U.S. patent application number 12/031625 was filed with the patent office on 2008-08-21 for device, array, and methods for disease detection and analysis.
Invention is credited to Matthew A. Coleman, Stephen M. Lane, Dennis L. Matthews, Rupa S. Rao.
Application Number | 20080200342 12/031625 |
Document ID | / |
Family ID | 39707197 |
Filed Date | 2008-08-21 |
United States Patent
Application |
20080200342 |
Kind Code |
A1 |
Rao; Rupa S. ; et
al. |
August 21, 2008 |
Device, Array, And Methods For Disease Detection And Analysis
Abstract
A device and array coupled to capture molecules are provided.
Specifically, the device and array can be used for detecting the
presence and concentration of biomarkers in a sample from a
subject. The device and array can also allow the use of a method
for scoring a sample for, e.g., the purpose of diagnosing a
disease. The method can also be advantageous to applications where
there is a need to accurately determine the disease stage of a
subject for the purpose of making therapeutic decisions.
Inventors: |
Rao; Rupa S.; (Stockton,
CA) ; Lane; Stephen M.; (Oakland, CA) ;
Matthews; Dennis L.; (Moss Beach, CA) ; Coleman;
Matthew A.; (Oakland, CA) |
Correspondence
Address: |
LLNL/FENWICK;JOHN H. LEE, ASSISTANT LABORATORY COUNSEL
LAWRENCE LIVERMORE NATIONAL LABORATORY, L-703, P.O. BOX 808
LIVERMORE
CA
94551
US
|
Family ID: |
39707197 |
Appl. No.: |
12/031625 |
Filed: |
February 14, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60902147 |
Feb 15, 2007 |
|
|
|
Current U.S.
Class: |
506/9 ; 506/39;
506/7 |
Current CPC
Class: |
B01J 2219/00385
20130101; B01L 3/50273 20130101; B01L 3/5025 20130101; B01J
2219/00725 20130101; B01J 19/0046 20130101; B01L 3/5027 20130101;
C40B 60/12 20130101; B01J 2219/00621 20130101; B01J 2219/00612
20130101; G01N 33/543 20130101; B01J 2219/00626 20130101; B01J
2219/00549 20130101; B01J 2219/00637 20130101 |
Class at
Publication: |
506/9 ; 506/39;
506/7 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 60/12 20060101 C40B060/12; C40B 30/00 20060101
C40B030/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The United States Government has rights in this invention
pursuant to Contract No. DE-AC52-07NA27344 between the United
States Department of Energy and Lawrence Livermore National
Security, LLC for the operation of Lawrence Livermore National
Laboratory.
Claims
1. A detection device, comprising: a solid support comprising a
plurality of distinct capture molecule groups, each distinct
capture molecule group comprising a plurality of capture molecules
specific for a biomarker, wherein the plurality of distinct capture
molecule groups is specific for a plurality of biomarkers; a cover
plate, wherein the cover plate forms an upper surface positioned
above the solid support; a vertical support, wherein the vertical
support forms a connection between the solid support and the cover
plate, the connection forming at least one channel around the
capture molecule groups, and wherein the channel comprises a first
end and a second end and wherein the first end of the channel
comprises an opening; and an absorbent material connected to the
second end.
2. The device of claim 1, wherein the solid support comprises
glass.
3. The device of claim 1, wherein the solid support comprises a
glass slide.
4. The device of claim 1, wherein the capture molecules comprise
antibodies.
5. The device of claim 1, wherein the capture molecules are
specific for biomarkers selected from the group consisting of:
Her-2, MMP-2, CA 15-3, VEGF, and OPN.
6. The device of claim 1, wherein the capture molecules are
specific for biomarkers selected from the group consisting of:
Her-2, MMP-2, CA 15-3, VEGF, OPN, p53, CA 125, and SER.
7. The device of claim 1, wherein the capture molecules are
specific for Her-2, MMP-2, CA 15-3, and OPN.
8. The device of claim 1, wherein the capture molecules are Clone
191924, Clone 36006.211, Clone M8071022, and Clone 190312.
9. The device of claim 1, wherein the capture molecules are blocked
by a blocking agent.
10. The device of claim 1, wherein the plurality of distinct
capture molecule groups of capture molecules is arranged in an
array format.
11. The device of claim 1, wherein the solid support comprises at
least two capture molecule groups, the at least two capture
molecule groups comprising identical capture molecules, and each of
the at least two capture molecule groups comprising a different
number of capture molecules.
12. The device of claim 1, wherein the cover plate comprises
glass.
13. The device of claim 1, wherein the cover plate comprises a
glass cover slip.
14. The device of claim 1, wherein the vertical support comprises
adhesive silicone.
15. The device of claim 1, wherein the absorbent material comprises
a Hi-Flow Plus Nitrocellulose Membrane HF240.
16. The device of claim 1, comprising a glass slide comprising an
array of a plurality of distinct groups of antibodies cross-linked
to the slide, each distinct group specific for a biomarker selected
from the group consisting of Her-2, MMP-2, CA 15-3, and OPN; a
glass cover slip positioned above the solid support; and a silicone
adhesive connection between the glass slide and the glass cover
slip forming at least one channel around the antibody groups, and
wherein the channel comprises a first open end and a second end
connected to a Hi-Flow Plus Nitrocellulose Membrane HF240.
17. The device of claim 1, further comprising a component for
detecting biomarkers bound to the solid support.
18. The device of claim 17, wherein said component comprises an
optical reader and a screen for displaying output from the optical
reader.
19. A method for determining the presence or absence of a plurality
of biomarkers in a sample, comprising: acquiring a liquid mixture,
wherein the mixture comprises the sample; applying the mixture to
the open first end of the at least one channel of the device of
claim 1; allowing the mixture to flow through the at least one
channel over the solid support; absorbing the mixture with the
absorbent material connected to the second end; and detecting the
presence of biomarkers on the solid support, wherein presence of
the biomarkers on the solid support indicates the presence of the
biomarkers in the sample.
20. The method of claim 19, wherein the device of claim 1 comprises
capture molecules comprising antibodies and the liquid mixture
comprises the sample, at least one detector antibody, and at least
one fluorescent reporter, and further comprising the steps of
analyzing the sample with an optical reader to determine the
presence or absence of the plurality of biomarkers in the sample;
and outputting the data, wherein the data comprise the presence or
absence of the plurality of biomarkers in the sample.
21. The method of claim 19, wherein the device of claim 1 comprises
capture molecules specific for a plurality of biomarkers selected
from the group consisting of CA 15-3, OPN, Her-2, and MMP-2.
22. The method of claim 19, wherein the sample comprises human
blood serum.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/902,147, filed Feb. 15, 2007, the entire
disclosure of which is hereby incorporated by reference in its
entirety for all purposes.
BACKGROUND
[0003] 1. Field of the Invention
[0004] The invention relates to the fields of biology and
chemistry.
[0005] 2. Description of the Related Art
[0006] Research over the past decade has focused on discovering new
biomarkers that provide accurate diagnosis of disease, guide
therapeutic decision making, and predict the future patterns of
disease. Cancer antigen (CA)-125 and Carcinoembryonic antigen (CEA)
have both shown promise as biomarkers for ovarian and colorectal
cancer, respectively (Fields M M, C. E. Ovarian cancer screening: a
look at the evidence. Clin J Oncol Nurs. 10, 77-81 (2006);
Hasholzner U, S. P., Reiter W, Zimmermann A, Hofmann K, Schalhorn
A. CA 242 in comparison with established tumour markers in
colorectal, pancreatic and lung cancer. Anticancer Res. 19,
2477-2480 (1999)). However, while these biomarkers have shown some
potential for possible specific detection of ovarian and colon
cancers, no single marker has yet been identified for breast
cancer. This can be due to the fact that breast cancer is not a
single disease, but a genetically heterogeneous set of diseases,
thus suggesting that it can not be possible for breast cancer to be
diagnosed with any single marker. The present invention addresses
this need by providing multiple, mutually complementary biomarkers
that provide a sensitive diagnostic assay for breast cancer.
[0007] Point of care (POC) devices and systems can process samples
for a number of different types of biomarkers in a variety of
settings, such as clinical laboratories, patients' bedside and
doctors' offices. Various forms of single biomarker POC
technologies are available including Lateral flow assays (LFA)
(Panteghini M, P. F. Characterization of a rapid
immunochromatographic assay for simultaneous detection of high
concentrations of myoglobin and CK-MB in whole blood. Clin Chem
Clin Biochem 42, 1292-1293. (1996); Millipore Corp A Short Guide
Developing Immuno-chromatographic Test Strips. (1996); Lou S C, P.
C., Ching S, Gordon J. One-step competitive immunochromatographic
assay for semiquantitative determination of lipoprotein(a) in
plasma. J. Clin Chem 39 (1993); Lee-Lewandrowski E, L. K. Selected
topics in point-of-care testing--Urinalysis, pregnancy testing,
microbiology, fecal occult blood, and other tests. Clin Lab Med 21,
389 (2001)), Disposable microchips (Pugia M J, B. G., Peters R P,
Profitt J A, Kadel K, Willms T, Sommer R, Kuo H H, Schulman L S.
Microfluidic tool box as technology platform for hand-held
diagnostics. Clin Chem. 51, 1923-1932 (2005)), the RAMP.TM.
platform (Donald E. Brooks, D. V. D., Paul C. Harris, Joanne E.
Harris, Mark E. Miller, Andrew D. Olal, Linda J. Spiller and Zongen
C. Xie RAMP: A Rapid, Quantitative Whole Blood
Immunochromatographic Platform for Point-of-Care Testing. Clinical
Chemistry 45, 1676-1678 (1999)), and the Dual Path Platform (DPP)
technology (Carlos Ponce, E. P., Elizabeth Vinelli, Alberto
Montoya, Vilma de Aguilar, Antonio Gonzalez, Bianca Zingales,
Rafael R. Aldao, Mariano J. Levin, Javan Esfandiari, Eufrosina S.
Umezawa, Alejandro O. Luquetti, and Jose Franco da Silveira.
Validation of a rapid reliable test for the diagnosis of Chagas'
disease in blood banks and medical emergencies in Central America.
The Journal of Clinical Microbiology, 5065-5068 (2005)). In
addition, multiplexed LFAs have also been developed (Jeong D S, C.
E. Simultaneous Quantitative Determination of Multiple Analytes
with Fluorescence-Tagged Probes by Immunochromatography. Korean J
Biol Sci 7, 89-92 (2003)).
[0008] Multiplexed LFAs, although sensitive and specific, require
elaborate imaging devices for sensitive quantification, thus
limiting application at the POC. Another form of multiplexed assay
are microfabricated flow channels which pass a sample over an
immobilized array (Delehanty J. B. Ligler F. S A microarray
immunoassay for simultaneous detection of proteins and bacteria.
Anal. Chem. 74, 5681-5687 (2002); C. R. Taitt, J. P. G., Y. S.
Shubin, L. C. Shriver-Lake, K. E. Sapsford3, A. Rasooly, F. S.
Ligler A Portable Array Biosensor for Detecting Multiple Analytes
in Complex Samples. Microbial Ecology 47, 175-185 (2004); Frances
S. Ligler, C. R. T., Lisa C. Shriver-Lake, Kim E, Sapsford, Yura
Shubin, Joel P. Golden Array biosensor for detection of toxins.
Anal Bioanal Chem 377, 469-477 (2003); Mark J. Feldstein, J. P. G.,
Chris A. Rowe, Brian D. MacCraith, Frances S. Ligler Array
Biosensor: Optical and Fluidics Systems. Journal of Biomedical
Microdevices 1, 138-153 (1999)). Use of these assays requires that
the sample, detection antibodies, and wash buffers be sequentially
introduced at one end of the chamber and drawn over the microarray
surface using a peristaltic pump. These assays demonstrate better
multiplexed sensitivities as compared to the LFAs, however, they
involve sequential detection along the length of the strip rather
than simultaneous detection, which limits the number of biomarkers
that can be simultaneously analyzed due to the number of capture
zones that can be created along the length of the strip. In
addition, the flow channels are made using polydimethylsiloxane
(PDMS) as the material which requires elaborate microfabrication
facilities to manufacture. Also, the fluid exchange through the
channels was achieved using a peristaltic pump, and the assay
involved multiple incubation and wash steps, making it challenging
to automate and reduce this device to a small, rugged portable
form.
[0009] The present invention addresses these problems by providing
a channel flow device that allows simple, rapid, and sensitive
detection of multiple biomarkers.
SUMMARY
[0010] Disclosed herein is a detection device. In one aspect, the
detection device includes a solid support including a plurality of
distinct capture molecule groups, each distinct capture molecule
group including a plurality of capture molecules specific for a
biomarker, wherein the plurality of distinct capture molecule
groups is specific for a plurality of biomarkers; a cover plate,
wherein the cover plate forms an upper surface positioned above the
solid support; a vertical support, wherein the vertical support
forms a connection between the solid support and the cover plate,
the connection forming at least one channel around the capture
molecule groups, and wherein the channel includes a first end and a
second end and wherein the first end of the channel includes an
opening; and an absorbent material connected to the second end. In
another aspect of the detection device, the solid support includes
glass. In another aspect of the detection device the solid support
includes a glass slide.
[0011] In one embodiment, the capture molecules include antibodies.
In another embodiment, the capture molecules are specific for
biomarkers selected from Her-2, MMP-2, CA 15-3, VEGF, and OPN. In
another embodiment, the capture molecules are specific for Her-2,
MMP-2, CA 15-3, VEGF, OPN, p53, CA 125, and SER. In another
embodiment, the capture molecules are specific for Her-2, MMP-2, CA
15-3, and OPN. In another embodiment, the capture molecules are
Clone 191924, Clone 36006.211, Clone M8071022, and Clone 190312. In
another embodiment, the capture molecules are blocked by a blocking
agent. In another embodiment, the plurality of distinct groups of
capture molecules is arranged in an array format. In another
embodiment, the solid support includes at least two capture
molecule groups including identical capture molecules, and each of
the at least two capture molecule groups including a different
number of capture molecules.
[0012] In one embodiment, the cover plate includes glass. In
another embodiment, the cover plate is a glass cover slip. In one
embodiment, the vertical support includes adhesive silicone. In one
embodiment, the absorbent material includes a Hi-Flow Plus
Nitrocellulose Membrane HF240.
[0013] In another aspect, the detection device includes a glass
slide including an array of a plurality of distinct groups of
antibodies cross-linked to the slide, each distinct group specific
for a biomarker selected from Her-2, MMP-2, CA 15-3, and OPN; a
glass cover slip positioned above the solid support; and a silicone
adhesive connection between the glass slide and the glass cover
slip forming at least one channel around the antibody groups, and
wherein the channel includes a first open end and a second end
connected to a Hi-Flow Plus Nitrocellulose Membrane HF240.
[0014] In one embodiment, the detection device further includes a
component for detecting biomarkers bound to the solid support. In a
related embodiment, the component includes an optical reader and a
screen for displaying output from the optical reader.
[0015] Also disclosed herein is a method for determining the
presence or absence of a plurality of biomarkers in a sample,
including: acquiring a liquid mixture, wherein the mixture includes
the sample; applying the mixture to the open first end of the at
least one channel of the device, described above; allowing the
mixture to flow through the at least one channel over the solid
support; absorbing the mixture with the absorbent material
connected to the second end; and detecting the presence of
biomarkers on the solid support, wherein presence of the biomarkers
on the solid support indicates the presence of the biomarkers in
the sample.
[0016] In one embodiment of the method for determining the presence
or absence of a plurality of biomarkers in a sample, the detection
device described above, is used, the device includes capture
molecules including antibodies and the liquid mixture includes the
sample, at least one detector antibody, and at least one
fluorescent reporter, and the method further including the steps of
analyzing the sample with an optical reader to determine the
presence or absence of the plurality of biomarkers in the sample;
and outputting the data, wherein the data include the presence or
absence of the plurality of biomarkers in the sample. In another
embodiment of the method for determining the presence or absence of
a plurality of biomarkers in a sample, the detection device,
described above, includes capture molecules specific for a
plurality of biomarkers selected from the group consisting of CA
15-3, OPN, Her-2, and MMP-2. In another embodiment, the sample
includes human blood serum.
[0017] Also disclosed herein is an array of antibodies immobilized
on a solid support, the array including: a plurality of distinct
antibody groups, each distinct antibody group including a plurality
of antibodies specific for a biomarker, wherein the plurality of
distinct antibody groups is specific for a plurality of biomarkers,
and wherein the plurality of biomarkers include CA 15-3, OPN,
Her-2, and MMP-2.
[0018] Also disclosed herein is a method for determining protein
concentration data in a sample with an array, including: acquiring
a mixture, wherein the mixture is in a liquid state, and wherein
the mixture includes the sample from a mammalian subject, a
detector antibody, and a fluorescent reporter; applying the mixture
to the array described above; analyzing the sample with a reader to
determine the concentration of the plurality of biomarkers in the
sample; and outputting the data, wherein the data include protein
concentration data for the plurality of biomarkers, and wherein the
plurality of biomarkers include CA 15-3, OPN, Her-2, and MMP-2.
[0019] Also disclosed herein is a method of scoring a sample
acquired from a mammalian subject, including: obtaining a first
dataset including quantitative data associated with a plurality of
biomarkers associated with breast disease and the plurality of
biomarkers include CA 15-3, and OPN, wherein the data include
measured values obtained from the sample; analyzing the first
dataset against a second dataset to produce a score for the sample;
and outputting the score.
