describes a failure test for a
secondary antibody. Control strips with varying concentrations of
ER peptide; were subjected to IHC staining with various dilutions (
1/300, 1/1200, and 1/4800) of a biotinylated anti-mouse IgG
secondary antibody.
[0038] FIG. 6 is a list of peptides, using the single letter codes
for amino acids, that simulate the binding epitope of estrogen
receptor antibody 1D5.
[0039] FIG. 7 is a chemical representation of the surface of a
glass slide.
[0040] FIG. 8 is a chemical representation of the surface of a
glass slide after derivatization with 3-isocyanato
propyltriethoxysilane.
[0041] FIG. 9 is a chemical representation of the surface of a
glass slide after derivatization with
3-aminopropyltriethoxysilane.
[0042] FIG. 10 is a chemical representation of the surface of a
glass slide after initial derivatization as shown in FIG. 9 and
subsequent treatment of the glass slide with 1,4 phenyl
diisocyanate.
[0043] FIG. 11 is a chemical representation of the surface of a
glass slide after conjugation of a protein or DNA molecule
(represented as a jagged line) via an amino group on the DNA or
protein.
[0044] FIG. 12 is a chemical representation of the surface of a
glass slide after conjugation of a carbohydrate or DNA molecule
(represented as a jagged line) via a hydroxyl group (on the
carbohydrate or DNA molecule).
[0045] FIG. 13 is a chemical representation of a mildly stable
intermediate formed by the reaction of carbonyldiimidazole with an
amine bound to the glass surface.
[0046] FIG. 14 is a chemical representation of a mildly stable
intermediate formed by the reaction of methyl ethyl ketoneoxime
(MEKO) carbonate with an amine bound to the glass surface.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present invention as herein described addresses the
important quality control issues in cytochemical assays, and in
particular in immunohistochemical (IHC) assays. Most histopathology
laboratories use tissue samples previously documented to contain
particular antigens of interest (target antigen) as positive
controls.
[0048] One of the most important aspects that a positive control
should address is the early detection of reagent failure. The ideal
method of detecting early failure is to determine the level of
sensitivity of the working concentration of antibody. Sensitivity
is determined by titrating the antigen concentration until the
antigen is no longer detected. In this manner, the sensitivity of
the assay can be stated as the limit of the assay to detect a
certain amount of antigen, e.g., nanomoles or picomoles of antigen.
The limit of sensitivity should ideally be checked daily. In this
manner, long-term trends (towards decreasing or increasing
sensitivity) can be detected. It is impossible to perform this type
of analysis using tissue sections, since there is no practical
method for quantitation and titration of antigen in a tissue
section. Immunohistochemical assays are inherently more prone to
operator performance error than conventional immunoassays. This
creates an additional problem with the existing systems of quality
control. Namely, even if the control tissue slide appears
satisfactory, how is a performance error detected on another
(sample) slide?
[0049] There are several reasons that IHC assays are predisposed to
operator error. First, the fact that the IHC reaction is performed
on a microscope slide--a flat planar surface--allows for the
possibility that the reagent may fall off the edge of the slide or
evaporate, which would result in unsatisfactory staining due to
tissue drying. Conventional immunoassays, performed in test tubes,
cuvettes, microwells, or the like, have supporting walls for the
reagent. This physical constraint for the reagent prevents reagent
spillage.
[0050] Additionally, many clinical laboratories still perform IHC
staining manually. This inherently creates the possibility that the
technologist can place an incorrect reagent, or no reagent, during
one of the many steps in the procedure.
[0051] Even with an automated platform, which are recently gaining
market acceptance, machine malfunction is reported on an anecdotal
level. Each of the machine platforms assumes that an
electromechanical action translates into a dispensed volume of
reagent. This is not always the case. For example, an automatic
reagent dispenser can have several potential failure modes. These
include insufficient priming of the dispenser, a leaking dispenser
due to a faulty seal, and excessive friction (creating a drag
force) by the plunger in the reagent reservoir. Standard XYZ axis
pipettors depend upon the technician placing a sufficient amount of
antibody into a vial at the beginning of each run.
[0052] The final step in IHC staining is colorimetric development,
catalyzed by an enzyme such as peroxidase or alkaline phosphatase.
A colorless and soluble substrate is converted into a colored and
insoluble precipitate. The timing of the reaction is important. Too
short a development yields a poor signal while too long a
development time can increase non-specific background staining.
Consequently, this step is performed under the watchful eye of the
technologist, who examines the tissue sections for color
development under the microscope. When the color is optimally
developed, with low background, the technologist stops the reaction
by immersing the slide in buffer. In practice, this step is
labor-intensive, since each slide is examined individually.
[0053] The use of a quality control device with every assay, or on
every microscope slide, would solve the assay performance problem.
Described herein is a quality control device comprising multiple
quality control reagent moieties affixed to a matrix or membrane.
The quality control device can be affixed, or adhered, to a test
platform, such as a microscope slide, using an adhesive. These
reagent moieties comprise the target molecule (e.g., the antigen,
antibody or nucleic acid to be detected in the assay), or a target
molecule mimic, defined herein as a molecule that mimics the
characteristics, or properties (e.g., physical structure or
antigenic properties) of the target molecule sufficiently so that
the mimic molecule reacts substantially the same as the target
molecule in the cytochemical assay. In one embodiment of the
present invention, the target molecule mimic is a short synthetic
peptide that mimics the antigen site of the target molecule and
therefore, binds to antibody specific for the target molecule (also
referred to herein as a synthetic antigen or synthetic
control).
[0054] In one embodiment of the present invention, the quality
control device comprises a membrane strip, approximately
0.8.times.0.2 inches, with an adhesive backing that can be applied
to a microscope slide. FIG. 1 demonstrates a recommended position
on the microscope slide (1) for such a control strip (2). At one
end, a label (3) with patient information is typically applied. The
information on the label typically includes the type of stain,
surgical accession number, patient name, and occasionally a bar
code. The tissue section (5) is typically placed in the middle of
the slide. Immunohistochemical reactions are performed by the
sequential incubation and removal of a series of reagents to and
from the tissue section (5). By placing the control strip (2) near
the tissue section (5), the strip will also contact the same
reagents, as they are applied and removed.
[0055] FIG. 2 demonstrates the placement of antigen on a
representative antigen control strip. A series of spots of antigen
(7-12) are evenly spaced across the length of the strip. Each spot
comprises a macromolecule, preferably a peptide, immobilized onto
the membrane matrix. In a preferred embodiment, spots 8-12 comprise
the same peptide at different molar concentrations immobilized onto
the membrane matrix. Therefore, if an immunohistochemical assay is
known to have a threshold of sensitivity of 10 nanograms per
mm.sup.3 of antigen X, then spot 10 would ideally have such a
concentration. Spots 11 and 12 would have progressively increasing
concentrations (e.g., 30 ng/mm.sup.3 and 90 ng/mm.sup.3,
respectively). Spots 9 and 8 would have progressively decreasing
concentrations (3 and 1 ng/mm.sup.3). Spot 7 is designed to test
specificity rather than sensitivity. Therefore, spot 7 would
comprise an irrelevant antigen (antigen Y-) immobilized onto the
membrane matrix. Ideally, the concentration of antigen Y in spot 7
will be higher than the threshold concentration for detecting
antigen X (10 ng/mm.sup.3).
[0056] FIG. 3 demonstrates the expected calorimetric result of the
antigen spots after an immunohistochemical assay. The assay
sensitivity and peptide concentrations are as stated in the above
paragraph. Spots 10-12 are increasingly dark colored, owing to the
increasing concentrations of peptide antigen. Spots 8 and 9 are
colorless, since their concentration is stated to be below the
level of detection. Spot 7 is colorless, since the antibody to
antigen X does not bind to antigen Y (comprising spot 7).
[0057] Matrix
[0058] The quality control reagent moieties are affixed to a matrix
or membrane as described herein. The matrix can be manufactured of
materials capable of binding macromolecules. Suitable matrix
materials include glass, nylon, nitrocellulose, and
polyvinylidenedifluoride (PVDF). Nitrocellulose is commonly used
for Western (protein), Northern (RNA) and Southern (DNA) blots, and
is capable of binding macromolecules through non-covalent bonds.
PVDF and nylon have greater tensile strength than nitrocellulose
and can be derivatized to allow covalent coupling of
macromolecules. Such matrices are commercially available and sold
as Immobilon.TM. AV (Millipore Corporation, Bedford, Mass.) or
Immunodyne membrane (Pall Corporation, East Hills, N.Y.). Both
matrices are derivatized by the manufacturer so that there is an
abundance of free carboxyl groups on their surface. This allows for
the coupling of macromolecules to the carboxyl groups through an
amine or a sulfhydryl group. Macromolecules are coupled to the
matrix in the present invention by placing (e.g., spotting) a small
aliquot (typically less than one microliter) of the
macromolecule-containing solution on top of the activated matrix.
The macromolecule, such as the antigen or peptide, is dissolved in
a coupling buffer. The coupling buffer is preferably at a pH that
maximizes the reaction of the macromolecule to the derivatized (or
"activated") matrix. A preferred buffer is 0.5M Potassium phosphate
pH 7.4. The reaction is performed at room temperature. Five minutes
to an hour is a preferred period of time for coupling, for
convenience of manufacture and typically results in approximately
80% coupling efficiency. The matrix is then rinsed with buffer to
remove any unbound macromolecules.
