U.S. patent application number 10/035368 was filed with the patent office on 2002-11-07 for microarrays and uses therefor.
Invention is credited to Fernandez, Joseph M., Hoeffler, James P., Nasoff, Marc S..
Application Number | 20020164656 10/035368 |
Document ID | / |
Family ID | 22114700 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020164656 |
Kind Code |
A1 |
Hoeffler, James P. ; et
al. |
November 7, 2002 |
Microarrays and uses therefor
Abstract
Methods of using microarrays to simplify analysis and
characterization of genes and their function are provided. Such
methods can be used to identify and characterize antibodies having
binding affinity for a specific target antigen. A method of
determining gene expression at the protein level by contacting an
array of characterized or uncharacterized antibodies on a solid
surface with one or more proteins and identifying the antibodies to
which said protein(s) binds also is provided. This method can be
used to compare the protein expression in two different populations
of cells, such as normal cells and cancer cells or resting cells
and stimulated cells. In addition, a method of determining gene
expression at the protein level by contacting a microarray of
nucleic acid samples derived from a variety of different sources
with one or more nucleic acid probes then identifying the sample or
samples to which the probe binds is provided.
Inventors: |
Hoeffler, James P.;
(Carlsbad, CA) ; Fernandez, Joseph M.; (Carlsbad,
CA) ; Nasoff, Marc S.; (San Diego, CA) |
Correspondence
Address: |
GARY CARY WARE & FRIENDENRICH LLP
4365 EXECUTIVE DRIVE
SUITE 1600
SAN DIEGO
CA
92121-2189
US
|
Family ID: |
22114700 |
Appl. No.: |
10/035368 |
Filed: |
October 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10035368 |
Oct 26, 2001 |
|
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09245615 |
Feb 4, 1999 |
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60073605 |
Feb 4, 1998 |
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Current U.S.
Class: |
435/7.2 ;
435/287.2; 435/7.9 |
Current CPC
Class: |
G01N 33/6854 20130101;
B01J 2219/00637 20130101; B01J 2219/0061 20130101; B01J 2219/00612
20130101; G01N 33/548 20130101; B01J 2219/00605 20130101; G01N
33/54393 20130101; B01J 2219/00626 20130101; B01J 2219/00608
20130101; B01J 2219/00641 20130101; G01N 33/6845 20130101; B01J
2219/00659 20130101; C12Q 1/6837 20130101; G01N 33/543
20130101 |
Class at
Publication: |
435/7.2 ;
435/7.9; 435/287.2 |
International
Class: |
G01N 033/53; G01N
033/567; G01N 033/542; C12M 001/34 |
Claims
That which is claimed is:
1. A method of identifying antibodies having binding affinity for
an antigen, said method comprising: (a) contacting an array of
uncharacterized antibodies on a solid surface with at least one
antigen; and (b) identifying the antibodies to which the antigen
binds.
2. A method according to claim 1 wherein the antigen is a
protein.
3. A method according to claim 1 wherein the antigen is an intact
cell.
4. A method according to claim 1 wherein the antigen is a cell
lysate.
5. A method according to claim 2 wherein the protein is
recombinant.
6. A method according to claim 5 wherein the protein is
full-length.
7. A method according to claim 5 wherein the protein is a protein
fragment.
8. A method according to claim 7 wherein the protein fragment is
encoded by an EST fragment.
9. A method according to claim 1 wherein the antibodies are
monoclonal antibodies.
10. A method according to claim 1 wherein the antibodies are
polyclonal antibodies.
11. A method according to claim 1 wherein the antibodies are
antibody fragments.
12. A method according to claim 11 wherein the antibody fragments
are single chain antibodies.
13. A method according to claim 1 wherein the antibodies are
recombinant antibodies.
14. A method according to claim 1 wherein the antigen is detectably
labeled.
15. A method according to claim 14 wherein the detectable label is
a fluorescent moiety, avidin, streptavidin, or biotin.
16. A method according to claim 1 wherein the antigen is a fusion
protein comprised of an epitope tag or a fluorescent protein.
17. A method according to claim 1 wherein the binding affinity of
said antibody for said antigen is determined by iterative washing
of said solid surface with a suitable diluent and detecting when
antigen is no longer released therefrom.
18. A method of comparing protein expression in two or more
populations of cells, said method comprising: (a) contacting an
array of antibodies on a solid surface with a cell lysate of a
first cell population, generating a first binding pattern; (b)
contacting a duplicate array of antibodies on a solid surface with
a cell lysate of a second cell population, generating a second
binding pattern; and (c) comparing the binding pattern of the first
cell lysate with the binding pattern of the second cell lysate.
19. A method according to claim 18 wherein the antibodies are
uncharacterized antibodies.
20. A method according to claim 18 wherein the antibodies are
recombinant antibodies.
21. A method according to claim 18 wherein the first cell lysate is
derived from normal cells and the second cell lysate is derived
from abnormal cells.
22. A method according to claim 21 wherein the abnormal cells are
cancer cells.
23. A method according to claim 18 wherein the first cell lysate is
derived from normal cells in a resting state and the second cell
lysate is derived from normal cells in a stimulated state.
24. A method according to claim 18 wherein the difference between
the first and second set of cells is the presence of a different
detectable label.
25. A method for determining the effect of varying binding
conditions on the binding affinity of antibodies to a specific
antigen, said method comprising: (a) contacting an array of
antibodies on a solid surface with at least one antigen under a
first set of binding conditions, generating a first binding
pattern; (b) contacting a duplicate array with the antigen under a
second set of binding conditions, generating a second binding
pattern; (c) comparing the first and second binding patterns.
26. A method according to claim 21 wherein said varying binding
conditions comprise varying pH, temperature, salt concentration,
and/or duration.
27. A method for characterizing a cell, based on the pattern of
protein expression produced thereby, said method comprising: (a)
contacting an array of antibodies on a solid surface with a cell
lysate; and (b) identifying the profile of antibodies to which
components of the lysate binds.
28. A method of diagnosing a disorder, said method comprising: (a)
contacting an array of antibodies specific for one or more antigens
characteristic of a disorder with a biological sample obtained from
a subject under conditions suitable for the formation of an
antigen:antibody complex, wherein the presence of the antigens in
the biological sample would be indicative of the disorder; and (b)
detecting the formation of any antibody: antigen complexes.
29. A method according to claim 28 wherein the biological sample is
cerebral spinal fluid, blood, plasma, urine, feces, saliva, tears,
or extracted tissue.
30. A method according to claim 29 wherein the disorder is stroke,
cerebral hemorrhage, myocardial infarction, peripheral blood clots,
diabetes, cancer, Alzheimer's disease, and sepsis.
31. A kit comprising: (a) an array of uncharacterized antibodies on
a solid surface; and (b) instructions for using the array.
32. A kit according to claim 31 wherein the instructions are for
identifying antibodies to a specific antigen, comparing protein
expression in two or more populations of cells, characterizing a
cell based on the pattern of protein expression produced thereby,
or determining the effect of varying binding conditions on the
binding affinity of the antibodies.
33. A kit according to claim 31 wherein the antibodies are
monoclonal antibodies, polyclonal antibodies or antibody
fragments.
34. A kit according to claim 33 wherein the antibody fragments are
single chain antibodies.
35. A kit according to claim 31 wherein the antibodies are
recombinant antibodies.
36. A kit according to claim 31 further comprising reagents for
detecting an antigen and instructions for use thereof.
37. A kit comprising: (a) an array of antibodies on a solid
surface; and (b) instructions for using the array; wherein the
instructions are for diagnosing a disorder, characterizing a cell
based on the pattern of protein expression produced thereby, or
comparing protein expression in two or more populations of
cells.
38. A kit according to claim 37 further comprising reagents for
detecting an antigen and instructions for use thereof.
39. A kit according to claim 37 wherein the antibodies are
recombinant antibodies.
40. A kit according to claim 37 wherein the antibodies are single
chain antibodies.
41. A method of comparing protein expression patterns, said method
comprising: (a) contacting a microarray of nucleic acid samples
derived from different sources with one or more nucleic acid probes
and (b) identifying the sample or samples to which the probe(s)
binds.
42. A method according to claim 41 wherein the microarray comprises
nucleic acid samples derived from a single tissue type but from
different species.
43. A method according to claim 41 wherein the microarray comprises
nucleic acid samples derived from a single species but from
different tissue types.
44. A method according to claim 41 wherein the microarray comprises
nucleic acid samples derived from the same tissue type at different
developmental stages.