[0020] In one embodiment, the plurality of biomarkers includes
Her-2. In another embodiment, the plurality of biomarkers includes
MMP-2. In another embodiment, the plurality of biomarkers includes
Her-2 and MMP-2. In another embodiment, the plurality of biomarkers
includes Her-2, MMP-2, VEGF, p53, CA 125, and SER. In another
embodiment, the quantitative data includes protein concentrations.
In another embodiment, the data is immunoassay data. In another
embodiment, the protein concentrations are obtained using an
immunoassay including antibodies. In a related embodiment, the
immunoassay is a sandwich immunoassay. In another embodiment, the
protein concentrations are obtained using a multiplexed channel
flow-based device. In another embodiment, the antibodies of the
immunoassay are Clone 191924, Clone 36006.211, Clone M8071022,
Clone 190312, and Clone A183C-13G8.
[0021] In another embodiment, the analyzing step includes use of a
predictive model. In a related embodiment, the predictive model is
developed using principal component analysis. In another related
embodiment, the predictive model is developed using linear
discriminant analysis. In another embodiment, the analyzing step
includes categorizing the sample into categories according to a
score produced with the predictive model. In a related embodiment,
the categorization is selected from the group consisting of: a
healthy categorization, an early-stage disease categorization, and
a late-stage disease categorization. In another related embodiment,
a probability that the categorization is correct is at least 60%,
at least 70%, at least 80%, at least 87%, at least 90%, and at
least 95%.
[0022] In another embodiment, the method further includes comparing
the score to a second score determined for a second sample obtained
from the mammalian subject. In a related embodiment, wherein a
difference between the first score and the second score indicates a
disease stage of breast cancer. In another embodiment, wherein the
mammalian subject is a human subject. In another embodiment,
wherein the score is used to diagnose a neoplastic breast disease.
In another embodiment, wherein the breast disease is breast
cancer.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0023] These and other features, aspects, and advantages of the
present invention will become better understood with regard to the
following description, and accompanying drawings, where:
[0024] FIG. 1 is a schematic representation of the point of care
channel flow-based immunoassay detection device.
[0025] FIG. 2 is a schematic of the benchtop imaging system.
[0026] FIG. 3 shows testing for the specificity of the tested
antibodies using western blotting with five antigens.
[0027] FIG. 4 shows optimization of capture and detector antibody
concentrations for developing standard curves using enzyme linked
immunosorbent assay (ELISA).
[0028] FIG. 5 are standard curves using ELISA.
[0029] FIG. 6 is a schematic of the sandwich immunoassay format
used in protein microarrays.
[0030] FIG. 7 shows optimization of capture and detector antibody
concentrations for developing standard curves using the antibody
microarray immunoassay.
[0031] FIG. 8 is visualization of standard curves using the
antibody microarrays.
[0032] FIG. 9 is a comparison of standard curves obtained in a
single plex and a multiplex format. using the antibody microarrays,
with phosphate buffered saline (PBS) and Serum as the medium.
[0033] FIG. 10 is a comparison of standard curves obtained using
antibody microarrays with the ELISA.
[0034] FIG. 11 shows multiplexed assays on protein microarrays.
[0035] FIG. 12 shows the experimental layout of the breast cancer
sample pilot study.
[0036] FIG. 13 shows the pilot studies with 30 breast cancer
patient samples.
[0037] FIG. 14 shows the experimental layout of the breast cancer
study.
[0038] FIG. 15 shows the measurement of biomarkers in breast cancer
patient sera.
[0039] FIG. 16 is the standard curves for biomarkers in multiplex
format.
[0040] FIG. 17 shows clustering and linearization of the four
dimensional data obtained using the multiplex assay and principle
component analysis.
[0041] FIG. 18 shows classification error estimate using linear
discriminant analysis; H=Her-2; M=MMP-2; O=OPN; C=CA 15-3.
[0042] FIG. 19 is a schematic representation of a lateral flow test
strip.
[0043] FIG. 20 shows fluorescence images of the lateral flow assay
(LFA).
[0044] FIG. 21 shows fluorescence images of the sandwich LFA.
[0045] FIG. 22 shows the results of the proof of principle on
microarray channels.
[0046] FIG. 23 shows standard curves using Quantum Dots on
microarray channels.
[0047] FIG. 24 shows the multiplex assay using Quantum Dots on
microarray channels.
[0048] FIG. 25 shows the results of troubleshooting the Quantum Dot
assay on microarray channels.
[0049] FIG. 26 is the standard curves obtained using microarray
channels in 15 min.
[0050] FIG. 27 is a demonstration of multiplexed immunoassays on
microarray flow channels.
[0051] FIG. 28 is a determination of the best combination of assay
speed and sensitivity for the microarray flow channels.
[0052] FIG. 29 shows biomarker concentration in patient serum
samples.
[0053] FIG. 30 shows imaging system standard curves.
[0054] FIG. 31 is a comparison of the imaging system and a
photomultiplier tube (PMT).
DETAILED DESCRIPTION
[0055] Briefly, and as described in more detail below, described
herein is a channel flow based immunoassay detection device for
determining the presence and/or concentration of a plurality of
biomarkers in a sample. Also disclosed are methods of using the
device, and arrays of capture molecules, e.g., antibodies, for use
with the device. In one embodiment, the device is used to detect a
plurality of biomarkers related to breast cancer. Described herein
are methods of scoring a sample using data associated with the
breast cancer biomarkers.
[0056] Advantages of this approach are numerous. The device
provides the ability to perform multiplexed analysis of multiple
biomarkers in a format that is simple to use, amenable to
automation, and in a small, rugged format. The device has been
developed in a point of care (POC) format, allowing for rapid
diagnostic assay, and facilitating faster therapeutic decisions and
possible increased patient survival rates.
[0057] The device can be used to diagnose and prescribe treatment
for a wide variety of medical conditions, especially cancers, heart
diseases, respiratory diseases, and microbial infections. In one
embodiment, the device is used to diagnose breast cancer.
[0058] Also disclosed is a multiplexed immunoassay to detect a set
of biomarkers associated with breast cancer. The immunoassay can
accurately detect a panel of two, three, four, five, six, seven, or
eight biomarkers from the sera of breast cancer patients and
distinguish between control, early stage, and metastatic breast
cancer populations. The immunoassay was shown to predict the stage
of unknown sample. The assay can be used can be used along with
mammography for result validation and in between annual mammograms
to diagnose rapidly-growing tumors. The advantage of the multiplex
assay is the ability to determine the levels of these markers
simultaneously, thus reducing time, effort, overall volume of
reagent and patient sample. Such a panel can offer a complete range
of tests such as diagnosis, prognosis, treatment options and
treatment monitoring in a single assay, providing additional
information enabling rapid diagnosis and improved patient survival
rates.
DEFINITIONS
[0059] Terms used in the claims and specification are defined as
set forth below unless otherwise specified.
[0060] A "capture molecule" is a molecule that is immobilized on a
surface. The capture molecule generally, but not necessarily, binds
to a target or target molecule, e.g., a biomarker. The capture
molecule is typically an antibody, a peptide, or a protein. In the
case of a solid-phase immunoassay, the capture molecule is
immobilized on the surface of a solid support and is an antibody
specific to the target, an antigen or epitope, to be detected. The
capture molecule can be labeled, e.g., a fluorescently labeled
antibody or protein. The capture molecule can or can not be capable
of binding to just the target. Capture molecules can include e.g.,
RNA, DNA, peptides, antibodies, aptamers, and protein-based
aptamers. In one embodiment the capture molecule is an
antibody.
[0061] A "biomarker" is a molecule of interest that is to be
detected and/or analyzed, e.g., a peptide, or a protein. Typically
a biomarker is associated with a particular physical condition,
e.g., a disease or disease state, e.g., late stage breast
cancer.
[0062] A biomarker that "binds" to a capture molecule is a term
well understood in the art, and methods to determine such specific
or preferential binding are also well known in the art. A molecule
is said to exhibit "binding" if it reacts or associates more
frequently, more rapidly, with greater duration and/or with greater
affinity with a particular target than it does with alternative
substances. A capture molecule "binds" to a target if it attaches
with greater affinity, avidity, more readily, and/or with greater
duration than it attaches to other substances. For example, a
capture molecule that specifically or preferentially binds to a
target is an antibody that binds this target with greater affinity,
avidity, more readily, and/or with greater duration than it binds
to other substances. It is also understood by reading this
definition that, for example, a capture molecule that specifically
or preferentially binds to a first target may or may not
specifically or preferentially bind to a second target. As such,
"binding" does not necessarily require (although it can include)
exclusive binding. Generally, but not necessarily, reference to
"binding" means preferential binding. The concept of "binding" also
is understood by those of skill in the art to include the concept
of specificity. Specific binding can be biochemically characterized
as being saturable, and binding for specific binding sites can be
biochemically shown to be competed.
[0063] An "antibody" is an immunoglobulin molecule capable of
specific binding to a target, such as a carbohydrate,
polynucleotide, lipid, polypeptide, etc., through at least one
antigen recognition site, located in the variable region of the
immunoglobulin molecule.
[0064] The terms "polypeptide," "oligopeptide," "peptide," and
"protein" are used interchangeably herein to refer to polymers of
amino acids of any length. The polymer can be linear or branched,
it can comprise modified amino acids, and it can be interrupted by
non-amino acids. The terms also encompass an amino acid polymer
that has been modified naturally or by intervention; for example,
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component. Also included within the
definition are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other modifications known in the art.
[0065] An "array" format is a known or predetermined and ordered
spatial arrangement of one or more capture molecules on a solid
support. A "multiplexed array" format is an ordered spatial
arrangement of two or more capture molecules on a solid support. In
one embodiment, row and column arrangements are used due to the
relative simplicity in making and assessing such arrangements. The
spatial arrangement can, however, be essentially any form selected
by the user, and preferably, but need not be, in a pattern. Array
formats are characterized by the use of spatial location within the
array to identify the feature present at that location.
[0066] "Detect" refers to identifying the presence, absence and/or
amount of protein to be detected. Detection can be done visually or
using a device, e.g., a scanner and detector.
[0067] The term "mammal" as used herein includes both humans and
non-humans and include but is not limited to humans, non-human
primates, canines, felines, murines, bovines, equines, and
porcines.
[0068] "Solid support" refers to a material or group of materials
having a rigid or semi-rigid surface or surfaces. In some aspects,
at least one surface of the solid support will be substantially
flat, although in some aspects it can be desirable to physically
separate regions for different molecules with, for example, wells,
raised regions, pins, etched trenches, or the like.
[0069] To "analyze" includes determining a set of values associated
with a sample by measurement of constituent expression levels in
the sample and comparing the levels against constituent levels in a
sample or set of samples from the same subject or other
subject(s).
[0070] A "predictive model" is a mathematical construct developed
using an algorithm or algorithms for grouping sets of data to allow
discrimination of the grouped data. As will be apparent to one of
ordinary skill in the art, a predictive model can be developed
using e.g., principal component analysis (PCA), and linear
discriminant analysis (LDA).
[0071] A "score" is a value or set of values selected or used to
discriminate a subject's condition based on, for example, a
measured amount of sample constituent from the subject.
[0072] The term percent "identity," in the context of two or more
nucleic acid or polypeptide sequences, refers to two or more
sequences or subsequences that have a specified percentage of
nucleotides or amino acid residues that are the same, when compared
and aligned for maximum correspondence, as measured using one of
the sequence comparison algorithms described below (e.g., BLASTP
and BLASTN or other algorithms available to persons of skill) or by
visual inspection. Depending on the application, the percent
"identity" can exist over a region of the sequence being compared,
e.g., over a functional domain, or, alternatively, exist over the
full length of the two sequences to be compared.
[0073] For sequence comparison, typically one sequence acts as a
reference sequence to which test sequences are compared. When using
a sequence comparison algorithm, test and reference sequences are
input into a computer, subsequence coordinates are designated, if
necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0074] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally Ausubel et al., infra).
[0075] One example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in Altschul et al., J. Mol. Biol.
215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information, most conveniently at its website.
[0076] Abbreviations used include: human estrogen receptor-2
(Her-2), matrix metallopeptidase-2 (MMP-2), cancer antigen 15-3 (CA
15-3), osteopontin (OPN), tumor protein 53 (p53), vascular
endothelial growth factor (VEGF), cancer antigen 125 (CA 125),
Serum Estrogen Receptor (SER)
[0077] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Devices of the Invention
[0078] The invention provides a POC, multiplexed, channel
flow-based immunoassay detection device as shown in FIG. 1. The
device is comprised of a solid support, a cover plate, a vertical
support, and an absorbent material. The solid support has an array
of capture molecules, e.g., antibodies specific for set of
biomarkers, immobilized on its upper surface. One or more flow
channels are formed by coupling the upper surface of the solid
support to the lower surface of the cover plate with the vertical
support. Coupling is performed using, e.g., an adhesive. When
coupled with the vertical support, the device has an open end for
administration of a fluid mixture and a closed end opposite of the
open end that is closed with the absorbent material. The flow
channel is designed in a manner that allows for flow of the fluid
mixture over the immobilized array of capture molecules and that is
preferably driven by capillary action, although in certain
embodiments involving relatively larger sample volumes, bulk flow
forces may operate the device.
[0079] The fluid mixture is typically a sample, e.g., a blood
sample that includes the biomarkers of interest. Often the fluid
mixture includes additional reagents, e.g., detection antibodies,
for detection of the biomarkers bound to the capture molecules
immobilized on the solid support. The detection antibodies can be
labeled for detection, e.g., fluorescently labeled.
[0080] In use, the fluid mixture is added to the open end of the
device. This fluid is drawn into the channel and over the
immobilized array by force preferably produced by capillary action.
The fluid is then wicked from the opposite end of the channel by
the absorbent material. Flow through the flow channel is
unidirectional due to the absorbent material. Protein biomarkers in
the fluid mixture that are bound by the capture molecules are
quantified, e.g., by an optical reader that detects the
fluorescently labeled detection antibodies bound to the biomarkers
bound to the capture molecules immobilized to the array. The
fluorescence of the array is proportional to the biomarker
concentration in the fluid mixture.
[0081] Solid Support
[0082] The device includes a solid support comprising immobilized
capture molecules, e.g., antibodies. Solid supports suitable for
immobilizing, binding and/or linking antibodies (and modifications
to render solid supports suitable for immobilizing capture
molecules) are well known in the art. Examples of a solid support
include: a microwell plate and a protein microarray (e.g.,
technology owned by Zyomyx, Inc. See, e.g. U.S. Pat. No.
6,365,418). In addition, pads, film, nanowells, or microfluid
channels can also serve as a solid support. In some embodiments,
the capture molecules are immobilized, bound, or linked on a solid
support surface such as polyvinylidene difluoride, nitrocellulose,
agarose, and/or polyacrylamide gel pads. In other embodiments, the
solid support can be made of glass or include a glass slide. Glass
slides activated with aldehyde, polylysine, or a homofunctional
cross-linker can also been used. In yet other embodiments, the
capture molecules can be arranged in a three-dimensional array, for
example in the three dimensional polyacrylamide gel pad microarray
described in Mirzabekov et al., Nucleic Acids Res 24(15): 2998-3004
(1996).
[0083] The invention provides a solid support, wherein capture
molecules are immobilized. For the purposes of the invention, the
term "immobilized" includes immobilized, bound, or linked to the
solid support. Linking can be covalent or noncovalent. Methods of
linking capture molecules to the solid support are well known in
the art. See, e.g. Kennedy et al. (Clin. Chim. Acta 70:1-31
(1976)), and Schurs et al. (Clin. Chim. Acta 81:1-40 (1977))
(describing coupling techniques, including the glutaraldehyde
method, the periodate method, the dimaleimide method, the
m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which
methods are incorporated by reference herein).
[0084] The capture molecules can be bound to many different solid
support materials. Examples of well-known materials include
polypropylene, polystyrene, polyethylene, polymers, dextran, nylon,
amylases, glass, natural and modified celluloses, polyacrylamides,
agaroses, silicone, and magnetite. Other materials are well known
in the art. See, e.g., Angenendra et al., Next generation of
protein microarray support materials: Evaluation for protein and
antibody microarray applications; Journal of Chromatography A;
Volume 1009, Issues 1-2, 15 Aug. 2003, Pages 97-104.
[0085] Preferably, the capture molecules are arranged on the solid
support in an array format. More preferably, the capture molecules
are arranged on the solid support in a multiplex array format.
Alternatively, the capture molecules can be arranged on the solid
support in ordered, sequential lines. Capture molecules and
detection agents, including suitable labels, are further described
herein.
[0086] Capture Molecules
[0087] The invention further provides a plurality or set of capture
molecules, wherein the set comprise at least about 2 distinct
capture molecules, wherein each distinct capture molecule
recognizes a different biomarker, e.g., peptide or target. In some
embodiments, the set comprises at least about 3, 4, 5, 6, 7, 8, or
more distinct capture molecules.
[0088] A capture molecule can encompass monoclonal antibodies,
polyclonal antibodies, antibody fragments (e.g., Fab, Fab',
F(ab').sub.2, Fv, Fc, etc.), chimeric antibodies, single chain Fvs
(ScFvs), mutants thereof, fusion proteins comprising an antibody
portion, and any other polypeptide that comprises an antigen
recognition site of the required specificity (including antibody
mimetics. See, e.g., Xu et al, Chem. Biol. 2002 Aug. 9(8):933-42).
The antibodies can be murine, rat, rabbit, chicken, human, or any
other origin, including humanized antibodies. Capture molecules,
such as antibodies, can be made recombinantly and expressed using
any method now known or later discovered in the art. In addition,
antibodies can be made recombinantly by phage display technology.