[0059] The matrix is spotted with a series of dilutions of the
relevant target molecule or target molecule mimic (e.g., synthetic
peptide antigen). Different concentrations of the antigen
(peptide), will be useful in quickly estimating the extent of
colorimetric development without the need for microscopic
examination. For example, the synthetic peptide control
concentrations can be appropriately calibrated so that a 3+ control
reaction will yield a strong tissue signal with minimal background.
The amount of antigen blotted onto the matrix would need to
approximate the amount of antigen present in the tissue section.
This can be empirically determined by coupling sufficient antigen
to the matrix so that the color development on the matrix
approximately parallels the intensity of the color development in
the tissue section itself.
[0060] Typically, the first spot is the antigen at the highest
concentration, and is expected to yield a strong positive reaction
(e.g., detectable signal) after IHC staining. Subsequent serial
dilutions vary from two to twenty-fold in concentration. It is
desirable to have a clearly defined limit of sensitivity.
Specifically, the user of this control strip should be able to view
the immunologic reactions that occur on the strip, and
unequivocally identify an endpoint of sensitivity. The calorimetric
IHC reaction product is strong above the limit of sensitivity and
significantly weaker or absent below it. Therefore, the antigen
dilutions are sufficiently different from each other so that there
is a sharp falloff in immunoreactivity, creating a clearly-defined
limit of sensitivity. A preferred embodiment of the invention is to
spot the series of dots with antigen at approximately 3-6 fold
serial antigen dilutions. An irrelevant antigen of similar size and
charge characteristics is also spotted at the end of the strip. The
irrelevant antigen provides a control for specificity of the
immunologic reaction.
[0061] Additionally, a semi-quantitative determination of the
concentration of a target molecule in a biological sample can be
ascertained by comparing the detectable signal (e.g., color)
generated from the antigen present in the biological sample and the
detectable signal generated from the antigen in the quality control
reagent moiety. The closest match of detectable signals between the
sample and the control is indicative of the approximate relative
concentration of the antigen in the sample.
[0062] The relevant antigens are spotted on the matrix surface
using micropipettes or precision syringes. This can be accomplished
in an automated fashion using an array of micropipettes that
repetitively apply a defined number of microliters to defined
positions on the quality control strips (matrix).
[0063] An alternative embodiment of the invention is to place
multiple, distinct antigens onto the matrix. In this alternative, a
single quality control device might have all of the relevant
antigens for a particular disease state. For example, one device
might be for a lymphoma IHC panel and contain antigens for many of
the relevant antigens for a lymphoma diagnostic work up. Another
device can be for a soft tissue tumor IHC panel, another for an
undifferentiated "round, blue-cell" tumor work up, etc. In this
fashion, the synthetic control devices are more broadly applicable
to many different monoclonal primary antibodies.
[0064] After the coupling procedure, the "activated" (derivatized)
matrix still contains a high density of available active surface
groups (e.g, carboxyl groups) for coupling to macromolecules.
According to one manufacturer (Millipore Corporation, Bedford,
Mass.), a carboxyl group is present every seven Angstroms on the
matrix. Therefore, only a small proportion of the active carboxyl
sites on the matrix are typically occupied by a macromolecule after
coupling. The remaining carboxyl groups must be "quenched" (or
"capped"), blocking any future potential reactivity. The matrix is
quenched with any of a variety of small molecules that have free
amino groups. Suitable quenching agents include ethanolamine,
ethylenediamine, dithiothreitol, aminoethanediol, and
aminopropanesulfonate. Alternatively, proteins such as gelatin or
casein or amino acids such as glycine, can quench the matrix. A
recommended quenching procedure is to cover the membrane with 10%
v/v monoethanolamine in 1.0 M Sodium Bicarbonate buffer, pH 9.5.
The matrix is incubated with the quenching solution for few minutes
to an hour at room temperature with constant agitation. The
quenching solution is then aspirated or drained and the matrix
rinsed with 0.01 M Sodium Phosphate, 0.14 M NaCl, pH 7.4, 0.1% v/v
Tween 20 (PBS-Tween). Each wash is for five to 30 minutes with
constant agitation. The wash solution is then aspirated or drained,
and the matrix is allowed to dry.
[0065] Antigen
[0066] Two forms of antigen can be used as a synthetic positive
tissue control. First, the whole antigen can be coupled to the
membrane. A list of some of the most commonly used antigens for
clinical IHC diagnosis is shown in Table 1. Some of these antigens
are available commercially. Examples include human immunoglobulins,
prostate specific antigen, S-100 antigen, alpha-fetoprotein, and
carcinoembryonic antigen, available from Fitzgerald Industries
International, Inc., Concord, Mass. Other antigens can be isolated
by solubilization of cultured cells or tissue homogenates and
purification by affinity chromatography. Affinity purification
methods are well known to those skilled in the art.
[0067] Antigens can also be obtained by recombinant DNA methods.
cDNA constructs encoding the desired antigen can be transfected
into suitable host cells, expressed and purified using techniques
well-known to those of skill in the art.
[0068] Alternatively, a short peptide can serve as the antigen.
Suitable peptides are typically approximately ten amino acids in
length, but can be longer or shorter in length and still
specifically bind antibody. Antibodies only bind to a small region
of an antigen, typically comprising a few amino acids. Antibody
epitopes of protein antigens can be broadly classified as
sequential ("linear") or conformational ("discontinuous"). This
classification is based on whether or not the amino acids that
interact with the antibody are positioned immediately adjacent to
each other in the linear amino acid sequence of the native protein.
The surface of the antigen that interacts with the antibody can
thus either consist of amino acids that are adjacent to each other
or of amino acids that are separated in the primary sequence but
brought together as a result of the natural folding (conformation)
of the protein to its native shape. Epitopes consisting of residues
close together in the primary sequence are called linear,
continuous, or sequential epitopes, whereas epitopes consisting of
residues separated in the primary sequence are called discontinuous
or conformational epitopes.
[0069] Short peptides can substitute for antigens when monoclonal
antibodies are used. Monoclonal antibodies have a single, defined
binding epitope. Different monoclonal antibodies often bind to
distinct epitopes of an antigen. Therefore, when short peptides are
used as antigens in the synthetic positive control device, they are
specific to the monoclonal antibody that is being used. Each
monoclonal antibody requires a distinct peptide sequence as a
surrogate antigen. Therefore, the synthetic positive controls,
using short peptides as antigen, are both antigen-specific as well
as antibody-specific. Several of the relevant peptides for antigens
in Table 1 have been identified for specific monoclonal antibodies.
For example, the 20 amino acid tandem repeat in the extracellular
domain of polymorphic epithelial mucin (cancer-associated antigen
CA15-3) is shown below, each letter representing an amino acid.
TABLE-US-00001 TABLE 1 COMMON ANTIGEN TARGETS FOR CLINICAL IHC
ASSAYS PROGNOSTIC MARKERS Estrogen receptor Progesterone receptor
p53 protein Ki-67 protein Proliferating cell nuclear antigen (PCNA)
HEMATOLOGIC MARKERS CD3 CD15 CD20 CD30 CD34 CD45 CD45RO CD99 Kappa
light chain Lambda light chain Factor VIII EPITHELIAL
DIFFERENTIATION MARKERS Prostate specific antigen (PSA) Prostate
specific alkaline phosphatase (PSAP) Cytokeratin Epithelial
membrane antigen (EMA) Carcinoembryonic antigen (CEA) Polymorphic
epithelial mucin Mesenchymal differentiation markers Desmin
Vimentin Actin Collagen type IV MELANOCYTIC MARKERS S-100 HMB45
MISCELLANEOUS Neuron-specific enolase Glial fibrillary acidic
protein Chromogranin Synaptophysin
[0070] The binding sites for the DF3 (DAKO Corporation,
Carpinteria, Calif.) and B27.29 monoclonal antibodies are shown
above and below the sequence (SEQ ID NO:1). The relevant region is
indicated by the dotted lines. (Reference: Bon G G, von
Mensdorff-Pouilly S, Kenemans P, van Kamp G J, et. al. Clinical and
technical evaluation of ACS BR serum assay of MUCI gene-derived
glycoprotein in breast cancer, and comparison with CA15-3 assays.
Clin. Chem. 1997 43:585-593). TABLE-US-00002 ------DF3------ -P A H
G V T S A P D T R P A P G S T A P- ------B27.29-------
[0071] Two general methods for identifying relevant peptides are
known to those skilled in the art. One of these methods is the use
of fragments of the protein antigen that are identical to one or
more portions of the linear sequence. Sequence mapping technologies
such as Multipin Peptide Synthesis Technology (Chiron Mimotopes,
Victoria, Australia) enable linear sequence mapping by creating
overlapping series of peptides on a 96 pin plastic structure. In
this method, the primary sequence of the protein must be known.
Using automated peptide-synthesis equipment, overlapping fragments
of the protein are synthesized on the various pins. For example,
pin #1 might have the first ten amino acids. Pin #2 would have
amino acids 2-11, pin #3 amino acids 3-12, etc. If the monoclonal
antibody recognizes a linear epitope, then it will probably
recognize and bind to one or more of the protein fragments on the
pins.
