U.S. patent application number 10/627352 was filed with the patent office on 2004-04-29 for methods and arrays for detecting biomolecules.
This patent application is currently assigned to 20/20 GeneSystems, Inc.. Invention is credited to Emmert-Buck, Michael R., Knezevic, Vladimir.
Application Number | 20040081987 10/627352 |
Document ID | / |
Family ID | 22513857 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040081987 |
Kind Code |
A1 |
Knezevic, Vladimir ; et
al. |
April 29, 2004 |
Methods and arrays for detecting biomolecules
Abstract
The present invention is directed to a device and a method for
detecting biomolecules in a tissue section or other two-dimensional
sample by creating "carbon copies" of the biomolecules eluted from
the sample and visualizing the biomolecules on the copies using
antibodies or DNA probes having specific affinity for the
biomolecules of interest. Thin membranes in a stacked or layered
configuration are applied to the sample, such as a tissue section,
and reagents and reaction conditions are provided so that the
biomolecules are eluted from the sample and transferred onto each
of the stacked membranes thereby producing multiple replicas of the
biomolecular content of the sample.
Inventors: |
Knezevic, Vladimir; (Silver
Spring, MD) ; Emmert-Buck, Michael R.; (Silver
Spring, MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
One World Trade Center
Suite 1600
121 S.W. Salmon Street
Portland
OR
97204
US
|
Assignee: |
20/20 GeneSystems, Inc.
The Govt. of the USA as represented by the Secretary of the
Dept. of Health and Human Sevs.
|
Family ID: |
22513857 |
Appl. No.: |
10/627352 |
Filed: |
July 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10627352 |
Jul 25, 2003 |
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09718990 |
Nov 20, 2000 |
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6602661 |
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09718990 |
Nov 20, 2000 |
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PCT/US00/20354 |
Jul 26, 2000 |
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60145613 |
Jul 26, 1999 |
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Current U.S.
Class: |
435/6.11 ;
435/6.1; 435/91.2 |
Current CPC
Class: |
G01N 33/57434 20130101;
C40B 30/04 20130101; G01N 33/54386 20130101; G01N 33/6845 20130101;
G01N 2333/4742 20130101; C12Q 1/6841 20130101; G01N 2333/96455
20130101; G01N 2333/7051 20130101; G01N 33/6803 20130101; G01N
2333/96486 20130101; G01N 2333/4712 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
What is claimed is:
1. A method of making multiple substantial copies of a biological
sample, comprising: providing a stack of layered membranes, wherein
said membranes permit biomolecules applied to said stack to move
through multiple of said membranes, while directly capturing said
biomolecules on multiple membranes; and applying said biological
sample to said stack, under conditions that allow said multiple
membranes to directly capture said biomolecules from said sample
and form said multiple substantial copies of the biological
sample.
2. The method of claim 1, further comprising detecting biomolecules
of interest on said multiple membranes.
3. The method of claim 2, wherein detecting biomolecules of
interest comprises exposing said multiple membranes to a
detector.
4. The method of claim 3, wherein the biological sample is a tissue
specimen that is placed on said stack of layered membranes, and
biomolecules from said tissue specimen are directly captured by
said layered membranes as said biomolecules from said tissue
specimen move through said multiple membranes.
5. The method of claim 3, further comprising separating said
multiple membranes prior to detecting said biomolecules of
interest.
6. The method of claim 1, wherein said biomolecules applied to the
stack are themselves detectors that are exposed to a biological
specimen to be analyzed, and the method further comprises exposing
one or more of said multiple membranes to said biological specimen
under conditions that allow said biological specimen to be analyzed
by the detectors.
7. The method of claim 6, wherein said biomolecules on said
multiple membranes are nucleic acid molecules, and detecting
biomolecules of interest comprises exposing said nucleic acid
molecules on said multiple membranes to said biological specimen to
be analyzed, under conditions that allow hybridization between said
nucleic acid molecules on said membranes and nucleic acid molecules
in said biological specimen.
8. A method of detecting biomolecules in a sample comprising: a.
providing a stack of layered membranes; b. applying said sample to
said stack under conditions that permit movement of said
biomolecules through multiple layered membranes of said stack, and
allow direct capture of said biomolecules on said membranes; and c.
detecting said biomolecules on one or more of said multiple
membranes.
9. The method according to claim 8 wherein said membranes comprise
a plurality of porous substrates each having a thickness of less
than 30 microns.
10. The method according to claim 9 wherein one or more of said
substrates comprise a material for increasing the affinity of the
membrane to the biomolecules.
11. The method of claim 10, wherein said material is coated on the
one or more of said membranes.
12. The method of claim 9 wherein said porous substrates comprise a
material selected from the group consisting of polycarbonate,
cellulose acetate, and mixtures thereof.
13. The method of claim 12, wherein said porous substrate is a
polycarbonate substrate.
14. The method of claim 10, wherein said material for increasing
affinity is selected from the group consisting of nitrocellulose,
poly-L-lysine, and mixtures thereof.
15. The method according to claim 8 wherein said sample is a tissue
section.
