U.S. patent application number 10/931015 was filed with the patent office on 2005-02-03 for suppression of cross-reactivity and non-specific binding by antibodies using protein a.
Invention is credited to Shao, Weiping.
Application Number | 20050025773 10/931015 |
Document ID | / |
Family ID | 25461263 |
Filed Date | 2005-02-03 |
United States Patent
Application |
20050025773 |
Kind Code |
A1 |
Shao, Weiping |
February 3, 2005 |
Suppression of cross-reactivity and non-specific binding by
antibodies using protein A
Abstract
The structure, formation and use of blocked antibodies,
especially those blocked with Protein A, or active fragments of
Protein A, are disclosed as well as processes of producing such
antibodies. The uses of such blocked antibodies to achieve
significant reduction in both specific cross-reaction and
non-specific interaction thereby increasing specificity and
reactivity with targeted antigenic sites is also described.
Inventors: |
Shao, Weiping; (Cheshire,
CT) |
Correspondence
Address: |
CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART & OLSTEIN
5 Becker Farm Road
Roseland
NJ
07068
US
|
Family ID: |
25461263 |
Appl. No.: |
10/931015 |
Filed: |
August 31, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10931015 |
Aug 31, 2004 |
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09931736 |
Aug 17, 2001 |
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Current U.S.
Class: |
424/178.1 ;
435/320.1; 435/328; 435/69.1; 530/391.1; 536/23.53 |
Current CPC
Class: |
G01N 2035/00158
20130101; G01N 33/543 20130101; C07K 2319/00 20130101; G01N 33/6857
20130101; C07K 16/00 20130101; C07K 14/31 20130101 |
Class at
Publication: |
424/178.1 ;
435/069.1; 435/320.1; 435/328; 530/391.1; 536/023.53 |
International
Class: |
C12Q 001/70; C07H
021/04; A61K 039/395; C07K 016/46 |
Claims
What is claimed is:
1. A blocked immunoglobulin comprising an antibody portion and a
Protein A portion.
2. The blocked immunoglobulin of claim 1 wherein said antibody
portion comprises at least one light chain variable region of an
antibody.
3. The blocked immunoglobulin of claim 1 wherein said antibody
portion comprises at least one heavy chain variable region of an
antibody.
4. The blocked immunoglobulin of claim 1 wherein said antibody
portion comprises at least one light chain variable region and at
least one heavy chain variable region of an antibody.
5. The blocked immunoglobulin of claim 1 wherein said antibody
portion comprises two light chain variable regions of an
antibody.
6. The blocked immunoglobulin of claim 1 wherein said antibody
portion comprises two heavy chain variable regions of an
antibody.
7. The blocked immunoglobulin of claim 1 wherein said antibody
portion comprises two light chain variable regions and two heavy
chain variable regions of an antibody.
8. The blocked immunoglobulin of claim 1 wherein said antibody
portion comprises at least one antigen-reactive fragment of an
antibody.
9. The blocked immunoglobulin of claim 1 wherein said Protein A
portion comprises at least one protein A compound.
10. The blocked immunoglobulin of claim 9 wherein said Protein A
compound is a fragment of Protein A.
11. The blocked immunoglobulin of claim 1 further comprising a
solid support to which said immunoglobulin is attached.
12. The blocked immunoglobulin of claim 11 wherein said
immunoglobulin is attached to said solid support through a covalent
linkage.
13. The blocked immunoglobulin of claim 11 wherein the antibody
portion of said immunoglobulin is attached to said solid
support.
14. The blocked immunoglobulin of claim 11 wherein the antibody
portion of said immunoglobulin is attached to said solid support
through a tether.
15. The blocked immunoglobulin of claim 11 wherein the Protein A
portion of said immunoglobulin is attached to said solid
support.
16. The blocked immunoglobulin of claim 11 wherein the Protein A
portion of said immunoglobulin is attached to said solid support
through a tether.
17. The blocked immunoglobulin of claim 11 wherein the solid
support is made of a material selected from the group consisting of
acrylamide, agarose, cellulose, nitrocellulose, glass, polystyrene,
polyethylene vinyl acetate, polypropylene, polymethacrylate,
polyethylene, polyethylene oxide, polysilicates, polycarbonates,
teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides,
polyglycolic acid, polylactic acid, polyorthoesters,
polypropylfumerate, collagen, glycosaminoglycans, and polyamino
acids.
18. The blocked immunoglobulin of claim 11 wherein the solid
support further comprises a member selected from the group
consisting of thin film, membrane, bottles, dishes, fibers, woven
fibers, shaped polymers, particles, beads, microparticles, and a
combination of the foregoing.
19. A composition comprising at least one blocked immunoglobulin of
claim 1 in a suitable carrier.
20. The composition of claim 19 comprising at least two blocked
immunoglobulins of claim 1 in a suitable carrier.
21. The composition of claim 20 wherein said blocked
immunoglobulins have different specificities.
22. A composition comprising at least one blocked immunoglobulin of
claim 11 in a suitable carrier.
23. The composition of claim 22 comprising at least two blocked
immunoglobulins of claim 11 in a suitable carrier.
24. The composition of claim 23 wherein said blocked
immunoglobulins have different specificities.
25. An microarray comprising a solid support attached to a
plurality of blocked immunoglobulins of claim 1.
26. The microarray of claim 25 wherein the antibody portion of each
of said blocked immunoglobulins has the same antigenic
specificity.
27. The microarray of claim 25 wherein the antibody portion of at
least two of said blocked immunoglobulins have different antigenic
specificities.
28. The microarray of claim 25 wherein the antibody portion of each
of said blocked immunoglobulins has a different antigenic
specificity.
29. The microarray of claim 25 wherein said solid support is made
of a member selected from the group consisting of acrylamide,
agarose, cellulose, nitrocellulose, glass, polystyrene,
polyethylene vinyl acetate, polypropylene, polymethacrylate,
polyethylene, polyethylene oxide, polysilicates, polycarbonates,
teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides,
polyglycolic acid, polylactic acid, polyorthoesters,
polypropylfumerate, collagen, glycosaminoglycans, and polyamino
acids.
30. The microarray of claim 25 wherein said microarray is a bead or
microparticle.
31. The microarray of claim 25 wherein the solid support is
porous.
32. A process for forming a blocked immunoglobulin comprising
contacting an antibody with a Protein A compound under conditions
promoting the binding of said Protein A to said antibody.
33. The process of claim 32 wherein said protein A compound is
Protein A.
34. The process of claim 32 wherein said protein A compound is a
fragment of Protein A.
35. The process of claim 32 wherein said antibody forms a covalent
linkage with said Protein A compound.
36. The process of claim 32 wherein said antibody is attached to a
solid support prior to contacting with said Protein A compound.
37. The process of claim 36 wherein said solid support is
porous.
38. The process of claim 36 wherein said solid support is in the
form of beads or microparticles.
39. The process of claim 36 wherein said solid support is composed
of a material selected from the group consisting of acrylamide,
agarose, cellulose, nitrocellulose, glass, polystyrene,
polyethylene vinyl acetate, polypropylene, polymethacrylate,
polyethylene, polyethylene oxide, polysilicates, polycarbonates,
teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides,
polyglycolic acid, polylactic acid, polyorthoesters,
polypropylfumerate, collagen, glycosaminoglycans, and polyamino
acids.
40. The process of claim 36 further comprising quenching the solid
support prior to contacting the antibody with the Protein A
compound.
41. The process of claim 40 wherein said antibody and said Protein
A are contacted at a temperature of at least about 37.degree.
C.
42. The process of claim 41 wherein said contacting occurs for at
least about 30 minutes.
43. The process of claim 42 wherein following said contacting with
Protein A the blocked immunoglobulin is further contacted with a
blocking agent other than a Protein A compound.
44. The process of claim 43 wherein said blocking agent other than
a Protein A compound is bovine serum albumin (BSA).
45. The process of claim 36 wherein said Protein A compound is
Protein A.
46. The process of claim 45 wherein said Protein A is present at a
concentration of at least about 0.5 mg/ml.