[0072] From general experience with peptide mapping, a minority of
monoclonal antibodies bind to linear epitopes. The remaining
epitopes are conformational (discontinuous). Therefore, an
alternative method of identifying antibody-binding peptides is
required. To identify discontinuous epitopes, the preferred method
of peptide identification is known as phage display. Phage display,
the display of genetically encoded diversity on the surface of M13
filamentous bacteriophage, allows the production and screening of
tens of millions of proteins and peptides in a few weeks (Ladner,
R. and S. Guterman, in W090/02809 (1990), the teachings of which
are herein incorporated in their entirety by reference.; Ladner,
R., et al., Directed Evolution of Novel Binding Proteins, (1993)
U.S.; Wells, J. and H. Lowman, Curr. Op. Struct. Biol., (1992)
3(4): p. 355-362) (McLafferty, M., et al., Gene, (1993) 128:
p.29-36; Clackson, T. and J. Wells, TIBTECH, (1994) 12: p.
173-184), the teachings of which are herein incorporated in their
entirety by reference. The organisms containing those peptides that
have the desired binding characteristics can be replicated,
allowing repeated screening with increased stringency and
amplification of the ligands. After a few rounds of screening and
amplification, the peptides that remain are the higher affinity
binders to the target molecule. Phage display is recognized as an
efficient method of producing proteins and peptides that bind to
targets of interest. (Ladner, R. and S. Guterman, in International
patent application W090/02809 (1990); Ladner, R., et al., Directed
Evolution of Novel Binding Proteins, (1993) U.S.; Roberts, B., et
al., Proc. Natl. Acad. Sci. USA, (1992) 89: p. 2429-2433; Markland,
W., A. Ley, and R. Ladner, Biochemistry, (1996) 35: p. 8058-67;
Markland, W., et al., Biochemistry, (1996) 35: p. 8045-57; Ley, A.,
W. Markland, and R. Ladner, Mol Divers, (1996) 2: p. 119-24, the
teachings of which are herein incorporated in their entirety by
reference.
[0073] Adhesive Backing
[0074] One embodiment for the synthetic antigen control strips is
that they peel off from an 8.5.times.11 inch sheet. Each sheet
would contain a plurality of control strips. The user simply peels
off the appropriate antigen control from the sheet and applies it
to the end of the slide, Therefore, the patient's name and
identifying information would be at one end of the slide, and the
antigen control at the other. Each strip would be spotted with
several different concentrations of antigen.
[0075] Commercially available nitrocellulose, nylon or PVDF
membranes can be used to create adhesive strips using techniques
established in the printing industry. Specifically, a
pressure-sensitive adhesive is applied to the back of the membrane.
A suitable adhesive is FASSON S727 acrylic adhesive, because it is
waterproof and permanent. Backing paper is then applied to the
adhesive-coated nylon membrane. A suitable backing paper is 50
pound Kraft paper with a silicone liner. The purpose of the backing
and liner is to provide a surface from which the membrane strips
can be peeled. When the membrane strips are peeled away from the
backing, the adhesive largely remains with the membrane. The
application of adhesive and backing paper is typically performed by
machinery that starts with two rolls (one roll of nylon membrane
and backing paper, each) and ends with one roll of the
nylon-adhesive-backing paper sandwich.
[0076] An alternative method of fabricating a membrane with an
adhesive backing is to apply a double-faced tape to the back of the
membrane. The double-faced tape comprises a film with adhesive
coatings on both sides. A release liner covers the adhesive on one
side and the other adhesive side is applied to the back of the
membrane. Suitable double-sided tape can be obtained commercially,
such as from Adhesives Research, Inc., Glen Rock, Pa. Suitable
tapes generally use acrylic adhesives and polyester films. Acrylic
adhesives are preferred because they exhibit a high degree of
solvent resistance and are biologically inert. Examples of suitable
tapes include their model ARCARE.TM. 7737, 8570, 7840, and 7841.
The membrane and double-sided tape are applied to each other by
machinery well known in the printing and labels industry, where
each starts out as a separate roll. Each spool feeds its film or
membrane into a roller that compresses the tape against the
membrane. The hybrid sandwich of membrane and tape is then taken up
on a third spool.
[0077] By placing the control strip against a liquid impermeable
surface (an adhesive backing on top of a glass slide), vertical
(downward) flow of reagent from the reagent surface of the strip
inwards is prevented. The adhesives often have a hydrophobic
character preventing water penetration. If double-sided tape is
used, the tape described herein is also water impermeable. The
glass slides are also impermeable to water or other liquids. This
feature is in contradistinction to many other blotting techniques
where vertical flow through the membrane into adsorbent paper
towels is helpful. By preventing vertical flow of reagent, the
strips exhibit an increased sensitivity of antigen detection,
reasonably attributed to a longer incubation of the reagent with
the quality control moieties immobilized to the reagent surface of
the matrix.
[0078] Use of the Quality Control Device
[0079] In practice, the technologist typically mounts a tissue
section, cytospin, or cellular smear on a glass microscope slide so
that the stained specimen can be visualized with a microscope.
These techniques are well-known to those of skill in the art. The
quality control strip is applied-to the same glass slide, near or
adjacent to the specimen. In this manner, the strip receives the
same series of reagents, for the same incubation times,
temperatures, and washing conditions as the biologic specimen. For
immunohistochemical assays, a series of antibodies are applied to a
biologic sample, such as a tumor section. The first antibody is for
the purpose of conveying the assay specificity. A list of many
commonly used antibody targets is listed in Table 1. Typically, the
antibodies will be monoclonal, of murine origin. The primary
antibody is commonly incubated with the tissue section for a period
of about 15-60 minutes.
[0080] The remaining reagents in the assay are considered detection
reagents. They demonstrate where in the biologic specimen the first
(primary) antibody bound, by causing that site to become colored.
After the primary antibody incubation, the slide is rinsed with a
buffer, commonly phosphate buffered saline, in order to remove any
unbound antibody molecules from the surface of the slide. Then, a
secondary antibody is applied. A common secondary antibody is a
polyclonal anti-murine IgG or IgM recognizing unique epitopes
present on murine immunoglobulins. This secondary antibody is
commonly coupled (covalently) with biotin. Consequently, wherever
the primary antibody bound to the biologic specimen, biotin
molecules are now found. After the secondary antibody incubation,
excess unbound antibody reagent is rinsed off with a buffer. The
third step in the reaction sequence provides for coupling of an
enzyme, typically peroxidase, to the site where the primary
antibody bound. This is commonly performed by incubating the slide
with avidin (or streptavidin) covalently coupled to peroxidase.
Avidin (and streptavidin) has a high binding affinity to biotin.
Therefore, peroxidase will be immobilized at sites of biotin. After
completion of this third step in the reaction, the excess unbound
enzyme--avidin reagent is rinsed off with buffer. The final step is
the addition of an enzyme substrate. Substrates are chosen so that
they are soluble and relatively colorless. However, after action of
the enzyme upon the substrate, they become colored and insoluble.
Therefore, the substrate precipitates wherever the enzyme is found.
A commonly used peroxidase substrate is 3,3-diaminobenzidine.
[0081] Numerous other variations of the immunohistochemistry
procedure exist and are described in the relevant literature. This
quality control strip is designed so that it will serve as a
positive control regardless of the detection system used. Whatever
series of reactions that occur on the biologic sample will also
occur on the quality control strip. The use of more sensitive
detection systems will correspondingly be reflected in the
increased color intensity of both the biologic sample and the
quality control strip. Moreover, a more sensitive detection system
will detect the lower concentration spots of antigen. The opposite
will be true of less sensitive detection methods. In this manner,
the quality control strips can verify and quantify the performance
characteristics of immunohistochemical assays.
[0082] Although immunohistochemical assays have been specifically
referenced in the examples, the same quality control method can
also be applied to other types of assays performed on microscope
slides. For example, nucleic acid targets can be immobilized onto
the matrix and serve as positive controls for assays such as in
situ hybridization. In this configuration, probes that detect
specific nucleic acid sequences will also hybridize with
complementary sequences on, the quality control strip. Such assays
are well known to practitioners in the field and published in
scientific literature. Commonly performed in situ hybridization
assays test for the presence of viruses. As a control, dsDNA
containing the desired double-stranded sequence is covalently
coupled to the matrix. A convenient method for coupling dsDNA is to
derivatize it at one end with a free amino group. The free amino
group can then be coupled to the carboxy-derivatized matrix in the
same fashion as already described.
[0083] This quality control method can also be applied to the field
of histochemical stains. Classes of compounds that are detected by
histochemical methods can be immobilized onto the matrix. Examples
include carbohydrates for the Periodic acid-Schiff (PAS) stain,
mucins for the mucicarmine stain, extracellular matrix components
for the reticulin stain, etc. Another application of this quality
control method can be in the diagnosis of microbes or parasites.
Whole microbes, or purified or recombinant microbial or parasite
specific antigens or antigen-mimics can be similarly used as
quality control antigen strip on the slide. Such microbial controls
can serve as controls for cytochemical stains used in microbial
detection, such as in the Gram, Steiner, or methenamine silver
stains. Alternatively, macromolecules from these microorganisms (or
antigen mimics) can serve as controls in immunohistochemical or
nucleic acid diagnostic assays for these microbes.