45. A method according to claim 41 wherein the nucleic acid samples
are comprised of mRNA or cDNA.
46. A method according to claim 41 wherein the probe is detectably
labeled.
47. A method according to claim 46 wherein the detectable label is
a fluorescent label.
48. A method according to claim 18 wherein the first and second
cell lysates are derived from cells from a single tissue type but
from different species.
49. A method according to claim 18 wherein the first and second
cell lysates are derived from cells from a single species but from
different tissue types.
50. A method according to claim 18 wherein the first and second
cell lysates are derived from cells from the same tissue type at
different developmental stages.
Description
[0001] This application is a divisional application of U.S. Ser.
No. 09/245,615, filed Feb. 4, 1999, which claims the benefit of
priority under 35 U.S.C. .sctn. 119 of U.S. Ser. No. 60/073,605,
filed Feb. 4, 1998 (now abandoned), the entire contents of each of
which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention disclosed herein relates to new methods of
using microarray technologies. The methods are useful for
identifying and characterizing specific antibodies as well as the
characterization of different tissues or cells by protein or
nucleic acid analysis.
BACKGROUND OF THE INVENTION
[0003] Recent breakthroughs in nucleic acid sequencing technology
have made possible the sequencing of entire genomes from a variety
of organisms, including humans. The potential benefits of a
complete genome sequence are many, ranging from applications in
medicine to a greater understanding of evolutionary processes.
These benefits cannot be fully realized, however, without an
understanding of how and where these newly sequenced genes
function.
[0004] Traditionally, functional understanding started with
recognizing an activity, isolating a protein associated with that
activity, then isolating the gene, or genes, encoding that protein.
The isolated protein was also used to generate antibody reagents.
Specific antibodies and fragments of the isolated gene were both
employed to study tissue expression and function.
[0005] Several methods have been used to study protein expression
patterns including in situ hybridization studies of tissue sections
and northern blots. These methods are both time consuming and
require relatively large amounts of material to perform
successfully.
[0006] Antibodies that bind to specific antigens have been produced
by a variety of methods including immunization of animals, fusion
of mammalian spleen cells to immortalized cells to produce
hybridomas, random peptide generation using phage or bacterial
display and constrained peptide libraries. Regardless of how the
desired antibody is generated, the methods currently available to
identify one with a particular binding specificity are generally
laborious and incapable of the simultaneous testing of large
numbers of unknowns.
[0007] One method involves binding the antigen to a porous
membrane, such as nitrocellulose, contacting the membrane with a
source of test antibodies, then determining whether or not any of
the test antibodies has bound to the antigen. This method only
allows the testing of one source of test antibodies per piece of
porous membrane, making the method both inconvenient and wasteful
of materials.
[0008] Antibody/antigen reactions can also be evaluated in plastic
plates, such as 96-well microtiter plates, using methods similar to
those described above. This method is likewise limited in the
number of samples that can be tested in any one assay, thus
requiring many assays to fully evaluate a large number of antibody
unknowns. Chang (U.S. Pat. No. 4,591,570, issued 5/27/86) describes
an array of a limited number of characterized antibodies to known
antigens on a glass surface that can be used to bind to specific
antigens on the surface of whole cells.
[0009] Recently new technologies have arisen that allow the
creation of microarrays containing thousands or millions of
different elements. Such array technology has been applied mainly
to forming arrays of individual nucleic acids (see, for example,
Marshall and Hodgson, Nature Biotech. 16:27-31, 1998; Ramsay,
Nature Biotech. 16:40-44, 1998), in particular short
oligonucleotides synthesized in situ.
[0010] Methods are needed to simply and rapidly screen very large
numbers of uncharacterized antibodies for those specific for a
given antigen as well as for the characterization of tissues and
cells by nucleic acid and/or protein analysis. The invention
described herein addresses that need.
BRIEF DESCRIPTION OF THE INVENTION
[0011] The invention disclosed herein comprises methods of using
microarrays to simplify analysis and characterization of genes and
their function. In one aspect of the invention the methods are used
to identify and characterize antibodies having binding affinity for
a specific target antigen. This method comprises contacting an
array of uncharacterized antibodies bound to a solid surface with
at least one target antigen and identifying the antibodies to which
the target antigen binds. The method can be performed under a
variety of conditions to identify antibodies with a range of
binding affinities.
[0012] A second aspect of the invention comprises a method of
determining gene expression at the protein level comprising
contacting an array of characterized or uncharacterized antibodies
on a solid surface with one or more proteins and identifying the
antibodies to which said protein(s) binds. This method can be
further used to compare the protein expression in two different
populations of cells, such as normal cells and cancer cells or
resting cells and stimulated cells. A related embodiment can be
used as a tool in the diagnosis of various disorders.
[0013] A further aspect of the invention comprises a method of
determining gene expression at the protein level comprising
contacting a microarray of nucleic acid samples derived from a
variety of different sources with one or more nucleic acid probes
then identifying the sample or samples to which the probe
binds.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIGS. 1A, 1B, and 1C show microarrays of antibodies bound to
positively charged nylon, reacted with antigen and detected by
non-fluorescent means.
[0015] FIG. 2 shows a microarray produced using a robotic arraying
apparatus. Antigen binding is detected by non-fluorescent
means.
[0016] FIG. 3 shows the ability of the antibody microarrays to
evaluate relative binding affinities to a specific antigen.
[0017] FIG. 4 shows a microarray of polyclonal antibodies in
comparison to a microarray of monoclonal antibodies.
[0018] FIGS. 5A, 5B and 5C show a microarray of antibodies reacted
with a cell lysate under conditions that vary the amount of
background binding.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention discloses methods of using microarrays
to simplify analysis and characterization of genes and their
function. In a first aspect of the invention the methods are used
for identifying and characterizing antibodies having binding
specificity to a particular antigen or set of antigens. This method
utilizes microarray technology to create ordered matrices of large
numbers of uncharacterized antibodies which can then be contacted
with antigen under a variety of conditions. The method is rapid and
simple to perform and is applicable to the simultaneous screening
of very large numbers of antibodies.
[0020] Briefly, uncharacterized antibodies are bound to a solid
surface in an array format consisting of discrete spots whose
spatial location can be easily identified. Each location represents
an antibody from a known source, such as a particular hybridoma
growing in a well in a 96-well microtiter plate. The space between
the antibody spots is treated to minimize non-specific binding to
the solid support. The arrayed antibodies are then contacted with
an antigen, or a set of antigens, for which specific antibodies are
sought. The antigen solution is left in contact with the array for
an amount of time sufficient to allow antigen:antibody complexes to
form (generally 10 minutes to 2 hours), then the unbound antigen is
washed away under suitable conditions. Bound antigen is detected at
a particular antibody spot using one of a variety of detection
methods, thus identifying the source of an antibody specific for
the particular antigen.
[0021] The term "antibody" is used herein in the broadest sense and
specifically includes intact monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g. bispecific antibodies)
formed from at least two intact antibodies, and antibody fragments,
including single chain antibodies, so long as they exhibit the
desired binding properties as described herein.
[0022] Various procedures well-known in the art may be used for the
production of polyclonal antibodies to an epitope or antigen of
interest. A host animal of any of a number of species, such as
rabbits, goats, sheep, horse, cow, mice, rats, etc. is immunized by
injection with an antigenic preparation which may be derived from
cells or microorganisms, or may be recombinantly or synthetically
produced. Various adjuvants well known in the art may be used to
enhance the production of antibodies by the immunized host, for
example, Freund's adjuvant (complete and incomplete), mineral gels
such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, liposomes,
potentially useful human adjuvants such as BCG (Bacille
Calmette-Guerin) and Propionibacterium acanes, and the like.
[0023] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. Preferred antibodies are mAbs, which
may be of any immunoglobulin class including IgG, IgM, IgE, IgA,
and any subclass or isotype thereof.
[0024] In addition to their specificity, monoclonal antibodies are
advantageous in that they are synthesized by hybridoma culture,
uncontaminated by other immunoglobulins. The modifier "monoclonal"
indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any
particular method. For example, the monoclonal antibodies to be
used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Pat. No. 4,816,567, incorporated by reference herein). The
"monoclonal antibodies" may also be isolated from phage antibody
libraries using the techniques described in Clackson et al.,
Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,
222:581-597 (1991), for example.