16. The method of claim 8, wherein detecting said biomolecules
comprises separating one or more of said membranes from said stack,
and detecting said biomolecules on the one or more of the separated
membranes.
17. The method of claim 8, wherein said conditions that permit
movement of said biomolecules through said multiple membranes
comprises passing a transfer liquid through said layered
membranes.
18. The method of claim 8, wherein said conditions that permit
movement of said biomolecules through one or more of said membranes
comprises providing a wick that encourages movement of said
biomolecules through said stack of layered membranes in a desired
direction of movement.
19. The method of claim 8, wherein said stack of layered membranes
comprises 50 or more of said membranes.
20. The method of claim 8, wherein said sample is a DNA sample.
21. The method of claim 8, further comprising correlating said
biomolecules detected on said one or more membranes with a
biological characteristic of said sample.
22. A kit comprising: a. a membrane array for detecting
biomolecules in a sample, said array comprising a plurality of
membranes, wherein each of said plurality of membranes have
substantially a same affinity for said biomolecules; and b.
containers of antibodies or probes for detecting biomolecules
captured on each membrane.
23. The kit according to claim 22 wherein said membranes comprise a
polymer substrate coated with a material for increasing an affinity
of said substrate to said biomolecules.
24. The kit according to claim 23 wherein said coating material is
nitrocellulose.
25. The kit according to claim 22 said antibodies or probes are
specific capture molecules for biomolecules sought to be detected
on particular membranes of said array.
26. The kit according to claim 25 wherein each container contains
an antibody cocktail.
27. The kit according to claim 22 wherein said plurality of
membranes have a low capacity for said biomolecules.
28. The kit according to claim 22 wherein said plurality of
membranes each have a thickness of less than about 30 microns.
29. A method of creating a set of microarray copies comprising: a.
providing a stack of layered membranes; and b. applying a plurality
of DNA probes, antibodies, or a combination thereof, to said stack
of layered membranes, wherein said stack of layered membranes
comprises a plurality of substrates through which said probes or
antibodies move, and in which a portion of said probes or
antibodies are directly captured by one or more of said
substrates.
30. The method of claim 29, further comprising separating said
substrates to provide corresponding substrates having a plurality
of said DNA probes, antibodies or combination thereof, in
corresponding positions of each of said substrates.
31. The method of claim 29, wherein applying said plurality of DNA
probes, antibodies, or combination thereof, is applied to said
stack from a plate having a plurality of wells each containing a
different DNA probe or antibody, and said DNA probes or antibodies
are transferred from said wells to said stack so as to create a set
of substantially replicate microarrays.
Description
STATEMENT OF GOVERNMENT RIGHTS
[0001] At least one of the inventors is an employee of an agency of
the Government of the United States, and the government may have
certain rights in this invention.
FIELD OF THE INVENTION
[0002] The present invention is directed to arrays for identifying
large numbers of biomolecules in a biological sample so as to help
determine their function and role in disease. More particularly,
the invention relates to arrays of membranes for detecting and
identifying large numbers of biomolecules in a multiplex manner.
The application is a continuation-in-part of PCT application
US00/20354 entitled Method and Production of Layered Expression
Scans For Tissue and Cell Samples, which is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] Now that the 100,000 or so genes that make up the human
genome have been sequenced, new tools are needed to determine when
and in what type of tissue those genes are active so as to
ascertain their function and role in disease. This effort, often
referred to as "functional genomics" and "proteomics," is
especially important in efforts to discover new drugs since most
new pharmaceutical agents are being designed to target enzymes,
receptors, and other proteins. Eventually, this information will be
used in clinical diagnostics to help guide treatment selection in
the emerging era of "personalized medicine."
[0004] Some believe that the 100,000 human genes may turn out to
produce up to a million different protein variants. Of these, it is
estimated that about 10,000 proteins will be identified over the
next ten years as targets of pharmaceutical intervention. However,
only a small fraction of these proteins are expressed in any
particular tissue type. For example, a very different set of genes
is expressed in brain tissue from those expressed in kidney even
though cells in both organs have the same set of genes. Moreover,
the subset of genes expressed in a kidney tumor differ from those
active in healthy tissue from that organ. It is clear, therefore,
that tools are needed to identify the activity of large numbers of
genes in tissue samples removed from subjects.
[0005] To meet this need a number of "multiplex" assays have been
introduced. Among the most common type of assays for surveying the
expression of large numbers of genes in parallel are DNA
microarrays (a/k/a "biochips"). Most microarrays consist of a glass
slide or other solid surface upon which thousands of cDNA probes
are anchored. With these devices DNA probes are arrayed in a
grid-like format. Messenger RNAs are isolated from the samples of
interest and allowed to hybridize to the probes anchored to the
biochip revealing the profile of the genes expressed. Various
scanners and software programs are used to profile the patterns of
genes that are "turned on." Representative of this biochip approach
is the GeneChip.RTM. system from Affymetrix, Inc. (Santa Clara,
Calif.).