[0084] With respect to the five criteria previously mentioned, the
quality control devices described herein are antigen-specific,
easily manufactured in large quantities, inexpensive, stable and
standardized. The antigenic specificity is conferred by virtue of
the antigen that is immobilized onto the antigen strip or glass
slide. Short peptides (e.g., approximately 5-50 amino acids long,
and more typically approximately 10-20 amino acids long) are easily
manufactured at low cost. Moreover, they tend to be quite stable,
especially if stored at 4 degrees C. By applying a calibrated
amount of antigen to the strip, under reproducible coupling
conditions, the amount of antigen bound can be standardized.
[0085] The synthetic controls described herein provide an absolute
quantitative standard for immunohistochemical reactions. The
antigen strips or glass slides are manufactured so that a series of
known concentrations of peptide are deposited, as previously
described. This provides laboratories the means to verify that they
each have comparable assay sensitivity. For example, estrogen
receptor analysis is commonly performed by visually quantifying
(under microscopic examination) the percentage of tumor cells that
stain positively using an antibody to estrogen receptor. However, a
significantly more sensitive assay will be expected to detect more
positive cells than a less sensitive assay. Therefore, assay
sensitivity standardization is important for laboratories to
meaningfully communicate their results. Since the synthetic antigen
control strips are manufactured in a standardized and reproducible
fashion, each laboratory has a reproducible assay standard.
[0086] In summary, the quality control devices described herein can
serve three functions:
[0087] 1. Validation of Proper Assay Performance.
[0088] The devices contain analytes that are identical to or mimic
the proteins, peptides, nucleic acids, carbohydrates, lipids, etc.
found in the cell or tissue sample that is being analyzed. These
analytes produce a substantially similar colorimetric signal as the
signal produced by the staining reaction on the cells or tissue
section. Therefore, if the quality control device yields a positive
signal, it serves as an indicator that the staining procedure was
performed correctly. This is particularly useful in instances where
the analytes being tested for in the cell or tissue sample are not
present, e.g., it is useful in assays that produce a negative
tissue reaction. A positive reaction on the quality control device
establishes that the negative result in the tissue sample is a true
negative rather than being due to errors in the staining
procedure.
[0089] 2. Determination of Assay Sensitivity.
[0090] The devices that contain varying quantities of the analytes
provide a method for establishing the endpoint of sensitivity of
the assay, e.g., the lowest detectable concentration of analyte on
the control device that is detectable represents the threshold of
sensitivity.
[0091] 3. Quantitation of Analytes in the Cell or Tissue
Sample.
[0092] The devices that contain varying quantities of the analytes
can provide a concentration reference standard that can be used for
analyte quantification. Specifically, the calorimetric signals can
be expected to vary according to the analyte concentration. Low
analyte concentrations will yield weak calorimetric signals; high
concentrations will yield strong calorimetric signals. The
intensity of the signal can either be estimated visually or
directly quantified using computer-assisted image analysis
techniques. With either method of quantification, the signal
intensity on the quality control device can be correlated with the
signal intensity found in the tissue section. In this manner, the
approximate amount of analyte in the cell or tissue section can be
estimated. If quantitative signal measurement methods are used
(image cytometry), then a calibration curve can be established
every time the assay is run using the quality control device, e.g.,
the signal intensity is plotted (y axis) against the analyte
concentration (x axis). The signal intensity in the cells or tissue
section can then be interpolated on this graph to deduce the
analyte concentration. The following examples will further
illustrate the claimed invention.
EXAMPLE 1
[0093] Select Phage that Mimic Target Epitopes from Monoclonal
Antibodies Commonly used in Clinical Immunohistochemical
Laboratories
[0094] For the device and methods describe herein, it is desirable
to identify peptides that will mimic the antigen-specific
interaction of antibody with the native antigen. These peptides
will be suitable for use on either the quality control adhesive
strips or glass slides described herein. Suitable peptide sequences
identified as described herein are more amenable to consistent
manufacture than the native protein itself. The monoclonal antibody
clones suitable-for use as described herein are those that
recognize the antigen even after the tissue has been fixed with
formalin and embedded in paraffin wax. These monoclonal antibody
clones represent some of the most commonly used primary antibodies
in the clinical IHC laboratory. The cyclic peptide libraries
comprise a random assortment of peptides that have two cysteine
residues that are linked by a disulfide bond. This intramolecular
bond imparts the advantage of a better-defined and more stable
three-dimensional structure. The DNA sequence coding for the random
peptides are inserted into the Gene 3 phage protein, available from
Dyax Corp., Cambridge, Mass.
[0095] (a) Screening of Peptide and Protein Display Libraries.
[0096] The following procedure is used to select phage from
libraries that bind specifically to antibody combining sites.
Selection of phage is effected through binding of phage to target
mouse monoclonal antibody that is captured onto human anti-mouse
IgG-conjugated paramagnetic beads (CELLectin Pan Mouse IgG
Dynabeads, available from Dynal Inc., Lake Success, N.Y.).
[0097] The human anti-mouse IgG-conjugated magnetic beads were
capable of capturing approximately 0.5 to 2 .mu.g of mouse IgG from
a hybridoma cell culture supernatant. Captured antibody can be
eluted with 0.1 M HCl. Peptides displayed on phage particles that
mimic the epitope of specific target molecule recognized by a
specific monoclonal antibody are screened. In essence the peptide
binds to the antibody combining site in a similar fashion as would
the target antigen.
[0098] At the start of the panning procedure, non-specifically
binding phage are initially removed by five rounds of negative
selection against magnetic beads coated with normal mouse IgG. This
step removes phage that bind to framework determinants of murine
IgG. Approximately 100 microliters of CELLectin Pan Mouse IgG
Dynabeads coated with normal mouse IgG are resuspended in 500 .mu.l
of a PBS-Tween 20 (0.05%) buffer with 10.sup.11 phage particles
that comprise the starting phage library. The beads and phage
particles are incubated at 25 degrees C. for 1 hour, with gentle
tumbling.
[0099] After the initial rounds of negative selection, the positive
selection of desired phage particles is then performed. The target
mouse monoclonal antibody is captured onto Pan Mouse IgG Dynabeads
(human anti-mouse IgG-conjugated magnetic beads). The negatively
depleted phage particles are then incubated with the
antibody-coated beads. Bound phage are eluted by acidification (pH
2, by addition of 150 .mu.l of 50 mM glycine-HCl) of the bead
pellet. The beads are incubated for 15 minutes at 25 degrees C. in
the pH 2 buffer. The acid-eluted phage are harvested from the
supernatant and the pH neutralized by addition of 150 .mu.l of 200
mM NaPO.sub.4 buffer, pH 7.5. The positively-selected phage are
then grown for further rounds of panning.
[0100] (b) Enrichment for Clones that Contain High Affinity Binders
to Target Antibody.
[0101] The phage libraries contain between 10.sup.8-10.sup.9
distinct phage particles. At the outset, there are approximately
100-1000 copies of each phage particle. Therefore, there are a
total of approximately 10.sup.10-10.sup.11 phage particles in the
first screen. The goal of enrichment is to select only those phage
particles that bind specifically and with high affinity to the
target antibody. Typically, between 10.sup.5-10.sup.6 binding phage
are recovered during the first round of screening. Many of the
phage often represent redundant particles.
[0102] This pool of phage is then amplified and further screened.
The enriched phage library is much less complex than at the outset,
since this second round is pre-selected for binding phage.
Therefore, the fraction of binding phage is usually to be greater
than in the first round. Since this second round of amplification
starts with the same number of total phage (after amplification of
the first round of selected phage), up to 10.sup.7-10.sup.8 phage
particles are expected to be obtained after the second round. Each
round of screening should yield a greater number of binding phage,
indicating enrichment. When the number of phage recovered has
leveled off, this is an indicator that no further enrichment is
occurring.
[0103] The eluted phage are used to inoculate 5 ml of a 1:100
dilution of an overnight culture of TG1 or XL-1 Blue E. coli in 2X
YT media, which are then grown for no more than 8 hours at 37
degrees C., shaking at 220 rpm. The cells are pelleted by
centrifugation at 5000 rpm in a Sorvall Superspeed T21 tabletop
centrifuge, and the phage in the supernatant are recovered by
precipitation with polyethylene glycol. The selection is then
repeated. Four rounds of selection are usually sufficient to enrich
the pool for phage that contain high affinity binders to the
antibody (though some phage screens gave high binders after only
two rounds of selection). For increased selection pressure, the
stringency conditions for binding phage can be increased (e.g., by
increasing the salt concentration or adding non-ionic detergent or
gradually decreasing the concentration of target antibody
molecule).
[0104] From section (b), it is expected to obtain between 10 to 20
phage clones that exhibit high affinity binding, to each of the
target antibody molecules. Some of these phage will be identical to
each other. Ultimately, the aim is to generate approximately five
distinct peptide sequences that bind to the antibody. Each of these
clones are amplified and sequenced so that peptides can be
generated using solid phase chemical synthesis.