For examples of these expression and production methods see e.g.,
U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743 and 6,265,150; and
Winter et al., Annu. Rev. Immunol. 12:433-455 (1994).
[0089] As used herein, the term "antibody" encompasses not only
intact polyclonal or monoclonal antibodies, but also fragments
thereof (such as Fab, Fab', F(ab').sub.2, Fv), single chain (ScFv),
mutants thereof, fusion proteins comprising an antibody portion,
and any other modified configuration of the immunoglobulin molecule
that comprises an antigen recognition site of the required
specificity. An antibody includes an antibody of any class, such as
IgG, IgA, or IgM (or sub-class thereof), and the antibody need not
be of any particular class. Depending on the antibody amino acid
sequence of the constant domain of its heavy chains,
immunoglobulins can be assigned to different classes. There are
five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM,
and several of these can be further divided into subclasses
(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The
heavy-chain constant domains that correspond to the different
classes of immunoglobulins are called alpha, delta, epsilon, gamma,
and mu, respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0090] "Fv" is an antibody fragment that contains a complete
antigen-recognition and -binding site. In a two-chain Fv species,
this region consists of a dimer of one heavy and one light chain
variable domain in tight, non-covalent association. In a
single-chain Fv species, one heavy and one light chain variable
domain can be covalently linked by a flexible polypeptide linker
such that the light and heavy chains can associate in a dimeric
structure analogous to that in a two-chain Fv species. It is in
this configuration that the three complementarity determining
regions (CDRs) of each variable domain interact to define an
antigen-binding specificity on the surface of the VH-VL dimer.
However, even a single variable domain (or half of a Fv comprising
only 3 CDRs specific for an antigen) has the ability to recognize
and bind antigen, although generally at a lower affinity than the
entire binding site.
[0091] A "monoclonal antibody" refers to a homogeneous antibody
population wherein the monoclonal antibody is comprised of amino
acids (naturally occurring and non-naturally occurring) that are
involved in the selective binding of an antigen. A population of
monoclonal antibodies (as opposed to polyclonal antibodies) are
highly specific, in the sense that they are directed against a
single antigenic site. The term "monoclonal antibody" encompasses
not only intact monoclonal antibodies and full-length monoclonal
antibodies, but also fragments thereof (such as Fab, Fab',
F(ab').sub.2, Fv), single chain (ScFv), mutants thereof, fusion
proteins comprising an antibody portion, and any other modified
configuration of the immunoglobulin molecule that comprises an
antigen recognition site of the required specificity and the
ability to bind to an antigen (see definition of antibody). It is
not intended to be limited as regards to the source of the antibody
or the manner in which it is made (e.g., by hybridoma, phage
selection, recombinant expression, transgenic animals, etc.).
[0092] Capture molecules can be blocked using blocking agents such
as, e.g., serum or serum diluted in phosphate buffered saline (PBS)
and other blocking agents known in the art.
[0093] The choice of capture molecules depends on the application
and the biomarkers to be detected in the sample. In one embodiment,
the biomarkers to be detected are associated with breast cancer and
include, e.g., include Her-2, MMP-2, CA 15-3, OPN, p53, VEGF, CA
125, and Serum Estrogen Receptor (SER). These biomarkers can
include known fragments, splice variants, and full length peptides
as well as other variations that are not currently known. Examples
of sequence identifiers as of Feb. 11, 2008 at the HUGO on-line
database for these markers include, but are not limited to, Her-2
(X03363), MMP-2 (NM.sub.--004530), OPN (NM.sub.--001040058), p53
(NM.sub.--000546), VEGF (MGC70609), CA 125 (Q8WX17), SER
(NP.sub.--000116.2), and CA 15-3 (NM.sub.--002456). Additional sets
of biomarkers can be chosen for other diseases including prostate
cancer, ovarian cancer, and heart disease. Biomarker targets for
prostate cancer can include prostate specific antigen (PSA).
Biomarker targets for ovarian cancer can include CA 125. Biomarker
targets for heart disease can include Troponin T, Troponin I,
C-reactive protein (CRP), Homocysteine, Myoblobin, and Creatine
kinase. In addition, capture molecules specific for biomarker
associated with respiratory diseases can be chosen, including
biomarkers associated with influenza A, influenza B, Anthrax,
Plague, and allergens.
[0094] In one embodiment, the capture molecules are capture
antibodies specific for breast cancer markers. Capture antibodies
specific for breast cancer markers can include: 1) anti-Her-2
(R&D systems; Monoclonal Anti-human ErbB2 Antibody; MAB-1129;
Clone 191924), 2) anti-Matrix metallopeptidase (MMP)-2 (R&D
systems; Monoclonal Anti-human MMP-2 Antibody; MAB-902; Clone
36006.211), 3) anti-CA 15-3 (Fitzgerald; Monoclonal Anti-human CA
15-3 Antibody; 10-C03; Clone M8071022), 4) anti-Osteopontin (OPN)
(R & D Systems; Monoclonal Anti-human Osteopontin Antibody;
MAB-1433; Clone 190312), and 5) anti-Vascular Endothelial Growth
Factor (VEGF) (Biosource; VEGF purified mouse anti-human; AHG011;
Clone A183C-13G8).
[0095] As described herein, a capture molecule can bind a peptide
epitope of 2 or more consecutive (i.e., sequential) amino acids. It
is understood that the amino acid(s) forming the target epitope can
be linear or branched, and can comprise an amino acid(s) that has
been modified naturally or by intervention; for example, disulfide
bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component. The amino acid(s) forming
the target epitope can further encompass, for example, one or more
analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other modifications known in the art. In
some embodiments the target is a protein biomarker. Protein
biomarkers are further described herein.
[0096] In some embodiments, the capture molecule binds its cognate
target epitope with an affinity of binding reaction of at least
about 10.sup.-7 M, at least 10.sup.-8 M, or at least about
10.sup.-9 M, or tighter. In some embodiments, a binding interaction
will discriminate over adventitious binding interactions in the
reaction by at least two-fold, at least five-fold, at least 10- to
at least 100-fold or more.
[0097] It is understood that other target binding agents can be
used, in addition to the capture molecule sets described herein. In
addition, it is evident that the number of capture molecules in the
capture molecule set depends on the contemplated uses and
applications of the set, complexity of the sample, average size of
the proteins in the sample, frequency that the cognate target
epitope is present or predicted to be present in a sample, binding
affinity and/or specificity of the capture molecules, knowledge of
target protein(s), and stability of the capture molecules. Such
factors, and others, are well known in the art.
[0098] Detection of Biomarkers
[0099] Detection of target, e.g., biomarker, binding to a capture
molecule can be made using detection molecules and detection
agents. In one embodiment, detection molecules are detection
antibodies. Detection antibodies are specific for a biomarker
target, similar to capture antibodies or capture molecules.
[0100] Detection agents can be labeled using methods well-known to
one of skill in the art, e.g., detection agents can be fluorescent
agents, colorimetric agents, and magnetic agents. Fluorescent
agents can include e.g., quantum dots and fluorophores, e.g., ALEXA
546.
[0101] In one embodiment, the detection antibodies include: 1)
anti-Her-2 (R & D Systems; Polyclonal Goat Anti-human ErbB2
Antibody; AF-1129), 2) anti-MMP-2 (R & D Systems; Polyclonal
Goat Anti-human MMP-2 Antibody; AF-902), 3) anti-CA 15-3
(Fitzgerald; Monoclonal Anti-human CA 15-3 antibody; 10-C03B; Clone
M8071021), 4) anti-Osteopontin (R & D Systems; Polyclonal Goat
Anti-human Osteopontin Antibody; AF-1433), and 5) anti-VEGF
(Biosource; Polyclonal Rabbit Anti-human VEGF Biotin Conjugated
Antibody; AHG9119).
[0102] Cover Plates
[0103] The device includes a cover plate coupled to the upper
surface of the solid support via vertical supports. The cover plate
provides an upper surface of the channel through which fluid flows.
In some embodiments, the cover plate is positioned a fixed distance
above the solid support for allowing the entrance of fluids into a
channel formed by the coupling. The cover plate can be made of any
material suitable for the application, e.g., polypropylene,
polystyrene, polyethylene, polymers, dextran, nylon, plastic,
amylase, silicone, glass, natural and modified celluloses,
polyacrylamides, agaroses, and magnetite. In one embodiment, the
cover plates are made of a hydrophilic material. In another
embodiment, the cover plates are made of an optically transparent
material.
[0104] In another embodiment, the cover plate is a cover slip made
of glass. In another embodiment, the cover plate is a Corning.RTM.
cover glass rectangle, Cat # 2935-244 size 24 mm (W).times.40 mm
(H).times.0.13 mm (Thick).
[0105] Vertical Supports
[0106] The cover plate is connected to the solid support using a
vertical support. The vertical support can be singular or, the
device can include a plurality of vertical supports. The vertical
support can be curved or bent to facilitate connection with the
solid support. The vertical support can be arranged on the solid
support to surround or outline the plurality of capture molecules
immobilized on the solid support. The vertical supports can be made
of e.g., polypropylene, polystyrene, polyethylene, polymers,
dextran, nylon, amylase, silicone, glass, natural and modified
celluloses, polyacrylamides, agaroses, and magnetite.
[0107] In one embodiment, the vertical support includes an
adhesive. The adhesive can be positioned on the solid support.
After the vertical supports are placed on the adhesive on the solid
support another adhesive can be applied to the upper edges of the
vertical supports. The cover plate can then be placed upon the
vertical supports. The vertical supports inhibit the cover plate
from contacting the solid support. In this manner, a channel can be
formed between the solid support and the cover plate which allows
fluid to pass into the channel.
[0108] The channel or channels formed by the solid support,
vertical support, and cover plate can be round, trapezoidal,
triangular or other geometric shapes as required. Channel sizes are
optimally determined by the application. Channels can be from 0.01
mm to several millimeters deep and from 0.01 mm to several
millimeters wide. Channels can be straight, curved, zig-zag, or
U-shaped depending upon the application and specific function of
the channel. Channels can be from 0.05 mm to several millimeters
deep and from 0.1 to a centimeter or more in diameter. Capacity of
the channels can range from nanoliters to 1 mL or more depending
upon the application. In another embodiment, the vertical supports
are SA2260, (Grace Biolabs); 1.5 mm (Wide).times.1 mm
(Thick).times.65 mm (Long). In a related embodiment, the top
adhesive layer of the SA2260 is removed and the two sides are cut
out from the product and used separately.
[0109] Absorbent Material
[0110] The device comprises an absorbent material at one end of the
channel or channels. The absorbent material comprises a material
pervious to the passage of fluid and is absorbent. Absorbent
materials can include e.g., plastics, polymers, acrylics, nylon,
paper, cellulose, nitrocellulose, and ceramics. Other examples of
absorbent materials include membranes available from the Pall
Corporation (East Hills, N.Y.). The absorbent material may or may
not comprise pores. In one embodiment, the absorbent material has
pores. The pore sizes (cross-sectional dimension) of the absorbent
material can range between and including about 1 nanometer to about
1 centimeter. Pore size can be adjusted according to the properties
of the sample and to control the rate of fluid movement or flow
over the solid support. Preferably, the absorbent material provides
for wicking (i.e., drawing in of fluid by capillary action or
capillarity) of the fluid into the absorbent material. In order to
promote wicking of the fluid into the absorbent material, the
absorbent material can also comprise a hydrophilic material, which
can be provided, for example, by the absorbent material itself with
or without post treating (e.g., plasma surface treatment such as
hypercleaning, etching or micro-roughening, plasma surface
modification of the molecular structure, surface chemical
activation or crosslinking), or by a coating provided thereto, such
as a surfactant. In another embodiment, the absorbent material used
in a device of the invention results in a linear flow rate on the
order of approximately 1 centimeter/minute. Additionally, flow
rates can be adjusted (i.e., increased or decreased) by adding or
subtracting material from the absorbent material.
[0111] In another embodiment, the absorbent material is Hi-Flow
Plus Nitrocellulose Membrane HF240 (Millipore; Billerica,
Mass.).
[0112] Detection of Biomaker Bound to the Capture Molecules
[0113] In another embodiment, the device comprises a detection
component for detecting the biomarker bound to the sold support via
the capture molecules. The detection component can include a reader
and a screen for displaying output from the reader. The reader can
be optical. One embodiment of a detection component of the
invention is the ScanArray.TM. 5000 XL (PerkinElmer, Inc.;
Wellesley, Mass.). This is a benchtop, laser-based confocal
scanning device with a photomultiplier tube (PMT) for sensitive
fluorescence detection. Images collected onto a computer can be
analyzed by QuantArray.TM. software. Raw intensities for each spot
can then be computed by taking the average of the logarithm of the
intensity over all pixels in the region of interest that were
greater than zero for quadruplicate spots on a slide and across
duplicate chambers.
[0114] Another embodiment of a detection component of the invention
is a imaging system that comprises a charge coupled device (CCD)
camera. The arrangement of this imaging system is shown in FIG. 2
and it consists of a scientific-grade 16-bit, 1392.times.1040 pixel
CCD camera (Lumenera Corp. MA), which is configured for Kohler
epi-illumination of the sample microarray. The imaging system
allows the sample to be illuminated from the front, while
simultaneously being imaged from the same side by the CCD camera.
Excitation light from a full-field White Lite.RTM. light 300 W
xenon arc lamp can be bandpass-filtered using a 525 nm excitation
filter (Omega Optical Inc, Vt.) and focused uniformly on a sample
using a set of two optic fiber cables (mellesgriot) held at an
angle of 45 degrees. The spots can be focused onto the CCD using a
camera lens (Infinimite.RTM. alpha, Edmund Optics) and filtered
using a 600 nm longpass filter. Custom algorithms, built within the
Lumenera camera software correct for CCD dark noise. Images saved
in tiff format can be analyzed using the Scanarray Express.TM.
software (Perkin Elmer, Wellesley, Mass.). Output from the imaging
system can be displayed on a computer screen or other viewing
apparatus, including e.g., a liquid crystal display (LCD)
device.
Arrays of the Invention
[0115] As describe herein, the device of the invention includes an
array comprising a solid support, e.g., a glass slide and a
plurality of capture molecules immobilized on the solid support. In
one aspect, the invention provides an array comprising a plurality
of capture molecules specific for biomarkers associated with breast
cancer. The biomarkers can include, e.g., Her-2, MMP-2, CA 15-3,
OPN, p53, VEGF, CA 125, and Serum Estrogen Receptor (SER). In one
embodiment, the array includes capture molecules specific for
CA-15-3 and OPN; in another embodiment the array includes capture
molecules specific for Her-2, MMP-2, CA 15-3, and OPN.
[0116] In a related aspect, the plurality of capture molecules are
a plurality of capture antibodies. The plurality of capture
antibodies can include at least two of the following capture
antibodies: 1) anti-Her-2 (R&D systems; Monoclonal Anti-human
ErbB2 Antibody; MAB-1129; Clone 191924), 2) anti-MMP-2 (R&D
systems; Monoclonal Anti-human MMP-2 Antibody; MAB-902; Clone
36006.211), 3) anti-CA 15-3 (Fitzgerald; Monoclonal Anti-human CA
15-3 Antibody; 10-C03; Clone M8071022), 4) anti-Osteopontin (R
& D Systems; Monoclonal Anti-human Osteopontin Antibody;
MAB-1433; Clone 190312), and 5) anti-VEGF (Biosource; VEGF purified
mouse anti-human; AHG011; Clone A183C-13G8). In one embodiment, the
array includes 1) anti-Her-2 (R&D systems; Monoclonal
Anti-human ErbB2 Antibody; MAB-1129; Clone 191924), 2) anti-MMP-2
(R&D systems; Monoclonal Anti-human MMP-2 Antibody; MAB-902;
Clone 36006.211), 3) anti-CA 15-3 (Fitzgerald; Monoclonal
Anti-human CA 15-3 Antibody; 10-C03; Clone M8071022), 4)
anti-Osteopontin (R & D Systems; Monoclonal Anti-human
Osteopontin Antibody; MAB-1433; Clone 190312).
Methods for Categorizing a Sample
[0117] In one embodiment, the invention provides a method for
scoring a sample from a subject, e.g., categorizing a human sample
using quantitative data associated with a plurality of biomarkers
wherein the biomarkers are associated with breast cancer.
[0118] Samples
[0119] A sample can be derived from any subject of interest,
including mammalian subjects and, e.g., human subjects, e.g.,
patients. A sample can include blood and other liquid samples of
biological origin, solid tissue samples such as a biopsy specimen
or tissue cultures or cells derived therefrom, and the progeny
thereof. A sample can be of cancerous origin, e.g., breast cancer.
A sample can comprise a single cell or more than a single cell.
Samples can also have been manipulated in any way after their
procurement, such as by treatment with reagents, solubilization, or
enrichment for certain components, such as proteins or
polynucleotides. Sample also encompasses a clinical sample, and
also includes cells in culture, cell supernatants, and cell
lysates.
[0120] Quantitative Data
[0121] A "dataset" of "quantitative data" is a set of numerical
values resulting from evaluation of a sample (or population of
samples) under a desired condition. In one embodiment, the
quantitative data is protein concentration data. The values of the
protein concentration data can be obtained, for example, by
experimentally obtaining measures from a sample and constructing a
dataset from the measurements. In another embodiment, the protein
concentration data is obtained using an enzyme linked immunosorbent
assay (ELISA) format. In another embodiment, the protein
concentration data is obtained using a sandwich immunoassay format.