[0006] While there are many uses for the aforementioned DNA
microarrays, there are several limitations. First, they do not
detect proteins, only nucleic acids. Since mRNA and protein levels
do not always correlate in the cell and many regulatory processes
occur after transcription, a direct measure of proteins is more
desirable. Thus, since mRNA and protein levels do not always
correlate in the cell and many functional protein modifications
occur after translation, a direct way to monitor proteins is
needed.
[0007] Another disadvantage of the microarrays known in the art is
the fact that the sample being tested is disassociated from the
tissue from which it was isolated. Disease is the result of
disturbed biological equilibrium in groups of cells. Thus, it is
often desirable to observe gene expression patterns in the context
of the tissue in which the genes are active. In situ detection and
visualization of proteins traditionally has been accomplished
through immuno-histochemistry (IHC). This technique involves
mounting a thin tissue section on the glass slide and visualizing a
protein of interest with a detectable antibody that has specific
binding affinity for the target protein. Because of certain
technical limitations of IHC, only one or two proteins from a
single tissue section can be analyzed. Also, proteins are still
embedded in the tissue and are not presented to the antibodies in
the most appropriate way (proteins are not highly denatured)
lowering the success rate of the antibody reactivity.
[0008] Additionally microarrays known in the art require that in
order to collect enough of the material for analysis, the sample
being tested be a mixture of a number of different cell types
(diseased tissue and adjacent normal cells) that are disassociated
together and used for biomolecule extraction. As the result of this
approach, biomolecules originating in the diseased tissue (e.g.
tumor) are diluted and harder to detect and characterize. Since the
morphological relationship is not preserved, it is hard to know
what component of the sample is responsible for the changes
observed in gene expression.
[0009] It is therefore desirable to have a method and device that
combines the morphological advantages of IHC and other in-situ
approaches with the multiplex and high-throughput characteristics
of DNA microarrays.
[0010] To meet this need, Englert, et al. describe a very
innovative technique which they refer to as "layered expression
scanning" for molecular analysis of tumor samples that uses a
layered array of capture membranes coupled to antibodies or DNA
sequences to perform multiplex protein or mRNA analysis. Cancer
Research 60, 1526-1530, Mar. 15, 2000. With this technique cell or
tissue samples are transferred through a series of individual
capture layers, each linked to a separate antibody or DNA sequence.
As the biomolecules traverse the membrane set, each targeted
protein or mRNA is specifically captured by the layer containing
the corresponding antibody or cDNA sequence. The two-dimensional
relationship of the cell populations is maintained during the
transfer process thereby producing a molecular profile of each cell
type present.
[0011] It would be desirable to supplement and enhance the layered
expression scanning technique described by Englert, et al. with an
approach that utilizes a stack of "blank" membranes that are not
specific for any particular target. Instead, such membranes would
ubiquitously bind to all (or a subset) of the biomolecules in a
sample so as to give the user the flexibility of detecting a wide
variety of biomolecules in an open format.
SUMMARY OF THE INVENTION
[0012] The present invention is directed to a device and a method
for detecting biomolecules in a tissue section or other
two-dimensional sample by creating "carbon copies" of the
biomolecules eluted from the sample and visualizing the
biomolecules on the copies using detectors, for example antibodies
or DNA probes, having specific affinity for the biomolecules of
interest.
[0013] Thin membranes in a stacked or layered configuration are
applied to the sample, such as a tissue section, and reagents and
reaction conditions are provided so that the biomolecules are
eluted from the sample and transferred onto each of the stacked
membranes thereby producing multiple substantial replicas of the
biomolecular content of the sample. The treated membranes (or
layers) are then separated. Each membrane is incubated with one or
more different detectors (for example antibodies) specific for a
particular biomolecule (such as a protein) of interest. The
detectors employed are labeled or otherwise detectable using any of
a variety of techniques such as chemiluminescence.
[0014] In an example in which proteins are detected, each membrane
has essentially the same pattern of proteins bound to it, but
different combinations of proteins are made visible on each
membrane due to the particular antibodies selected to be applied.
For example, one membrane layer may display proteins involved in
programmed cell death (apoptosis) while an adjacent layer may
display enzymes involved in cell division such as tyrosine kinases.
In addition to proteins, nucleic acids may be targeted by using
labeled DNA probes in lieu of antibodies. Moreover, different types
of target biomolecules may be detected in different layers. For
example, both protein and nucleic acid targets can be detected in
parallel by applying both antibodies and probes to different layers
of the array.
[0015] Another feature of the present invention is providing a kit
that includes a group of membranes in a stack or other
configuration that permits them to be stacked, and different
detectors, such as cocktails of antibodies or probes, to be applied
to the treated membranes for biomolecule detection.
[0016] With the foregoing and other objects, advantages and
features of the invention that will become hereinafter apparent,
the nature of the invention may be more clearly understood by
reference to the following detailed description of the invention,
the appended claims and to the several views illustrated in the
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a perspective view of the membrane array according
to the present invention shown transferring molecules from a tissue
section.
[0018] FIG. 2 is a longitudinal sectional view of an individual
membrane according to the present invention.
[0019] FIG. 3 is a schematic illustration comparing direct and
indirect capture.