[0105] (c) Phase Clone Amplification, Sequence Identification and
Peptide Production
[0106] Large quantities of each of the selected phage are grown and
nucleic acid extracted for sequence analysis. Based on the
nucleotide sequence coding for the inserted ten-mer peptide,
corresponding peptides are synthesized. Numerous commercial vendors
provide peptide synthesis capabilities. The binding affinity of
each of the selected peptide sequences for their antibodies is
determined using Scatchard analysis. The percentage of antibodies
binding to the peptide can be ascertained using fluorescence
polarization analysis, provided that the peptide is fluorescently
labeled. The specificity of binding for each peptide antigen mimic
is tested using irrelevant, isotype-matched antibodies.
[0107] At the end of the above process, peptide antigens are
identified that mimic the binding, characteristics of the
monoclonal antibody to the real antigen.
EXAMPLE 2
[0108] Peptide (Antigen Mimic)--Matrix Optimization
[0109] (a) Overview
[0110] Once an appropriate peptide sequence is identified, the next
step is to determine the linear range of the peptide concentration
on the control strips. In other words, how much peptide should be
placed onto the nylon strip so as to be most sensitive to early
reagent failure.
[0111] Carboxy-derivatized nylon or PVDF matrices allow high
capacity covalent immobilization of proteins to the surface of the
matrix while retaining biological activity. High capacity
nitrocellulose membranes also serve to immobilize proteins or
peptides in a manner adaptable to this invention. Processing steps
include the covalent or non-covalent binding of protein to the
membrane and a blocking step to quench the remaining covalent
binding capacity, as described herein. Standardization is more
readily achieved with proteins that are covalently, rather than
non-covalently, adsorbed to matrices. Other desirable
characteristics of nylon or PVDF membranes include moderate cost,
resistance to microbial attack, and mechanical strength.
[0112] (b) Maximizing Sensitivity to Early Reagent Failure.
[0113] Described herein is a preferred method for determining the
optimal concentration of peptide on the quality control strip.
Based on the manufacturers' specifications (Millipore Inmobilon-AV
Affinity Membrane or Pall Biodyne D membrane), the coupling
capacity far exceeds the likely desired peptide concentration. In
order to serve as a useful absolute quantitative standard, it is
important to precisely control the amount of peptide that is bound
to the membrane.
[0114] Different laboratories use slightly different concentrations
of primary antibody, depending upon the sensitivity of their
detection systems. If the purpose of the control strip is to
monitor assay sensitivity, then this invention will be configured
to bracket a range of peptide concentrations. A series of two to
five-fold dilutions for spots of peptide on a single control strip
is ideal. The control strip will include a spot with an irrelevant
peptide. If the control strips are configured with a series of
peptides at the correct concentrations, then they will be highly
sensitive to a decrement in antibody activity. To achieve maximal
sensitivity, it is important to identify the dynamic (linear) range
for the IHC assay for each peptide/primary antibody
combination.
[0115] For each peptide/primary antibody combination, dynamic
ranges are determined as follows. A series of spots are placed onto
a derivatized matrix. Each spot represents a two to five fold
dilution of peptide, so that the peptide concentration varies over
a broad range. The spots are then detected using primary antibody
concentrations already optimized for tissue IHC staining.
Typically, primary antibodies in IHC assays are used at a 1-10
microgram/ml concentration. The, intensity of the spots can be
visually estimated on a 1-4+ scale. Alternatively the spots can be
scanned using a flat bed scanner and quantified by densitometry
using ImageQuaNT software (Molecular Dynamics Inc.). The staining
intensity, or "dot density," is quantified on a numeric scale
(arbitrary units).
[0116] FIG. 4 demonstrates results using the ER peptide mimic (as
described herein) and the 1D5 anti-human estrogen receptor
antibody. The 1D5 antibody (purchased from DAKO Corporation,
Carpinteria, Calif.) was diluted 1:50 in phosphate buffered saline
and incubated for 30 minutes at 37 degrees C. The binding of the
1D5 antibody to the ER peptide mimic was detected using a standard
immunohistochemical detection kit. The kit comprises a secondary
antibody (anti-mouse IgG-biotin conjugate) and a
peroxidase-straptavidin conjugate. This type of detection kit is
widely understood to those skilled in the art.
[0117] The ideal mean peptide concentration for the center spot on
the control strip is that concentration near the juncture of the
linear and plateau portions of the graph. From FIG. 4, that point
is the 4 micromolar peptide concentration. In this particular assay
setting, the signal intensity beyond a peptide concentration of
approximately 6 .mu.M seems to plateau off, while the lowest
threshold of detection appears to be approximately 2 .mu.M. It is
at this peptide concentration that the control strips of the
present invention will be most able to detect early reagent
failure. If a reagent begins to experience diminished activity, the
calorimetric intensity (y axis) will correspondingly diminish
(linear portion of curve). On the other hand, operating on the
plateau portion of the curve will cause a lack of colorimetric
decrement if mild reagent degradation occurs.
EXAMPLE 3
[0118] Validation of Synthetic Control Strips
[0119] Each peptide mimic or antigen comprises a somewhat unique
and independent product. Therefore, each needs to be independently
validated. This section describes the process of validating the
synthetic control strips to verify that (a) each synthetic control
strip has a high signal to noise ratio, (b) the synthetic control
strip is antibody-specific, and (c) each synthetic control strip is
capable of detecting early reagent failure.
[0120] (a) Measurement of the Signal to Noise Ratio
[0121] The synthetic control strip contains two distinct areas: (1)
the reagent surface that is devoid of bound peptide, and (2) the
test area with specific peptide. The test strip containing no
peptide should be used as a test for non-specific binding of
antibody to the membrane itself. Excessive background to the
negative control (surface without peptide) indicates the need to
investigate the blocking procedure, buffer composition, and/or the
washing procedure during the assay.
[0122] In order to conduct the validation, control strips can be
generated with three portions, or regions, on the strip: (1) a
portion that contains the relevant specific peptide, (2) a portion
that contains an irrelevant peptide, and (3) a portion that does
not contain any peptide. The latter two are both negative
background controls. Each of the three regions are blocked and
washed in an identical fashion. Namely, the strips are blocked in
4% blotto or 10 mg/ml casein) in PBS and washed with PBS with 0.05%
Brij 35. The strips are then stained using a conventional
immunostaining procedure.
[0123] At the end of the immunostaining procedure (but before the
addition of the calorimetric substrate), the three sections of the
control strip are separated from each other with a razor blade and
placed into microtitre wells. A soluble peroxidase substrate is
added to each well (o-phenylenedihydrochloride) with 0.3%
H.sub.2O.sub.2. After the color develops, the strips are removed
from the wells with a forceps. Color development is measured
spectrophotometrically with a microtitre plate spectrophotometer. A
signal to noise ratio is then calculated. The first portion of the
strip (relevant peptide) represents the signal. The other two
portions are both negative controls. A S/N ratio greater than 10
should be obtained for acceptable performance.
[0124] (b) Testing for Non-specific Binding of Peptides to Other
Antibodies.
[0125] Before settling upon a specific candidate peptide for use as
an antigen mimic, it is important to verify that it binds only to
the desired antibody. Therefore, it is desirable to test the
peptide against an array of 5-10 different primary antibodies. The
experimental method is nearly identical to that described above,
for determining the signal to noise ratio. Namely, replicate spots
of the peptide sequence are coupled onto a matrix (by methods
previously described). The spots are separated from each other and
placed into microtiter wells, as previously described. Different
primary antibodies, each at the appropriate working concentration
(usually 1-10 micrograms/ml), are added to the wells. In this
manner, the same peptide target is tested against a variety of
different primary antibodies. After incubation and removal of the
primary antibodies, the remainder of the detection procedure is
carried out. An antigen-specific mimic causes a colormetric
reaction to occur following incubation with the specific antibody
but not by other non-specific primary antibodies.
[0126] Table 2 shows representative data obtained during testing of
the ER peptide mimic. The peptide was detected using the 1D5
anti-ER antibody (as previously described). In addition, the
indicated other primary antibodies were also tested for
immunoreactivity to the ER peptide mimic. The color intensity was
scored on a semi-quantitative 1-4 scale, 4 representing intense
staining. TABLE-US-00003 TABLE 2 Immunoreactivity of monoclonal
antibodies to ER peptide mimic Staining intensity Antibody (1-4+)
1D5 (estrogen receptor) 4 PR636 (progesterone receptor) 0 PD7/26 +
2B11 (CD45) 0 AE1/AE3 (cytokeratin) 0 Normal mouse IgG 0 MIB-1
(Ki-67 antigen) 0
[0127] (c) Validating Sensitivity of the Control Strips to
Detecting Early Reagent Failure.
[0128] For optimal performance, the range of concentrations of
antigen or peptide mimic (coupled onto the matrix) should be such
that it will be most useful in detecting early decrements in
reagent performance. This is in contradistinction to standard
tissue controls in that the amount of an analyte in a tissue
control can not be varied at will. The control strip is most
sensitive to detecting early reagent failure when the amount of
antigen or antigen mimic on the strip brackets the threshold of
detection.