[0025] The monoclonal antibodies contemplated for use herein
specifically include "chimeric" antibodies (immunoglobulins) in
which a portion of the heavy and/or light chain is identical with
or homologous to corresponding sequences in antibodies derived from
a particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired biological activity (U.S. Pat. No. 4,816,567; Morrison
et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
[0026] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementarity-determining region (CDR)
of the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues which are found neither
in the recipient antibody nor in the imported CDR or framework
sequences. These modifications are made to further refine and
maximize antibody performance. In general, the humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin. For further
details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et
al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992). The humanized antibody includes a
PRIMATIZED.TM. antibody wherein the antigen-binding region of the
antibody is derived from an antibody produced by immunizing macaque
monkeys with the antigen of interest.
[0027] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies
(Zapata et al. Protein Eng. 8(10):1057-1062 (1995)); single-chain
antibody molecules; and multispecific antibodies formed from
antibody fragments.
[0028] Particularly preferred in the practice of the invention are
single-chain antibodies. "Single-chain" or "sFv" antibodies are
antibody fragments comprising the V.sub.H and V.sub.Ldomains of an
antibody, wherein these domains are present in a single polypeptide
chain. Preferably, the Fv polypeptide further comprises a
polypeptide linker between the V.sub.H and V.sub.L domains which
enables the sFv to form the desired structure for antigen binding.
For a review of sFvs see Pluckthun in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
[0029] Large quantities of single chain antibodies with
uncharacterized randomized binding specificity can be produced
using a number of methodologies known in the art. Recombinant
antibody libraries can be created in filamentous phage particles
(Daniels and Lane, Methods 9(3):494-507, 1996; Reichmann and Weill,
Biochemistry 32(34):8848-8855; Rader and Barbas, Curr Opin
Biotechnol 9(4):503-508, 1997; Iba and Kurosawa, Immunol Cell Biol
75(2):217-221, 1997, WO 90/05144, WO 92/01047, WO 92/20791, WO
93/19172, GB 9722131.8, GB9810228.8 and GB 9810223.9, all of which
are incorporated by reference herein in their entirety), for
example, or similarly in yeast, bacteria, and the like. Other
methods for creating random libraries of sFvs include various solid
state synthesis methods.
[0030] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[0031] The antibodies employed in the invention can be isolated
prior to creating a microarray. An "isolated" molecule, whether an
antibody, antigen or nucleic acid, is one which has been identified
and separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with particular uses for the
molecule, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, a protein will be purified (1) to greater than 95% by
weight of protein as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated protein
includes the protein in situ within recombinant cells since at
least one component of the protein's natural environment will not
be present. Ordinarily, however, isolated protein will be prepared
by at least one purification step. Unpurified antibodies, such as
those found in serum, can also be employed in the present
invention.
[0032] By "isolated" in reference to nucleic acid is meant a
polymer of 14, 17, 21 or more contiguous nucleotides, including DNA
or RNA that is isolated from a natural source or that is
synthesized. The isolated nucleic acid of the present invention is
unique in the sense that it is not found in a pure or separated
state in nature. Use of the term "isolated" indicates that a
naturally occurring sequence has been removed from its normal
cellular (i.e., chromosomal) environment. Thus, the sequence may be
in a cell-free solution or placed in a different cellular
environment. The term does not imply that the sequence is the only
nucleotide sequence present, but that it is essentially free (about
90-95% pure at least) of non-nucleotide material naturally
associated with it and thus is meant to be distinguished from
isolated chromosomes.
[0033] One particularly useful method of isolating antibodies, such
as single chain antibodies from a cell extract, is affinity
purification. Resins suitable for antibody purification are well
known in the art, for example, protein A SEPHAROSE.TM.. A
recombinant antibody can be engineered to contain an affinity
purification tag to facilitate its purification. Resins suitable
for antibody purification are well known in the art, for example,
protein A SEPHAROSE.TM. resin.
[0034] Affinity purification tags are generally peptide sequences
that can interact with a binding partner immobilized on a solid
support. Synthetic DNA sequences encoding multiple consecutive
single amino acids, such as histidine, when fused to the expressed
protein, may be used for one-step purification of the recombinant
protein by high affinity binding to a resin column, such as nickel
SEPHAROSE.TM. resin. An endopeptidase recognition sequence can be
engineered between the polyamino acid tag and the protein of
interest to allow subsequent removal of the leader peptide by
digestion with enterokinase, and other proteases. Sequences
encoding peptides such as the chitin binding domain (which binds to
chitin), biotin (which binds to avidin and strepavidin), and the
like can also be used for facilitating purification of the protein
of interest. The affinity purification tag can be separated from
the protein of interest by methods well known in the art, including
the use of inteins (protein self-splicing elements, Chong, et al,
Gene 192:271-281, 1997).
[0035] By using an amount of resin with binding sites sufficient
for only a small portion of the antibody present in the unpurified
mixture, the process of isolation can be used to simultaneously
normalize yield and isolate the antibody. For example, although
each sample will contain a different and unknown amount of antibody
protein, the samples can be contacted with an amount of resin whose
maximum binding capacity is 10 mgs. Thus any antibody greater than
this amount will pass through the resin unbound. The maximum bound
amount can then be eluted from the resin.
[0036] Methods for creating microarrays are known in the art
including printing on a solid surface using pins (passive pins,
quill pins, and the like) or spotting with individual drops of
solution. Passive pins draw up enough sample to dispense a single
spot. Quill pins draw up enough liquid to dispense multiple spots.
Bubble printers use a loop to capture a small volume which is
dispensed by pushing a rod through the loop. Microdispensing uses a
syringe mechanism to deliver multiple spots of a fixed volume. In
addition, solid supports, can be arrayed using piezoelectric (ink
jet) technology, which actively transfers samples to a solid
support.
[0037] One method is described in Shalon and Brown (WO 95/35505,
published 12/28/95) which is incorporated herein by reference in
its entirety. The method and apparatus described in Shalon and
Brown can create an array of up to six hundred spots per square
centimeter on a glass slide using a volume of 0.01 to 100 nl per
spot. Suitable concentrations of antibody range from about 1
ng/.mu.l to about 1 .mu.g/.mu.l. In the present invention, each
spot can contain one or more than one distinct antibody.
[0038] Other methods of creating arrays are known in the art,
including photolithographic printing (Pease, et al, Proc. Natl.
Acad. Sci. USA, 91(11):5022-5026, 1994) and in situ synthesis.
While known in situ synthesis methods are less useful for
synthesizing polypeptides long enough to be antibodies, they can be
used to make polypeptides up to 50 amino acids in length, which can
serve as binding proteins as described below.
[0039] The microarrays can be created on a variety of solid
surfaces such as plastics (e.g. polycarbonate), complex
carbohydrates (e.g. agarose and SEPHAROSE.TM.), acrylic resins
(e.g. polyacrylamide and latex beads), and nitrocellulose.
Preferred solid support materials include glass slides, silicon
wafers, and positively charged nylon. Specific examples of suitable
solid supports are described in the Examples below.
[0040] Methods for covalent attachment of antibodies to a solid
support are known in the art. Examples of such methods are found in
Bhatia, et al, Anal. Biochem. 178(2):408-413, 1989; Ahluwalia, et
al, Biosens. Bioelectron. 7(3):207-214, 1992; Jonsson, et al,
Biochem. J. 227(2):373-378, 1985; and Freij-Larsson, et al,
Biomaterials 17(22):2199-2207, 1996, all of which are incorporated
by reference herein in their entirety. Proteins may additionally be
attached to a solid support using methods described in the examples
below.
[0041] Methods of reducing non-specific binding to a solid surface
are well known in the art and include washing the arrayed solid
surface with bovine serum albumin (BSA), reconstituted non-fat
milk, salmon sperm DNA, porcine heparin, and the like (see Ausubel,
et al., Short Protocols in Molecular Biology, 3rd ed. 1995).
[0042] The arrays used to identify antigen-specific antibodies are
contacted with a solution containing one or more known antigens in
order to identify antibodies in the array with binding specificity
for the antigen. The antigens are often proteins, although they may
also be organic chemical compounds, carbohydrates, nucleic acids,
and the like. They may be isolated or semi-isolated, recombinant or
naturally occurring. The amount of antigen used can vary from about
1-100 ng/.mu.l. The antigen is left in contact with the array for
an amount of time sufficient for antibody:antigen complexes to
form, should one of the antibodies in the array be specific for the
antigen. The amount of time sufficient for this purpose will range
from 5 minutes to 24 hours, and will generally be from 0.5 to 2
hours.