In a related embodiment, the protein concentration data is obtained
using methods described herein, including sandwich immunoassay
formats, and the following antibodies: Capture antibodies 1)
anti-Her-2 (R&D systems; Monoclonal Anti-human ErbB2 Antibody;
MAB-1129; Clone 191924), 2) anti-MMP-2 (R&D systems; Monoclonal
Anti-human MMP-2 Antibody; MAB-902; Clone 36006.211), 3) anti-CA
15-3 (Fitzgerald; Monoclonal Anti-human CA 15-3 Antibody; 10-C03;
Clone M8071022), 4) anti-Osteopontin (R & D Systems; Monoclonal
Anti-human Osteopontin Antibody; MAB-1433; Clone 190312), and 5)
anti-VEGF (Biosource; VEGF purified mouse anti-human; AHG011; Clone
A183C-13G8); Detection antibodies 1) anti-Her-2 (R & D Systems;
Polyclonal Goat Anti-human ErbB2 Antibody; AF-1129), 2) anti-MMP-2
(R & D Systems; Polyclonal Goat Anti-human MMP-2 Antibody;
AF-902), 3) anti-CA 15-3 (Fitzgerald; Monoclonal Anti-human CA 15-3
antibody; 10-C03B; Clone M8071021), 4) anti-Osteopontin (R & D
Systems; Polyclonal Goat Anti-human Osteopontin Antibody; AF-1433),
and 5) anti-VEGF (Biosource; Polyclonal Rabbit Anti-human VEGF
Biotin Conjugated Antibody; AHG9119). In another embodiment, the
data are obtained using a multiplexed channel flow-based device,
e.g., the device described above (FIG. 1).
[0122] Alternatively, quantitative data can be obtained from a
service provider such as a laboratory, or from a database or a
server on which the data has been stored.
[0123] Plurality of Biomarkers
[0124] The method of categorizing sample uses data associated with
a plurality of biomarkers associated with breast cancer. In one
aspect, the plurality of biomarkers associated with breast cancer
includes CA 15-3 and OPN. In another aspect, additional biomarkers
can include Her-2. In another aspect, additional biomarkers can
include MMP-2. In another aspect, additional biomarkers can include
VEGF. In another aspect, additional biomarkers can include Her-2
and MMP-2. In yet another aspect, additional biomarkers can further
include p53, CA 125, and Serum Estrogen Receptor (SER). In one
embodiment of the invention, the method of categorizing a sample
uses data associated with the following biomarkers: CA 15-3, OPN,
Her-2, and MMP-2.
[0125] Scoring the Sample
[0126] In one embodiment, scoring the sample comprises analyzing
the data and outputting a score. Analysis of the data can include
use of a predictive model. Predictive models can be developed
using, e.g., principal component analysis (PCA), and linear
discriminant analysis.
[0127] PCA is a technique used to reduce multidimensional data sets
to lower dimensions for analysis. Mathematically, PCA is defined as
an orthogonal linear transformation that transforms the data to a
new coordinate system such that the greatest variance by any
projection of the data comes to lie on the first coordinate (called
the first principal component), the second greatest variance on the
second coordinate, and so on. PCA can be used as a tool in
exploratory data analysis and for making predictive models. PCA can
also involve the calculation of the eigenvalue decomposition of a
data covariance matrix or singular value decomposition of a data
matrix, usually after mean centering the data for each attribute.
The results of a PCA are usually discussed in terms of component
scores and loadings.
[0128] Linear discriminant analysis is a method used to find the
linear combination of features which best separate two or more
classes of objects or events. The resulting combination can be used
as a linear classifier, or, alternatively, for dimensionality
reduction before later classification.
[0129] In another embodiment, the analysis can include categorizing
the sample according to a predictive model. The probability that
categorization is correct is model- and biomarker-dependent and can
be at least 60%, at least 70%, at least 80%, at least 87%, at least
90%, or at least 95% correct. Categories can include a healthy
categorization, i.e. disease-free, an early-stage disease
categorization, and a late-stage disease categorization.
[0130] In another embodiment, the score can be compared to a second
score determined for a second sample from the mammalian subject.
This comparison can be used e.g., to determine the progress of
therapy for the treatment of disease. In yet another embodiment,
the score can be used to diagnose a neoplastic breast disease. A
neoplastic breast disease can include e.g., breast cancer.
EXAMPLES
[0131] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way. Efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperatures,
etc.), but some experimental error and deviation should, of course,
be allowed for.
[0132] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of protein chemistry,
biochemistry, recombinant DNA techniques and pharmacology, within
the skill of the art. Such techniques are explained fully in the
literature. See, e.g., T. E. Creighton, Proteins: Structures and
Molecular Properties (W.H. Freeman and Company, 1993); A. L.
Lehninger, Biochemistry (Worth Publishers, Inc., current addition);
Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd
Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan
eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences,
18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey
and Sundberg Advanced Organic Chemistry 3.sup.rd Ed. (Plenum Press)
Vols A and B (1992).
Example 1
Development of Multiplexed Assays
[0133] A multiplexed immunoassay for the measurement of breast
cancer biomarkers using the protein microarray format was
developed.
[0134] Protein microarrays have the potential to be used to
simultaneously analyze large numbers of serum proteins in a rapid
and reproducible manner. In recent years, protein microarrays have
evolved as powerful tools to address high-throughput requirements
by performing "traditional" biochemistry in an
ultra-high-throughput and miniaturized format. On protein
microarrays, purified capture molecules are immobilized in unique
locations on the surface of the substrate that allow for
recognizing the target under study. Since each capture molecule is
immobilized in a precise, predetermined spot, the protein
microarrays achieve multiplexing capability based on this unique
location of the capture molecule and therefore the target protein
bound to it (assuming high specificity of the antibody-antigen
reaction). In the case of a reverse assay on the microarray, the
immobilized molecule includes large collections of purified
proteins or samples such as sera and lysates, which is then probed
with a single antibody.
Materials
[0135] A multiplexed breast cancer assay based on the protein
microarray was developed after validating the reagents using 2d Gel
Electrophoresis and ELISA techniques. This example presents the
assay development materials and methods on these platforms.
[0136] Common Reagents
[0137] Recombinant proteins, capture, and biotinylated detection
antibodies for Her-2, MMP-2 and Osteopontin were purchased from
R&D systems (Minneapolis, Minn.). Other reagents used in the
assay include: VEGF antigen and capture and biotinylated detection
antibodies (Biosource International Camarillo, Calif.), CA 15-3
antigen and anti-CA 15-3 capture and detection antibodies
(Fitzgerald, Concord, Mass.). The CA 15-3 detection antibody was
biotinylated using a kit and according to the manufacturer's
(Pierce, Rockford, Ill.) instructions. All other detection
antibodies were purchased as biotin conjugates. Lyophilized human
serum was purchased from Rockland Immunochemicals (Gilbertsville,
Pa.). Capture antibodies used were: 1) Her-2 (R&D systems;
Monoclonal Anti-human ErbB2 Antibody; MAB-1129; Clone 191924), 2)
MMP-2 (R&D systems; Monoclonal Anti-human MMP-2 Antibody;
MAB-902; Clone 36006.211), 3) CA 15-3 (Fitzgerald; Monoclonal
Anti-human CA 15-3 Antibody; 10-C03; Clone M8071022), 4)
Osteopontin (R & D Systems; Monoclonal Anti-human Osteopontin
Antibody; MAB-1433; Clone 190312), and 5) VEGF (Biosource; VEGF
purified mouse anti-human; AHG011; Clone A183C-13G8). Detection
antibodies used were: 1) Her-2 (R & D Systems; Polyclonal Goat
Anti-human ErbB2 Antibody; AF-1129), 2) MMP-2 (R & D Systems;
Polyclonal Goat Anti-human MMP-2 Antibody; AF-902), 3) CA 15-3
(Fitzgerald; Monoclonal Anti-human CA 15-3 antibody; 10-C03B; Clone
M8071021), 4) Osteopontin (R & D Systems; Polyclonal Goat
Anti-human Osteopontin Antibody; AF-1433), and 5) VEGF (Biosource;
Polyclonal Rabbit Anti-human VEGF Biotin Conjugated Antibody;
AHG9119).
[0138] Reagents for Western Blotting
[0139] Anti-goat, anti-rabbit and anti-mouse secondary antibodies
were conjugated to horseradish peroxidase (HRP) (EMD Biosciences,
San Diego, Calif.). Laemmli sample buffer, precision plus dual
molecular weight standard and 7.5% and 10% ready gels were
purchased from BioRad Inc (Hercules, Calif.), enhanced
chemiluminescence (ECL) detection kits and hyperfilm were obtained
from Amersham Biosciences (Piscataway, N.J.) and PVDF transfer
membrane from (Millipore, Bedford, Mass.). Tris buffered saline
(TBS) and Tris buffered saline with 0.05% tween (TBS-T), was
purchased from Sigma-Aldrich (St. Louis, Mo.).
[0140] Reagents for ELISA
[0141] Antibody biotinylation kit and Blotto were purchased from
Pierce (Rockford, Ill.). Vectastain kit was purchased from Vector
Labs (Burlingame, Calif.), Nunc ELISA 96 well plates was obtained
from Nalge Nunc International, (Rochester, N.Y.) and Hanks buffer
was purchased from Invitrogen--GIBCO (Carlsbad, Calif.). Phosphate
buffered saline with 0.05% tween (PBS-T), Phosphate buffered saline
(PBS) and TMB (3,3'.5,5'-tetramethylbenzidine) were purchased from
Sigma-Aldrich (St. Louis, Mo.).
[0142] Reagents for Protein Microarrays
[0143] Streptavidin conjugated Alexa 546 was purchased from
Invitrogen--Molecular Probes (Carlsbad, Calif.), GAPS II.TM. slides
were purchased from Corning LifeSciences (Corning, N.Y.) and BSA
was purchased from Sigma-Aldrich (St. Louis, Mo.).
[0144] Methods
[0145] Reagent Quality Validation Using Gel Electrophoresis and
Western Blotting
[0146] Gel electrophoresis for each biomarker was performed in
non-reducing conditions with 7.5% (for Her-2 (185 kD) and CA 15-3
(250 kD)) and 10% (for OPN (66 kD), MMP-2 (72 kD) and VEGF (38 kD))
polyacrylamide gels. Samples were solubilized in Laemmli sample
buffer and boiled for 3 minutes before loading into the sample
wells. Approximately 150 .mu.g of each protein was loaded into each
well. Precision plus dual standard was used as the molecular weight
marker. After electrophoresis for 2 h at 120V, the proteins were
transferred from the gel on to the PVDF membrane under an electric
field, using a fully immersed wet unit (BioRad, Hercules, Calif.),
for 1 h at room temperature with ice in the unit. The membrane was
then immersed in wash buffer (TBS-T) for 15 minutes and blocked
overnight at 4.degree. C., with 5% w/v milk (BioRad, Hercules,
Calif.) prepared in wash buffer. Following the blocking step, the
membrane was washed in TBS-T for 30 minutes and incubated with
primary antibody at 500 ng/ml (Mouse and Goat anti Her-2 antibody,
Mouse and Goat anti MMP-2 antibody, Mouse and Goat anti-Osteopontin
antibody, Mouse and Rabbit anti-VEGF antibody and two Mouse anti-CA
15-3 antibodies) for 1 hour at room temperature. After a 30 minute
wash in TBS-T, the membrane was incubated for 1 hour at room
temperature with the secondary antibody conjugated to HRP
(Anti-Goat, Anti-Rabbit or Anti-Mouse as applicable) at 500 ng/ml.
The protein bands in the gel were finally visualized using the ECL
kit and a sensitive photo film.
[0147] Selection of Antibody Pairs: Enyzme Linked Immunosorbent
Assays (ELISA)
[0148] The concentrations of the capture and detector antibodies
for all five biomarkers, to be used in a sandwich format, were
optimized using standard two antibody sandwich ELISA. 100 .mu.L of
capture antibody solution in ELISA coating buffer (Hanks buffer,
with 0.375% NaHCO.sub.3) was added in duplicate to the wells of the
96-well plate in a series of four dilutions (0.1 .mu.g/ml, 0.3
.mu.g/ml, 1 .mu.g/ml and 3 .mu.g/ml). These dilutions were added
across the rows of the plate. After overnight incubation at
4.degree. C., the coating antibody solution was aspirated from the
wells and the plates were rinsed 6 times in PBS-T and then blocked
for 2 h at room temperature in 200 .mu.l of BLOTTO. The solution
was then aspirated from the wells, and the plate was washed 6 times
in PBS-T. 100 .mu.L of the recombinant antigen was added to the
appropriate wells in dilutions representing the middle portion of
the clinical range for the biomarkers. The concentrations of
antigens used for this assay were 10 ng/ml for Her-2, 600 ng/ml for
MMP-2, 130 U/ml for CA 15-3, 700 ng/ml for OPN, and 550 pg/ml for
VEGF, respectively. Buffer without antigen was used to represent
background signal. The plate was sealed and incubated at room
temperature for 2 hours followed by 6 washes with PBS-T. 100 .mu.l
of biotinylated detection antibody solutions were then added down
the columns of the plate in a series of four dilutions (0.36
.mu.g/ml, 1.1 .mu.g/ml, 3.3 .mu.g/ml and 9.9 .mu.g/ml). For VEGF,
biotinylated antibody was used at higher concentrations of 3
.mu.g/ml, 8 .mu.g/ml, 25 .mu.g/ml and 75 .mu.g/ml according to
manufacturer's (R&D systems) suggestions. Following a 1-hour
incubation at room temperature, the plates were washed 6 times and
100 .mu.l of Vectastain solution (prepared according to the
manufacturer's instructions) was added for 30 minutes to probe for
the detection antibodies. The plates were then washed 6 times and
incubated with 100 .mu.l of TMB for 30 minutes at room temperature.
The reaction was stopped by adding 50L of 1N H.sub.2SO.sub.4 to
each well before reading the absorbance at 485 nm in the microplate
reader (BioRad, Hercules, Calif.). After color development and
measurement of absorbance, the capture and detector antibody
concentrations yielding the best signal to noise ratio were
selected for further ELISA development.
[0149] Standard curves of each of the biomarkers were then obtained
by performing the ELISA as outlined in the basic protocol above
except that optimized concentrations of capture and detector
antibodies were used with 8 serial dilutions of recombinant antigen
standards to obtain a standard titration curve. The concentrations
of biomarkers used to establish this standard curve were 1
ng/ml-128 ng/ml for Her-2, 10 ng/ml-1280 ng/ml for MMP-2, 2
U/ml-256 U/ml for CA 15-3, 10 ng/ml-1280 ng/ml for OPN and 1
pg/ml-1280 pg/ml for VEGF.
[0150] Microarray Spotting and Assay Protocol
[0151] Concentrations of capture and detector antibody pair for
each of the five biomarkers were optimized for use in a sandwich
assay format on the microarray similar to the ELISA optimizations.
Aminosilanated, GAPS II.TM. barcoded glass slides were spotted with
optimized dilutions of capture antibodies using a robotic arrayer
(Norgen Systems Inc.; Mountain View, Calif.). Four print heads were
used to deposit approximately 1 nl of capture antibody solution,
generating a total of 8 arrays per slide with 250 .mu.m diameter
spots with a spot-to-spot distance of 350 .mu.m. The layout of each
8.times.12 array of printed antibody spots corresponded to one spot
per well in a standard 8.times.12 (96-well) format. These capture
antibodies were printed in a series of four dilutions (1000
.mu.g/ml, 500 .mu.g/ml, 250 .mu.g/ml and 125 .mu.g/ml). Also
printed on each slide were two controls. Bovine serum albumin (BSA)
served as negative control (NC). Alexa 546 spots were used as
position controls (PC), which served as reference points when the
slides were imaged. The spotted slides were cross-linked under
ultraviolet light for 5 minutes and were stored in the dark at
4.degree. C.
[0152] The eight arrays were separated using silicone gasket
chambers (Schleicher & Schuell Bioscience, Keene N.H.) and were
blocked with 1 mg/ml BSA solution for 30 minutes. The protein
microarrays were then washed for 15 minutes and incubated with 100
.mu.l of target antigen at dilutions representing the middle
portion of the clinically significant ranges for the biomarkers.
The concentrations of antigens used for this assay were 10 ng/ml
for Her-2, 600 ng/ml for MMP-2, 130 U/ml for CA 15-3, 700 ng/ml for
OPN and 550 pg/ml for VEGF respectively. Buffer without any added
recombinant antigen was used to determine the background due to
non-specific binding on the arrays. The solution was aspirated and
washed with PBS-T for 15 minutes and the wells were then incubated
with 100 .mu.l of a series of four dilutions of biotinylated
antibody solution (1.8 .mu.g/ml, 3.75 .mu.g/ml, 7.5 .mu.g/ml and 15
.mu.g/ml) in PBS for 30 minutes. Following a 15 minute wash, the
arrays were incubated with 100 .mu.l streptavidin conjugated Alexa
546 for 10 minutes. The chambers were then removed and the slides
were agitated in PBS-T for 10 minutes and dried by centrifugation
prior to scanning.
[0153] Two sets of standard curves were obtained. One, using PBS as
the diluting medium for the recombinant antigens and one using
human serum as the medium. The protein microarray standard
titration curves were obtained as outlined in the basic protocol
except that serial dilutions of recombinant antigen standards in
PBS and human serum were used as analytes and the optimized
concentrations of capture and detector antibodies were used for
detection. The concentrations of biomarkers used to establish this
standard curve were 1 ng/ml-128 ng/ml for Her-2, 10 ng/ml-1280
ng/ml for MMP-2, 2 U/ml-256 U/ml for CA 15-3, 10 ng/ml-1280 ng/ml
for OPN, and 1 pg/ml-1280 pg/ml for VEGF.