47. The process of claim 45 wherein said Protein A is present at a
concentration of about 0.5 mg/ml.
48. A process for detecting an analyte in a sample comprising
contacting an analyte with a blocked immunoglobulin of claim 1
wherein the antibody portion of said blocked immunoglobulin is
specific for said analyte and detecting the binding of said analyte
to said blocked immunoglobulin.
49. The process of claim 48 wherein said sample comprises at least
two antigenically different analytes.
50. The process of claim 48 wherein said analyte is contacted with
more than one blocked immunoglobulin.
51. The process of claim 50 wherein the antibody portion of at
least two of said blocked immunoglobulins exhibits a different
antigenic specificity.
52. The process of claim 48 wherein said sample comprises a
plurality of analytes contacted with a plurality of blocked
immunoglobulins comprising antibody portions having at least two
different antigenic specificities.
53. The process of claim 48 wherein said blocked immunoglobulin is
attached to a solid support.
54. The process of claim 53 wherein said solid support is
porous.
55. The process of claim 53 wherein said solid support is in the
form of beads or microparticles.
56. The process of claim 53 wherein said solid support is composed
of a material selected from the group consisting of acrylamide,
agarose, cellulose, nitrocellulose, glass, polystyrene,
polyethylene vinyl acetate, polypropylene, polymethacrylate,
polyethylene, polyethylene oxide, polysilicates, polycarbonates,
teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides,
polyglycolic acid, polylactic acid, polyorthoesters,
polypropylfumerate, collagen, glycosaminoglycans, and polyamino
acids.
57. A process for detecting an analyte in a sample comprising
contacting an analyte with the microarray of claim 25 wherein the
antibody portion of at least one of the blocked immunoglobulins on
said microarray is specific for said analyte and detecting binding
of an analyte to at least one blocked immunoglobulin on said
microarray.
58. The process of claim 57 wherein the sample contains a plurality
of antigenically different analytes.
59. The process of claim 57 wherein said microarray comprises a
plurality of blocked immunoglobulins comprising antibody portions
exhibiting a plurality of different antigenic specificities.
60. The process of claim 59 wherein said process is part of an
antibody sandwich assay, an enzyme-linked immunosorbent assay, an
antibody dipstick assay, an antibody microarray assay, a
radioimmunoassay, or a rolling circle amplification assay.
61. The process of claim 48 wherein said process is part of an
antibody sandwich assay, an enzyme-linked immunosorbent assay, an
antibody dipstick assay, an antibody microarray assay, a
radioimmunoassay, or a rolling circle amplification assay.
62. The process of claim 48 wherein said process occurs on a
column, a plate, a microtitre dish, a dipstick, a cell sample or a
tissue sample.
63. The process of claim 48 wherein said process occurs in
situ.
64. The process of claim 48 wherein said analyte comprises a
rolling circle replication primer and wherein detection of binding
of analyte to blocked immunoglobulin is accomplished by contacting
said bound analyte with an amplification target circle (ATC)
comprising a primer complementary sequence complementary to a
portion of said primer under conditions promoting rolling circle
amplification and wherein the production of tandem sequence DNA
(TS-DNA) indicates the presence of said analyte.
65. The process of claim 57 wherein said analyte comprises a
rolling circle replication primer and wherein detection of binding
of analyte to blocked immunoglobulin is accomplished by contacting
said bound analyte with an amplification target circle (ATC)
comprising a primer complementary sequence complementary to a
portion of said primer under conditions promoting rolling circle
amplification and wherein the production of tandem sequence DNA
(TS-DNA) indicates the presence of said analyte.
66. The process of claim 64 or 65 wherein said analyte comprises a
protein.
67. The process of claim 48 wherein said blocked immunoglobulin
comprises a rolling circle replication primer and wherein detection
of binding of analyte to blocked immunoglobulin is accomplished by
contacting said bound analyte with an amplification target circle
(ATC) comprising a primer complementary sequence complementary to a
portion of said primer under conditions promoting rolling circle
amplification and wherein the production of tandem sequence DNA
(TS-DNA) indicates the presence of said analyte.
68. The process of claim 57 wherein said blocked immunoglobulin
comprises a rolling circle replication primer and wherein detection
of binding of analyte to blocked immunoglobulin is accomplished by
contacting said bound analyte with an amplification target circle
(ATC) comprising a primer complementary sequence complementary to a
portion of said primer under conditions promoting rolling circle
amplification and wherein the production of tandem sequence DNA
(TS-DNA) indicates the presence of said analyte.
69. The process of claim 64 or 65 wherein said analyte comprises a
protein.
70. A process for detecting analytes, the method comprising an
amplification operation, wherein an amplification target circle is
coupled to a blocked antibody composition, wherein the blocked
antibody composition can interact with an analyte, wherein the
amplification operation comprises rolling circle replication of the
amplification target circle to produce tandem sequence DNA.
71. The process of claim 70 wherein said analyte is a protein.
72. A process for detecting analytes in a sample, comprising: a DNA
ligation operation and an amplification operation, wherein the DNA
ligation operation comprises circularization of an open circle
probe, wherein circularization of the open circle probe is
dependent on hybridization of the open circle probe to a target
sequence, wherein the target sequence is coupled to a blocked
antibody composition, wherein the blocked antibody composition can
interact with an analyte, wherein the amplification operation
comprises rolling circle replication of the circularized open
circle probe to produce tandem sequence DNA.
73. The process of claim 72 wherein said analyte is a protein.
74. A process for detecting analytes, comprising: (a) contacting a
blocked antibody composition with a target sample comprising an
analyte wherein a target sequence is coupled to the blocked
antibody composition, wherein the blocked antibody composition
binds to the analyte, (b) contacting an open circle probe with the
target sample, to produce an OCP-target sample mixture, and
incubating the OCP-target sample mixture under conditions that
promote hybridization between the open circle probe and the target
sequence in the OCP-target sample mixture, (c) contacting a ligase
with the OCP-target sample mixture, to produce a ligation mixture,
and incubating the ligation mixture under conditions that promote
ligation of the open circle probe to form an amplification target
circle, (d) contacting a rolling circle replication primer with the
ligation mixture, to produce a primer-ATC mixture, and incubating
the primer-ATC mixture under conditions that promote hybridization
between the amplification target circle and the rolling circle
replication primer in the primer-ATC mixture, and (e) contacting
DNA polymerase with the primer-ATC mixture, to produce a
polymerase-ATC mixture, and incubating the polymerase-ATC mixture
under conditions that promote replication of the amplification
target circle, wherein replication of the amplification target
circle results in the formation of tandem sequence DNA.
75. The process of claim 74 wherein said analyte is a protein.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of
antibody-antigen interactions and the use of blocked antibodies to
effect significant reduction in both specific cross-reaction and
non-specific interaction thereby increasing specificity and
reactivity with targeted antigenic sites.
BACKGROUND OF THE INVENTION
[0002] Antibodies have found great use in the area of diagnostics
and for assaying for the presence of antigenic materials in samples
such as those derived from biological fluids and for research
purposes. A drawback to such successes, however, has been the
occurrence of unwanted side reactions in addition to the intended
specific reactions of the antibody, which lead to the localization
of the antigens under investigation by the specific antibodies used
in the assays and the like.
[0003] Such unwanted side reactions can include unintended specific
reactions, which are frequently considered as cross-reactivity, and
which, under certain circumstances, may provide useful information
regarding the variations of the structures of the materials under
study. Here, the specific binding agent (e.g., the specific
antibody used in the assay) recognizes epitopes in molecules other
than the antigen under investigation. In addition, there is the
often more troublesome unintended non-specific reactions governed
by general physico-chemical properties, such as hydrophobic and
electrostatic interactions between the antigen and the antibody.
Methods to prevent cross-reactivity and non-specific reactions are
essential to achieve the lowest background and so to optimize assay
performance.