[0043] One antigen of particular interest in the practice of the
invention is recombinant protein, either a full-length gene product
or a fragment thereof, for example an Expressed Sequence Tag (or
EST fragment). EST fragments are relatively short cDNA sequences
that have been randomly generated and sequenced, generally as part
of an ongoing effort to map an entire genome (Adams, et al, Science
252(5013):1651-1656, 1991). Large numbers of these sequences are
available in public databases. The identity of the proteins encoded
by the vast majority of these sequences is unknown. The following
discussion, although directed to the expression of EST-encoded
peptides, is equally applicable to any expressed product of a
nucleic acid sequence, including full-length proteins.
[0044] Techniques are available in the art by which cells can be
genetically engineered to express the peptide encoded by a given
EST fragment. The methods of the invention can then be used to
identify antibodies specific for the peptide. These antibodies are
then useful as reagents that can be employed in purification and
identification of the full-length protein, and in other
experimental procedures designed to elucidate the protein's
location and function.
[0045] Prokaryotic hosts are, generally, very efficient and
convenient for the production of recombinant proteins and are,
therefore, one type of preferred expression system for EST
fragments. Prokaryotes most frequently are represented by various
strains of E. coli. However, other microbial strains may also be
used, including other bacterial strains.
[0046] In prokaryotic systems, plasmid vectors that contain
replication sites and control sequences derived from a species
compatible with the host may be used. Examples of suitable plasmid
vectors may include pBR322, pUC 118, pUC 119, and the like;
suitable phage or bacteriophage vectors may include .lambda.gt10,
.lambda.gt11, and the like; and suitable virus vectors may include
pMAM-neo, PKRC and the like. Preferably, the selected vector of the
present invention has the capacity to replicate in the selected
host cell.
[0047] Recognized prokaryotic hosts include bacteria such as E.
coli and those from genera such as Bacillus, Streptomyces,
Pseudomonas, Salmonella, Serratia, and the like. However, under
such conditions, the polypeptide will not be glycosylated. The
prokaryotic host selected for use herein must be compatible with
the replicon and control sequences in the expression plasmid.
[0048] To express an EST fragment in a prokaryotic cell, it is
necessary to operably link the gene sequence to a functional
prokaryotic promoter such as the T7 promoter or RSC promoter. Such
promoters may be either constitutive or, more preferably,
regulatable (i.e., inducible or derepressible). Examples of
constitutive promoters include the int promoter of bacteriophage
.lambda., the bla promoter of the .beta.-lactamase gene sequence of
pBR322, the CAT promoter of the chloramphenicol acetyl transferase
gene sequence of pPR325, and the like. Examples of inducible
prokaryotic promoters include the major right and left promoters of
bacteriophage (P.sub.L and P.sub.R), the trp, reca, lacZ, LacI, and
gal promoters of E. coli, the .alpha.-amylase (Ulmanen et al., J.
Bacteriol. 162:176-182, 1985) and the sigma-28-specific promoters
of B. subtilis (Gilman et al., Gene sequence 32:11-20(1984)), the
promoters of the bacteriophages of Bacillus (Gryczan, In: The
Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)),
Streptomyces promoters (Ward et at., Mol. Gen. Genet. 203:468-478,
1986), and the like. Exemplary prokaryotic promoters are reviewed
by Glick (J. Ind. Microbiol. 1:277-282, 1987); Cenatiempo
(Biochimie 68:505-516, 1986); and Gottesman (Ann. Rev. Genet.
18:415-442, 1984).
[0049] Proper expression in a prokaryotic cell also requires the
presence of a ribosome binding site upstream of the gene
sequence-encoding sequence. Such ribosome binding sites are
disclosed, for example, by Gold et at. (Ann. Rev. Microbiol.
35:365-404, 1981). The selection of control sequences, expression
vectors, transformation methods, and the like, are dependent on the
type of host cell used to express the gene.
[0050] Host cells which may be used in the expression systems of
the present invention are not strictly limited, provided that they
are suitable for use in the expression of the peptide of interest.
Suitable hosts may often include eukaryotic cells. Preferred
eukaryotic hosts include, for example, yeast, fungi, insect cells,
and mammalian cells either in vivo, or in tissue culture. Mammalian
cells which may be useful as hosts include HeLa cells, cells of
fibroblast origin such as VERO, 3T3 or CHOK1, HEK 293 cells or
cells of lymphoid origin (such as 32D cells) and their derivatives.
Preferred mammalian host cells include SP2/0 and JS58L, as well as
neuroblastoma cell lines such as IMR 332 and PC12 which may provide
better capacities for correct post-translational processing.
[0051] In addition, plant cells are also available as hosts, and
control sequences compatible with plant cells are available, such
as the cauliflower mosaic virus 35S and 19S, nopaline synthase
promoter and polyadenylation signal sequences, and the like.
Another preferred host is an insect cell, for example the
Drosophila larvae. Using insect cells as hosts, the Drosophila
alcohol dehydrogenase promoter can be used. Rubin, Science
240:1453-1459, 1988). Alternatively, baculovirus vectors can be
engineered to express large amounts of peptide encoded by an EST
fragment in insects cells (Jasny, Science 238:1653, 1987); Miller
et al., In: Genetic Engineering (1986), Setlow, J. K., et al.,
eds., Plenum, Vol. 8, pp. 277-297).
[0052] Any of a series of yeast gene sequence expression systems
can be utilized which incorporate promoter and termination elements
from the actively expressed gene sequences coding for glycolytic
enzymes which are produced in large quantities when yeast are grown
in media rich in glucose. Known glycolytic gene sequences can also
provide very efficient transcriptional control signals. Yeast
provides substantial advantages in that it can also carry out
post-translational peptide modifications. A number of recombinant
DNA strategies exist which utilize strong promoter sequences and
high copy number of plasmids which can be utilized for production
of the desired proteins in yeast. Yeast recognizes leader sequences
on cloned mammalian gene sequence products and secretes peptides
bearing leader sequences (i.e., pre-peptides). For a mammalian
host, several possible vector systems are available for the
expression of and EST fragment
[0053] A wide variety of transcriptional and translational
regulatory sequences may be employed, depending upon the nature of
the host. The transcriptional and translational regulatory signals
may be derived from viral sources, such as adenovirus, bovine
papilloma virus, cytomegalovirus, simian virus, or the like, where
the regulatory signals are associated with a particular gene
sequence which has a high level of expression. Alternatively,
promoters from mammalian expression products, such as actin,
collagen, myosin, and the like, may be employed. Transcriptional
initiation regulatory signals may be selected which allow for
repression or activation, so that expression of the gene sequences
can be modulated. Of interest are regulatory signals which are
temperature-sensitive so that by varying the temperature,
expression can be repressed or initiated, or are subject to
chemical (such as metabolite) regulation.
[0054] Expression of an EST fragment in eukaryotic hosts involves
the use of eukaryotic regulatory regions. Such regions will, in
general, include a promoter region sufficient to direct the
initiation of RNA synthesis. Preferred eukaryotic promoters
include, for example, the promoter of the mouse metallothionein I
gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982);
the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982);
the SV40 early promoter (Benoist et al., Nature (London)
290:304-310, 1981); the yeast gal4 gene sequence promoter (Johnston
et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982); Silver et
al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984), the CMV
promoter, the EF-1 promoter, and the like.
[0055] An EST fragment and an operably linked promoter may be
introduced into a recipient prokaryotic or eukaryotic cell either
as a nonreplicating DNA (or RNA) molecule, which may either be a
linear molecule or, more preferably, a closed covalent circular
molecule (a plasmid). Since such molecules are incapable of
autonomous replication, the expression of the gene may occur
through the transient expression of the introduced sequence.
Alternatively, permanent or stable expression may occur through the
integration of the introduced DNA sequence into the host
chromosome.
[0056] A vector may be employed which is capable of integrating the
desired gene sequences into the host cell chromosome. Cells which
have stably integrated the introduced DNA into their chromosomes
can be selected by also introducing one or more markers which allow
for selection of host cells which contain the expression vector.
The marker may provide for prototrophy to an auxotrophic host,
biocide resistance, e.g., antibiotics, or heavy metals, such as
copper, or the like. The selectable marker gene sequence can either
be directly linked to the DNA gene sequences to be expressed, or
introduced into the same cell by cotransfection. Common selectable
marker gene sequences include those for resistance to antibiotics
such as ampicillin, tetracycline, kanamycin, bleomycin,
streptomycin, hygromycin, neomycin, Zeocin.TM., and the like.