[0154] Microarray Spotting and Assay Protocol: Multiplex Curves
[0155] This multiplexed assay was performed essentially as
described for the standard titration curves. Capture antibodies for
Her-2, MMP-2, OPN, CA 15-3 and VEGF were spotted in quadruplicate
at 500 .mu.g/ml on the GAPS II.TM. slides to form a 4.times.5 array
grid. Ten different multiplex samples were prepared. Five samples
were prepared with a mixture of all but one antigen and five of the
remaining samples contained only one antigen each. The
concentrations of recombinant antigens used in this assay were 20
ng/ml Her-2, 800 ng/ml MMP-2, 130 U/ml CA 15-3, 900 ng/ml OPN and
950 pg/ml VEGF. A second set often slides was prepared in a similar
manner, but using human serum as the medium instead of PBS. In this
case, only four biomarkers were used (Her-2, MMP-2, CA 15-3 and
Osteopontin). Antibody microarrays were incubated with these
antigen samples in duplicate, followed by incubation with a
detector antibody "cocktail" containing biotinylated antibodies for
all five biomarkers at a concentration of 3.75 .mu.g/ml.
Streptavidin conjugated Alexa 546 was used as the reporter at a
concentration of 5 .mu.g/ml. The chambers were then removed and the
slides were agitated in PBS-T for 10 minutes and dried by
centrifugation prior to scanning.
[0156] Microarray Imaging and Analysis Protocol
[0157] Slides were imaged with ScanArray 5000 XL (Perkin Elmer,
Wellesley, Mass.), which is a laser-based confocal scanner, at 543
nm excitation. Images collected onto a PC were analyzed by
Scanarray Express.TM. software (Perkin Elmer, Wellesley, Mass.).
Raw intensities for each spot were computed by taking the average
of the logarithm of the intensity over all pixels in the region of
interest that were greater than zero for the spot. A median of all
quadruplicate spots across 2 wells (resulting in a total of eight
spots per sample) was computed and plotted against concentration
for a titration curve.
[0158] Results
[0159] Western Blotting
[0160] Antigens purchased from various sources were tested for
purity by performing SDS-PAGE, which demonstrated the specificities
of the antibodies as well as tested the purity of the recombinant
antigens. This was followed by detection with monoclonal antibodies
(FIG. 3A) (to be employed as capture antibodies in the ELISA)
polyclonal antibodies (FIG. 3B) (to be employed as detector
antibodies in the ELISA) using Western Blotting. Approximately 150
ng of protein was loaded in each lane and the gels were run under
non-reducing conditions to mimic the detection of proteins in
serum. Distinct bands are observed for four of the biomarkers,
MMP-2 (72 kDa), CA 15-3 (250 kDa), OPN (66 kDa) and VEGF (38 kDa).
For the biomarker, Her-2 (185 kDa) distinct, multiple bands were
seen due to the phosphorylated forms of the protein, all of which
are recognized by the antibody.
[0161] To demonstrate the specificity of these antibodies, all five
recombinant protein biomarkers were run in neighboring wells in the
gel, transferred them onto the PVDF membrane and incubated the
membrane with one single antibody. As can be seen in FIG. 3, the
capture (A-E) and detector (F-J) antibodies showed high specificity
to their respective antigens. Using this method, the most specific
capture (monoclonal) antibodies and detector (monoclonal and
polyclonal) for all five biomarkers from a total of 30 antibody
pairs and 5 recombinant antigens were selected.
[0162] ELISA
[0163] The selected antibodies were then characterized for dynamic
range and sensitivity in the clinically-relevant concentrations (as
observed in normal and breast cancer patient sera) using ELISA as
the validation method. The monoclonal antibodies were used as
capture and the biotinylated polyclonal antibodies were used as
detector (except in the case of CA 15-3, where both antibodies were
monoclonal). ELISA plates were coated with four different
concentrations of the capture antibodies in the ELISA coating
buffer as described in materials and methods. Two dilutions of
recombinant antigen were used, one on either end of the
clinically-relevant range. Sample with no antigen added was used as
negative control for the assay. Biotinylated detector antibodies
were then added in four different dilutions to the wells. FIG. 4
shows the results obtained from these optimizations. The
concentrations of capture and detector antibodies that yielded a
good signal (O. D between 1.0 and 2.0, which was neither too low
nor saturated) were chosen for the assays. Since the detector
antibodies were more expensive than the capture antibodies (because
of their biotin-conjugation), a combination of capture and
detection concentration was chosen that gave a good signal, but
nevertheless used the minimum amount of detector antibody. Table 1
lists the final chosen concentrations of both the antibodies using
this criterion and which were used to develop ELISA curves.
[0164] Standard curves for all five biomarkers were developed on
the ELISA platform. 96 well polystyrene plates were coated with
capture antibody at optimized concentration, followed by incubation
with 8 serial dilutions of recombinant antigen in PBS. The
concentrations of biomarkers used to establish this standard curves
were 1 ng/ml-128 ng/ml for Her-2, 10 ng/ml-1280 ng/ml for MMP-2, 2
U/ml-256 U/ml for CA 15-3, 10 ng/ml-1280 ng/ml for OPN and 1
pg/ml-1280 pg/ml for VEGF. The wells were then probed with
optimized concentration of biotinylated detector antibody. Enzyme
based detection was used in this assay in which, wells were
interrogated with a spectrophotometer to obtain intensities of
substrate color. Increased color intensity was observed with
increased protein concentration. These intensities are quantified
and plotted as a function of antigen concentration to obtain a
standard curve. FIG. 5 demonstrates that the ELISA assay curves are
linear over the clinically-relevant ranges for these biomarkers and
span normal as well as elevated levels as seen in cancer. Data
points for each curve represent the average intensities of two
replicate samples. Reproducibility was determined from the
coefficient of variation, which was approximately 5% for all
protein biomarker curves. The background signal, which is a measure
of non-specific binding, was considered to be the signal from the
wells in which no antigen was added. Each point on the ELISA curve
plotted below represents the signal from the wells minus this
background.
[0165] Standard Curves were Established on Protein Microarrays
[0166] Western blotting was used to validate the specificity of the
reagents selected and ELISA was used to characterize the
performance of these antibodies in a sandwich immunoassay format.
These antibody pairs were then employed on the protein microarray
platform to generate standard curves in singleplex as well as
multiplex format. Similar to the ELISA, concentrations of these
antibodies were optimized on the protein microarray prior to
establishing the standard curves. A schematic diagram of the
microarray assay approach used in this study is shown in FIG.
6.
[0167] Capture antibodies immobilized on modified glass slides are
probed with sample containing antigen. A second biotinylated
antibody then binds to the antigen on the array and a
streptavidin-linked fluorescent dye was used for detection. For the
optimization studies, amine modified microarray slides were printed
with four distinct dilutions of the capture antibodies in PBS
buffer as described in materials and methods. The spot size was
approximately 250 .mu.m in diameter. Two dilutions of recombinant
antigen were used, one on either end of the clinically-relevant
range. Sample with no antigen added was used as negative control
for the assay. A second, biotinylated antibody recognizing a
different epitope on the same antigen was used in three different
dilutions for detection. This "sandwich" approach favors
specificity in analyte detection, since the two separate antibodies
sequentially enable selective detection. A streptavidin-Alexa 546
fluorescent reporter was then used to bind to the biotin moiety of
the detection antibody which then produced fluorescent signals
proportional to the amount of antigen bound on the array.
[0168] These fluorescent spots were then quantified using a
fluorescence microarray reader. FIG. 7 illustrates the signals
obtained from these optimizations and Table 2 demonstrates the
final chosen concentrations of both the antibodies that were used
to develop microarray curves. Similar to the ELISA optimization
experiments, the combination of capture and detector antibody that
yielded maximum response with minimum amount of detector antibody
was chosen as the optimized concentration.
[0169] The optimized concentrations of capture antibodies were
printed on modified microarray slides using a robotic arrayer such
that each antibody was present in quadruplicate. The capabilities
of the microarray were further tested by analyzing single
biomarkers over a range of concentrations in a multiplexed format,
using all five capture and detector antibodies (see FIGS. 8 and 9).
The goal here was to measure the effect of the presence of other
capture agents and varying antibody affinities on the multiplexed
detection of five proteins. Protein microarrays were incubated with
8 serial dilutions of recombinant antigen diluted in PBS. The
concentration ranges used were 1 ng/ml-128 ng/ml for Her-2, 10
ng/ml-1280 ng/ml for MMP-2, 2 U/ml-256 U/ml for CA 15-3, 10
ng/ml-1280 ng/ml for OPN and 10 pg/ml-1280 pg/ml for VEGF. Standard
curves were obtained on the protein microarray format for each
biomarker in four formats. First, only one capture antibody
(Singleplex format) was used and second, the all five capture
antibodies were spotted on the array and all five detector
antibodies were used (Multiplex format). In both cases the antigens
were diluted in PBS. This method was most similar to the ELISA. The
third and fourth formats are similar to the first and second,
except that human serum was used as the diluting medium instead of
PBS. This method enabled the quantification of effect of serum on
background noise and thus simulated detection of these proteins in
patient sera.
[0170] FIG. 8 shows a composite image of eight different arrays
with each column representing a different array that was incubated
with increasing antigen concentration from right side of the FIG.
to the left side of the FIG. Slides shown in Panel A were incubated
with Her-2 at concentrations ranging from 0 ng/ml (right) to 128
ng/ml (left). Slides shown in Panel B were incubated with MMP-2
with a concentration range of 0 ng/ml (right) to 1280 ng/ml (left).
Slides shown in Panel C were incubated with CA 15-3 at
concentrations ranging from 0 U/ml (right)-256 U/ml (left) and
those shown in Panel D were incubated with Osteopontin at
concentrations from 0 ng/ml (right) to 1280 ng/ml (left). Finally,
Panel E shows slides incubated with VEGF in the concentration range
of 0 pg/ml (right)-1280 pg/ml (left). The results show increased
fluorescence intensity with increased protein concentration.
[0171] The fluorescence from these spots is quantified using the
Scanarray software and plotted as a function of antigen
concentration in FIG. 9 (A-E) for the experiments with PBS as the
medium and in FIG. 9(F-J) for the experiments in which human serum
was used as the diluent. The background signal, which is a measure
of non-specific binding, was considered to be the signal from the
arrays in which no antigen was added. Data points for each curve
represent the average intensities of eight replicates (background
subtracted) obtained using quadruplicate spots in two replicate
arrays. Reproducibility was determined by the coefficient of
variation, which was approximately 15% for all protein biomarker
curves.
[0172] The multiplex assays have a higher background noise level
than the singleplex assays probably due to the presence of
additional detector antibodies in the assay. However, the shapes of
the intensity curves compare well to the assays in the singleplex
format validating the use of multiplex microarray assays. A higher
background in the serum assays as compared to the PBS based assays
was observed, likely due to the presence of other proteins in the
serum that bind non-specifically to the slide.
[0173] Comparison of ELISA and Protein Microarray Formats
[0174] The developed microarray immunoassay offers many advantages
over the traditional ELISA technique, including smaller size, lower
costs, and multiplexing capability. Similar to the ELISA, we
carefully optimized the concentrations of antibodies on the
microarray to yield sensitive dose response curves. FIG. 10 shows
the comparison of the standard curves obtained for each of the
biomarkers using microarray technology and the ELISA in a scatter
plot. Protein microarray shows similar dynamic range. Data from the
two methods showed a linear relationship with a correlation
coefficient (r.sup.2) of greater then 0.97, indicating that both
methods produce similar results.
[0175] The background noise due to non-specific adsorption of
protein on the microarray glass surface increases with increasing
protein concentration. Therefore, the detection of a specific
target protein is limited not only by its concentration, but also
by the concentration of the other proteins in the mixture. Serum is
a complex mixture of many proteins with a very high concentration
(.about.85 mg/ml) compared to PBS (0 mg/ml) causing significantly
higher background levels in the assays developed using serum
instead of PBS as the medium. The four biomarkers were detectable
above this noise level in this assay. However, for proteins present
in very small amounts (pM or fM) in serum, the high background can
reduce the sensitivity of the assay. In that case, the abundant
proteins (such as albumin) could be filtered out and the serum
could be concentrated to enhance the detection limit. The signal
can be amplified using specific secondary antibody molecules.
[0176] Microarray Multiplexed Assays
[0177] To demonstrate that this array technology could be used to
simultaneously detect multiple biomarkers, all five protein
biomarkers were analyzed in one single microarray. In this
experiment, twelve identical slides were printed with capture
antibodies to the five protein biomarkers. One of these slides was
incubated with a mixture of all five antigens in the high
concentrations observed in cancer, while one slide was incubated
with a mixture of all five antigens in the lower concentrations
observed in normal sera. Five slides were incubated with the same
mixture of proteins containing all but one biomarker and the other
five slides were incubated with only one antigen. The specificity
of our immunoassays is illustrated in the FIG. 11(A).
[0178] Four (Her-2, MMP-2, CA 15-3 and OPN) of the five biomarkers
diluted in PBS were able to be detected, simultaneously with high
sensitivity and specificity in the assays. However no signal was
observed for VEGF even at high concentrations. While VEGF could be
sensitively detected in a singleplex assay (FIGS. 8E and 9E) very
strong laser settings (power=100% and PMT gain 95%) had to be used.
This posed a problem in the multiplexed format where the other
biomarkers were sensitively detected at lower laser settings (70%
laser power and 75% PMT gain). Therefore, two scans were performed,
one at the lower setting to obtain fluorescence signals from the
four biomarkers and one at the higher setting to obtain fluorescent
signals from VEGF. No VEGF signal could be observed at the low
laser setting and at the higher laser setting, tremendously high
backgrounds were observed with reduced the signal to noise ratios
for all the biomarkers. Therefore, in a further effort to
selectively increase signal strength from VEGF, a biotinylated goat
anti-rabbit secondary antibody specific to the biotinylated rabbit
anti-VEGF antibody in the mixture was used. In this procedure, the
microarray was first incubated with the antigen mixture followed by
incubation with biotinylated antibodies to the five biomarkers,
Her-2, CA 15-3, MMP-2, OPN and VEGF. The microarray was
subsequently incubated with biotin-anti rabbit secondary antibody.
This step was introduced to specifically amplify the VEGF signal
since the biotinylated antibodies for the other 4 biomarkers were
prepared in goat or mouse as the host. However, this antibody
cross-reacted with the monoclonal capture antibodies for all the
biomarkers, thus generating very high background noise. Recombinant
antibodies engineered for affinity and specificity can in the
future improve the multiplexing capability of assays by eliminating
cross-reactivity and by increasing sensitivity. This would offer
the antibody arrays a flexible, quantitative range and thereby
increasing the pool of biomarkers that can be potentially assayed
simultaneously.
[0179] A multiplexed assay similar to the one described above was
developed, but replacing PBS with human serum as the medium. The
results from this assay are shown in FIG. 11 (B). In assays where
only one antigen was preset it was observed that the signal from
the other spots is not negative. This is also observed when a
particular antigen is left out of the mixture, as serum contains
these proteins in low concentrations unlike PBS, which contains no
protein at all. Also the overall background noise arising from
serum on the slide is much higher than the PBS arrays, similar to
the effect observed in the standard curves using serum.
Example 2
Sensitive Multiplexed Diagnostic Test for Breast Disease
[0180] The multiplexed protein microarray assay developed
previously is applied to the differential detection of four
biomarkers in breast cancer patient sera.
[0181] Materials and Methods
[0182] Microarray assay reagents and protocols are essentially
similar to those described above in Example 1. Recombinant
proteins, capture and biotinylated detection antibodies for Her-2,
MMP-2 and Osteopontin were purchased from R&D systems
(Minneapolis, Minn.). Other reagents used in the assay include:
VEGF antigen and capture and biotinylated detection antibodies
(Biosource International Camarillo, Calif.), CA 15-3 antigen and
anti-CA 15-3 capture and detection antibodies (Fitzgerald, Concord,
Mass.). The CA 15-3 detection antibody was biotinylated using a kit
and according to the manufacturer's (Pierce, Rockford, Ill.)
instructions. All other detection antibodies were purchased as
biotin conjugates. Lyophilized human serum was purchased from
Rockland Immunochemicals (Gilbertsville, Pa.). Streptavidin
conjugated Alexa 546 was purchased from Invitrogen--Molecular
Probes (Carlsbad, Calif.), GAPS II.TM. slides were purchased from
Corning LifeSciences (Corning, N.Y.) and BSA was purchased from
Sigma-Aldrich (St. Louis, Mo.). Sera from 41 metastatic breast
cancer patients, 33 breast cancer patients with early stage disease
and 39 controls were obtained from the Breast Cancer Serum
Resource, Lombardi Cancer Center (Washington, D.C.). Capture
antibodies used were: 1) Her-2 (R&D systems; Monoclonal
Anti-human ErbB2 Antibody; MAB-1129; Clone 191924), 2) MMP-2
(R&D systems; Monoclonal Anti-human MMP-2 Antibody; MAB-902;
Clone 36006.211), 3) CA 15-3 (Fitzgerald; Monoclonal Anti-human CA
15-3 Antibody; 10-C03; Clone M8071022), 4) Osteopontin (R & D
Systems; Monoclonal Anti-human Osteopontin Antibody; MAB-1433;
Clone 190312), and 5) VEGF (Biosource; VEGF purified mouse
anti-human; AHG011; Clone A183C-13G8). Detection antibodies used
were: 1) Her-2 (R & D Systems; Polyclonal Goat Anti-human ErbB2
Antibody; AF-1129), 2) MMP-2 (R & D Systems; Polyclonal Goat
Anti-human MMP-2 Antibody; AF-902), 3) CA 15-3 (Fitzgerald;
Monoclonal Anti-human CA 15-3 antibody; 10-C03B; Clone M8071021),
4) Osteopontin (R & D Systems; Polyclonal Goat Anti-human
Osteopontin Antibody; AF-1433), and 5) VEGF (Biosource; Polyclonal
Rabbit Anti-human VEGF Biotin Conjugated Antibody; AHG9119).