[0004] Fragments of immunoglobulins lacking the Fc region (e.g. Fab
fragments) have been produced for diagnostic immunoassays for a
variety of reasons. The most common reason is to eliminate
interference from rheumatoid factor or other heterophilic antibody
activity (cross linking), which most often occurs at the Fc region
of the antibody. The Fc portion of IgG is also hydrophobic, which
has high non-specific binding potential. None of the antigen
binding domain of the immunoglobulin resides in the Fc portion;
consequently, its removal has little or no effect on antigen
binding affinity. One drawback of this approach is that preparation
of purified Fab fragments is costly and time-consuming.
[0005] Protein A consists of a single polypeptide chain with little
or no carbohydrate. The molecule is relatively heat stable and
retains its native conformation even after exposure to denaturing
reagents. The present invention solves the aforementioned problems
by taking advantage of the discovery that Protein A is able to bind
specifically to the Fc region of immunoglobulin molecules, which is
distant from the antigen binding sites of such immunoglobulins.
This invention discloses for the first time an immunoglobulin
blocked with Protein A as well as processes using such a blocked
structure in the immunological assay of selected analytes. Thus,
the present invention relies on using protein A to block the Fc
region in such a way as to suppress both cross-reactivity and the
non-specific binding associated with this region, and to eliminate
the need to generate Fab or other active fragments.
[0006] The present invention finds use in immunoassays carried out
on microarrays of immobilized antibodies. Measurement of multiple
antigens on these microarrays involves use of complex mixtures of
antibodies for detection steps in the immunoassay. The use of such
mixtures greatly increases the likelihood of non-specific signals,
which can significantly reduce the sensitivity and dynamic range of
the assay. We have shown that Protein A reduces the non-specific
signals associated with the use of complex mixtures of
antibodies.
[0007] In the past, a number of different molecular weight protein
block such as BSA, gelatin, casein, non-fat dry milk, skim milk,
normal serum, etc. have been used to prevent non-specific
binding.
[0008] Protein A not only functions as a normal protein for
blocking, but also has a number of advantages over the normal
protein block:
[0009] 1. By specifically binding the Fc portion of antibodies
(which causes unintended specific binding) Protein A suppresses
cross-reactivity without affecting antigen-antibody binding
sites.
[0010] 2. By specifically binding the Fc portion of antibodies,
protein A changes the Fc region's physico-chemical properties (such
as hydrophobicity) thereby reducing non-specific binding without
affecting antigen-antibody binding sites.
[0011] 3. With protein A blocking, the Signal/Noise ratio in
immunoassays is significantly improved.
BRIEF SUMMARY OF THE INVENTION
[0012] In one aspect, the present invention relates to a blocked
immunoglobulin comprising an antibody portion and a Protein A
portion. In specific embodiments, such blocked immunoglobulin may
comprise in its antibody portion one or more light chain variable
regions of an antibody with specificity for a given analyte and/or
at least one heavy chain variable region of an antibody. In a
preferred embodiment, the present invention provides a blocked
immunoglobulin wherein the antibody portion comprises two light
chain variable regions and two heavy chain variable regions of an
antibody. The protein A portion of said blocked immunoglobulins may
contain Protein A or an active fragment thereof.
[0013] The present invention also relates to such blocked
immunoglobulins that are attached to a solid support, such as
through a covalent linkage or other chemical adduct or attachment,
including via a tether. Such attachment could be through either the
antibody portion or the Protein A portion of the blocked
immunoglobulin.
[0014] In particular embodiments, the solid support is made of a
material such as acrylamide, agarose, cellulose, nitrocellulose,
glass, polystyrene, polyethylene vinyl acetate, polypropylene,
polymethacrylate, polyethylene, polyethylene oxide, polysilicates,
polycarbonates, teflon, fluorocarbons, nylon, silicon rubber,
polyanhydrides, polyglycolic acid, polylactic acid,
polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans,
or polyamino acids, especially glass or plastic.
[0015] In other embodiments, the blocked immunoglobulins of the
invention are attached to a solid support that further comprises a
thin film, membrane, bottles, dishes, fibers, woven fibers, shaped
polymers, particles, beads, microparticles, and a combination of
the foregoing.
[0016] In other embodiments, blocked immunoglobulins of the
invention are present in a composition, such as where they are
dissolved or suspended in a suitable carrier, including any
appropriate diluent or excipient.
[0017] The antibody portion of a blocked immunoglobulin of the
invention may have structures with different specificities for
different analytes. In addition, compositions of the invention may
comprise more than one blocked immunoglobulin, some of which may
have the same antigenic specificities and some of which may have
different antigenic specificities.
[0018] The present invention also relates to an array, such as a
microarray comprising a solid support attached to a plurality of
blocked immunoglobulins of the invention. In some embodiments, the
microarray comprises blocked immunoglobulins wherein the antibody
portion of each of said blocked immunoglobulins has the same
antigenic specificity or has different antigenic specificities,
including embodiments wherein the antibody portion of each of said
blocked immunoglobulins has a different antigenic specificity. Such
microarrays may be present as beads or microparticles and may be
composed of porous or non-porous materials.
[0019] The present invention also provides for a process for
forming a blocked immunoglobulin comprising contacting an antibody
with a Protein A compound under conditions promoting the binding of
said Protein A compound to said antibody. In preferred embodiments,
the protein A compound is protein A or an active fragment thereof.
The resulting linkage may be covalent.
[0020] In other preferred embodiments of this process, the antibody
is attached to a solid support prior to contacting with said
Protein A compound. In an especially preferred embodiment of this
process, the solid support is chemically quenched prior to
attaching the Protein A compound to the antibody.
[0021] The present invention also relates to a process for
detecting an analyte in a sample comprising contacting an analyte
with a blocked immunoglobulin of the invention wherein the antibody
portion of said blocked immunoglobulin is specific for said analyte
and detecting the binding of said analyte to said blocked
immunoglobulin. In a preferred embodiment of such a process, the
sample comprises at least two antigenically different analytes. In
other preferred embodiments, the analyte is contacted with more
than one blocked immunoglobulin, especially where the antibody
portion of at least two of said blocked immunoglobulins exhibits a
different antigenic specificity.
[0022] In an especially preferred embodiment, the sample comprises
a plurality of analytes contacted with a plurality of blocked
immunoglobulins comprising antibody portions having at least two
different antigenic specificities. Such process may take place on a
solid support as disclosed herein, including use of a
microarray.
[0023] In other embodiments of this process, the process may be
part of an antibody sandwich assay, an enzyme-linked immunosorbent
assay, an antibody dipstick assay, an antibody microarray assay, a
radioimmunoassay, or a rolling circle amplification assay or such
process may be part of an antibody sandwich assay, an enzyme-linked
immunosorbent assay, an antibody dipstick assay, an antibody
microarray assay, a radioimmunoassay, or a rolling circle
amplification assay. The processes of the invention may also occur
on a column, a plate, a microtitre dish, a dipstick, a cell sample
or a tissue sample, or in situ.
[0024] The present invention also provides a process for detecting
an analyte in a sample wherein said analyte comprises a rolling
circle replication primer and wherein detection of binding of
analyte to blocked immunoglobulin is accomplished by contacting
said bound analyte with an amplification target circle (ATC)
comprising a primer complementary sequence complementary to a
portion of said primer under conditions promoting rolling circle
amplification and wherein the production of tandem sequence DNA
(TS-DNA) indicates the presence of said analyte. Such process may
occur on a microarray.
[0025] The present invention also provides a process of detecting
an analyte wherein a blocked immunoglobulin comprises a rolling
circle replication primer and wherein detection of binding of
analyte to blocked immunoglobulin is accomplished by contacting
said bound analyte with an amplification target circle (ATC)
comprising a primer complementary sequence complementary to a
portion of said primer under conditions promoting rolling circle
amplification and wherein the production of tandem sequence DNA
(TS-DNA) indicates the presence of said analyte. Such process may
also be part of a microarray. Such analyte may comprise a
protein.
BRIEF DESCRIPTION OF THE DRAWING
[0026] FIG. 1 shows reduction of non-specific signals for a
cross-reactivity experiment using Protein A-blocked immunoglobulin
versus non-protein-A blocked antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention relates to a blocked immunoglobulin
comprising an antibody portion and a Protein A portion. In specific
embodiments, the antibody portion comprises at least one light
chain variable region of an antibody and/or at least one heavy
chain variable region of an antibody. Commonly, there will be at
least two light chain and two heavy chain regions, which may
include both constant and variable regions.