Selectable auxotrophic gene sequences include, for example, hisD,
which allows growth in histidine free media in the presence of
histidinol.
[0057] Additional elements may also be needed for optimal synthesis
of single chain binding protein mRNA. These elements may include
splice signals, as well as transcription promoters, enhancers, and
termination signals. cDNA expression vectors incorporating such
elements include those described by Okayama, Mol. Cell. Bio. 3:280,
1983.
[0058] The recombinant antigen may be produced as a fusion protein.
When two protein-coding sequences not normally associated with each
other in nature are in the same reading frame the resulting
expressed protein is called a "fusion protein" as two distinct
proteins have been "fused" together. Fusion proteins have a wide
variety of uses. For example, two functional enzymes can be fused
to produce a single protein with multiple enzymatic activities or
short peptide sequences, such as epitope tags or affinity
purification tags (see above), can be fused to a larger protein and
serve as aids in purification or as means of identifying the
expressed protein by serving as epitopes detectable by specific
antibodies.
[0059] Epitope tags are short peptide sequences that are recognized
by epitope-specific antibodies. A fusion protein comprising a
recombinant protein and an epitope tag can be simply and easily
purified using an antibody bound to a chromatography resin. The
presence of the epitope tag furthermore allows the recombinant
protein to be detected in subsequent assays, such as Western blots,
without having to produce an antibody specific for the recombinant
protein itself. Examples of commonly used epitope tags include V5,
glutathione-S-transferase (GST), hemagglutinin (HA), the peptide
Phe-His-His-Thr-Thr, chitin binding domain, and the like.
[0060] A fusion protein may be a means by which the recombinant
antigen protein can be easily detected. For example, the fusion
component can itself be a detectable moiety, such as fluorescent
protein (fluorescent green protein, fluorescent yellow protein, and
the like), or alternatively can be one member of a specific binding
pair (such as biotin and streptavidin, for example) which can be
detected by reacting with the other member conjugated to a
detectable substance.
[0061] The foregoing elements can be combined to produce vectors
suitable for use in the methods of the invention. Those of skill in
the art would be able to select and combine the elements suitable
for use in their particular system.
[0062] The introduced nucleic acid molecule can be incorporated
into a plasmid or viral vector capable of autonomous replication in
the recipient host. Any of a wide variety of vectors may be
employed for this purpose. Factors of importance in selecting a
particular plasmid or viral vector include: the ease with which
recipient cells that contain the vector may be recognized and
selected from those recipient cells which do not contain the
vector; the number of copies of the vector which are desired in a
particular host; and whether it is desirable to be able to
"shuttle" the vector between host cells of different species.
[0063] Suitable prokaryotic vectors include plasmids such as those
capable of replication in E. coli (for example, pBR322, ColEl,
pSC101, PACYC 184, itVX, pRSET, pBAD (Invitrogen, Carlsbad,
Calif.), and the like). Such plasmids are disclosed by Sambrook
(cf. "Molecular Cloning: A Laboratory Manual", second edition,
edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor
Laboratory, (1989)). Bacillus plasmids include pC194, pC221, pT127,
and the like, and are disclosed by Gryczan (In: The Molecular
Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329).
Suitable Streptomyces plasmids include plJlOl (Kendall et al., J.
Bacteriol. 169:4177-4183,1987), and Streptomyces bacteriophages
such as .phi.C31 (Chater et al., In: Sixth International Symposium
on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary
(1986), pp. 45-54). Pseudomonas plasmids are reviewed by John et
al. (Rev. Infect. Dis. 8:693-704, 1986), and Izaki (Jpn. J.
Bacteriol. 33:729-742, 1978).
[0064] Suitable eukaryotic plasmids include, for example, BPV,
vaccinia, SV40, 2-micron circle, pCDN3.1 (Invitrogen), and the
like, or their derivatives. Such plasmids are well known in the art
(Botstein et al., Miami Wntr. Symp. 19:265-274, 1982); Broach, In:
"The Molecular Biology of the Yeast Saccharomyces: Life Cycle and
Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., p. 445-470 (1981); Broach, Cell 28:203-204, 1982); Dilon et
at., J. Clin. Hematol. Oncol. 10:39-48, 1980); Maniatis, In: Cell
Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence
Expression, Academic Press, NY, pp. 563-608 (1980).
[0065] Once antibody:antigen complexes have been formed and unbound
antigen washed away under suitable conditions, the antibody:antigen
complexes can be detected using one of several techniques known in
the art. Suitable washing conditions are known to those skilled in
the art (see, for example, Ausubel, et al, Short Protocols in
Molecular Biology, 3rd ed. 1995). Exemplary washing conditions are
shown in the examples below.
[0066] For detection in the case of recombinant antigens,
expression vectors can be used that form chimeric fusion peptides
as described above. The epitope tagged antigen can be 55464
detected using an antibody specific for the tag sequence. This
antibody may be itself detectably labeled or can be detected with a
third detectably-labeled antibody. Alternatively, the antigen can
be complexed with biotin and detected using detectably-labeled
avidin or streptavidin. The antigen itself can also be detectably
labeled, such as with a fluorescent dye compound.
[0067] The term "detectably labeled" as used herein is intended to
encompass antigen directly coupled to a detectable substance, such
as a fluorescent dye, and antigen coupled to a member of binding
pair, such as biotin/streptavidin, or an epitope tag that can
specifically interact with a molecule that can be detected, such as
by producing a colored substrate or fluorescence.
[0068] Substances suitable for detectably labeling proteins include
fluorescent dyes such as fluorescein isothiocyanate (FITC),
fluorescein, rhodamine, tetramethyl-rhodamine-5-(and
6)-isothiocyanate (TRITC), Texas red, cyanine dyes (Cy3 and Cy5,
for example), and the like; and enzymes that react with
colorimetric substrates such as horseradish peroxidase. The use of
fluorescent dyes is generally preferred in the practice of the
invention as they can be detected at very low amounts. Furthermore,
in the case where multiple antigens are reacted with a single
array, each antigen can be labeled with a distinct fluorescent
compound for simultaneous detection. Labeled spots on the array are
detected using a fluorimeter, the presence of a signal indicating
an antigen bound to a specific antibody.
[0069] The formation of antibody:antigen complexes can be performed
under a variety of conditions to identify antibodies with varying
binding characteristics. Antigen-containing reaction solutions can
contain varying degrees of salt or be conducted at varying pH
levels. In addition, the binding reaction can be carried out at
varying temperatures. Each set of conditions will identify
antibodies with different affinity for the antigen. For example,
antibodies that bind at pH 2 may have utility under highly acidic
conditions such as those that exist in the stomach. Similarly,
antibodies that bind at temperatures near boiling may be useful in
studying thermophilic organisms. In general pH conditions will
range from 2-10 (most preferably around pH 8), temperatures from
0.degree. C.-100.degree. C. and salt conditions from 1 .mu.M to 5 M
(in the case of NaCl).
[0070] Affinity constants are a measure of the interaction between
a particular ligand and its cognate receptor. The "binding
affinity" or the measure of the strength of association between a
particular antibody:antigen interaction is generally measured by
affinity constants for the equilibrium concentrations of associated
and dissociated configurations of the antibody and its antigen.
Preferably the binding of the antigen should occur at an affinity
of about k.sub.a=10.sup.-6M or greater to be useful for the present
invention, with greater than about 10.sup.-7M being more
preferable, and most preferably between about 10.sup.-8M and about
10.sup.-11M. Antibody fragments will generally have affinities in
the range of about 10.sup.-6M to 10.sup.-7M.
[0071] In another embodiment of the invention, microarrays of
uncharacterized antibodies are used to compare the protein
expression profiles of cells. For example, comparisons can be made
between a population of cells from one tissue, such as arterial
endothelial cells, and a second tissue, such as venous endothelial
cells or from cells derived from a particular tissue but from
different species. Comparisons can be made between normal cells and
cells from the same tissue type that originate from an individual
with a pathogenic disorder. For example, comparisons can be made
between normal cells and cancer cells. Comparisons can additionally
be made between cells in a resting state and cells in an activated
state, for example, resting T-cells and activated T-cells.
[0072] In another example, the disclosed arrays are useful for
evaluating the expression of proteins by pathogens, such as, for
example, bacteria, parasites, viruses, and the like. A solution
(such as a lysate) made from the pathogen which represents all
proteins expressed by the pathogen can be used to contact an
antibody array to identify antibodies recognizing
pathogen-expressed proteins. These antibodies have utility as
diagnostic agents as well as potential therapeutics.