[0129] To test the control strips, reagent failure is simulated in
a typical immunohistochemical staining procedure. This is
accomplished by serially diluting the primary antibody. The limit
of sensitivity is measured by visually noting the lowest peptide
concentration that will yield a 1+ calorimetric signal. This type
of visual quantitation is similar to that used for hemagglutination
reactions. The intensity of the reaction (1-4+) is graphed against
the concentration of the primary antibody. The goal is to identify
the smallest increment in primary antibody dilution that can be
detected using the control strips. The sensitivity of the controls
strips is quantified as the smallest increment (change in
concentration) in primary antibody dilution that can be reliably
detected. (See, for example, Table 3). TABLE-US-00004 TABLE 3
Primary antibody concentration Colorimetric intensity 40 mcg/ml 4+
20 mcg/ml 4+ 10 mcg/ml 4+ 5 mcg/ml 3+ 2.5 mcg/ml 2+ 1.0 mcg/ml 1+
0.6 mcg/ml 0 (negative result)
[0130] In this example, the control strip is able to detect a
two-fold dilution of primary antibody between 1 and 10 mcg/ml of
primary antibody.
[0131] The signal intensity on the control strips can also be
monitored quantitatively using an image analysis software program,
as described for FIG. 4. Such a quantitative analysis was performed
and the data are shown in. FIG. 5. FIG. 5 summarizes the data from
a representative experiment of reagent failure, testing the primary
and secondary antibody reagents. FIG. 5A illustrates the dot
density (y axis) on a series of spots (x axis) containing varying
concentrations of ER peptide (to be described later). The control
strips are stained with three different dilutions of primary
antibodies in FIG. 5A. FIG. 5B illustrates the signal intensity
using three different dilutions of the secondary antibody. In each
case, decreasing the antibody concentration (increasing dilution)
results in a diminished signal, providing a visual cue that the
antibody reagent is less potent. Moreover, the endpoint of
detection is also sometimes affected. For example, if the cutoff
for positivity (a 1+ visual determination) is a 20,000 dot signal
density, then the endpoint of detection in FIG. 5B, 1:300 dilution
is 1 micromolar peptide concentration. Using the same standard, the
1:1200 dilution of antibody falls below this point at the 2
micromolar concentration. The 1:4800 concentration never attains
even this level of signal intensity (dot density). According to
this method, early reagent failure can be monitored by the downward
deviation of the spots' color intensity.
EXAMPLE 4
[0132] Identification of Peptide Mimic of Estrogen Receptor
(ER)
[0133] Monoclonal antibody clone 1D5 is a widely used antibody that
is used for identification of ER in tissue sections by
immunohistochemistry. Antibody 1D5 binding peptides (ER epitope
mimics) were obtained by screening the cyclic peptide library that
is displayed by M13 filamentous phage. The phage library was
purchased from Dyax Corp., Cambridge, Mass. FIG. 6 shows the
peptides (SEQ ID NOS: 2-29) and consensus sequences (SEQ ID NOS:
32-42; the underlined amino acids of SEQ ID NOS: 2-29 shown in FIG.
6 are referred to herein as SEQ ID NOS: 32-42, respectively) that
specifically binds to ER monoclonal antibody 1D5. These ER peptides
mimic the three-dimensional structure (conformational epitope) of
ER that is recognized by the 1D5 monoclonal antibody. However,
other peptides e.g., about 10-20 amino acids long, that comprise
(contain) these consensus sequences, SEQ ID NOS: 32-42, be used in
the described assays.
[0134] Appropriate alterations of the peptides described herein,
which retain the peptides's functionality will be clear to a person
of ordinary skill in the art. Such alterations include, for
example, conservative substitution of amino acid residues, e.g.,
replacement of a hydrophobic amino acid with a different
hydrophobic amino acid, e.g., glycine, valine, leucine or
isoleucine. Similarity, negatively charged residues may be
replaced, e.g., aspartic acid for glutamic acid, or vice versa. In
the case of positively charged residues, arginine may be replaced
with leucine, or vice versa. Such alterations do not change the
biological activity of the peptide, e.g., the ability of a peptide
to bind specific antibody, or the usefulness of the peptide as a
quality control moiety in the assays described herein.
[0135] The synthetic ER peptide sequences that were deduced from
the selected peptide phage library were further labeled with
fluorescein tag for quantitative and qualitative evaluative
purposes.
[0136] Screening for a Peptide Mimic for Anti-ER Antibody.
[0137] Two cyclic libraries (TN6 and TN10, purchased from Dyax
Corp.) were used to screen for phage that bound specifically to the
ID5 mouse monoclonal antibody (ER-specific antibody). The screening
procedure is the same as one described in earlier sections. After
two rounds of positive selection, individual phage clones were
amplified and used in a phage ELISA. The DNA of representative
phage clones (that had the highest ELISA O.D. readings i.e., about
80-90% of the clones in our screen) were sequenced. As shown in
FIG. 6, clones from each library had a consensus sequence.
[0138] Three synthetic peptides (designated as ER peptide 3, ER
peptide 4, and ER peptide 6), that are representative of above
consensus phage sequence, were synthesized (by SynPep Labs, Dublin,
Calif. and Bachem Labs, King of Prussia, Pa.) and tested for
specificity/affinity to 1D5 monoclonal antibody. Peptides 3 and 6
are shown in Table 4. In a peptide ELISA, ER peptide 3 the highest
signal. In a fluorescence polarization assay, ER peptide 3
displayed a Kd of about 10.sup.-9 respectively. TABLE-US-00005
TABLE 4 Synthetic ER Peptide Mimics Peptide #3
Ac-DFQCPYVECVVNAPGGK(FITC)GK-CONH.sub.2 (SEQ ID NO:30) Peptide #6
Ac-HSHCQAPYLSMACLPPAGK(FITC)GK-CONH.sub.2 (SEQ ID NO:31)
[0139] Notes:
[0140] The "(FITC)" group represents a fluorescein group attached
to an epsilon side chain of lysine.
[0141] The carboxy termini of both peptides are comprised of a
lysine (K) whereby the carboxy terminus is modified to an amide
group.
[0142] 1. The amino termini of both peptides is acetylated.
[0143] 2. The consensus regions of both peptides is underlined.
EXAMPLE 5
[0144] Attachment of Peptide Antigen Quality Control Moieties
Directly to Glass
[0145] In an alternative embodiment of the present invention,
antigen is immobilized directly onto the surface of a microscope
slide. This is accomplished by placing a small spot of soluble
antigen onto one or more spatially discrete regions of the glass
surface. In order for the antigen to bind to the glass, the glass
is chemically derivatized ("activated") so as to be capable of
binding to macromolecules. The front surface of the glass slide
(typically the side with a frosted-end on one side of the
microscope slide) is referred to herein as the reagent surface.
This is the same surface to which a matrix with an adhesive backing
would have been attached in the previous embodiment. The reagent
surface has one, or more, quality control moieties immobilized to
it. The biological sample (e.g., tissue section) to be tested is
also mounted to the reagent surface of the slide, preferably
adjacent to the control moieties. By being placed adjacent to each
other, the tissue section and the quality control moieties are
treated with the same reagents, at the same temperature and for the
same period of time.
[0146] The clinical uses of this alternative embodiment (quality
control moieties attached directly to glass slides) are similar to
those already described for the previous embodiment (quality
control moieties attached to a matrix with an adhesive backing).
These uses include detection of early reagent failure, verification
of proper assay performance, determination of assay sensitivity,
and quantitation of an analyte in a tissue section or cell
sample.
[0147] There are advantages of attaching the quality control
moieties directly to glass slide (second embodiment) instead of a
matrix with an adhesive backing (first embodiment). First, it is
possible that immersion of the slide into xylene or alcohol will
cause damage to the adhesive backing. This can occur because many
adhesives are at least partially soluble in xylene or alcohol.
Xylene and alcohol is used for removing paraffin wax from tissue
sections and for dehydration before coverslipping. Placing the
quality control moieties directly onto the glass circumvents this
problem. Second, many IHC procedures require a pretreatment step,
often termed "antigen-retrieval" or "epitope retrieval", that
involves immersing the microscope slide in a boiling water
temperature aqueous buffer. This boiling can also potentially
damage the matrix and adhesive. This problem is also circumvented
by attaching the quality control moieties directly to glass, since
glass is impervious to the effects of high temperature. Third, the
matrix and adhesive backing typically have a small but defined
thickness. This thickness causes the reagent surface of the
membrane to be at a slightly higher elevation than the glass slide.
In order for the reagents to contact the surface of the matrix,
there must be a sufficient amount of reagent so that the fluid
layer height is greater than the thickness of the matrix and
adhesive backing. Many of the assays are performed using only a few
drops of reagent. Such limiting volumes of reagent can sometimes be
insufficient to contact the matrix surface. Attaching the quality
control moieties directly to the glass overcomes this problem as
well, since no physical height barrier separates the tissue section
and the quality control moieties.
[0148] The Chemistry of Covalently Coupling Macromolecules to a
Class Surface.
[0149] It is desirable to create a covalent linkage between the
silica (glass) surface and the macromolecule serving as a quality
control reagent. A covalent linkage is likely to be more stable
over a long period of time. Moreover, it is more likely to
withstand treatment with solvents and elevated temperatures.
Covalent coupling to glass is often accomplished by use of silanes.
The silanes of interest are trialkoxy(triiminoxy) silanes and
dialkoxy(diiminoxy) silanes. The iminoxy silanes are also called
oximinosilanes. The rest of the valency of silicon is substituted
with functionalized alkyl or aryl groups. The dialkoxy (diiminoxy)
or the trialkoxy (triiminoxy) groups of the silane are replaced
with the Si--OH groups of the glass surface, resulting in the
anchoring of the new functionalized silanes. Functionalized
trialkoxy (triiminoxy) silanes are preferred since they can
crosslink using their three arms and adhere to the glass surface.