[0028] With the advent of methods of molecular biology and
recombinant technology, it is now possible to produce antibody
molecules by recombinant means and thereby generate gene sequences
that code for specific amino acid sequences found in the
polypeptide structure of the antibodies. Such antibodies can be
produced by either cloning the gene sequences encoding the
polypeptide chains of said antibodies or by direct synthesis of
said polypeptide chains, with in vitro assembly of the synthesized
chains to form active tetrameric (H.sub.2L.sub.2) structures with
affinity for specific epitopes and antigenic determinants. This has
permitted the ready production of antibodies having sequences
characteristic of neutralizing antibodies from different species
and sources.
[0029] Regardless of the source of the antibodies, or how they are
recombinantly constructed, or how they are synthesized, in vitro or
in vivo, using transgenic animals, such as cows, goats and sheep,
using large cell cultures of laboratory or commercial size, in
bioreactors or by direct chemical synthesis employing no living
organisms at any stage of the process, all antibodies have a
similar overall 3 dimensional structure. This structure is often
given as H.sub.2L.sub.2 and refers to the fact that antibodies
commonly comprise 2 light (L) amino acid chains and 2 heavy (H)
amino acid chains. Both chains have regions capable of interacting
with a structurally complementary antigenic target. The regions
interacting with the target are referred to as "variable" or "V"
regions and are characterized by differences in amino acid sequence
from antibodies of different antigenic specificity.
[0030] The variable regions of either H or L chains contains the
amino acid sequences capable of specifically binding to antigenic
targets. Within these sequences are smaller sequences dubbed
"hypervariable" because of their extreme variability between
antibodies of differing specificity. Such hypervariable regions are
also referred to as "complementarity determining regions" or "CDR"
regions. These CDR regions account for the basic specificity of the
antibody for a particular antigenic determinant structure.
[0031] The CDRs represent non-contiguous stretches of amino acids
within the variable regions but, regardless of species, the
positional locations of these critical amino acid sequences within
the variable heavy and light chain regions have been found to have
similar locations within the amino acid sequences of the variable
chains. The variable heavy and light chains of all antibodies each
have 3 CDR regions, each non-contiguous with the others (termed L1,
L2, L3, H1, H2, H3) for the respective light (L) and heavy (H)
chains. The accepted CDR regions have been described by Kabat et
al, J. Biol Chem. 252:6609-6616 (1977). The numbering scheme is
shown in the figures, where the CDRs are underlined and the numbers
follow the Kabat scheme.
[0032] In all mammalian species, antibody polypeptides contain
constant (i.e., highly conserved) and variable regions, and, within
the latter, there are the CDRs and the so-called "framework
regions" made up of amino acid sequences within the variable region
of the heavy or light chain but outside the CDRs.
[0033] The antibodies disclosed according to the invention may also
be wholly synthetic, wherein the polypeptide chains of the
antibodies are synthesized and, possibly, optimized for binding to
the polypeptides disclosed herein as being receptors. Such
antibodies may be chimeric or humanized antibodies and may be fully
tetrameric in structure, or may be dimeric and comprise only a
single heavy and a single light chain. Such antibodies may also
include fragments, such as Fab and F(ab.sub.2)' fragments, capable
of reacting with and binding to any of the polypeptides disclosed
herein as being receptors.
[0034] The blocked immunoglobulin of the invention may also have an
antibody portion that comprises at least one antigen-reactive
fragment of an antibody. However, while the presence of such a
fragment, as disclosed herein, does not in any way limit the
invention, it should be understood that a major advantage of the
invention is that it obviates the need to form such active
fragments in order to avoid the unwanted side reactions produced by
the presence of an Fc region. Thus, by using the blocked
immunoglobulins of the invention there is little or no need to
generate immunologically active fragments of antibodies although
these may find use in some instances, such as where a blocked
immunoglobulin comprises a whole light and heavy chain as a dimer
and the Fc portion of the heavy chain constant region has been
blocked with a protein A compound.
[0035] As used herein the term "Protein A compound" refers to
protein A, a fragment of protein A, or a variant of protein A that
can interact with the Fc region of an antibody. This includes
recombinant forms of protein A and proteolytic fragmens of protein
A (as described, for example, in J. Chromat. 597:527-562
(1992)).
[0036] In preferred embodiments, the protein A compound is protein
A itself. Protein A has two different forms, the native one which
is from Staphylococus aureus, and the recombinant one which is a
genetically truncated version. Both forms of protein A exhibit the
same affinity for Ig G molecules, and have been proved effective
for blocking cross-reactivity and non-specific binding. Besides
microarry-based assay, this invention can also be applied in other
solid-phase and antibody based protein assays or immunoassays. In
addition to the procedures described herein, the blocking
procedures and conditions of protein A with antibodies can be
changed or optimized for specific applications. Proteolytic
treatment of Protein A to generate univalent fragments (J. Chromat.
597, 257-62 (1992)
[0037] In accordance with the foregoing, the present invention
relates to a blocked immunoglobulin of the invention wherein said
Protein A portion comprises at least one protein A compound. In
specific embodiments, the Protein A compound is a fragment of
Protein A so long as said fragment is able to bind to the Fc region
of the antibody portion of the immunoglobulins of the
invention.
[0038] In a preferred embodiment, the present invention is directed
to a blocked immunoglobulin as disclosed herein and further
comprising a solid support to which this blocked immunoglobulin is
attached. Commonly, but not exclusively, this attachment is through
a covalent linkage but other types of bonds are possible so long as
they do not defeat the utility of the invention or interfere with
the ability of Protein A to bind the antibody portion of the
immunoglobulin. Such attachment may optionally include the use of a
tether. Such antibody readily serves as the capture antibody in a
typical sandwich assay.
[0039] Commonly, the blocked immunoglobulins of the invention are
attached to the solid support through a linkage of the antibody
portion of the blocked immunoglobulin but attachment through the
protein A portion is possible so long as this does not compromise
the increased specificity of the blocked immunoglobulin. Such
attachment may or may not include the use of a tether.
[0040] In specific embodiments of the invention, the blocked
immunoglobulins disclosed herein are attached to a solid support
made of a material including any of the following non-limiting or
exhaustive list. Thus, such materials may include acrylamide,
agarose, cellulose, nitrocellulose, glass, polystyrene,
polyethylene vinyl acetate, polypropylene, polymethacrylate,
polyethylene, polyethylene oxide, polysilicates, polycarbonates,
teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides,
polyglycolic acid, polylactic acid, polyorthoesters,
polypropylfumerate, collagen, glycosaminoglycans, polyamino acids,
or combinations of these, with glass and plastic being highly
preferred.
[0041] The solid supports useful in the present invention may be in
the form of a thin film, membrane, bottles, dishes, fibers, woven
fibers, shaped polymers, particles, beads, microparticles, or any
combination of the foregoing.
[0042] The present invention also relates to compositions
comprising at least one blocked immunoglobulin of the invention in
a suitable carrier, which includes all suitable diluents or
excipients. The blocked immunoglobulins of the invention may be
suspended, dissolved or otherwise contained in such carrier and the
specific form of the composition or identity of the carrier is in
no way limiting of the invention so long as it in no way detracts
from the essential structure and function and other identifying
characteristics of the invention.
[0043] Of course, such compositions need in no way contain only one
blocked immunoglobulin or one kind of blocked immunoglobulin but
may contain any number of range of blocked immunoglobulins
encompassed by the invention disclosed herein. The blocked
immunoglobulins contained in such a composition may be blocked
immunoglobulins having the same or different specificities and may
include compositions wherein one or more of the blocked
immunoglobulin entities in the composition have the same
specificity and wherein one or more of the blocked immunoglobulin
entities in the composition have different specificities.