[0020] FIG. 4 is a schematic illustration comparing the binding
capacity of membranes constructed of nitrocellulose and
polycarbonate, both coated and uncoated. FIG. 4A shows scanned
images of the membranes incubated in protein comparing the
intensity of signal and FIG. 4B is a chart plotting the amount of
protein bound to different membrane materials.
[0021] FIG. 5 are images of tissue sections that show that portions
of total biomolecules can be successfully transferred through a
stack of polycarbonate (PC) layers onto the trap. FIG. 5A shows
transfer through polycarbonate membranes. FIG. 5B shows transfer
through polycarbonate coated with nitrocellulose. FIG. 5C shows
transfer through polycarbonate coated with poly-L-lysine
membranes.
[0022] FIG. 6 is a series of images showing immunodetection of
different proteins from two regions (healthy and cancerous) of a
breast tissue using the membrane array according to the present
invention.
[0023] FIG. 7 is a perspective view of the array according to the
present invention shown in use with a microtiter plate.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0024] "Biomolecules" means molecules typically produced by living
organisms including peptides, proteins, glycoproteins, nucleic
acids, fatty acids, and carbohydrates.
[0025] "Sample" means a material which contains biomolecules
including tissue, gels, bodily fluids, and individual cells in
suspensions or in pellet, as well as materials in containers of
biomolecules such as microtiter plates.
[0026] "Captor" means a molecule, such as an antibody or DNA probe,
that is anchored to a membrane and has an affinity (such as a
specific affinity) for one of the biomolecules.
[0027] "Direct capture" means the conjugation or binding of a
biomolecule directly onto the surface of the membrane without the
aid of a captor antibody or the like.
[0028] "Indirect capture" means the conjugation or binding of a
biomolecule onto a captor antibody or the like which in turn is
bound to the surface of the membrane. Thus, with indirect capture
the biomolecule is not directly conjugated to the membrane.
[0029] "Array" means two or more.
[0030] "Affinity" means the chemical attraction or force between
molecules.
[0031] "Capacity" means the ability to receive, hold, or absorb
biomolecules from the sample.
[0032] "Detector" means a molecule, such as an antibody or DNA
probe, that is free in solution (i.e. not anchored to a membrane)
and has an affinity for one of the sample components.
[0033] "Antibody cocktails" means mixtures of between two to about
100 different detector antibodies.
[0034] "Identical" means having substantially the same affinity for
biomolecules.
[0035] "Membrane" means a thin sheet of natural or synthetic
material that is porous or otherwise at least partially permeable
to biomolecules.
[0036] "Stack" refers to adjacent substrates, whether stacked
horizontally, vertically, at an angle, or in some other direction.
The substrates may be spaced or touching, for example
contiguous.
[0037] The specific example illustrated in FIG. 1 shows a device
and a method for detecting biomolecules in a tissue section 11 or
other two-dimensional sample by creating "carbon copies"
(substantial copies that are not necessarily identical copies, they
may have slight differences but can be identical or nearly
identical) of the biomolecules eluted from the sample, and
visualizing the biomolecules on the copies using antibodies or
other molecules having specific affinity for the biomolecules of
interest. Thin membranes 12 in a stacked or layered configuration
are brought into contact with the sample and reagents, and reaction
conditions are provided so that the biomolecules are eluted from
the sample onto the membranes, whereupon the biomolecules can be
visualized using a variety of techniques, as set forth herein.
[0038] Certain examples of the invention include a method of
detecting an analyte in a biological sample using stacked
contiguous layered membranes that permit biomolecules to move
through a plurality of the membranes, while directly capturing the
biomolecules on one or more of the membranes. Biomolecules from the
sample are moved through the membranes under conditions that allow
one or more of the membranes to directly capture the biomolecules,
and biomolecules of interest are concurrently or subsequently
detected on the membranes, for example by exposing the biomolecules
of interest to a detector, such as a specific capture molecule (for
example an antibody or a nucleic acid probe).
[0039] Alternatively, the biomolecule itself may be a detector
(such as a nucleic acid probe) to which a sample is exposed. In
this case, the biological sample is one or more purified nucleic
acid probes placed in assigned locations on a surface of the stack,
which are allowed to migrate through membranes (for example in a
direction of movement transverse to the layers) to produce multiple
substantial "copies" of the original probes in corresponding
locations on the multiple membranes. The layers can then be
separated and exposed to a target biological specimen which may
have nucleic acid molecules that hybridize to the probes.
[0040] In some examples, the biological sample is a tissue specimen
that is placed on the stack of layered membranes, and biomolecules
from the tissue specimen are directly captured by the membranes as
the biomolecules move through the membranes. The membranes may, for
example, be separated prior to detecting the biomolecules of
interest, and the separated membranes are exposed to the detectors.
Alternatively, the biological molecules of interest may be
contained in a biological specimen to which the membranes are
exposed. For example, the biomolecules directly captured by the
membranes may themselves be nucleic acid probes or antibodies, and
the membranes may be exposed to a biological specimen in which a
nucleic acid or peptide (such as a protein) is to be detected.
[0041] In particular embodiments, the membranes comprise a material
that non-specifically increases the affinity of the membranes to
the biological molecules (or a class of biomolecules such as
proteins or nucleic acids) that are moved through the membranes.