In order to attach macromolecules, we placed a reactive group on
the free end of these functionalized silanes. The glass surface
before any chemical reaction is represented in FIG. 7. Free
hydroxyl groups are attached to silicon atoms that form the glass
surface.
[0150] The first step in chemically activating glass slides is to
clean them. Slides can be cleaned by one of several alternative
methods: incubation in acid (e.g., 2N HCl or 2N H.sub.2SO.sub.4),
or incubation in a base (e.g., 10% NaOH or 10% KOH), or incubation
in a cleaning solution (10% Aquet in water, VWR Scientific) for
10-15 minutes. Glass slides were further rinsed by dipping ten
times in distilled water and then dried for 2-5 minutes in a 60
degrees C. oven. Slides treated in this fashion are referred to
herein as "cleaned" slides.
[0151] In a one-step method of activating the glass surface (for
attachment to macromolecules), an isocyanate end-capped trialkyl
siloxane (e.g., 3-isocyanato propyl triethoxysilane) is incubated
with the glass slide. This molecule has both a siloxane attachment
site (for attachment to glass) and an isocyanate group (for
attachment to a macromolecule). A chemical representation of the
isocyanate end-capped trialkoxylsilane-derivatized glass surface is
shown in FIG. 8. The free isocyanate groups are highly reactive and
serve to immobilize macromolecules to the glass surface.
[0152] To perform the one step activation of glass slides,
3-isocyanato propyl triethoxysilane is dissolved in acetone,
toluene, or ethanol at a concentration of 2-10%. The cleaned slides
are then immersed in the 3-Isocyanato propyl triethoxysilane
solution for 30-60 minutes with occasional stirring. The slides are
incubated at 37-45 degrees C. which functions well to activate the
glass. The coated glass slides are then dipped three times in
acetone and dried in a 60 degrees C. oven for 5-10 minutes. A
potential drawback to this method is that the free isocyanate group
can compete with the alkoxy group of the silane for attachment to
the glass. If this occurs, the isocyanate group is not available
for attachment to the macromolecule, since it is bound to the glass
surface. This can ultimately lead to poor incorporation of the
macromolecule onto the glass surface. Therefore, a two step
sequence for incorporating the isocyanate groups on to glass
surface is also described herein.
[0153] In the two step method of chemically activating glass
slides, the glass surface is first derivatized to form an amino
triethoxysilane, forming an amino end-capped glass surface. This
amino end-capped silane-derivatized glass surface is shown in FIG.
9. Cleaned and dried slides are coated with a commercially
available silane solution (e.g., 3-aminopropyltriethoxysilane or
N'-(2-aminoethyl)-3-aminopropyltriethoxysilane or cyanosilane
etc.,) at a 2-10% concentration (in any of the following solvents:
acetone, a 95:5 ratio of acetone-ethanol, toluene, di or
tri-chloromethane, ethanol, or methanol). The reaction of the glass
slides with the silane solution occurs at 37-45 degrees C. for 30
minutes, with occasional mixing. Silane-coated glass slides are
then dipped three times in acetone and dried in a 60 degrees C.
oven for 5-10 minutes.
[0154] The amino groups on the glass slide are then subsequently
converted to reactive isocyanate groups by one of two methods. In
the first method, the amino group can be reacted with a molecule
containing -diisocyanate. One cyanate group (of the diisocyanate)
reacts with the amino end-cap to form a urethane bond. The other
isocyanate group is available for reacting with the quality control
macromolecule. The following diisocyanates will work for this
purpose: phenylene diisocyanate, 1,4-diisocyanato butane,
4,4'-diisocyanato dicyclohexyl methane (See e.g., FIG. 10). For
example, incubation with a 0.5-1% solution of 1,4 phenyl
diisocyanate, dissolved in acetone or toluene, for 30 minutes at
37-42 degrees C. will react with the amino groups on the glass. One
possible problem that can arise with a homobifunctional
cross-linker (such as a diisocyanate) is that both reactive
(isocyanate) groups react with the surface. Cross-linking
surface-bound amino groups (by the two arms of the diisocyanate) is
avoided by adding the diisocyanate in molar excess. The resulting
mono isocyanate end-capped glass surface is useful due to its
reactivity towards coating of biological molecules especially
proteins (or peptides), nucleic acids (DNA) and polysaccharides.
Two modes of chemical mechanism of attachment, via the isocyanate
group, are shown in FIGS. 11 and 12.
[0155] Instead of using a diisocyanate, the amino end-capped glass
surface can also be reacted with monomeric poly-isocyanates such as
triisocyanatobenzene or tetraisocyanatobenzene or their homologues
such as tetraisocyanato toluene. In this way, an even greater
number of active isocyanate groups are incorporated on to the glass
surface.
[0156] The previously described methods converted the amino
end-capped silane to a cyanate by using either a diisocyanate or a
poly-isocyanate. In an alternative method, the amino end-capped
silanes on glass can be converted to an isocyanate group by
treating the amino end-capped glass surface with phosgene
equivalents, like 1,1'-carbonyl diimidazole or oxime carbonates
(e.g., methyl ethyl ketone oxime carbonate). This latter
alternative (with phosgene equivalents) is preferred over the
former alternative (adding a diisocyanate) because of its higher
reactivity under milder conditions and avoidance of the possibility
of crosslinking the surface amino groups. In addition, the
materials are safer to handle, an advantage in manufacturing.
[0157] The isocyanate groups on the glass will readily react with
an amine (e.g., found in short peptides, proteins, and nucleic
acids), carboxyl group (e.g., found in short peptides and proteins)
or hydroxyl group (e.g., found in carbohydrates and sugars). Since
the isocyanate groups on activated slides are highly reactive, the
treated glass slides should be stored in a anhyhydrous environment.
Alternatively, the glass incorporated isocyanate groups can be
blocked with an oxime such as methyl ethyl ketoneoxime (MEKO). The
labile MEKO-blocked isocyanates lose MEKO to reliberate activated
isocyanates in the presence of amine-bearing peptides to form
stable urethanes at room temperature. Alternatively, MEKO can be
removed at elevated temperatures (80-120 degrees C.), liberating
the glass-attached free isocyanate groups.
[0158] These glass activation chemistries also provide for the
possibility of creating "blocked" isocyanate groups on the glass
slides. This was previously briefly mentioned with respect to
methyl ethyl ketoneoxime (MEKO). If the isocyanate groups are not
blocked, then the isocyanate group is so reactive that it can
potentially react with water. Over time, even the humidity in the
air can react, degrading the reactivity of the isocyanate groups.
To maximize the stability of slides with unblocked reactive
isocyanate groups, it will be ideal to store such slides in a
hygroscopic environment. The advantage of the blocked reactive
group is that it will provide for a longer shelf life. The blocked
isocyanate groups that we will describe are unreactive with water
at room temperature but will react with groups such as amines, as
would be found in proteins, carbohydrates, or nucleic acids.
[0159] Three blocking methods are described. In all three methods,
the glass is first reacted with 3-aminopropyltriethoxysilane, as
previously described. The triethoxysilane functionality reacts with
the silicon atoms of the glass, leaving a free primary amine group
covalently attached to the glass. In the first method, the
amine-derivatized glass is then reacted with
1,1-carbonyldiimidazole (10%) at 40 degrees C. for 30 minutes. In
the upper half of FIG. 13, the derivatized glass surface is shown,
bearing a free amino group. The bottom half of FIG. 13 shows the
structure of the product after the reaction with the
carbonyldiimidazole. The 1,1-carbonyldiimidazole reacts with the
amino group on the glass, forming a mildly stable intermediate
shown in FIG. 13. The imidazole-protected isocyanate is mildly
stable at room temperature. Namely, the attached imidazole group
will not leave in the presence of water (unless the temperature is
raised above 60 degrees C.). However, the protecting group will
leave in the presence of an amine. Thus, the glass surface is
reactive towards biologically-relevant macromolecules but does not
have the problem of instability to water, such as humidity. After
reacting with an amine, the final product appears as in FIG. 11 or
12.
[0160] A second method forming a protected isocyanate group is to
modify the glass-bound amine group with MEKO Carbonate or oxime
carbonate. The upper half of FIG. 14 shows a glass-bound amine (on
the end of aminopropylsilane) reacting with MEKO Carbonate. The
lower half of FIG. 14 shows the oxime-blocked isocyanate group.
This oxime-blocked isocyanate group is reactive towards amines, as
described previously for the imidazole-blocked isocyanate, but
towards water at room temperature. Upon contact with an amine
group, the oxime group is displaced to form a urethane linkage. In
this manner, the activated surface is unreactive towards water but
reactive with a biologically relevant macromolecule, such as a
protein or nucleic acid. After reacting with an amine, the final
product appears as in FIG. 11 or 12.
[0161] Coupling the macromolecule that serves as the quality
control reagent is accomplished by incubating a small aliquot
(approximately 1 microliter) of the macromolecule dissolved in an
appropriate coupling buffer (e.g., 0.1 M potassium phosphate
buffer, pH 7.5) on the activated glass surface for 5-30 minutes at
room temperature or 37 degrees C. Typically, small spots containing
the macromolecule are placed onto the surface of the glass. The use
of multiple discrete spots positioned on the glass slide is similar
to that already described for conjugation to matrices such as
membranes. The spotting of the antigen could be done either by
manually, with a pipette, or with a commercially available liquid
dispenser.