[0044] As used herein, the term "specificity" means antigenic
specificity and serves to denote the immunological characteristics
of the antibody portion of the blocked immunoglobulins of the
invention. Thus, where the antibody portion of a blocked
immunoglobulin of the invention binds more tightly to a particular
substrate, or analyte, or antigen, or antigenic determinant, as
those terms are used in the art, such as where the latter is a
protein or polypeptide, than it does to, say, a protein or
polypeptide with different antigenic properties, as where the
proteins or polypeptides differ in amino acid sequence so as to
produce different antigenic determinants, a different blocked
immunoglobulin molecule of the invention that binds preferentially
to that same protein or polypeptide is deemed to have the same or
similar specificity while a blocked immunoglobulin of the invention
that does not bind preferentially to the same polypeptide or
protein as another blocked immunoglobulin of the invention is
deemed to have a different specificity.
[0045] The processes of the present invention greatly facilitate
the use of blocked immunoglobulins with multiple analytes in a
sample and are particularly useful when presented as part of an
array, such as a microarray, using multiple blocked immunoglobulins
attached to a surface or other substrate and then contacted with a
sample containing one or more analytes to be detected. Thus, the
present invention specifically contemplates a microarray comprising
a solid support attached to a plurality of blocked immunoglobulins
according to the disclosure herein. In a particular embodiment
thereof, the microarray of the invention has the antibody portion
of each of the blocked immunoglobulins attached directly to the
substrate or else attached through a tether, such as a molecule
polymer of varying length that effective holds the blocked
immunoglobulins to the array.
[0046] In one such embodiment, the whole procedure may be part of a
sandwich assay, wherein the capture antibody is a blocked
immunoglobulin of the invention that reacts with an analyte that is
itself then contacted with an additional antibody that may
optionally be attached to an oligonucleotide primer for use in
rolling circle amplification as a means of detecting the detection
and binding of the analyte by the capture antibody.
[0047] In specific embodiments of such an array, the blocked
immunoglobulins attached thereto may have the same antigenic
specificity or may be of differing specificity, just as described
for blocked immunoglobulins of the invention that are not so
attached to an array or other substrate.
[0048] In accordance with the foregoing, the present invention
provides methods and compositions for the preparation and use of a
substrate having a plurality of structures like the blocked
immunoglobulins, acting as probes, in predefined regions of the
substrate, such as a solid substrate or solid support. This
substrate with attached probes or blocked immunoglobulins, is
called a "microarray" or "chip," and is used in screening a variety
of structures as ligands for binding with specific probes (i.e.,
the blocked immunoglobulins of the invention). In forming such an
array, it may be necessary to attach said blocked immunoglobulins
to the substrate using other linking structures, such as various
linking molecules, or "tethers," but in all cases such linkers will
in no way detract from the ability of blocked immunoglobulins to
bind the analyte to be determined. Methods of forming such arrays
are described in the literature (see the list or protein array
references below). As used in the present invention, such
microarrays will commonly be of the order of about 1 square
centimeter. Larger or smaller arrays are technologically possible
and may find use where the analytes to be determined are varying in
number and can conveniently be determined simultaneously or in
sequence.
[0049] In accordance with the foregoing, the microarray of the
present invention includes blocked immunoglobulins wherein the
antibody portion of at least two of the blocked immunoglobulins
have different antigenic specificities. This is readily extended to
three, four or more using the technology provided herein. Thus, the
present invention readily includes embodiments wherein the antibody
portion of each of the blocked immunoglobulins has a different
antigenic specificity.
[0050] The microarrays of the present invention comprise substrates
made of acrylamide, agarose, cellulose, nitrocellulose, glass,
polystyrene, polyethylene vinyl acetate, polypropylene,
polymethacrylate, polyethylene, polyethylene oxide, polysilicates,
polycarbonates, teflon, fluorocarbons, nylon, silicon rubber,
polyanhydrides, polyglycolic acid, polylactic acid,
polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans,
or polyamino acids, including combinations of these. Where the
substrate is composed of glass, prior chemical modification of the
surface may be required but will generally involve only procedures
well known in the art for preparing glass surfaces for binding to
macromolecules.
[0051] In addition, such arrays need not be flat surfaces but may
include such structures as beads or microparticles and the surfaces
of such microarrays may be porous or otherwise.
[0052] The present invention also relates to a process for forming
a blocked immunoglobulin comprising contacting an antibody with a
Protein A compound under conditions promoting the binding of said
Protein A compound to said antibody. In a preferred embodiment, the
Protein A compound is Protein A. In other preferred embodiments,
the protein A compound is a fragment of Protein A.
[0053] In another preferred embodiment, the present invention
comprises a process wherein the antibody forms a covalent linkage
with the Protein A compound. The process of the invention also
includes cases where the antibody is attached to a solid support
prior to contacting with said Protein A compound to form the
blocked immunoglobulins of the invention. As before, such solid
support may be porous and may be in the form of beads or
microparticles.
[0054] A preferred process for forming the blocked immunoglobulins
of the invention is to pre-block antibodies involved in the assays
using protein A for the blocking agent. For example, in using
solid-phase-based assays, the following procedures could be
advantageously included in the assay procedures:
[0055] (1) block the solid phase sequentially:
[0056] (a) Quench the activated groups with small molecules such
as:
[0057] Glycine, Lysine or NaBH4 depending on activation chemistry
of solid surface.
[0058] (b) Incubate the primary antibodies which are immobilized on
the solid phase with 0.5 mg/ml protein A at 37.degree. C. for 30
min.
[0059] (c) Incubate the primary antibodies which are immobilized on
the solid phase with normal blocking buffer (50 mM Glycine, pH 9.0,
2 mg/ml BSA, 5% non fat dry milk) containing 0.5 mg/ml protein A at
37.degree. C. for 30 min before reaction with antigen.
[0060] (2) Pre-incubate the secondary antibodies with 0.5 mg/ml
protein A at 37.degree. C. for 30 min before adding to react with
antigen.
[0061] (3) Pre-incubate the antibody conjugate which is used for
detection with 0.5 mg/ml protein A at 37.degree. C. for 30 min
before adding to react with the secondary antibodies.
[0062] Procedures 1, or 2, or 3 could each be used in the
alternative to provide the appropriate blocking of the
immunoglobulin. Here, the secondary antibody might be part of a
partial sandwich assay. In one non-limiting example, the capture
antibody is a blocked anti-body that binds to an analyte, such as a
polypeptide whose presence in a sample is to be detected and/or
quantitated. A secondary antibody (which also may be a blocked
immunoglobulin within the invention) is then contacted with said
analyte bound to the capture antibody to complete the sandwich.
This secondary antibody is itself optionally attached to a primer
oligonucleotide for use in a rolling circle amplification (RCA) as
disclosed herein by contacting said primer with an amplification
target circle (ATC) and enzymes that perform rolling circle
amplification and under conditions (including the presence of
appropriate and optionally labeled deoxynucleoside triphosphates
(dNTPs)) promoting said amplification. In another embodiment, the
secondary antibody may be attached to a ligand, such as where the
second antibody is biotinylated, and reacted with an anti-biotin
conjugate, such as a streptavidin and an anti-streptavidin
antibody, that is itself attached to an RCA primer for rolling
circle amplification. Such antibodies may include anti-mouse
antibodies and anti-rabbit antibodies from sera or may be some form
of recombinant or monoclonal antibody. In performing such assays,
the capture antibody is conveniently attached to some type of solid
support, possibly one made of glass or plastic, and which may be in
the form of an array, such as a microtitre plate, or beads,
microspheres, and the like.
[0063] By way of example only, for the results of FIG. 1, the
following procedure was used:
[0064] Procedure:
[0065] 1. Block: (1) Glycine, 37.degree. C., 30 min; (2) 0.5 mg/ml
protein A, 37.degree. C., 30 min; (3) normal blocking buffer+0.5
mg/ml protein A, 37.degree. C., 30 min.
[0066] 2. Secondary antibodies: (1) pre-incubate mixture of 20
biotinylated secondary antibodies with 0.5 mg/ml protein A at
37.degree. C., 30 min; (2) incubate at 37.degree. C. for 30
min.