[0183] Microarray Spotting Protocol
[0184] Aminosilanated, GAPS II.TM. barcoded glass slides were
spotted with capture antibodies using a robotic arrayer (Norgen
Systems Inc.; Mountain View, Calif.) in quadruplicate at 500
.mu.g/ml to form a 4.times.4 array grid. Four print heads were used
to deposit approximately 1 nl of capture antibody solution,
generating a total of 8 arrays per slide with 250 .mu.m diameter
spots and with a spot-to-spot distance of 350 .mu.m. The spotted
slides were cross-linked under ultraviolet light for 5 minutes and
were stored in the dark at 4.degree. C.
[0185] Multiplexed Microarray Assay Protocol with Patient Sera:
Pilot Study
[0186] The eight arrays were separated from each other using
silicone gasket chambers (Schleicher & Schuell Bioscience,
Keene N.H.) and were blocked with 1 mg/ml BSA solution for 30
minutes followed by incubation with 50 .mu.l of patient serum
sample for 60 min. A total of 30 different patient serum samples
were used in this study (10 metastatic, 10 early stage and 10
control). This experiment was performed in duplicate, for control
and early stage patients, but replicates were not used for
metastatic patients due to limited availability of samples. Arrays
were then washed in PBS-T for 15 min followed by incubation with
detector antibody "cocktail" containing biotinylated antibodies for
all four biomarkers at a concentration of 3.75 .mu.g/ml for 60 min.
Streptavidin conjugated Alexa 546 was used as the reporter at a
concentration of 5 .mu.g/ml for 10 min. The chambers were then
removed and the slides were agitated in PBS-T for 10 minutes and
dried by centrifugation prior to scanning.
[0187] Multiplexed Microarray Assay Protocol with Patient Sera:
Larger Sample Set
[0188] Multiplexed capture antibody arrays were printed as
described in the above section. In this assay, two control
molecules were also included on the slide. Bovine serum albumin
(BSA) served as negative control (NC). Alexa 546 spots were used as
positive controls (PC). A total of 87 patient samples were used in
this study, 29 of which were metastatic, 29 were early stage and
the other 29 were control samples. The experiment was also planned
such that each slide contained all the three sample categories
(Control, Early stage and Metastatic) and each sample was split
into two aliquots, which were placed on different slides. This
arrangement accounted for inter-slide technical variation. The
microarray assay was performed in a manner similar to Example
1.
[0189] Microarray Assay Protocol: Standard Curves
[0190] In this case, the protein microarrays were incubated with 50
.mu.l of recombinant antigen diluted in human serum for 60 min,
after the blocking step with BSA. The concentrations of biomarkers
used to establish these standard curves were 1 ng/ml-128 ng/ml for
Her-2, 10 ng/ml-1280 ng/ml for MMP-2, 2 U/ml-256 U/ml for CA 15-3,
10 ng/ml-1280 ng/ml for OPN, and 1 pg/ml-1280 pg/ml for VEGF. Serum
without any added recombinant antigen was used to determine the
baseline levels of the biomarkers. The solution was aspirated and
washed with PBS-T for 15 minutes and the wells were then incubated
with 100 .mu.l of biotinylated antibody solution for 60 minutes.
Following a 15 minute wash, the arrays were incubated with 100
.mu.l streptavidin conjugated Alexa 546 for 10 minutes. The
chambers were then removed and the slides were agitated in PBS-T
for 10 minutes and dried by centrifugation prior to scanning.
[0191] Microarray Imaging and Analysis Protocol
[0192] Slides were imaged with ScanArray 5000 XL (Perkin Elmer,
Wellesley, Mass.), which is a laser-based confocal scanner, at 543
nm excitation. Images collected onto a PC were analyzed by
Scanarray Express.TM. software (Perkin Elmer, Wellesley, Mass.).
Raw intensities for each spot were computed by taking the average
of the logarithm of the intensity over all pixels in the region of
interest that were greater than zero for the spot. Median
fluorescent intensities of all four spots for each biomarker were
then computed for all patient samples. Principal component analysis
was used to linearize the four dimensional data obtained from the
measurement of four biomarker levels. Linear discriminant analysis
was used to measure the accuracy of classification of unknown
samples into appropriate disease categories (control, early stage
or metastatic) based on biomarker levels. Both of these statistical
functions were carried out using MATLAB.
[0193] Results
[0194] Antibody arrays were prepared by spotting multiple capture
antibodies on amine-modified glass slides. These arrays were
incubated with 50 .mu.l breast cancer patient serum samples that
were arranged on the slides as shown in FIG. 12. The notation C
stands for control populations, E for early stage patients and M
for patients with metastatic disease. A total of 30 patient sera
were used; 10 controls, 10 early stage and 10 metastatic. The
control and early stage samples were used in duplicate aliquots and
the metastatic samples were used in single aliquots due to limited
amount of sample.
[0195] FIG. 13 shows the quantification of the fluorescent
intensities using box plots, which is an efficient method for
displaying a five-point summary of the data. Median fluorescent
signals were obtained for each of the biomarkers and their log (to
the base 2) values were plotted in the FIG. for each disease state.
This demonstrated the effect of disease state on individual marker
concentrations in serum. The upper boundary or hinge of the box
represents the 75.sup.th percentile of the data and the lower
boundary represents the 25.sup.th percentile of the data. Thus, the
box represents the middle 50% of the data and this region is called
the inter-quartile range. The dot inside the box represents the
median value of the data. In this case, the data is skewed since
the median value is not equidistant from the hinges. The ends of
the vertical lines or whiskers indicate 1.5 times the value of the
inter-quartile range, while the spots outside the whiskers denote
the outliers.
[0196] An increasing trend of fluorescent signal from control
samples to metastatic samples for the two biomarkers CA 15-3 and
Osteopontin was observed. For the other two biomarkers, however,
this trend is more subtle. This assay was planned such that each
slide contained all control, early stage, or metastatic samples.
This allowed measurement of technical variations within a slide
only for one data set, and not across the three cancer groups.
Since this assay did not include any controls on the slides, it was
difficult to measure and account for slide-slide variation.
Therefore, an accurate measure of fluorescent response to disease
state could not be made. Using a total of 30 patient samples,
however, showed promise for at least two of the biomarkers in as a
diagnostic test. We expanded this study by including more samples
in hopes of improving the sensitivity for Her-2 and MMP-2 as well
as to confirm the results obtained for CA 15-3 and Osteopontin.
[0197] Multiplex Biomarker Study Using Patient Sera
[0198] In the breast cancer patient pilot study, the layout of the
samples on the slides made it difficult to separate the effects of
slide-to-slide variation from true differences in biomarker levels
among normal subjects and patients with early or metastatic breast
cancer. Differences were seen between metastatic and control sera
for CA 15-3 and OPN only upon ignoring the effect of slide-slide
variation. Thus, an additional study was designed in a different
manner, to account for these variables. In this assay, antibody
arrays were prepared similar to those described above, except that
two additional controls were included on the slide. Bovine serum
albumin (BSA) served as negative control (NC). Alexa 546 spots were
used as positive control (PC). The sample size was increased to 87
patient samples in this study to include 31 metastatic, 23 early
stage and 29 control samples. FIG. 14 shows the layout of this
experiment, which was planned such that each slide consisted of a
combination of control, early stage, and late stage samples. The
sample was split into two aliquots, assigned to two arrays, on
different slides. The samples were randomly assigned to the eight
wells. On the last two slides (21 and 22) blank samples were
included to account for the background fluorescent signals. This
arrangement helped to account for the various technical variations
that were not calculated in the pilot study.
[0199] FIG. 15 shows box plot representations of the fluorescent
signals obtained for the four biomarkers as well as controls on the
22 experimental slides. The positive control (Alexa-546) spot
fluorescence remained at a constant high level across all the
experimental slides and the negative control (BSA) spot
fluorescence was consistently low across all 22 slides. An
increasing trend of fluorescent signal from control samples to
metastatic samples was observed for all of the four biomarkers. The
multiplexed microarray assay was able to distinguish between
control, early stage, and metastatic populations for all the
biomarkers with a fairly high accuracy (p value<0.05). Samples
which contained only blank blocking grade human serum (slides 21
and 22) were used to measure the biomarker background noise levels.
In the FIG., this background value matches that observed for the
negative control (BSA) spots. NC=Negative control, O=OPN,
PC=Positive control, C=CA 15-3, H=Her-2, and M=MMP-2.
[0200] Her-2 Status of Patients
[0201] Her-2 gene is amplified in about 30% of all breast cancers.
HER2-positive breast cancers tend to be more aggressive than other
types of breast cancer and they are also less responsive to hormone
treatment. Trastuzumab (Herceptin) is a monoclonal antibody drug
that targets HER2 and is used as an effective form of treatment for
Her-2 positive breast cancer patients. This is shown to slow the
growth of the cancer and even decrease its size. Herceptin can be
used as a treatment by itself or combined with chemotherapy.
Herceptin is also shown to reduce breast cancer recurrence by as
much as 50 percent, thereby demonstrating a high rate of success in
the patient survival. Detecting the Her-2 status of breast cancer
patients, therefore, has become a routine and crucial step in
treatment decision making. The standard procedure for determining
whether a patient is Her-2 positive involves the use of
Fluorescence In Situ Hybridization (FISH) to detect the over
amplified Her-2 gene. Sometimes, an ELISA is performed to measure
serum Her-2 levels, in which case, the patient is said to be Her-2
positive if the serum Her-2 level is higher than 15 ng/ml. The
Her-2 status information was obtained for some of the patient serum
samples from the Lombardi cancer center's serum repository and
compared it to the measured Her-2 levels in sera of the same
patients using the multiplexed microarray assay. Using the same
rule as the ELISA, a patient serum sample was declared as Her-2
positive if the Her-2 levels were measured above 15 ng/ml. The
results (shown in Table 1) show a 100% correlation in the
conclusions of Her-2 status derived from our multiplexed assay and
by traditional FISH or ELISA methods (as obtained from the Lombardi
Cancer Center). While both these methods consume large (a few
hundred microliters) quantities of sample and reagents and require
anywhere between 8-24 hours to produce results, the microarray
assay was performed with 50 ml of serum sample in 3 hours. The
value of the multiplexed assay lies in the fact that the panel of
biomarkers could not only be used for disease diagnosis, but also
to simultaneously provide valuable information about treatment
options for the patient.
TABLE-US-00001 TABLE 1 Her-2 status of sample Sample Stage of
Lombardi protein Measured # Cancer Cancer Center microarray Her-2
level 1 Early stage - - 9.62 2 Early stage - - 12.54 3 Early stage
- - 12.38 4 Early stage - - 13.47 5 Early stage - - 7.42 6 Early
stage - - 5.37 7 Early stage - - 9.03 8 Early stage - - 10.21 9
Early stage + + 17.44 10 Early stage + + 19.77 11 Early stage + +
24.07 12 Early stage + + 23.62 13 Early stage + + 19.32 14
Metastatic - - 11.62 15 Metastatic - - 13.79 16 Metastatic + +
45.73 17 Metastatic + + 46.82 18 Metastatic + + 49.15 19 Metastatic
+ + 13.43
[0202] Quantitation of Biomarker Levels in Patient Sera from
Fluorescent Signals
[0203] Antibody microarrays were used to generate standard curves
to quantify the biomarkers in patient serum samples. These curves
were obtained in a similar manner to the experiment described
above, except that these curves were obtained on the same day and
using the same set of slides as the patient samples, to minimize
technical error. Capture antibodies to the four biomarkers were
printed on modified microarray slides using a robotic arrayer such
that each antibody was present in quadruplicate. Slides were then
incubated with 8 serial dilutions of recombinant antigen diluted in
human serum. The concentration ranges used were 1 ng/ml-128 ng/ml
for Her-2, 10 ng/ml-1280 ng/ml for MMP-2, 2 U/ml-256 U/ml for CA
15-3 and 10 ng/ml-1280 ng/ml for OPN. All four detector antibodies
were used in this experiment to simulate the multiplexed assay with
the patient samples. Standard curves were obtained for each
biomarker by quantifying the fluorescence from the arrays and
plotting the median values as a function of antigen concentration.
This can be seen in FIG. 16 (A-D). The standard curves were used to
quantify the fluorescent intensities from patient serum samples
assayed on the multiplexed arrays. This demonstrates the
sensitivity and accuracy of our multiplexed microarray assay.
[0204] Relationship Between Use of Multiple Biomarkers and Accuracy
of Diagnosis
[0205] Although box plots shown above provide information about the
distribution of the data for each individual biomarker, they are
unable to provide information about all four biomarkers
simultaneously. This method also does not address the hypothesis
that multiple markers working in synergy provide a more sensitive
diagnosis of breast cancer. Thus, Principal Component Analysis
(PCA) was used to visualize the data from all four biomarkers in
one single plot. PCA is a classical statistical method that reduces
the dimensionality of a data set while retaining as much
information as is possible. It performs a linear transformation
that chooses a new coordinate system for the data set such that the
greatest variance of the data set lies on the first axis (called
the first principal component), the second greatest variance on the
second axis, and so on. It can be viewed as a rotation of the
existing axes to new positions in the space defined by the original
variables. There can be as many principal components as there are
variables. The first principal component accounts for much of the
variability in the data, and each succeeding component accounts for
the remaining variability. Principal components were computed for
the data set that contains fluorescence signals from four
biomarkers. FIG. 17 shows a scatter plot of first principal
component on the X-Axis and the second principal component on the
Y-Axis. Each vector of the principal components corresponds to a
patient serum sample, displayed in the FIG. as control populations
in green, early stage patients in pink and metastatic patients in
blue. It can be seen that the three groups are well separated with
little or no overlap. This demonstrates that the four biomarkers
are able to distinguish accurately between control populations and
patients with early stage and late stage breast cancer.
[0206] For a diagnostic test, accuracy of classification is an
important factor in its success. To measure the accuracy of
classification of the test and to study the effect of number of
biomarkers on this accuracy, Linear Discriminant Analysis was
performed on the data set. This technique is useful for detecting
the variables that allow the discrimination between different
naturally occurring groups (e.g., breast cancer stages), and for
predictive classification of unknown cases into their correct
groups, establishing the robustness of this discrimination. This
analysis was performed by dividing the data set into two parts. The
first half was used as a training set and the second part was used
as a test set. A linear discriminant function was built for the
training set of the data and used the test set for cross-validation
of predictive classification. This was used to produce unbiased
estimates of chance of misclassifying the test set into the
biological groups defined by the training set. This test was
performed for all possible combinations of the four biomarkers to
see the effect of number of biomarkers in the panel on the accuracy
of classification. FIG. 18 illustrates the percentage of
misclassifications occurred when the data was analyzed using
various combinations of biomarkers. O=OPN, C=CA 15-3, H=Her-2, and
M=MMP-2. It is observed that when only one single biomarker is
used, the error of classification can be as high as 30%. When two
biomarkers are used together, this error is reduced to
approximately 24.35%. Adding another biomarker further reduces this
error to approximately 18.5% and finally including all four
biomarkers results in a 16.5% error. The reduction in error of
classification is drastic from one to two biomarkers, but this
difference starts to plateau when three and four biomarkers are
included. Thus, it is possible that while multiple biomarkers do
improve the accuracy of a diagnostic test, too many markers, can
not provide any additional information. This is however, also
determined by the biological nature of the biomarker and its
physiological role in breast cancer. It should be noted that in
particular the errors for CA 15-3 and Osteopontin are much less
than those for Her-2 and MMP-2.
Example 3
Development of a Multiplexed Flow-Based Detector
[0207] A flow based platform was built with a unique custom flow
channel device onto which, the multiplexed assay was translated
from the protein microarrays. Below, the development of the device
is described. A new flow-based immunoassay system for the
simultaneous and rapid quantification of multiple analytes without
several processing steps is described. This example also discusses
the details of the device design, which is based on the standard
microarray sandwich immunoassay format, except that the static
incubation step was replaced with flow of the analyte mixture over
the antibody array. Multiple steps have also been eliminated
(including washing) by mixing all the assay reagents in
predetermined concentrations in one single sample. In addition, a
benchtop version of a portable imaging system was developed,
comprising a miniature uncooled CCD camera and a Xenon arc lamp.
This method demonstrated rapid quantitative measurement and
specific identification of analytes in complex samples with minimal
intervention.
[0208] Materials and Methods
[0209] Microarray Flow Channel Assay and Lateral Flow Assay
Components
[0210] Her-2 and Osteopontin protein, capture and biotinylated
detection antibodies as well as MMP-2-specific capture and
biotinylated detection antibodies were purchased from R&D
systems (Minneapolis, Minn.). Other reagents used in the assay
include: CA 15-3 antigen and anti-CA 15-3 capture and detection
antibodies (Fitzgerald, Concord, Mass.), MMP-2 Proenzyme (EMD
Biosciences, San Diego, Calif.), biotin-BSA (Pierce Biotechnology,
Rockford, Ill.) and Streptavidin Quantum Dot 605, Streptavidin
Quantum Dot 585, Goat-anti-mouse-Quantum Dot 605 and Quantum Dot
incubation buffer (Quantum Dot Corp (Invitrogen), Hayward, Calif.)
and Strepavidin Alexa 546 (Molecular Probes Invitrogen Corp.