[0073] Cellular lysates can be used as "antigens" as described
above and reacted with two identical microarrays. Antibodies
reactive in one array but not the other would indicate the presence
of a differentially expressed protein. This antibody is then useful
for the subsequent isolation and identification of those proteins
that are different in two populations of cells. In the case of
normal and cancer cells, for example, one may be able to identify
proteins expressed in the cancer cell that contribute to its
malignant state.
[0074] In a further aspect of the invention, microarrays can be
composed of previously characterized antibodies. These microarrays
have a variety of uses, one of which is cell profiling. For
example, an array can be composed of antibodies that recognize a
set of antigens known to be present in activated T-cells but not in
resting T-cells. A population of T-cells can then be lysed and the
lysate contacted with the array to determine if the population has
the profile of activated or resting T-cells.
[0075] Microarrays and the methods disclosed herein can be used in
methods of diagnosing particular disorders. For example, a
collection of antibodies specific for a range of antigens
associated with one or more disorders can be arrayed and contacted
with a bodily fluid containing antigens whose presence, or absence,
would indicate a particular disorder. The advantage of using a
microarray over a conventional immunoassay is the ability to
include a population of antibodies diagnostic for a variety of
disorders on a single surface, significantly reducing time, costs
and materials needed to effect a diagnosis.
[0076] For example, if a patient presents with symptoms that are
characteristic of several distinct disorders which can be
distinguished on the basis of the presence or absence of one or
more proteins, a single microarray assay could be used to make a
specific diagnosis, thus allowing the patient to be properly
treated. Patients suffering from stroke or brain infarcts release
several proteins into cerebrospinal fluid, examples of which are
neuron specific enolyse (NSE) from neuronal cells and S-100 from
glial cells and astrocytes. Such proteins are not released in
conditions that may have similar symptoms, such as drug reactions,
making proper diagnosis more difficult. A diagnostic array could
readily detect these and other proteins in the CSF, leading to a
rapid clinical diagnosis and treatment.
[0077] In another aspect of the invention microarrays are employed
to characterize protein expression patterns using nucleic acid
samples. Briefly, nucleic acid molecules from a whole cell or
tissue are applied to a solid support using a microarray format.
The arrayed nucleic acid samples are then contacted with a nucleic
acid probe specific for a gene encoding a known protein. The probe
solution is left in contact with the array for an amount of time
sufficient to allow sample:probe complexes to form, then the
unbound probe is washed away under suitable conditions (see, for
example, Ausubel, et al, Short Protocols in Molecular Biology, 3rd
ed. 1995 and the examples below). Bound probe is detected at one or
more nucleic acid sample spots using one of a variety of detection
methods.
[0078] This aspect of the invention has a variety of uses. For
example, the microarray can be constructed from nucleic acid
samples isolated from a single tissue type but from a large number
of species, with each spot representing a particular species. Thus
in a single assay format one can determine the evolutionary
development of the protein represented by the probe. Similarly, the
microarray can be constructed of multiple tissue types from a
single species, or from different developmental stages of a single
species (or multiple species) thus simply and efficiently
determining tissue expression of the protein represented by the
probe. For example, a microarray can be constructed with arrayed
samples representing all the developmental stages of Drosophila, a
well known organism the study of which has led to a greater
understanding of mammalian physiology and development.
[0079] The nucleic acid sample can be messenger ribonucleic acid
(mRNA) or can be complementary deoxyribonucleic acid (cDNA),
including EST fragments. Methods for extracting and isolating
nucleic acids from cells are well known in the art (for example
phenol extraction/ethanol precipitation, ammonium acetate
precipitation, cesium chloride gradients, and the like), as are
methods for generating cDNA (see, for example, "Molecular Cloning:
A Laboratory Manual," second edition, edited by Sambrook, Fritsch,
& Maniatis, Cold Spring Harbor Laboratory, 1989; and Ausubel,
et al, Short Protocols in Molecular Biology, 3rd ed. 1995, both of
which are incorporated by reference herein). Microarrays of these
nucleic acids are created using the methods described above.
Techniques for coupling nucleic acids to solid supports used to
construct microarrays are well known in the art, including the
poly-L-lysine and phenylboronic acid methods described in the
Examples below.
[0080] The nucleic acid probes used in the invention methods can be
designed based on the sequence of a gene encoding a known protein
or can be an EST fragment, as described above. One skilled in the
art can readily design such probes based on the known sequence
using methods of computer alignment and sequence analysis known in
the art (e.g., "Molecular Cloning: A Laboratory Manual", second
edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring
Harbor Laboratory, 1989; Ausubel, et al, Short Protocols in
Molecular Biology, 3rd ed. 1995). The probe can comprise any number
of nucleotides but will preferably be not fewer than 10 nucleotides
and preferably not more than about 300 nucleotides in length.
[0081] The probes of the invention can be labeled by standard
labeling techniques such as with a radiolabel, enzyme label,
fluorescent label, biotin-avidin label, chemiluminescent label, and
the like. After hybridization, the probes may be detected using
known methods. Preferred labels are fluorescent labels, as
described above.
[0082] The nucleic acid probes of the present invention include RNA
as well as DNA probes and nucleic acids modified in the sugar,
phosphate or even the base portion as long as the probe still
retains the ability to specifically hybridize under conditions as
disclosed herein. Such probes are generated using techniques known
in the art.
[0083] The term "hybridize" as used herein refers to a method of
interacting a nucleic acid sequence with a DNA or RNA molecule in
solution or on a solid support, such as cellulose or
nitrocellulose. If a nucleic acid sequence binds to the DNA or RNA
molecule with sufficiently high affinity, it is said to "hybridize"
to the DNA or RNA molecule. The strength of the interaction between
the probing sequence and its target can be assessed by varying the
stringency of the hybridization conditions. Various low to high
stringency hybridization conditions may be used depending upon the
specificity and selectivity desired. Stringency is controlled by
varying salt or denaturant concentrations. Examples of
hybridization conditions are shown in the Examples below. Those
skilled in the art readily recognize how such conditions can be
varied to vary specificity and selectivity. For example, under
highly stringent hybridization conditions only highly complementary
nucleic acid sequences hybridize. Preferably, such conditions
prevent hybridization of nucleic acids having even one or two
mismatches out of 20 contiguous nucleotides.
[0084] In a further aspect of the invention, microarrays can be
composed of randomly generated polynucleotides (DNA or RNA) and
contacted with proteins to identify unique binding pairs.
Polynucleotides are now known to bind to proteins and may have
potential as diagnostics and therapeutics (see, for example, Allen,
et al, Virology 209(2):327-336, 1995; Binkley, et al, Nucleic Acids
Res. 23(16):3198-3205, 1995). Polynucleotides can be evaluated in
very large numbers using the methods disclosed herein thus
increasing the likelihood of identifying a useful binder.
[0085] The invention will now be described in greater detail by
reference to the following non-limiting examples.
EXAMPLES
Example I-Nucleic Acid Microarrays
[0086] The following procedures are conducted at room temperature
and using double distilled water unless otherwise noted. These
methods are applicable to arrays of polypeptides or polynucleic
acids.
[0087] Glass slides are prepared as follows: NaOH (50 g) is
dissolved in 150 ml of double distilled water (ddH.sub.2O), then
200 ml of 95% EtOH is added while stirring. If the solution becomes
cloudy, ddH.sub.2O is added until it becomes clear. Approximately
30 glass slides (Gold Seal, Cat. No. 3010) are soaked in the
NaOH/EtOH solution for 2 hours, shaking. The slides are then rinsed
three times with ddH.sub.2O. The slides are next soaked in a
poly-L-lysine solution (70 ml poly-L-lysine (Sigma Cat. No. 8920)
to 280 ddH.sub.2O) for 1 hour. Excess liquid is removed by spinning
the slides in a rack on a microtiter plate carrier at 500 rpm. The
slides are dried at 40.degree. C. for 5 minutes, then stored in a
closed box for at least 2 weeks prior to use.