[0067] 3. Antibody-RCA Conjugate: (1) pre-incubate anti-biotin
antibody conjugate with 0.5 mg/ml protein A; (2) pre-anneal with
ATC circle; (3) incubate at 37.degree. C. for 30 min.
[0068] 4. LRCA reaction/decoration: RCA reaction solution
containing T7 native DNA polymerase (0.01 units/ul)/1 mM dNTPs/0.03
mg/ml ssDNA-binding protein/1x sequenase/8% DMSO/0.05 uM
Cy5-labeled DNA decorator, incubate at 37.degree. C. for 30 min. 5.
Dry and scan slides for Cy5 fluorescence
[0069] In accordance with the foregoing, the present invention
further relates to a process comprising quenching the solid support
prior to contacting the antibody with the Protein A compound. In
preferred embodiments of such a process, the antibody and Protein A
are contacted at a temperature of at least about 37.degree. C.,
especially wherein said contacting occurs for at least about 30
minutes.
[0070] In other preferred embodiments thereof, following said
contacting with Protein A the blocked immunoglobulin is further
contacted with a blocking agent other than a Protein A compound.
The blocking agent may be other than a Protein A compound, such as
bovine serum albumin (BSA). In other preferred embodiments, the
Protein A compound is Protein A, especially where said Protein A is
present at a concentration of at least about 0.5 mg/ml, most
especially where the Protein A is present at a concentration of
about 0.5 mg/ml.
[0071] The present invention finds wide use in all antibody-based
applications such as protein assays in proteomics, immunodiagnostic
tests in clinical science, forensic science, and environmental
analysis.
[0072] In accordance with such uses, the present invention relates
to a process for detecting an analyte in a sample comprising
contacting an analyte with a blocked immunoglobulin as disclosed
herein where the antibody portion of said blocked immunoglobulin is
specific for said analyte and detecting the binding of said analyte
to said blocked immunoglobulin. In preferred embodiments, the
sample comprises at least two antigenically, possibly more,
different analytes and the analyte may be contacted with more than
one blocked immunoglobulin. Commonly, the antibody portion of at
least two, possibly more, of said blocked immunoglobulins exhibits
a different antigenic specificity. In a preferred embodiment, the
present invention relates to a process as disclosed herein where
the sample comprises a plurality of analytes contacted with a
plurality of blocked immunoglobulins comprising antibody portions
having at least two different antigenic specificities. Such a
process is extremely well suited to use of a microarray as
disclosed herein.
[0073] In such cases, the blocked immunoglobulins of the invention
may include multiple structures comprising more than one antibody
and more than one protein A compound in the same molecule, which
may or may not be attached to a solid support or substrate. In such
cases, a blocked immunoglobulin may exhibit multiple antigenic
specificities. In other cases, the property of multiple
specificities may depend on the presence, in composition or as part
of a solid support or substrate, of multiple blocked
immunoglobulins each of which has a single antigenic specificity
but where the specificity varies from one blocked immunoglobulin to
another. Thus, for example, in a given composition, or as part of a
given microarray, there may be a plurality of blocked
immunoglobulins contained on a single chip or microarray wherein
have a given antigenic specificity or differ in that some are
specific for a particular type of antigenic structure, such as a
given protein or polypeptide while one or more of the other blocked
immunoglobulins on the same chip, or in the same composition, or
solution, or suspension, are specific for a different antigenic
structure, such as a different protein or polypeptide and wherein
the differences between the antigenic structure may be slight or
great so long as the difference is sufficient to permit the blocked
immunoglobulins housed on a given array, or contained in a
different composition, to detect the difference in such
antigenically active analytes. Thus, any desired combination or
permutation of the blocked immunoglobulins of the invention may be
used, either attached to a solid support or substrate or not so
attached. This can include blocked immunoglobulins in which a given
molecule comprises an antibody portion that comprises more than one
antibody structure, wherein each of the antibodies in said antibody
portion has the same or different antigenic specificity. In the
same way, the Protein A portion may include more than one Protein A
structure, either Protein A itself or active fragments thereof
(meaning fragments capable of blocking the Fc portion of an
antibody). For example, two antibodies blocked with two Protein A
compounds would form two blocked immunoglobulins but the latter,
when linked together, such as by some type of tether or other
convenient molecular structure, could then form a single blocked
immunoglobulin within the present invention.
[0074] Thus, such blocked immunoglobulins may be attached to a
solid support, such support being a substrate as described herein
for solid supports and thus may be of a porous material or in the
form of beads and the like as disclosed elsewhere herein.
[0075] By way of non-limiting example, the present invention
relates to a process for detecting an analyte in a sample
comprising contacting an analyte with a microarray of the invention
wherein the antibody portion of at least one of the blocked
immunoglobulins on said microarray is specific for said analyte and
detecting binding of an analyte to at least one blocked
immunoglobulin on said microarray. In one such embodiment, the
sample contains a plurality of antigenically different analytes. In
another such embodiment, the microarray comprises a plurality of
blocked immunoglobulins comprising antibody portions exhibiting a
plurality of different antigenic specificities.
[0076] In accordance with the present invention, such processes,
whether they employ the blocked immunoglobulins of the invention as
part of a composition and suspended or dissolved in a suitable
carrier or whether the blocked immunoglobulins are attached to a
support, such as where they are part of a microarray, may be part
of an antibody sandwich assay, an enzyme-linked immunosorbent
assay, an antibody dipstick assay, an antibody microarray assay, a
radioimmunoassay, or a rolling circle amplification assay.
[0077] The processes of the invention for detecting an analyte may
occur on a column, a plate, a microtitre dish, a dipstick, a cell
sample or a tissue sample, and may even be carried out in situ. For
use with a microarray, one preferred type of substrate would be a
microtitre plate, such as a 96 well or other size dish.
[0078] Among the methods of detecting binding of analyte to a
blocked immunoglobulin of the invention are the use of any number
of detector molecules, including all types of labels, such as
fluorescent and radiolabels. The analyte or one or more of the
blocked immunoglobulins may be linked to such a label or may be
linked to a structure that itself may be used to detect the binding
of the analyte.
[0079] In one such embodiment, the analyte and/or the blocked
immunoglobulin is linked to an oligonucleotide that can serve as a
primer for oligonucleotide amplification, for example, roiling
circle amplification (RCA), the characteristics of which are well
known in the art.
[0080] In accordance therewith, the present invention relates to a
process for detecting an analyte wherein said analyte comprises a
rolling circle replication primer and wherein detection of binding
of analyte to blocked immunoglobulin is accomplished by contacting
said bound analyte with an amplification target circle (ATC)
comprising a primer complementary sequence complementary to a
portion of said primer under conditions promoting rolling circle
amplification and wherein the production of tandem sequence DNA
(TS-DNA) indicates the presence of said analyte.
[0081] Any form of RCA may be used with the processes disclosed
herein. For example, linear rolling circle amplification (LRCA)
uses a primer annealed to a circular target DNA molecule and DNA
polymerase is added. The amplification target circle (ATC) forms a
template on which new DNA is made, thereby extending the primer
sequence as a continuous sequence of repeated sequences
complementary to the circle but generating only about several
thousand copies per hour. An improvement on LRCA is the use of
exponential RCA (ERCA), with additional primers that anneal to the
replicated complementary sequences to provide new centers of
amplification, thereby providing exponential kinetics and increased
amplification. Exponential rolling circle amplification (ERCA)
employs a cascade of strand displacement reactions, also referred
to as HRCA (Lizardi, P. M. et al. Nature Genetics, 19, 225-231
(1998)). In accordance with the foregoing, any type of rolling
circle amplification may be utilized, including procedures
involving multiple primers attaching to the same ATC as well as
multiple layers and multiple rounds of rolling circle
amplification. Such methods of detection include those recited in
U.S. patent application Ser. No. 09/506,192, filed 19 Jun. 2000,
U.S. patent application Ser. No. 09/577,444, filed 24 May 2000, and
U.S. Patent Application No. 60/299,345, filed 19 June 2001, the
disclosures of which are hereby incorporated by reference in their
entirety.