[0162] Alternatively (instead of dots), the various peptide/protein
controls can be marked as letters indicating the test antigen. For
example, the progesterone receptor control spots can be
calligraphed as "PR" in place of dots.
[0163] After the coupling procedure, the remaining reactive sites
on the chemically activated glass slide must be "quenched" (or
"capped"), blocking any future potential reactivity. Reactive sites
could otherwise directly attach to components of the assay, such as
antibodies, causing background staining. It is possible to leave
the slide unquenched until after the tissue has been mounted onto
the glass. In this manner, the isocyanate groups can bind to amino
or carboxyl groups on tissue proteins. This may help anchor the
tissue to the glass slide. After the tissue is mounted on the
glass, the remaining unreacted isocyanate groups can then be
quenched.
[0164] The glass surface is quenched with any of a variety of small
molecules that have free amino groups. Suitable quenching agents
include ethanolamine, ethylenediamine, aminoethanediol,
aminopropanesulfonate and dithiothreitol. Alternatively, proteins
such as gelatin or casein or amino acids such as glycine, can
quench the active glass surface. A recommended quenching procedure
is to cover the glass slide with 10% v/v monoethanolamine in 1.0 M
Sodium Bicarbonate buffer, pH 9.5. The glass slide is incubated
with the quenching solution for 5-30 minutes at room temperature or
37 degrees C. The quenching solution is then drained and the glass
slide is rinsed with 0.01 M Sodium Phosphate, 0.14 M NaCl, pH 7.4,
0.1% v/v Tween 20 (PBS-Tween).
[0165] Equivalents
[0166] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims.
Sequence CWU 0
0
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 42 <210>
SEQ ID NO 1 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 1 Pro Ala
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly 1 5 10 15
Ser Thr Ala Pro 20 <210> SEQ ID NO 2 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 Ser Asp Trp Ala Cys Asp Gln Glu Pro Phe Phe
Thr Leu Cys Ser Tyr 1 5 10 15 His Ala <210> SEQ ID NO 3
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 3 Ser His Leu His Cys Gln Ala
Pro Tyr His Asn Glu Gly Cys His His 1 5 10 15 Phe Ala <210>
SEQ ID NO 4 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 Ser His
Ser His Cys Gln Ala Pro Tyr Leu Ser Met Ala Cys Leu Pro 1 5 10 15
Pro Ala <210> SEQ ID NO 5 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
5 Ser His His Ser Cys Gln Ala Pro Phe Tyr Asp Arg Asp Cys Arg Asn 1
5 10 15 Asn Ala <210> SEQ ID NO 6 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 6 Ser His Asp Phe Cys Gln Ala Pro Trp Phe Asp
Glu Asn Cys Asn Ser 1 5 10 15 Asn Ala <210> SEQ ID NO 7
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 7 Ser Asn His Asn Cys Asp Gln
Ser Pro Tyr Tyr Leu Ala Cys Val Asn 1 5 10 15 Pro Ala <210>
SEQ ID NO 8 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 8 Ser Ser
Leu Asn Cys His Gln Ser Pro Tyr Leu Ser Tyr Cys His Tyr 1 5 10 15
Pro Ala <210> SEQ ID NO 9 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
9 Ser Tyr Phe Asp Cys Gln Gln Ser Tyr Tyr Leu Pro Asn Cys Phe Asn 1
5 10 15 Asn Ala <210> SEQ ID NO 10 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 10 Ser His Ser His Cys Gly Ser Gln Ala Pro
Tyr Tyr Met Cys Ser Asp 1 5 10 15 His Ala <210> SEQ ID NO 11
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 11 Ser His Pro Phe Cys Asp Ser
Asn Gln Thr Pro Tyr Tyr Cys Phe Asn 1 5 10 15 Asn Ala <210>
SEQ ID NO 12 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 12 Ser His
Asp Leu Cys Thr His Asn Gln Val Pro Tyr Phe Cys Asp Asn 1 5 10 15
Asn Ala <210> SEQ ID NO 13 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
13 Ser Leu Ser Asp Cys Asp Lys Phe Gln Ala Pro Tyr Val Cys Ala Phe
1 5 10 15 Asn Ala <210> SEQ ID NO 14 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 14 Ser His Asp Ser Cys Ala Phe Asn Gln Ser
Pro Tyr Phe Cys Asp His 1 5 10 15 Asn Ala <210> SEQ ID NO 15
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 15 Ser Asn His His Cys Met Asn
Phe Gln Gln Pro Val Tyr Cys Asn Asn 1 5 10 15 Tyr Ala <210>
SEQ ID NO 16 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 16 Ser His
Leu Asp Cys Tyr His Tyr Ser Gln Ala Pro Tyr Cys Gln Ser 1 5 10 15
Tyr Ala <210> SEQ ID NO 17 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
17 Ser Asn Asp Asp Cys Tyr Val Asp Asn Gln His Pro Tyr Cys His Leu
1 5 10 15 Leu Ala <210> SEQ ID NO 18 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 18 Thr Gly Ser Asp Lys Gln Cys Pro Val Ile
Asp Cys Met Glu Tyr Ala 1 5 10 15 Pro Gly <210> SEQ ID NO 19
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 19
Thr Gly Ser Ser Trp Gln Cys Pro Phe Trp Asp Cys Gly Asp Ser Ala 1 5
10 15 Pro Gly <210> SEQ ID NO 20 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: VARIANT <222>
LOCATION: (1)...(18) <223> OTHER INFORMATION: Xaa = Any Amino
Acid <400> SEQUENCE: 20 Thr Gly Ser Xaa Met Gln Cys Pro Val
Leu Asn Cys Ser Gly Asp Ala 1 5 10 15 Pro Gly <210> SEQ ID NO
21 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 21 Thr Gly Ser Ala Gln
Gln Cys Pro Val Lys Asn Cys Gly Ile Asn Ala 1 5 10 15 Pro Gly
<210> SEQ ID NO 22 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 22 Thr
Gly Ser Ser His Gln Cys Pro Ala Leu Ser Cys Ala Val Ser Ala 1 5 10
15 Pro Gly <210> SEQ ID NO 23 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 23 Thr Gly Ser Leu Ile Gln Cys Pro Ala Phe
Phe Cys Asp Asn Ala Ala 1 5 10 15 Pro Gly <210> SEQ ID NO 24
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 24 Thr Gly Ser Asp Phe Gln Cys
Pro Tyr Val Glu Cys Val Val Asn Ala 1 5 10 15 Pro Gly <210>
SEQ ID NO 25 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 25 Thr Gly
Ser Val Ser Gln Cys Pro Tyr Trp Glu Cys Asp Asp Tyr Ala 1 5 10 15
Pro Gly <210> SEQ ID NO 26 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
26 Thr Gly Ser Phe Trp Gln Cys Pro Phe Phe Gly Cys Asp Asn Phe Ala
1 5 10 15 Pro Gly <210> SEQ ID NO 27 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 27 Thr Gly Pro Phe Glu Leu Cys Lys Glu Asn
Asp Cys Gln Ala Pro Ala 1 5 10 15 Pro Gly <210> SEQ ID NO 28
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 28 Thr Gly Ser Tyr Gln His Cys
Pro Tyr Tyr Asp Cys Asp Val Asp Ala 1 5 10 15 Pro Gly <210>
SEQ ID NO 29 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 29 Thr Gly
Ser Asn Gln His Cys Pro Ala Tyr Ala Cys Gln Lys Pro Ala 1 5 10 15
Pro Gly <210> SEQ ID NO 30 <211> LENGTH: 19 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Peptide Mimic
<400> SEQUENCE: 30 Asp Phe Gln Cys Pro Tyr Val Glu Cys Val
Val Asn Ala Pro Gly Gly 1 5 10 15 Lys Gly Lys <210> SEQ ID NO
31 <211> LENGTH: 21 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Peptide Mimic <400> SEQUENCE: 31
His Ser His Cys Gln Ala Pro Tyr Leu Ser Met Ala Cys Leu Pro Pro 1 5
10 15 Ala Gly Lys Gly Lys 20 <210> SEQ ID NO 32 <211>
LENGTH: 3 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 32 Gln Glu Pro 1 <210> SEQ ID NO 33
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 33 Gln Ala Pro Tyr 1 <210>
SEQ ID NO 34 <211> LENGTH: 3 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 34 Gln Ala
Pro 1 <210> SEQ ID NO 35 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
35 Gln Ser Pro Tyr 1 SEQ ID NO 36 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
36 Gln Ser Tyr Tyr 1 SEQ ID NO 37 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
37 Gln Thr Pro Tyr 1 SEQ ID NO 38 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
38 Gln Val Pro Tyr 1 SEQ ID NO 39 <211> LENGTH: 5 <212>
TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 39 Gln Gln
Pro Val Tyr 1 5 SEQ ID NO 40 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
40 Gln His Pro Tyr 1 SEQ ID NO 41 <211> LENGTH: 3 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
41 Gln Cys Pro 1 SEQ ID NO 42 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
42 Gln His Cys Pro 1
* * * * *