[0088] A cDNA microarray is prepared as follows: Total MRNA is
isolated from tissue (for example, nerve cells) of a variety of
species representative of different classes of organisms such as
Drosophila, nematode, salmon, clam, chicken, mouse, dog, goat,
spider monkey, chimpanzee, human, and the like, by the FastTrac
method (Stratagene, La Jolla, Calif.) or other common methods. mRNA
is also obtained from a variety of unicellular organisms such as E.
coli, yeast, B. subtilis, mycoplasma and the like. Eukaryotic mRNA
is enriched from total RNA using oligo(dT) cellulose (Ausubel, et
al, Short Protocols in Molecular Biology, 3rd ed. 1995, pgs
4-11-4-12). Equivalent amounts (for example, 1 .mu.g) of mRNA from
each source are placed in a separate well of one or more 96 well
microtiter plates and precipitated with cold EtOH. The precipitate
is rinsed with 70% EtOH and allowed to dry.
[0089] The dried mRNA is resuspended in 3.times. SSC (sodium
chloride/sodium citrate--20X solution is 3 M NaCl (175g/L0 0.3 M
trisodium citrate 2H.sub.2O (88g/L adjusted to pH 7.0 with 1 M HCl)
then spotted onto a previously prepared glass slide using an array
device (for example, Shalon and Brown (WO 95/35505, published
12/28/95)). The prepared array can be kept for a long period of
time before probing, however, if the slides are to be kept for long
periods of time, stability is increased by converting each mRNA
sample into cDNA using techniques known in the art, such as
PCR.
[0090] The array is rehydrated by suspending the slide over a dish
of ddH.sub.2O (50.degree. C.) for approximately one minute. The
slide is quickly (approximately 3 seconds) dried by placing it on a
surface heated to 100.degree. C. (mRNA side up). The mRNA is
crosslinked to the poly-L-lysine coating of the slide using
ultraviolet radiation using a Stratalinker.TM. UV device according
to the manufacturer's instructions (Stratagene) set at 60
milliJoules.
[0091] The slides are next soaked in a solution of 5 grams of
succinic anhydride (Aldrich Cat. No. 23,969-0) dissolved in 315 ml
of N-methyl-pyrrilidinone (Aldrich Cat. No. 32,863-4) plus 35 mls
of 0.2 M sodium borate (brought to pH 8.0 with NaOH) for 15 minutes
with shaking. The slide is then transferred to a 95.degree. C.
water bath for 2 minutes followed by 95% EtOH for 1 minute. Excess
liquid is removed from the slides by spinning a rack of slides on a
microtiter plate carrier at 500 rpm.
[0092] A probe sequence of a known protein (for example, human
nerve growth factor, GenBank Accession No. E03589) is labeled using
standard protocols, for example by using a CyDye.TM. Nick
Translation kit (Amersham). The labeled probe (approximately 1
.mu.g/ml) is resuspended in 4.times. SSC (10 .mu.l) to which is
added 0.2 .mu.l 10% sodium dodecyl sulfate (SDS). The probe is
boiled for 2 minutes, then cooled for 10 seconds and transferred to
the array by pipette. The array is covered by a 22 mm.times.22 mm
cover slip, and the slide is placed in a humid hybridization
chamber and submerged into a hot water bath (.gtoreq.75.degree.
C.).
[0093] The slide is left in the bath for 10-24 hours, then the
cover slip is removed and the slide rinsed in 0.2.times. SSC with
0.1% SDS several times. Excess wash buffer is removed by
centrifugation on a microtiter plate carrier as described above.
The slide is scanned using a spectrofluorometer, such as the
ScanArray 3000 (General Scanning Inc., Watertown, Mass.). For
probes labeled with Cy5, for example, fluorescence is measured at
670 nm. Localization of spots on the array to which the probe
hybridizes indicates that the species represented by the spot
expresses a protein similar or identical to the probe protein.
[0094] The procedure outlined below is an alternative method for
binding arrayed molecules to a solid support, using an
SA(OCH.sub.2CN)-X-NHS linkage (see, for example, U.S. Pat. No.
5,594,111, issued 1/14/97; U.S. Pat. No. 5,648,470, issued May 15,
1997; U.S. Pat. No. 5,623,055, issued Apr. 22, 1997; all of which
are incorporated by reference herein).
[0095] Glass slides (Fisher Catalog No. 12-544-4) are soaked in an
acid bath (1 hour in 0.1 M HCl), then washed with water and dried
at room temperature. The slides should not be aggressively dried,
such as in an oven. The slides are next soaked in a silane solution
overnight at room temperature (5% APTES
(3-aminopropyl-triethoxysilane, Aldrich 28,177-8), 0.3% DIEA
(Sigma) v/v in EtOH). The slides may be sonicated for 10-15 minutes
right after being placed in the APTES solution.
[0096] The slides are rinsed with isopropyl alcohol, then sonicated
in isopropyl alcohol for several minutes. Sonication should remove
any white silane residue on the slides. If the residue remains, the
slides should be discarded. After sonication, the slides are left
to cure/dry for at least 24 hours before use.
[0097] The cured slides are next soaked in a linker solution
overnight at room temperature. The linker solution is made by
dissolving 115 mg of 9Y SA(OCH.sub.2CN)-X-COOH (Prolix, Bothell,
Wash.) in 1 ml dimethylformamide (DMF) plus 60 .mu.l DIEA, then
adding 60 mg TSTU (Sigma) and leaving for 15 minutes at room
temperature. This stock is diluted in 270 ml of isopropyl alcohol
plus 270 .mu.l DIEA before using.
[0098] The slides are removed from the linker solution and soaked
in 1 M NH.sub.2OH, 1 mM EDTA, 0.1 M NaHCO.sub.3 (pH 10) for 4 hours
at room temperature. This solution is removed, the slides are
extensively washed with water then let air dry at room temperature.
The slides can be stored at room temperature away from light before
using to make arrays.
Example II
Determination of Optimal Concentrations of Antibody and Antigen
[0099] Various concentrations (1 .mu.g/.mu.l, 100 ng/.mu.l, 10
ng/.mu.l, 1 ng/.mu.l) of total mouse IgG or a mouse monoclonal
anti-PLC-gamma were spotted on aldehyde slides (Cel Associates,
Inc., Houston, Tex.), which allow non-covalent attachment of
proteins. Using a manual 8 pin hand arrayer the slides were blocked
for 1 hour with PBST (phosphate buffered saline and 0.10% Tween
20), and 3% milk protein. The slides were subsequently washed three
times, 15 minutes each, in PBST. Duplicate slides were incubated
with 50 .mu.l of goat anti-mouse IgG antibody (GAMG) conjugated
with CY3 or CY5 fluorescent dye compounds (Amersham, Arlington
Heights, Ill.) at 10 .mu.g/ml or 1 .mu.g/ml. Slides were then
washed for 15 minutes in PBST three additional times and dried by
centrifugation prior to scanning. Binding was detected as shown in
Table 1 below.
Example III
Comparison of Solid Supports
[0100] Serial dilutions (1 .mu.g/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml)
of mouse IgG or PLC-gamma were hand arrayed onto aldehyde,
polystyrene, nitrocellulose and Surmodics slides. Aldehyde,
nitrocellulose, polystyrene and Surmodics slides were purchased
from various outside vendors (aldehyde Slides--Cel Associates,
Inc., Houston, Tex.; nitrocellulose Slides --Molecular Probes,
Inc., Eugene, Oreg.; polystyrene Slides--Nunc, Inc., Naperville,
Ill.; Surmodics Slides--Surmodics, Inc., Eden Prairie, Minn.).
Surmodics slides have an undisclosed polymer on the glass surface
which forms a covalent linkage with proteins under the appropriate
conditions (described by the manufacturer).
[0101] Following hand arraying of the antibodies (approximately
20-30 nanoliters per spot), the nitrocellulose, aldehyde, and
polystyrene slides were immediately blocked for 1 hour with PBST
and 3% milk, washed 3 times with PBST, and hybridized with 50 .mu.l
of GAMG-CY3 for 30 minutes. Surmodics slides were incubated
overnight in a moist salt chamber as recommended by the
manufacturer. The following day, the Surmodics slides were
processed as described above. Following hybridization all of the
various slides were washed 3 times in PB ST, dried and scanned
using a Scan Array 3000 fluorescent scanner.