[0082] The oligonucleotide primers useful such processes can be of
any desired length so long as they can be bound to an analyte or to
an analyte or can be bound to an antibody structure or protein A
structure as disclosed herein. For example, such primers may be of
a length of from at least 2 to about 30 to 50 nucleotides long,
preferably about 2 to about 35 nucleotides in length, most
preferably about 5 to about 10 nucleotides in length, with hexamers
and octamers being specifically preferred embodiments. Such primers
as are used herein may equally be specific only, or random only, or
a mixture of both, with random primers being especially useful and
convenient to form and use.
[0083] In RCA, a primer bound to an ATC produces a replication fork
as it is extended by the DNA polymerase around the ATC. The larger
an ATC is, the more amplification forks that could be formed and
thus a given ATC may attract primers bound to different
analytes.
[0084] The oligonucleotide primers useful in the processes of the
present invention will have segments complementary to a portion of
the ATC. Amplification target circles (ATCs) useful in the
processes of the present invention are circular DNA or RNA
molecules, either single or double stranded, including DNA-RNA
hybrid molecules generally containing between 40 to 10,000
nucleotides. However, it is expected that there will be no upper
limit to the size of the ATC. Where the ATC is a duplex circle,
such numbers are intended to refer to base pairs rather than
individual nucleotide residues. The ATCs useful in the processes
disclosed herein may have functionally different portions, or
segments, making them particularly useful for different purposes.
At least one such portion will be complementary to an
oligonucleotide primers and, when present, is referred to as a
primer complementary portion or site.
[0085] Amplification target circles (ATCs) useful herein comprise
target sequences to be amplified as a means of detecting bound
analyte. Previous technologies for RCA are known and these have
utilized RCA for such signal detection [see, for example, Lizardi,
U.S. Pat. No. 5,854,033, the disclosure of which is hereby
incorporated by reference in its entirety]. The amplification
target circles utilized as templates for the amplification
disclosed for use with the present invention may be either single
stranded DNA circles or duplex (double stranded) DNA circles but
are commonly single stranded. Where said ATCs are duplex, it may be
desirable that at least one strand of said duplex contains a nick.
Such nicks are commonly present in duplex circles but they may also
be introduced into such circles, such as by enzymatic methods well
known in the art, if not already present therein. Where duplex
circles are employed, amplification will commonly occur from both
strands as templates. Simultaneous amplification of both circles
may or may not be desirable.
[0086] In some circumstances it may be desirable to quantitatively
determine the extent of amplification occurring and/or the amount
of TS-DNA being formed or, in some circumstances, to be able to
measure in a discriminating fashion the relative quantities of
amplification target circles being formed where the ATCs of the
starting mixture are not uniform in structure and/or size. In such
instances, the present invention works well with any number of
standard detection schemes, such as where special deoxynucleoside
triphosphates (dNTPs) are utilized that make it easier to do
quantitative measurements. The most common example is where such
nucleotide substrates are radiolabeled or have attached thereto
some other type of label, such as a fluorescent label or the like.
Again, the methods that can be employed in such circumstances are
many and the techniques involved are standard and well known to
those skilled in the art. Thus, such detection labels include any
molecule that can be associated with amplified nucleic acid,
directly or indirectly, and which results in a measurable,
detectable signal, either directly or indirectly. Many such labels
for incorporation into nucleic acids or coupling to nucleic acid
probes are known to those of skill in the art. General examples
include radioactive isotopes, fluorescent molecules, phosphorescent
molecules, enzymes, antibodies, and ligands.
[0087] In such cases, the dNTPs used to form the TS-DNA products
may contain structures capable of binding to detector molecules.
For example, said nucleotide units in the TS-DNA may include
decorator molecules that can bind to detectors useful in
determining the presence and amount of TS-DNA formed from the
rolling circle amplification procedure. For example, such
structures may bind haptens for which antibodies are specific anf
thus labeled antibodies can be used to attach to the haptens
thereby quantitating the amount of TS-DNA product produced since
this will be proportional to the amount of hapten bound to the
TS-DNA. The antibodies used to bind the haptens will themselves be
labeled, such as with a fluorescent label or by being biotinylated,
although the exact nature of such labeling is in no way limiting of
the present invention.
[0088] Examples of suitable fluorescent labels include CyDyes such
as Cy2, Cy3, Cy3.5, Cy5, And Cy5.5, available from Amersham
Pharmacia Biotech (U.S. Pat. No. 5,268,486). Further examples of
suitable fluorescent labels include fluorescein, 5,6-carboxymethyl
fluorescein, Texas red, nitrobenz-2-oxa-1,3-diazol-4-yl (NBD),
coumarin, dansyl chloride, and rhodamine. Preferred fluorescent
labels are fluorescein (5-carboxyfluorescein-N-hydroxysuccinimde
ester) and rhodamine (5,6-tetramethyl rhodamine). These can be
obtained from a variety of commercial sources, including Molecular
Probes, Eugene, Oreg. and Research Organics, Cleveland, Ohio.
[0089] Labeled nucleotides are a preferred form of detection label
since they can be directly incorporated into the products of RCA
during synthesis. Examples of detection labels that can be
incorporated into amplified DNA include nucleotide analogs such as
BrdUrd (Hoy and Schimke, Mutation Research, 290:217-230 (1993)),
BrUTP (Wansick et al., J. Cell Biology, 122:283-293 (1993)) and
nucleotides modified with biotin (Langer et al., Proc. Natl. Acad.
Sci. USA, 78:6633 (1981)) or with suitable haptens such as
digoxygenin (Kerkhof, Anal. Biochem., 205:359-364 (1992)). Suitable
fluorescence-labeled nucleotides are
Fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP
(Yu et al., Nucleic Acids Res., 22:3226-3232 (1994)). A preferred
nucleotide analog detection label for DNA is BrdUrd (BUDR
triphosphate, Sigma), and a preferred nucleotide analog detection
label is Biotin-16-uridine-5'-tri- phosphate (Biotin-16-dUTP,
Boehringher Mannheim). Radiolabels are especially useful for the
amplification methods disclosed herein. Thus, such dNTPs may
incorporate a readily detectable moiety, such as a fluorescent
label as described herein.
[0090] The present invention provides a means to achieve signal
amplification in a variety of methods. In this case, the goal is to
amplify a signal that allows detection of the binding or
non-binding of an analyte. In methods including, but not limited to
cases where a DNA is detected by annealing of a labeled probe or by
incorporation of a labeled nucleotide, or by labeling DNA product
after synthesis, for example, by covalent modifications or
intercalation of detectable molecules, the present invention
provides a way to amplify DNA product and thereby signal
intensity.
[0091] In one such embodiment, an analyte comprising (for example,
attached to) an oligonucleotide primer is to be detected, or
otherwise determined or quantitatively measured. A sample
containing such an analyte is contacted with one or more blocked
immunoglobulins of the invention, such as where these are present
on an array, and allowed to interact. The array can then be washed
to remove unbound analyte (for example, a protein present in the
sample) and the array then contacted with a source of amplification
target circles and under conditions (enzyme, dNTPs, Mg ions,
buffer, etc.) to promote rolling circle amplification which, when
it occurs, indicates the presence of oligonucleotide primers (and
bound analyte) on the array, thereby indicating the presence of
analyte in the sample previously contacted with the array.
[0092] In accordance with the foregoing, the present invention also
relates to such a process wherein said analyte comprises a rolling
circle replication primer and wherein detection of binding of
analyte to blocked immunoglobulin is accomplished by contacting
said bound analyte with an amplification target circle (ATC)
comprising a primer complementary sequence complementary to a
portion of said primer under conditions promoting rolling circle
amplification and wherein the production of tandem sequence DNA
(TS-DNA) indicates the presence of said analyte. Commonly, said
analyte will comprise a protein or polypeptide structure.
[0093] Alternatively, the present invention also comprises a
process wherein the blocked immunoglobulin comprises a rolling
circle replication primer and wherein detection of binding of
analyte to blocked immunoglobulin is accomplished by contacting
said bound analyte with an amplification target circle (ATC)
comprising a primer complementary sequence complementary to a
portion of said primer under conditions promoting rolling circle
amplification and wherein the production of tandem sequence DNA
(TS-DNA) indicates the presence of said analyte. In other
embodiments, the blocked immunoglobulins are attached to a
microarray as described herein.