Carlsbad, Calif.). Phosphate Buffered Saline (PBS; 50 mM potassium
phosphate, 150 mM NaCl; pH 7.4) and Phosphate Buffered Saline with
0.05% Tween 20 (PBS-T) and BSA were obtained from Sigma Aldrich
Corp. (St. Louis, Mo.). The CA 15-3 detection antibody was
biotinylated using a kit and according to the manufacturer's
instructions (Pierce Biotechnology, Rockford, Ill.). All other
detection antibodies were purchased as biotin conjugates.
Lyophilized human serum was purchased from Rockland Immunochemicals
(Gilbertsville, Pa.). Sera from metastatic and early stage breast
cancer patients and controls were obtained from the Breast Cancer
Serum Biomarkers Resource, Lombardi Cancer Center (Washington,
D.C.). The nitrocellulose membrane (NC) HF180, polystyrene clear
backing and conjugate pad were from Millipore Corp. (Watertown,
Mass., USA). GAPS II.TM. glass slides for the microarrays were
obtained from Corning Lifesciences (Corning, N.Y.) and the silicone
chambers from Grace Biolabs (Bend, Oreg.). Capture antibodies used
were: 1) Her-2 (R&D systems; Monoclonal Anti-human ErbB2
Antibody; MAB-1129; Clone 191924), 2) MMP-2 (R&D systems;
Monoclonal Anti-human MMP-2 Antibody; MAB-902; Clone 36006.211), 3)
CA 15-3 (Fitzgerald; Monoclonal Anti-human CA 15-3 Antibody;
10-C03; Clone M8071022), 4) Osteopontin (R & D Systems;
Monoclonal Anti-human Osteopontin Antibody; MAB-1433; Clone
190312), and 5) VEGF (Biosource; VEGF purified mouse anti-human;
AHG011; Clone A183C-13G8). Detection antibodies used were: 1) Her-2
(R & D Systems; Polyclonal Goat Anti-human ErbB2 Antibody;
AF-1129), 2) MMP-2 (R & D Systems; Polyclonal Goat Anti-human
MMP-2 Antibody; AF-902), 3) CA 15-3 (Fitzgerald; Monoclonal
Anti-human CA 15-3 antibody; 10-C03B; Clone M8071021), 4)
Osteopontin (R & D Systems; Polyclonal Goat Anti-human
Osteopontin Antibody; AF-1433), and 5) VEGF (Biosource; Polyclonal
Rabbit Anti-human VEGF Biotin Conjugated Antibody; AHG9119).
[0211] Preparation of LFA Strip
[0212] Similar to the conventional immunochromatographic strips,
the LFA comprised a nitrocellulose membrane for separation and
detection of analytes, a conjugate pad to collect the
antibody-antigen complex, a wicking pad for the generation of
capillary action, and a plastic backing card for the protection of
the strip (see FIG. 19). Nitrocellulose membrane, the conjugate
pad, and the absorption pad were laminated on the adhesive side of
the polyester backing card. Capture molecules were dispensed and
immobilized on the nitrocellulose membrane to form the detection
zone using a precision-dispenser (Bio-Dot, Irvine, Calif.). The
width of the dispensing line was 0.5-1 mm and the diameter of spots
was 0.5 mm (20 nl) and 0.25 mm (10 nl) with a vertical spacing of
1.5 mm and horizontal spacing of 4 mm. 5 mm strips were cut using
the guillotine cutter (Bio-Dot, Irvine, Calif.) to form individual
tests.
[0213] LFA Assay Protocol
[0214] We adapted the principle of immunochromatographic assay for
the analysis of multiple analytes in this study. Instead of 40-nm
gold particles as used in conventional immunochromatography,
Quantum Dots (QD) were used as the signaling molecule, which is
bound to the detector antibody through a biotin-streptavidin bond.
Fluorescent immunoassays were performed on these customized lateral
flow membranes. In order to demonstrate proof of principle in the
LFA system, biotin-BSA was spotted as a capture molecule onto
nitrocellulose strips in both line (1 mm) and spot (20 nl and 10
nl) formats. Streptavidin-conjugated quantum dot solution was then
wicked through the biotin-BSA.
[0215] To demonstrate the principal of multiplexed LFAs, the
nitrocellulose membrane was spotted with biotin-BSA (500 .mu.g/ml
and mouse IgG .mu.g/ml) as capture molecules and a mixture of
Streptavidin-conjugated QD 585 (10 nM) solution and Goat anti mouse
conjugated QD 605 (10 nM) solution was then wicked through the
membrane. Resulting fluorescent signals due to the accumulation of
quantum dots at these sites were observed on a UV transilluminator.
For membranes that were spotted with capture antibodies,
sandwich-type fluorescent immunoassays were performed on customized
lateral flow membranes. When an analyte is first mixed with the
quantum dot-linked detector antibody and then applied to the
nitrocellulose membrane, the analyte-detector antibody-quantum dot
complex moves from the sample pad toward absorption pad. During the
propagation, the complex encounters the immobilized capture
antibodies on the nitrocellulose membrane and forms sandwich-type
detector-analyte-capture antibody complexes. This fluorescence was
observed on the UV transilluminator.
[0216] Preparation of Microarray Channels
[0217] Unique flow channels were designed that allow for simple
capillary flow of assay reagents without the need for complex
fluidics and mechanical parts. Antibody microarrays were printed
similar to traditional microarrays using a robotic arrayer (Norgen
Systems Inc.; Mountain View, Calif.). Two print heads were used to
deposit approximately 1 mL of capture antibody solution, generating
a total of 2 arrays per slide with 225 .mu.m diameter spots with a
spot-to-spot distance of 350 .mu.m. The layout of each 8.times.12
array of printed antibody spots corresponded to one spot per well
in a standard 8.times.12 (96-well) format. These capture antibodies
were printed in quadruplicate at a concentration of 1 mg/ml. Also
printed on each slide were two controls. Bovine serum albumin (BSA)
served as negative control (NC) and Alexa 546 was used as positive
control (PC). The spotted slides were cross-linked under
ultraviolet light for 5 minutes and were stored in the dark at
4.degree. C. This scheme yields two channels per slide. As shown in
FIG. 1, the channels are created by using adhesive silicone
supports (0.5 mm height) and a glass cover slip facilitating fluid
exchange through an inlet and an outlet (length=3 cm and width=1
cm). This helps draw fluids onto the immobilized antibody
microarrays through capillary action. The fluid flow stops at the
end of the channels at which point, it can be wicked with an
absorbent material, the porosity of which can used to control the
flow rate through the channels and hence the assay time.
[0218] Channel Assay Protocol Using Recombinant Antigen
[0219] For demonstrating proof of principle of quantitative
detection on the microarray channels, protein microarray slides
were printed with biotin-BSA in a series of 4 dilutions from 500
.mu.g/ml to 62.5 .mu.g/ml. The two arrays on the glass slide were
separated using silicone channels as described above and were
blocked with 1 mg/ml of BSA solution in PBS for 30 minutes. This
solution was wicked and the arrays were allowed to air dry for two
minutes. 200 .mu.l of 10 nM streptavidin QD 605 was drawn into the
channels and the fluid was completely wicked at the other end. The
flow channels were removed and arrays were washed and air dried
prior to scanning.
[0220] To demonstrate the multiplexing capability of the channel
flow assays, two different capture molecules were immobilized;
biotin-BSA at 500 .mu.g/ml and mouse IgG at 500 .mu.g/ml.
Unconjugated BSA was used as a negative control. The microarrays in
the channels were blocked with BSA at 1 mg/ml for 30 minutes
followed by incubation with a streptavidin QD 605 and Goat
anti-mouse IgG--QD 605 for 2 minutes in four different
configurations. In the first set, no reporter was used, in the
second set, both streptavidin QD 605 and Goat anti-mouse IgG-QD 605
were; in the third set, only streptavidin QD 605 and in the final
set, only Goat anti-mouse IgG-QD 605 was used. The flow channels
were removed and arrays were washed and air dried prior to
scanning.
[0221] To demonstrate the sensitivity of the flow channels,
standard curves were obtained for the four biomarkers of interest
using sandwich immunoassays. A mixture of biotinylated detector
antibody (at 10 .mu.g/ml) and streptavidin-linked Alexa 546 (at 7
.mu.g/ml) was added to serial dilutions of recombinant antigens
prepared in phosphate buffered saline (PBS) and human serum. The
antigen concentration ranges used were 6.25-100 ng/ml for Her-2,
62.5-1000 ng/ml for MMP-2, 9.4-150 U/ml for CA 15-3 and 94-1500
ng/ml for OPN. 200 .mu.l of the sample mixture was added to the
entry of the channels. Capillary forces helped wick this fluid into
the channels over the printed capture antibodies for binding.
Samples were added to duplicate arrays across different slides to
account for technical variations. Following the appropriate
incubation time, the fluid was completely wicked and the flow
channels were removed. The chambers were then removed and the
slides were agitated in wash buffer for 5 minutes and air dried
prior to imaging.
[0222] To measure the specificity of the flow channel assays, a
multiplexed assay was performed essentially as described for the
standard titration curves. Capture antibodies for Her-2, MMP-2, CA
15-3 and OPN were spotted in quadruplicate at 500 .mu.g/ml on the
GAPS II.TM. slides to form a 4.times.4 array grid. A total of eight
different samples were prepared, four of which contained a mixture
of all but one antigen and four of the remaining samples contained
only one antigen each. The concentrations of recombinant antigens
diluted in human serum were 20 ng/ml Her-2, 800 ng/ml MMP-2, 130
U/ml CA 15-3 and 900 ng/ml OPN. Antibody microarrays were incubated
with these antigen samples mixed a detector antibody "cocktail"
containing biotinylated antibodies for all five biomarkers at a
concentration of 15 .mu.g/ml. Streptavidin conjugated Alexa 546 was
used as the reporter at a concentration of 10 .mu.g/ml. The samples
were assayed in duplicate. The chambers were then removed and the
slides were agitated in PBS-T for 10 minutes and dried by
centrifugation prior to scanning.
[0223] To determine the optimum speed of the assay without
compromising the assay sensitivity, a multiplexed assay was
performed, essentially as described above. Two sets of samples were
prepared. In the first case, the antigens were used at
concentrations known to be present in metastatic cancer patients
(30 ng/ml for Her-2, 850 ng/ml for MMP-2, 150 U/ml for CA 15-3 and
875 ng/ml for Osteopontin) and in the second case; the antigens
were used at concentrations known to be present in control patients
(8 ng/ml for Her-2, 600 ng/ml for MMP-2, U/ml for CA 15-3 and 440
ng/ml for Osteopontin). 4 aliquots of 200 .mu.l of the sample
mixture was added to duplicate arrays and incubated for 7 min, 15
min, 30 min and 60 min respectively before sample was wicked. The
chambers were then removed and the slides were agitated in wash
buffer for 5 minutes and air dried prior to imaging.
[0224] Channel Assay Protocol: Serum Samples
[0225] Two types of experiments were performed to measure the
response of the flow channels to the protein biomarkers in the sera
of patients with breast cancer. In the first study, 80 .mu.l of
sera from each of the 10 metastatic breast cancer patients and 80
.mu.l of sera from each of the 10 control subjects was pooled to
obtain a total of 800 .mu.l of metastatic breast cancer sample and
800 .mu.l of control sample. 80 .mu.l of this pooled sample was
wicked across 10 arrays in duplicate for 7 min and 15 min
respectively. The chambers were then removed and the slides were
agitated in wash buffer for 5 minutes and air dried prior to
imaging. This helped estimate the technical variations in the
assay. To measure the biological variation, 80 .mu.l of patient
sera from 6 metastatic breast cancer patients and 6 control
subjects was then mixed with 60 .mu.l of biotinylated detector
antibody cocktail and 60 .mu.l of streptavidin Alexa 546 reporter
to yield a final concentration of 10 .mu.g/ml for the antibodies
and 7 .mu.g/ml for the reporter. This sample mixture was added to
the arrays and incubated for 15 minutes before sample was wicked.
The chambers were then removed and the slides were agitated in wash
buffer for 5 minutes and air dried prior to imaging. Statistical
comparison of the biomarker levels in breast cancer patients and
controls was undertaken using a t-test and a probability value of
was obtained using MATLAB software.
Bench Top Imaging System
[0226] As a gold standard imaging system, the ScanArray.TM. 5000 XL
(PerkinElmer, Inc.; Wellesley, Mass.) was used at 543 nm
excitation. This is a benchtop, laser-based confocal scanning
device with a photomultiplier tube (PMT) for sensitive fluorescence
detection. Images collected onto a PC were analyzed by
QuantArray.TM. software. Raw intensities for each spot were
computed by taking the average of the logarithm of the intensity
over all pixels in the region of interest that were greater than
zero for quadruplicate spots on a slide and across duplicate
chambers resulting in a total of eight spots per sample for
analysis.
[0227] A imaging system was also developed that supports a charge
coupled device (CCD) camera as this technology is more amenable to
building smaller portable instruments. The arrangement of this
imaging system is shown in FIG. 2 and it consists of a
scientific-grade 16-bit, 1392.times.1040 pixel CCD camera (Lumenera
Corp. MA), which is configured for Kohler epi-illumination of the
sample microarray. In this case, the fluorescent sample is
illuminated from the front, while simultaneously being imaged from
the same side by the CCD camera. Excitation light from a full-field
White Lite.RTM. light 300 W xenon arc lamp was bandpass filtered
using a 525 nm excitation filter (Omega Optical Inc, Vt.) and
focused uniformly on the sample using a set of two optic fiber
cables (mellesgriot) held at an angle of 45 degrees. The
fluorescent spots were focused onto the CCD using a camera lens
(Infinimite.RTM. alpha, Edmund Optics) and filtered using a 600 nm
longpass filter. Custom algorithms, built within the Lumenera
camera software corrected for CCD dark noise. Images saved in tiff
format were analyzed using the Scanarray Express.TM. software
(Perkin Elmer, Wellesley, Mass.).
[0228] Results
[0229] A method was developed that requires the addition of only
one fluid sample and can therefore measure multiple biomarkers
simultaneously in one simple process. This technology showed a
potential to facilitate common use of antibody microarray in
medical and scientific field for high-throughput detection of a
wide variety of analytes.
[0230] Lateral Flow Assays
[0231] To achieve rapid, multiplexed detection, an LFA (as shown in
FIG. 19) was developed which combines the multiplexed, quantitative
advantages of the protein microarrays and the assay speed and
simplicity of traditional LFA. Multiple capture molecules were
spotted onto the nitrocellulose membrane, and the reporter mixture
was applied to the conjugate pad at one end of the membrane. This
complex was drawn through the membrane by capillary action, where
the markers were captured by their respective ligands. This simple,
yet rapid method required the addition of only one fluid sample
without the need for washes. Fluorescent QD nanocrystals were
employed as the reporter since they have an added advantage of
being multiplexed, yet quantitative. The use of spectrally
different QDs as well as spatial separation of these two capture
molecules enables reliable multiplexed detection in this lateral
flow format.
[0232] To demonstrate the proof of concept of multiplexed lateral
flow assays, two test analytes (Biotin-BSA and mouse IgG) were
detected on a single LFA. As shown in FIG. 20, when a mixture
containing streptavidin conjugated QD 585 and Goat anti-Mouse
conjugated QD 605 (both at 10 nM) is added to the conjugate pad
(left side of the FIG.), it flows through the pores of the
nitrocellulose membrane, to the capture zones where the
streptavidin conjugated QD 585 binds to the biotinylated BSA and
Goat anti-Mouse conjugated QD 605 binds to the mouse IgG
respectively. Since the capture molecules are fixed on the
membrane, the reporters continuously accumulate on the capture
zone. This generates a signal proportional to the amount of
immobilized capture molecules. FIG. 20(A) demonstrates a strip with
biotin-BSA spotted as two consecutive 1 mm (100 nl) lines along the
length of the nitrocellulose membrane at a concentration of 500
.mu.g/ml and at 125 .mu.g/ml in FIG. 20(B). In order to measure
multiple analytes along the strip, it is important that the flow
not be obstructed by the capture molecules. It is observed that
with a capture concentration of 500 .mu.g/ml very little signal is
obtained from the second line. However, when the concentration of
the capture molecule is reduced to 125 .mu.g/ml, some signal is
observed on the second line. When the concentration is kept high
(500 .mu.g/ml), but the dispensing volume is reduced to 20 nl, five
columns of capture molecule can be observed (FIG. 20(C)). However,
in this case, a reduction of signal intensity is observed in the
direction of sample flow. When this capture volume is reduced to 10
nl (FIG. 20(D)), this gradation disappears and the spots have
uniform fluorescence signal along the length of the membrane. The
result suggested the possibility that multiple target proteins
could be detected by adjusting the amount of capture antibody on
the strip. To demonstrate this multiplexing capability of the LFA,
one column (3 spots) containing biotin-BSA was immobilized and a
second column (3 spots) containing mouse IgG (FIG. 20(E)). Two
spectrally distinct sets of QDs were used for this assay and the
mixture containing the two different detectors is accurately
resolved on the LFA demonstrating its multiplexing capability.
[0233] The microarray LFA was then employed for the detection of
the panel of breast cancer biomarkers including Her-2, CA 15-3 and
Osteopontin. Monoclonal capture antibodies to the four antigens
were immobilized on the nitrocellulose membrane as 10 nl spots.