For example, the membranes may be dipped in, coated with, or
impregnated with nitrocellulose, poly-L-lysine, or mixtures
thereof. In certain examples the membranes are not treated with a
material that blocks the non-specific binding of the biomolecules
to the membranes, at least during transfer of the biomolecules
through the membranes. However, in other embodiments, some such
blocking agents can be added to the membranes, as long as the
amount of blocking agent minimizes the amount of biomolecules
bound, without blocking it altogether. In certain examples,
blocking agent may be added to the membranes after transfer of the
biomolecules through the membranes, but before or during exposure
to the detectors.
[0042] In particular examples, the membranes are sufficiently thin
to allow the biomolecules to move through the plurality of
membranes (for example 10, 50, 100 or more) in the stack. Such
membranes, for example, have a thickness of less than 30 microns.
The membranes may be made of a material that does not substantially
impede movement of the biomolecules through the membranes, such as
polycarbonate, cellulose acetate, or mixtures thereof.
[0043] The material of the membranes may maintain a relative
relationship of biomolecules as they move through the membranes, so
that the same biomolecule (or group of biomolecules) move through
the plurality of membranes at corresponding positions. In such
examples, this coherence of relative relationships allows the
different membranes to be substantial "copies" of one another, much
like a "carbon copy" would be. However, like a "carbon copy" there
may be some differences between the different "copies" present in
the different membranes.
[0044] In particular embodiments, a transfer liquid (such as a
buffer) is passed through the membranes to encourage movement of
the biomolecules through them. A distal or downstream wick may also
be provided to help move liquid (such as the buffer) through the
membranes in a desired direction of movement.
[0045] Biomolecules detected on the membrane copies may be
correlated with a biological characteristic of the sample. For
example, a tissue specimen may be placed in a position on top of
the stack, and a biomolecule of interest (such as a particular
protein) may be detected in one of the membrane copies at a
position that corresponds to the position in which the tissue
specimen (or one of its substructures such as an organelle) was
placed. The presence of that biomolecule in the tissue specimen can
then be correlated with a biological characteristic of the sample.
For example, a highly malignant tissue specimen may be found to
contain a protein, that may then be associated with the highly
malignant phenotype of the specimen.
[0046] Other embodiments of the invention can include kits which
contain a membrane array for detecting biomolecules (such as
proteins or nucleic acids) in a sample. The array includes a
plurality of membranes, each of which has a non-specific or
substantially same affinity for the biomolecules. The kit also
includes containers of antibodies or probes (or mixtures of
antibodies, mixtures of probes, or mixtures of the antibodies and
probes) for detecting biomolecules captured on each membrane. In
particular examples of the kit, the membranes are polymer
substrates containing or coated with a material (such as
nitrocellulose) for increasing an affinity of the substrate to the
biomolecules.
[0047] In particular examples, the method can be used to create a
set of microarray substantial "copies" by applying a plurality of
detectors, such as DNA probes, antibodies, or a combination
thereof, to the stack of layered membranes. The stack of layered
membranes provide a plurality of substrates through which the
probes or antibodies move, and in which a portion of the probes or
antibodies are directly captured by one or more of the substrates.
The substrates can be subsequently separated to provide
corresponding substrates having a plurality of DNA probes,
antibodies or a combination thereof, in corresponding positions of
each of said substrates. The multiple membranes maintain a
substantially coherent relationship between the probes and/or
antibodies as they move through the substrate. This coherent
relationship may or may not be a direct spatial correspondence, but
the relative relationship between the biomolecules may be
maintained in such a way that the identity of the biomolecules on
the membranes can be known from the relationship in which the
biomolecules were placed on the stack of layered membranes.
[0048] In particular embodiments, the plurality of DNA probes,
antibodies, or combination thereof, is applied to the stack of
membranes from a plate having a plurality of wells, each containing
a different DNA probe or antibody. The DNA probes or antibodies are
transferred from the wells to the stack so as to create a set of
substantially replicate microarrays.
[0049] Referring now in detail to the drawings of specific,
non-limiting detailed examples wherein like parts are designated by
like reference numerals throughout, there is illustrated in FIG. 1
a perspective view of the membrane array apparatus according to the
present invention designated generally by reference numeral 10.
Apparatus 10 includes a plurality of membranes 12 shown in a
layered or stacked configuration such as array 13. While only about
a dozen membranes are shown in FIG. 1 it should be appreciated that
many more membranes (e.g., 10, 50, 100 or more) may be employed
depending on the number of targets sought to be identified, the
quantity of biomolecules present in the sample, and the thickness
of the material employed to construct membranes 12. Optionally,
membranes 12 may be packaged in a suitable sealed enclosure or
frame (not shown) to maintain their integrity and prevent
contamination.
[0050] Membrane array 13 is placed atop a stack of blotting paper
14 that acts as a lower wick pulling buffer out of buffer chambers
18 though upper wicks 20 and membrane array 12 in the direction of
the arrows shown in FIG. 1. A biomolecule trap 22 is positioned
intermediate membrane array 12 and blotting paper 14 to help the
user ascertain whether proper transfer has occurred.