1TABLE 1 Detection Antibody Conc. Antigen Conc. Level PLC-gamma 1
.mu.g/.mu.l GAMG-CY3 10 .mu.g/ml +++ 100 ng/.mu.l 10 .mu.g/ml +++
10 ng/.mu.l 10 .mu.g/ml + 1 ng/.mu.l 10 .mu.g/ml - mouse IgG 1
.mu.g/.mu.l GAMG-CY3 10 .mu.g/ml +++ 100 ng/.mu.l 10 .mu.g/ml +++
10 ng/.mu.l 10 .mu.g/ml + 1 ng/.mu.l 10 .mu.g/ml - PLC-gamma 1
.mu.g/.mu.l GAMG-CY3 1 .mu.g/ml + 100 ng/.mu.l 1 .mu.g/ml + 10
ng/.mu.l 1 .mu.g/ml - 1 ng/.mu.l 1 .mu.g/ml - mouse IgG 1 ng/.mu.l
GAMG-CY3 1 .mu.g/ml + 100 ng/.mu.l 1 .mu.g/ml + 10 ng/.mu.l 1
.mu.g/ml - 1 ng/.mu.l 1 .mu.g/ml - PLC-gamma 1 .mu.g/gl GAMG-CY5 1
.mu.g/ml +++ 100 ng/.mu.l 1 .mu.g/ml +++ 10 ng/.mu.l 1 .mu.g/ml + 1
ng/.mu.l 1 .mu.g/ml mouse IgG 1 ng/.mu.l GAMG-CY5 1 .mu.g/ml +++
100 ng/.mu.l 1 .mu.g/ml +++ 10 ng/.mu.l 1 .mu.g/ml + 1 ng/.mu.l 1
.mu.g/ml - PLC-gamma 1 .mu.g/.mu.l GAMG-CY5 1 .mu.g/ml + 100
ng/.mu.l 1 .mu.g/ml + 10 ng/.mu.l 1 .mu.g/ml - 1 ng/.mu.l 1
.mu.g/ml - mouse IgG 1 .mu.g/.mu.l GAMG-CY5 1 .mu.g/ml + 100
ng/.mu.l 1 .mu.g/ml + 10 ng/.mu.l 1 .mu.g/ml - 1 ng/.mu.l 1
.mu.g/ml - +++ strong signal, ++ moderate signal + weak signal, -
no signal
[0102] All of the slides tested allowed for the detection of
antigen:antibody binding at higher concentrations of antibody. The
Aldehyde and Nitrocellulose treated slides were the most efficient
at binding antibody, and antibody:antigen interaction could be
detected at 1 ng/.mu.l.
Example IV
Detection of Binding Using Non-Fluorescent Methods
[0103] Positively charged nylon filters (Zeta Probe Membranes,
BioRad Laboratories, Hercules, Calif.) were hand arrayed using 1
.mu.l of anti-His, anti-V5, anti-thioredoxin (anti-Thio), anti-FOS,
anti-PLC-gamma and anti-CREB antibodies (Invitrogen, Carlsbad,
Calif.; all antibodies were approximately 1 mg/ml). Filters were
blocked for 1 hour with PBST and 3% milk, washed three times with
PBST, and incubated with 1 .mu.g/ml biotinylated D1 protein for
three hours at room temperature. D1 is a creatine kinase fusion
protein isolated from a human fetal heart cDNA library and cloned
into the pBAD-Thio-His-TOPO vector (Invitrogen, Carlsbad, Calif.)
to create a Thioredoxin-V5-His-creatine kinase fusion protein. D1
was biotinylated using the EZ-Link.TM. Sulfo-NHS-LC Biotinylation
Kit (Pierce, Rockford, Ill.) used according to the manufacturer's
instructions).
[0104] Following three additional washes with the same buffer,
filters were treated with streptavidin/alkaline phosphatase
conjugate or streptavidin/horseradish peroxidase conjugate
(Boehringer Mannheim, GmbH Germany) for 1 hour at room
temperature.
[0105] The filters were washed 5 times with PBST, dried, and
developed by immersion in ECL chemiluminescent substrate
(ECL--Amersham, Arlington Heights, Ill.) or the chromogenic
substrate BCIP/NBT (Sigma Chemicals, St. Louis, Mo.). Filters
developed with ECL were exposed to Kodak chemiluminescent film for
1 to 10 seconds.
[0106] The results are shown in FIGS. 1A, 1B, and 1C. In all cases,
only the antibodies specific for epitopes on the fusion protein
antigen were detectable, and only in the arrayed spots, showing
that the system has both good signal to noise ratio and
specificity.
[0107] The experiment was repeated using an array created with an
automated arrayer. Antibodies (1 mg/ml) were spotted using an
automated 96 pin microarrayer developed at Invitrogen. Fifteen
negative control antibodies (assorted mouse monoclonals) were
arrayed along with the three positive control antibodies (anti-His,
anti-Thio, anti-V5). Filters were treated as described above using
the alkaline phosphatase conjugate and the chromogenic substrate
BCIP/NBT.
[0108] As can been seen in FIG. 2, binding and detection of
antibody:antigen complexes was highly specific and sensitive.
Example V
Evaluation of Antibody Affinity
[0109] Anti-His, anti-V5, anti-FOS, anti-PLC-gamma, 25ClDG, and
anti-VEGF (vascular endothelial growth factor) antibodies were
arrayed on a nitrocellulose slide and reacted with biotinylated D1
protein as previously described. Binding was detected with
streptavidin-Cy3 as described above. The anti-V5 antibodies spots
showed red, the anti-His spots showed green, while the negative
controls were undetectable (see FIG. 3). When viewed in a black and
white drawing, relative increase in binding affinity is visualized
by an increase of white in a given area. The color of the spots
generally indicates a higher amount of fluorescently labeled
antigen present, and thus indicates relative binding affinity
between antibody and antigen. Colors, in descending order from
highest to lowest affinity, are white, red, yellow, green, and
blue. Using this technique, multiple antibodies can be tested for
their affinity to a single antigen.
Example VI
Polyclonal Antibody Microarrays
[0110] To demonstrate specific binding to polyclonal antibodies,
six antibodies were arrayed by hand on a nitrocellulose slide,
three polyclonal antibodies (anti-E12 (unpurified rabbit polyclonal
sera to a His-V5-thioredoxin-thymidine kinase fusion protein),
anti-lexA (lexA repressor protein), and anti-GFP(Green fluorescent
protein)) and three monoclonal antibodies (anti-V5, anti-His and
anti-GalU (a mammalian transcription factor). The slide was blocked
with PBST and 3% milk for 1 hour at room temperature, and incubated
with the E12-biotin conjugate, prepared according to the protocol
used for D1 protein. Following extensive washing with PBST, the
slides were incubated with streptavidin-CY3 conjugate (Amersham,
Arlington Heights, Ill.) for 1 hour at room temperature, washed 5
times with PBST and dried by centrifugation prior to scanning on
the Scan Array 3000.
[0111] As can be seen in FIG. 4, binding was detected with both the
antigen specific polyclonal antibody (anti-E12) and the antigen
specific monoclonal antibodies (anti-His, anti-V5) and not with any
of the negative control antibodies.
Example VII
Microarray Analysis of Labeled Cell Lysate
[0112] A series of experiments were conducted to determine if a
microarray of antibodies could specifically detect antigens in a
cell lysate.
[0113] CHO cells expressing high levels of beta-galactosidase were
grown to confluency in a T-175 flask. (Hams media with Pen/Strep,
and L-glutamine plus 10% FCS, at 37.degree. C. with 5% CO.sub.2)
Cells were harvested using Trypsin/EDTA. NP40 extracts were
prepared by pelleting the cells (10.sup.7 cells), washing once in
PBS and resuspending in 5% NP40. Cell debris was removed by
centrifugation. Soluble protein was biotinylated using a Pierce
biotinylation kit according to the manufacturer's instructions.
[0114] Nitrocellulose slides (see above) containing arrayed
monoclonal antibodies (anti-beta-gal, anti-His, anti-Thio, anti-V5,
anti-FOS, anti-PLC-gamma, anti-VEGF and 25C10G (an anti-CREB
antibody) were blocked, washed, hybridized and developed with
streptavidin-CY3 as described in Example VI supra. As can be seen
in FIG. 5A, beta-galactosidase binding was seen, however, some
non-specific binding was detected as well.
[0115] The experiment was repeated, except that after
centrifugation of the extract, soluble protein was dialyzed
overnight against 50 mM phosphate buffer at 4.degree. C. prior to
biotinylation. As can be seen in Figure 5B, much of the
non-specific binding seen in the previous experiment was
eliminated.
[0116] In the next experiment dialyzed extract containing the
biotinylated soluble proteins was adjusted to 10% glycerol to
reduce non-specific hydrophobic interactions. Furthermore, the
sodium chloride concentration was adjusted to 0.2 M NaCl to
increase specific ionic interactions. All other conditions remained
identical. As can be seen in FIG. 5C, all non-specific binding was
eliminated using this protocol.
* * * * *