This spotting was done in six schemes such that the sequence in
which the sample encountered the capture molecules was different in
each assay. A mixture of antigens and biotinylated detector
antibody labeled with streptavidin QD 605 as deposited on the
conjugate pad. The concentrations of reagents used in this assay
are Her-2 5 .mu.g/ml, CA 15-3 500 U/ml and Osteopontin 3 .mu.g/ml,
biotinylated detector antibodies 30 .mu.g/ml and strepavidin QD 605
10 nM. The solution was allowed to wick through the membrane and
the spot fluorescence was observed on the UV transilluminator.
Images were captured using a digital camera and displayed in FIG.
21, which shows that it is possible to observe signals from three
antigens in this multiplex format. The signal to noise ratio was
best for the case (C) and (E) where Her-2 is spotted at the far
right hand side of the membrane. Although high concentrations of
antigens were used, signal was barely observed above the high
background in the membrane.
[0234] Accumulation of fluorescent quantum dot in the pores of the
nitrocellulose membrane, as well as the high concentration of
reagents in this assay cause a high background noise in the LFA.
The LFAs had much lower sensitivity than that of the microarrays.
The concentrations of capture and biotinylated detector antibodies
were 8 fold and 6 fold higher than those used in the protein
microarrays respectively, making the assay very expensive. Although
the Her-2, CA 15-3 and OPN were detected in a multiplexed format,
MMP-2 assay did not produce any signals on the LFA. The detection
limits of the LFA for the antigens were approximately 10 fold
higher than protein microarrays.
[0235] The reagents for the multiplexed assay were optimized
specifically for the protein microarray platform as discussed
above. By optimizing a brand new set of reagents for the LFA, it
could improve the sensitivity of the assay as well as make MMP-2
work with the panel of biomarkers. The use of a membrane substrate
with high affinity to proteins made LFAs prone to very high
background noise. This is partially due to the fact that LFAs do
not involve wash steps, and that the flow of analyte solution is
through the membrane and not simply above the surface. This offers
a three dimensional matrix to which the analyte, detector antibody
and reporter complexes can bind non-specifically. Unlike the
traditional Western Blots, ELISAs or Microarrays, the blocking
agents employed to minimize the background levels bind to the
membrane matrix and offer resistance to the flow of analyte through
membrane. Additionally, such additives can displace capture
reagents from the membrane, thereby, reducing assay
sensitivity.
[0236] Channel Flow Assays
[0237] To design a new platform for rapid immunoassays, the
antibody arrays were printed on a glass slide and instead of the
static incubation chambers, unique flow channels were designed that
cover individual arrays, and allow for passive flow of analyte
mixture over the immobilized array. Channels are made by using
adhesive silicone supports and a glass cover slip. This helps draw
fluids onto the immobilized antibody microarrays through capillary
action. Fluid flow enhances the kinetic interaction between the
analyte and the immobilized ligand thereby overcoming the diffusion
limitation of the incubation assays and reducing assay time. Since
the flow is over the arrays and not through a three dimensional
matrix as in the case of LFAs, the arrays can be treated with
blocking agents to minimize background noise. This provides a
rapid, simple, yet multiplexed platform to measure protein
biomarkers in serum samples.
[0238] A mixture containing sample, detector antibody and
fluorescent reporter at predetermined concentrations was added to
the protein array. Capillary forces directed this fluid into a
chamber over the printed capture antibodies for binding. The fluid
was then wicked from the other end of the channel using absorbent
material. Flow rate through these capillary channels was controlled
by choosing appropriate wicking material. This also ensures
unidirectional flow of the sample. The protein biomarkers are
quantified by an optical reader as the fluorescence of the spots is
proportional to the analyte concentration. This technique enabled
the rapid measurement of multiple protein biomarkers under flow
conditions. The speed of the assay offers considerable advantages
over more conventional antibody microarrays that require long
incubation times.
[0239] To demonstrate the proof of principle of quantitation using
the channels, biotin-BSA was immobilized in a series of 4 dilutions
from 500 .mu.g/ml to 62.5 .mu.g/ml. Streptavidin QD at 10 nM was
used as reporter. The arrays were imaged and the fluorescent
intensities of the spots was quantified and plotted in the FIGS.
22(A and B, respectively). The spots show an increase in
fluorescent intensity with increased concentration. A linear
response was observed in this assay demonstrating the principle
that quantitative standard curves can be obtained using the
microarray channel flow device. To demonstrate the multiplexing
capability, two different capture molecules were used in
quadruplicate (shown as columns in the FIG. 22(C)); biotin-BSA at
500 .mu.g/ml and mouse IgG at 500 .mu.g/ml. BSA was used as the
negative control. Both spots and were washed with a mixture of
streptavidin QD 605 (10 nM) and Goat anti-mouse IgG--QD 605 (10 nM)
for 2 min. Observed in the FIG. 22(C) are four sets of spots. In
the first set on the left, no reporter was used and we see no
signal. In the second set, both streptavidin QD 605 and Goat
anti-mouse IgG--QD 605 were used and fluorescence is observed in
both the biotin-BSA and Mouse IgG spots. In the third set, only
streptavidin QD 605 and therefore only the biotin-BSA spots show
fluorescence. In this final set, only Goat anti-mouse IgG--QD 605
is used resulting in fluorescence only in the Mouse IgG spots. This
demonstrated the specificity of the microarray channels and its
ability to measure two different analytes simultaneously.
[0240] The biotin-BSA and Mouse IgG spots were observed with "comet
tails" or streaks in the direction of flow in the channel. Since
this assay was designed purely for the demonstration of proof of
principle, the concentrations of the reagents were not optimized.
As a result, too much capture molecule was deposited onto the glass
surface, resulting in excess unbound ligand, which bound to the
fluorescent reporter molecules in solutions and were smeared on the
glass surface generating a streaking effect. To optimize spot
morphology and minimize streaking, capture molecule concentrations
should be optimized.
[0241] Flow Channel Standard Curves with Quantum Dots
[0242] Standard curves were generated on flow channels by printing
capture antibodies to the four protein biomarkers such that each
antibody was present in quadruplicate within one channel. These
arrays were incubated with 6 serial dilutions of recombinant
antigen diluted in human serum. Standard curves were obtained on
the protein microarray format for each biomarker. FIG. 23 shows a
composite image of six different arrays with each column
representing a different array that was incubated with increasing
antigen concentration from right to left. Slides shown in Panel A
were incubated with Her-2 at concentrations ranging from 6.25 ng/ml
(right) to 100 ng/ml (left). Slides shown in Panel B were incubated
with MMP-2 with a concentration range of 62.5 ng/ml (right) to 1000
ng/ml (left). Slides shown in Panel C were incubated with
Osteopontin at concentrations from 94 ng/ml (right) to 1500 ng/ml
(left) and those shown in Panel D were incubated with CA 15-3 at
concentrations ranging from 9.4 U/ml (right)--150 U/ml (left).
Channels with no antigen added were treated as background. The
results show increased fluorescence intensity with increased
protein concentration for Osteopontin and CA 15-3. The fluorescence
from these spots is quantified using the Scanarray software and
plotted as a function of antigen concentration in FIG. 23 (E-F) for
these experiments. The standard curves were observed to be linear
for Osteopontin and CA 15-3 in the clinically-relevant ranges.
However, no signal from either Her-2 or MMP-2 was observed.
[0243] To further investigate and confirm these results, a
multiplex assay was performed in which all four protein biomarkers
were analyzed in one single microarray. In this experiment, four
identical channels were printed with capture antibodies to the four
protein biomarkers. As shown in FIG. 24, one of these slides was
incubated with only CA 15-3 antigen, the second slide was incubated
with only Osteopontin antigen, the third slide with all antigens
and the fourth with no antigen. All four biotinylated antibodies
were used in each assay. While the results for the assays involving
OPN and CA 15-3 yielded accurate and specific signals from the
correct capture antibody spots, no signal was obtained from Her-2
and MMP-2 spots in the case where all antigens were added. This
indicated that the assay for Her-2 and MMP-2 was not sensitive when
a rapid, one-step technique was employed.
[0244] In order to troubleshoot the failure of Her-2 and MMP-2
detection on channels, sandwich assays on Her-2 and MMP-2 antigens
were performed in three different formats. The first assay scheme
was a "wash assay" which represented sequential incubation of assay
reagents with washes in between, similar to a traditional
microarray assay. The second scheme called "long assay" involved a
one-step assay, where the antigen, biotinylated detector antibody
and streptavidin QD 605 were premixed and incubated on the array
for 60 min. The third assay scheme titled "short assay" was similar
to the "long assay" except that the arrays were incubated for 30
min. In FIG. 25, the results from this test for Her-2 (A) and MMP-2
(B) are observed. Although the spots showed bright fluorescence for
the wash assay, this signal was attenuated for the long assay in
which no washes were included. This signal reached background
levels for the short assay for both the antigens. The quantified
signals are observed on the plot on the right hand side. This
indicates that in order for the QD to be quantitative on the
channel assays for Her-2 and MMP-2, they either need a long
incubation time, or they need to be added individually to the
arrays and cannot be pre-mixed with the sample. Therefore, Alexa
546 was adopted as the reporter for the channel assays since it had
already been optimized in the above microarray assays.
[0245] Flow Channel Assays, Standard Curves Using Alexa 546
[0246] Standard curves were performed on all four antigens using
streptavidin Alexa 546 as the reporter instead of QD 605. FIG. 26
(A-D) shows the quantified fluorescence from the array spots
plotted as a function of antigen concentration. Channels with no
antigen added were treated as background. The mixture of antigen,
biotinylated antibody and streptavidin Alexa 546 was allowed to
incubate in the array in the channels for 15 min. Standard curves
were obtained for each biomarker which are seen in FIG. 26 A-E.
Data points for each curve represent the average intensities of two
replicate samples (and hence eight different spots) with a
coefficient of variation of approximately 15% for all protein
biomarker curves. The standard curves for all four biomarkers
appear to be linear in the clinically relevant ranges. Sensitive
and linear response is observed for all the four biomarkers
including Her-2 and MMP-2 in the concentration range selected. T
his therefore confirms that Alexa 546 works well as the molecular
reporter for our multiplexed assay on microarray channels.
[0247] Multiplexed Assay on the Flow Channels
[0248] Microarray flow channels were used to simultaneously detect
multiple biomarkers one single sample. In this experiment, eight
identical slides were printed with capture antibodies to the four
protein biomarkers printed in quadruplicate shown as columns in
FIG. 27. O=OPN, C=CA 15-3, H=Her-2, and M=MMP-2. Four arrays were
incubated with a mixture of all four but one biomarker (A) and the
other four slides were incubated with only one antigen (B).
Fluorescence was observed on the spots where the corresponding
antigens were added. Some background signal is observed from the
spots where no corresponding antigen was added to the mixture.
Since human serum was used as the medium of dilution, the non
specific binding of the serum proteins to the capture antibody
spots as well as the low, normal circulating levels of the
biomarkers generate this background signal from the spots even the
recombinant antigen was not added. This data therefore demonstrates
specific and sensitive detection of the four biomarkers in a
multiplex format on the microarray channel device.
[0249] The new device platform was designed to measure multiple
biomarkers and to produce rapid and reliable results in less than
15 minutes. Multiplexed assay with four different interaction times
were therefore performed. A set of eight arrays were printed with
all four capture antibodies. Four of these arrays were incubated
with a mixture of all antigens in the high concentrations as
observed in cancer, while the other set of four arrays was
incubated with a mixture of all four antigens in the lower
concentrations as observed in normal sera. The concentrations of
antigens used to represent metastatic cancer were 30 ng/ml for
Her-2, 850 ng/ml for MMP-2, 150 U/ml for CA 15-3 and 875 ng/ml for
Osteopontin and the concentrations of antigens used to represent
normal sera were 8 ng/ml for Her-2, 600 ng/ml for MMP-2, 15 U/ml
for CA 15-3 and 440 ng/ml for Osteopontin. One array from each of
the sets was incubated for 7 min, 15 min, 30 min and 60 min
respectively. The median fluorescence intensities of the four
biomarker spots were measured and are displayed in FIG. 28 (A-D).
The fluorescent signal increases were observed with a longer
incubation time for all the biomarkers. However, the difference
between a 30 min incubation and 60 min incubation is not as drastic
as the difference between a 7 min and 15 min incubation. This
indicates that the assay reaches equilibrium somewhere between 30
and 60 minutes. Differential signal between the high concentrations
(representing metastatic disease) and low concentrations
(representing normal sera) is tabulated in FIG. 28 (E). The ratio
of this differential signal shows that the ratio for the 7 minute
incubation is below 2.0 but above 1.5 for Her-2 and MMP-2, however
this ratio is very high for CA 15-3 and MMP-2. This implies that
only CA 15-3 and Osteopontin are appropriate for a 7 min diagnostic
test. However, all biomarkers have a ratio greater than 2 for a 15
min assay making this the best compromise between assay speed and
sensitivity
[0250] Flow Channel Patient Serum Immunoassays
[0251] To test the power of the flow channels to resolve signals
from cancer versus non-cancer samples accurately, breast cancer
patient serum samples were incubated on antibody arrays. Since the
serum samples were limited, the assay could not be performed with
duplicates, similar to the protein microarrays. Therefore, this
assay was designed in two parts. First, sera from 10 metastatic
breast cancer patients and 10 control subjects was pooled to obtain
a total of 800 .mu.l of metastatic breast cancer sample and 800
.mu.l of control sample. This assay eliminated the patient to
patient variation, but enabled the measurement of technical
variations across various channels and slides. An 80 .mu.l aliquot
of this pooled sample (mixed with biotinylated detector antibody
cocktail and streptavidin Alexa 546) was drawn across 10 replicate
arrays for 15 min. The resulting fluorescent intensities obtained
for the four biomarkers are plotted in FIG. 29 (A). Significant
differences between metastatic and control populations are observed
for all four biomarkers with minimal technical variations
(10%).
[0252] In the second part of this study, 80 .mu.l of patient sera
from 6 metastatic breast cancer patients and 6 control subjects was
mixed with biotinylated detector antibody cocktail and streptavidin
Alexa 546 reporter and incubated on the arrays for 15 minutes. The
fluorescent signals from the four biomarkers were quantified and
the median intensities were computed, which is shown in FIG. 29(B).
A t-test was performed on the two sample sets (metastatic and
control) for all four biomarkers and a p-value was generated to
estimate the resolving power of the system for accurately
identifying cancer vs. non-cancer samples. The table of these
p-values is listed in FIG. 29 (C). We observe that there is a
significant difference between the signals obtained for metastatic
and control samples for CA 15-3 (C) and Osteopontin (D). This
difference reduced for Her-2 (A) and MMP-2 (B), however, p value
table indicates that the channel assay is sensitively (p<0.05)
able to distinguish between metastatic and control populations for
all four.
[0253] Optical Reader
[0254] A benchtop was built of a rugged, portable fluorescence
imager, whose components include a miniature, megapixel CCD camera
and a high power xenon arc lamp white light generator. Arrays were
exposed to bandpass-filtered excitation light from the xenon
source. The resulting emitted light was bandpass-filtered and
collected by the CCD camera. The fluorescence images are exported
to Scanarray.TM. for subsequent analysis, where background is
calculated by taking into account the autofluorescence inherent to
the glass slide and the non-specific binding of fluorescence probe
material in the area surrounding the target spots on the array.
FIG. 30 shows images of microarray channels used to obtain standard
curves for all four biomarkers as captured by the CCD based imaging
system. Channels shown in FIG. 30(A) were incubated with Her-2 at
concentrations ranging from 6.25 ng/ml (right) to 100 ng/ml (left).
Channels shown in Panel B were incubated with MMP-2 with a
concentration range of 62.5 ng/ml (right) to 1000 ng/ml (left).
Channels shown in Panel C were incubated with CA 15-3 at
concentrations ranging from 9.4 U/ml (right)-150 U/ml (left) and
those shown in Panel D were incubated with Osteopontin at
concentrations from 94 ng/ml (right) to 1500 ng/ml (left). Human
serum was used as the diluting medium in these assays. The results
show increased fluorescence intensity with increased protein
concentration.
[0255] The fluorescence from these spots was quantified using the
Scanarray software and plotted as a function of antigen
concentration in FIG. 30 (E-H). Data points for each curve
represent the average intensities of eight replicates (background
subtracted) obtained using quadruplicate spots in two replicate
arrays. A linear relationship was observed between the
concentration and fluorescent intensities for all four
biomarkers.
[0256] FIG. 31 (A-D) shows the comparison between the CCD and PMT
based imaging systems for the quantification of dose response
fluorescence. Data from the two methods showed a linear
relationship with a correlation coefficient (r.sup.2) of greater
then 0.98, indicating that both methods produce similar results.
This result is further supported by the results obtained from the
multiplexed assays. The channels with arrays incubated with pooled
patient samples from 10 metastatic and 10 control populations were
imaged using the CCD system and compared the results to those
obtained using the PMT. A ratio of the median fluorescence
intensities obtained for metastatic populations to the median
fluorescence intensities obtained for control populations is
plotted in FIG. 31 (E) for the four biomarkers using both the CCD
and PMT based imaging systems. The results from the CCD system are
very similar to the ones obtained by using the PMT. A system was
thus developed in which the high sensitivities of PMTs used in the
large microarray scanners is matched by a miniature CCD camera by
controlling the excitation light intensity and the integration time
of the camera sensor.
[0257] While the invention has been particularly shown and
described with reference to a preferred embodiment and various
alternate embodiments, it will be understood by persons skilled in
the relevant art that various changes in form and details can be
made therein without departing from the spirit and scope of the
invention.
[0258] All references, issued patents and patent applications cited
within the body of the instant specification are hereby
incorporated by reference in their entirety, for all purposes.
* * * * *