[0051] With reference to FIG. 2, individual membranes 12 are
constructed of a porous substrate 30 coated with a material which
increases the affinity of the membrane to the biomolecules being
transferred. Substrate 30 is, for example, constructed of
polycarbonate or a similar polymeric material that maintains
sufficient structural integrity despite being made porous and very
thin. However, in lieu of polycarbonate the substrate 30 may for
example be constructed of cellulose derivatives such as cellulose
acetate, as well as polyolefins, (e.g. polyethelyle, polypropylene,
etc.). It is a particular feature of the present invention that
membranes 12 have a high affinity for proteins and other
biomolecules, but have a low capacity for retaining such molecules.
This feature permits the molecules to pass through the membrane
stack with only a limited number being trapped on each of the
successive layers thereby allowing multiple "carbon copies" to be
generated. In other words, the low capacity allows the creation of
multiple replicates as only a limited quantity of the biomolecules
are trapped on each layer. If a membrane were used that had a high
binding capacity for biomolecules-such as the transfer membranes
conventionally used with gel blotting-multiple replicas could not
be made.
[0052] To maintain the binding capacity of membrane 12 sufficiently
low to avoid trapping of too much of the sample, the thickness of
substrate 30 is, for example, less than about 30 microns, and in
particular embodiments is between about 4-20 microns, for example
between about 8 to 10 microns. The pore size of the substrate is,
for example, between about 0.1 to 5.0 microns, such as about 0.4
microns. Another advantage of using a thin membrane is that is
lessens the phenomenon of lateral diffusion. The thicker the
overall stack, the wider the lateral diffusion of biomolecules
moving through the stack.
[0053] The illustrated substrate 30 includes a coating 32 on its
upper and lower surfaces to increase non-specific binding of the
proteins or other targeted biomolecules. Although the binding to
the coating is "non-specific" in the sense that it does not
recognize particular proteins or other biomolecules, such as
particular nucleic acids, it may be specific in that it recognizes
and specifically binds classes of biomolecules, such as proteins or
nucleic acids, or combinations thereof. Coating 32 in the disclosed
embodiment is nitrocellulose, but other materials such as
poly-L-lysine may also be employed. Before being applied to
substrate 30, the nitrocellulose is dissolved in methanol or other
appropriate solvent in concentration from 0.1%-1.0%. The membranes
are immersed in this solution as described more fully in the
Examples, below. In lieu of coating 32, nitrocellulose or other
materials with an affinity for biomolecules can be mixed with the
polycarbonate before the substrate is formed thereby providing an
uncoated substrate having all of the desired characteristics of the
membrane. Alternative coating methods known in the art may be used
in lieu of dip coating including lamination. Alternatively, only
one surface may be coated, such as the surface that faces the
sample, instead of both surfaces.
[0054] It is a particular feature of the present invention that
membranes 12 have a high affinity for proteins and other
biomolecules, but have a low capacity for retaining such molecules.
This feature permits the molecules to pass through the membrane
stack with only a limited number being trapped on each of the
successive layers thereby allowing multiple "carbon copies" to be
generated. In other words, the low capacity allows the creation of
multiple substantial replicates as only a limited quantity of the
biomolecules are trapped on each layer. If a membrane were used
that had a high binding capacity for biomolecules-such as with
nitrocellulose membranes conventionally used with gel
blotting-multiple replicas could not as easily be made. More
specifically, the affinity and capacity of membrane 12 should be
such that when at least 5 and preferably more than 10 membranes are
stacked and applied to a sample according to the disclosed method,
most of the biomolecules of interest can be detected on any and all
of the membranes including those positioned furthest from the
sample.
[0055] With reference to FIG. 3, the aforementioned technique may
be described as "direct capture" since the target biomolecules 40
are captured directly on the surface of membranes (or within the
membrane), instead of being captured indirectly by a binding agent
(such as an antibody or nucleic acid probe) applied to the
membrane. During this disclosed process different components of the
sample bind to the membrane with the same affinity, but directly
proportional to their concentration in the sample (a component with
a higher concentration will leave more molecules on each membrane,
and a component with a lower concentration will leave less
molecules on each membrane). A detector molecule 42, such as a
labeled antibody that specifically binds to the biomolecule 40, may
be utilized to detect biomolecule bound to the membrane. In
examples in which the amount of a component bound to the membrane
is proportional to the amount of the component in the sample, an
amount of the detector molecule can be correlated to an amount (or
relative amount) of the biomolecule detected.
[0056] In order to achieve high affinity and high capacity for a
particular group of biomolecules from a sample, coating of the
membranes with a captor molecule 44 is performed in the method
described by Englert et al. (supra.). This may be referred to as
"indirect capture" and is illustrated in FIG. 4B. Captor 44 can be
cDNA, antibody, or any other protein specific for the target of
interest. Single-stranded cDNA molecules generated by number of
means (Polymerase Chain Reaction, nick translation, reverse
transcription, oligonucleotide synthesis) or proteins (like
immunoglobulin) can be directly attached to the membrane.
Alternatively, the linker-arms that would allow spatial control of
the captor binding could be directly attached to the membrane
followed by captor attachment to them. This will expose the
majority of the active target recognition sites increasing that way
capacity of the indirect capture. Streptavidin coated membranes may
be employed to bind end-biotinilated nucleic acids and randomly
biotinilated proteins, or protein A and protein G to bind
immunoglobulins.
[0057] In use and operation, apparatus 10 may be employed to create
"carbon copies" or substantial replicas of the biolmolecular
contents of the sample applied to the stack. Membranes 12 are
arrayed in a layered or stacked configuration as shown in FIG. 1 as
reference numeral 13. In a particular embodiment, a sample such as
a conventional frozen tissue section 11 is placed on a layer of
polycarbonate and then sandwiched between two slices of 2% agarose
(not shown). The entire preparation is positioned adjacent to the
membrane array. Buffer is applied using chambers 18 and wicks 20 to
elute and transfer proteins from the frozen section. About 50-100
milliliters of buffer per square centimeter are used in each
transfer with average length of the transfer being about 1-2 hours.
After transfer the membranes are separated and incubated with the
detector antibody. Antibodies are selected based on the types of
targets sought. Membranes are washed in a buffer, and the
protein/detector complex can be visualized using a number of
techniques such as ECL, direct fluorescence, or colorimetric
reactions. ECL is preferred. Commercially available flatbed
scanners and digital imaging software can be employed to display
the images according to the preference of the user.
[0058] It should be appreciated that because the size of the
membrane array can be varied, the user has the option of analyzing
a large number of different samples in parallel, thereby permitting
direct comparison between different patient samples. For example
different samples from the same patient at different stages of
disease can be compared in a side-by-side arrangement as can
samples from different patients with the same disease.
[0059] Another use of the membrane array according to the present
invention is to make multiple copies of a cDNA microarray in a
manner that is less expensive and labor-intensive than robotic
systems. With reference to FIG. 7, DNA sequences representing
different genes are placed into individual microtiter wells 52 of a
microtiter plate 54 (e.g. a 96-well plate). The microtiter plate 54
is placed adjacent to a stack of membranes 56 of the same
construction as membranes 12, to allow the contents of the
microtiter wells to be transferred from the respective wells to the
stack of membranes 50. In the illustrated embodiment, the contents
of the wells are transferred from the wells 52 to a top surface of
the stack of membranes 56, so that the contents are applied in a
pattern that corresponds to a pattern of the wells.
[0060] The DNA is transferred through the membranes in a direction
of movement from the wells toward a wick member 58, and the spatial
orientation of the samples is maintained. Because of the high
affinity, low capacity characteristics of membranes 56, as the
nucleic acids traverse the capture membrane set 56, a small
percentage of DNA hybridizes to each membrane, creating a series of
replicate copies, each one containing a grid of DNA spots that
match the orientation of the DNA samples in the microtiter plate.
Thus, a set of cDNA arrays may be created in a very rapid and
inexpensive fashion. Antibody and tissue lysate arrays can also be
created by this method.
EXAMPLES
Example #1
Construction of the Polycarbonate Membrane Suitable for the Protein
Binding
[0061] Native, non-coated polycarbonate membrane (Millipore, Mass.)
has low affinity and low binding capacity for proteins. To improve
its protein binding characteristics, polycarbonate membranes were
coated with either poly-L lysine (referred to as PC+Lysin in FIG.
4) or nitrocellulose (referred to as PC+NC in FIG. 4). Membranes
(177 square centimeters) were immersed for 1 minute in 5 ml of
either aqueous solution of 0.1% poly-L-lysine or 0.1-1.0%
nitrocellulose solution in 100% methanol. After coating, membranes
were suspended in vertical position and air-dried at room
temperature for 5-10 minutes. Poly-L-lysine treated membranes were
before use additionally baked for 2 hours at 50.degree. C. Small
squares (0.25 square centimeters) of both treated and non-treated
membranes were incubated in TBST solution (50 mM TRIS pH 8.0, 150
mM NaCl and 0.05% Tween-20) with 1.0-100.0 ng/ul of goat
immunoglobulin labeled with Cy3 fluorescent dye (Amersham Pharmacia
Biotech, USA) for 0.5-2 hours at room temperature. Membranes were
washed in TBST and examined on STORM scanner (Molecular Dynamics,
USA). The results are shown in FIG. 4A. The intensity of the signal
was quantified by ImageQuant (Molecular Dynamics, USA) and data
points from five different experiments were plotted using Microsoft
Excel. The results shown in FIG. 4B demonstrate that polycarbonate
membranes have a low protein binding potential that can be
considerably enhanced by coating the membrane with poly-L-lysine
(PC+Lysin) or nitrocellulose (PCNC).
Example #2
Testing the Porosity of Prepared Polycarbonate Layers
[0062] To demonstrate porosity of manufactured layers, native,
poly-L-lysine or nitrocellulose coated membranes were blocked in 5%
bovine serum albumen solution in 50 mM TRIS pH 8.0 to prevent any
protein binding. Fifty-one square centimeter pieces were cut out
and stacked together to make a pile. A non-blocked pure
nitrocellulose layer was used at the bottom to capture proteins
(NC-trap). Three adjacent 20 micrometer thick frozen sections of
normal breast tissue were collected on a polycarbonate membrane
with 5.0 um pore size and embedded in a 2% agarose gel and
transferred side by side through each stack. Between 50 and 100
milliliters of TBST buffer was used per square centimeter of the
membrane stack with average length of the transfer being 1 hour.
After transfer, proteins remaining in the tissue sections and total
proteins on the NC-trap were visualized by Ponceau S staining
(SIGMA, MO). As shown in FIG. 5, the outline of the total proteins
transferred through the stack and trapped on the nitrocellulose
layer very closely resembled the outline of the applied tissue
section suggesting that not only were membranes porous enough to
allow for the proteins to be transferred, but also that at least up
to 50 polycarbonate membranes can be used in this kind of assay
without apparent distortion of the image due to lateral
diffusion.
Example #3
Demonstration of Low Capacity Protein Binding to the Nitrocellulose
Coated Polycarbonate Layers
[0063] Examples #1 and #2 demonstrate that proteins in solution can
bind to a single nitrocellulose coated polycarbonate layer and that
complete saturation of the layer with proteins does not affect its
porosity. To ascertain how much of the total protein would be
trapped on each individual layer during the tissue section
transfer, 20 micron thick frozen sections of normal and tumor
breast tissue were placed adjacent to each other on a polycarbonate
membrane with 5.0 um pore size, embedded in 2% agarose gel and
transferred through 14 layers of nitrocellulose coated
polycarbonate to the NC-trap on the bottom, in 100 ml/cm2 of buffer
containing 25 mM TRIS pH 8.3, 192 mM glycine, 0.05% SDS and 20%
methanol. After transfer, proteins left over in the tissue sections
were visualized by Ponceau S staining (SIGMA, U.S.A.) and total
eluted proteins captured on the NC-trap were visualized by BLOT
FastStain (Chemicon, USA). The image formed on the trap
demonstrated successful transfer of the protein through the
membranes.
[0064] To determine whether sufficient total protein trapped on
each membrane during the transfer to perform immunological
detection 14 arbitrarily selected antibodies were used. Antibodies
were: Anti-GAPDH, 1:100 (Chemicon, MAB374); Anti-Rsk, 1:1,000
(Transduction Laboratories, R23820); Anti-Stat5a, 1:500 (Santa Cruz
Biotechnology, sc-1081); Anti-IFNalpha, 1:500 (Biosource, AHC4814);
Anti-RARalpha, 1:1,000 (Biomol, sa-178); Anti-phospho-EGFR, 1:1,000
(Upstate, 05483); Anti-non-phospho EGFR, 1:1,000 (Upstate, 05-484);
Anti-phospho-NR1, 1:500 (Upstate, 06-651); Anti-Stat1, 1:2,000
(Transduction Laboratories, G16920); Anti-Rb, 1:1,000 (Santa Cruz
Biotechnology, sc-50); Anti-Jak1, 1:500 (Santa Cruz Biotechnology,
sc-295); Anti-tubulin-alpha, 1:500 (Santa Cruz Biotechnology,
sc-5546); Anti-beta-actin, 1:2,000 (SIGMA, A5441).
[0065] Polycarbonate layers were first blocked in 1.times. casein
solution (Vector Labs, U.S.A.) for 1 hour at room temperature and
incubated overnight at +4.degree. C. in primary antibodies as
listed in FIG. 6 followed by TBST washes and incubation in alkaline
phosphatase (AP) conjugated secondary antibodies (1:2,000 dilution)
(Rockland, U.S.A.). Membranes were then incubated for 5 minutes in
enhanced chemiluminescence substrate (ECL, Vector Labs, U.S.A.)
followed by visualization of the protein by exposing the membranes
to X-ray film (Kodak, U.S.A.).
[0066] The results showed that the method of this invention allows
detection of number of different proteins. To ascertain how the
membranes performed with respect to the amount of total protein
captured, the membranes were each incubated with the same antibody,
allowing determination of the protein content on each of them.
Anti-GAPDH antibody was used for 3 hours at room temperature,
washed in TBST, incubated with anti-mouse secondary antibody
conjugated to horseradish peroxidase (HRP) and visualized in
enhanced chemiluminescence substrate specific only for HRP (PIERCE,
U.S.A.). After ECL reaction membranes were exposed to film as
stated before. The results confirmed that all of the membranes did
capture a similar portion of the total protein and differences seen
in the first part of the experiment are not the result of
differences in membrane "loading." For documentation purposes, the
X-ray film was scanned on the flat bed scanner (Lacie, USA) and
images were processed using ADOBE PhotoShop 4.0.
[0067] Although certain disclosed embodiments of the invention have
been described herein, it will be apparent to those skilled in the
art to which the invention pertains that variations and
modifications of the described embodiment may be made without
departing from the spirit and scope of the invention. Accordingly,
it is intended that the invention be limited only to the extent
required by the appended claims and the applicable rules of law.
The references cited above are hereby incorporated herein in their
entirety.
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