[0094] Thus, the process for analyzing for an analyte according to
the invention includes a method comprising the steps of:
[0095] (a) mixing or otherwise contacting a blocked antibody
composition with a target sample comprising an analyte wherein a
rolling circle replication primer is coupled to the blocked
antibody composition, wherein the blocked antibody composition
binds to the analyte,
[0096] (b) mixing or otherwise contacting the rolling circle
replication primer with an amplification target circle, to produce
a primer-ATC mixture, and incubating the primer-ATC mixture under
conditions that promote hybridization between the amplification
target circle and the rolling circle replication primer in the
primer-ATC mixture,
[0097] wherein the amplification target circle comprises a
single-stranded, circular DNA molecule comprising a primer
complement portion, wherein the primer complement portion is
complementary to the rolling circle replication primer, and
[0098] (c) mixing or otherwise contacting DNA polymerase with the
primer-ATC mixture, to produce a polymerase-ATC mixture, and
incubating the polymerase-ATC mixture under conditions that promote
replication of the amplification target circle,
[0099] wherein replication of the amplification target circle
results in the formation of tandem sequence DNA.
[0100] In a preferred embodiment, the present invention includes a
process for detecting proteins, the method comprising the steps
of:
[0101] (a) mixing or otherwise contacting a blocked antibody
composition with a target sample comprising a target molecule
wherein a rolling circle replication primer is coupled to the
blocked antibody composition, wherein the blocked antibody
composition binds to the target molecule,
[0102] wherein the target molecule is a protein,
[0103] (b) mixing or otherwise contacting the rolling circle
replication primer with an amplification target circle, to produce
a primer-ATC mixture, and incubating the primer-ATC mixture under
conditions that promote hybridization between the amplification
target circle and the rolling circle replication primer in the
primer-ATC mixture,
[0104] wherein the amplification target circle comprises a
single-stranded, circular DNA molecule comprising a primer
complement portion, wherein the primer complement portion is
complementary to the rolling circle replication primer, and
[0105] (c) mixing or otherwise contacting DNA polymerase with the
primer-ATC mixture, to produce a polymerase-ATC mixture, and
incubating the polymerase-ATC mixture under conditions that promote
replication of the amplification target circle,
[0106] wherein replication of the amplification target circle
results in the formation of tandem sequence DNA.
[0107] In accordance with the foregoing, the present invention
relates generally to a process for detecting analytes, the method
comprising
[0108] an amplification operation,
[0109] wherein an amplification target circle is coupled to a
blocked antibody composition, wherein the blocked antibody
composition can interact with an analyte,
[0110] wherein the amplification operation comprises rolling circle
replication of the amplification target circle to produce tandem
sequence DNA.
[0111] In a preferred embodiment of such a process, the present
invention contemplates a process for detecting a protein analyte,
the method comprising the steps of:
[0112] an amplification operation,
[0113] wherein an amplification target circle is coupled to a
blocked antibody composition, wherein the blocked antibody
composition can interact with a target molecule, wherein the target
molecule is a protein,
[0114] wherein the amplification operation comprises rolling circle
replication of the amplification target circle to produce tandem
sequence DNA.
[0115] In one such embodiment, the present invention relates
generally to a process for detecting analytes, the method
comprising the steps of:
[0116] (a) mixing or otherwise contacting a blocked antibody
composition with a target sample comprising an analyte wherein an
amplification target circle is coupled to the blocked antibody
composition, wherein the blocked antibody composition binds to the
analyte,
[0117] (b) mixing otherwise contacting a rolling circle replication
primer with the amplification target circle, to produce a
primer-ATC mixture, and incubating the primer-ATC mixture under
conditions that promote hybridization between the amplification
target circle and the rolling circle replication primer in the
primer-ATC mixture,
[0118] wherein the amplification target circle comprises a
single-stranded, circular DNA molecule comprising a primer
complement portion, wherein the primer complement portion is
complementary to the rolling circle replication primer, and
[0119] (c) mixing otherwise contacting a DNA polymerase with the
primer-ATC mixture, to produce a polymerase-ATC mixture, and
incubating the polymerase-ATC mixture under conditions that promote
replication of the amplification target circle,
[0120] wherein replication of the amplification target circle
results in the formation of tandem sequence DNA.
[0121] In a preferred embodiment, the present invention further
relates to a process for detecting proteins, the method comprising
the steps of:
[0122] (a) mixing or otherwise contacting a blocked antibody or
immunoglobulin composition with a target sample comprising a target
molecule wherein an amplification target circle is coupled to the
blocked antibody composition, wherein the specific binding molecule
binds to the target molecule,
[0123] wherein the target molecule is a protein,
[0124] (b) mixing or otherwise contacting a rolling circle
replication primer with the amplification target circle, to produce
a primer-ATC mixture, and incubating the primer-ATC mixture under
conditions that promote hybridization between the amplification
target circle and the rolling circle replication primer in the
primer-ATC mixture,
[0125] wherein the amplification target circle comprises a
single-stranded, circular DNA molecule comprising a primer
complement portion, wherein the primer complement portion is
complementary to the rolling circle replication primer, and
[0126] (c) mixing or otherwise contacting DNA polymerase with the
primer-ATC mixture, to produce a polymerase-ATC mixture, and
incubating the polymerase-ATC mixture under conditions that promote
replication of the amplification target circle,
[0127] wherein replication of the amplification target circle
results in the formation of tandem sequence DNA.
[0128] The present invention also relates to a process for
detecting analytes in a sample, the method comprising the steps
of:
[0129] a DNA ligation operation and an amplification operation,
[0130] wherein the DNA ligation operation comprises circularization
of an open circle probe, wherein circularization of the open circle
probe is dependent on hybridization of the open circle probe to a
target sequence, wherein the target sequence is coupled to a
blocked antibody composition, wherein the blocked antibody
composition can interact with an analyte,
[0131] wherein the amplification operation comprises rolling circle
replication of the circularized open circle probe to produce tandem
sequence DNA.
[0132] In a preferred embodiment, the present invention further
contemplates a process for detecting proteins, the method
comprising the steps of:
[0133] a DNA ligation operation and an amplification operation,
[0134] wherein the DNA ligation operation comprises circularization
of an open circle probe, wherein circularization of the open circle
probe is dependent on hybridization of the open circle probe to a
target sequence, wherein the target sequence is coupled to a
blocked antibody composition, wherein the specific binding molecule
can interact with a target molecule, wherein the target molecule is
a protein,
[0135] wherein the amplification operation comprises rolling circle
replication of the circularized open circle probe to produce tandem
sequence DNA.
[0136] The present invention also relates to a process for
detecting analytes, the method comprising,
[0137] (a) mixing or otherwise contacting a blocked antibody
composition with a target sample comprising an analyte wherein a
target sequence is coupled to the blocked antibody composition,
wherein the blocked antibody composition binds to the analyte,
[0138] (b) mixing or otherwise contacting an open circle probe
(OCP) with the target sample, to produce an OCP-target sample
mixture, and incubating the OCP-target sample mixture under
conditions that promote hybridization between the open circle probe
and the target sequence in the OCP-target sample mixture,
[0139] (c) mixing or otherwise contacting a ligase with the
OCP-target sample mixture, to produce a ligation mixture, and
incubating the ligation mixture under conditions that promote
ligation of the open circle probe to form an amplification target
circle,
[0140] (d) mixing or otherwise contacting a rolling circle
replication primer with the ligation mixture, to produce a
primer-ATC mixture, and incubating the primer-ATC mixture under
conditions that promote hybridization between the amplification
target circle and the rolling circle replication primer in the
primer-ATC mixture, and
[0141] (e) mixing or otherwise contacting DNA polymerase with the
primer-ATC mixture, to produce a polymerase-ATC mixture, and
incubating the polymerase-ATC mixture under conditions that promote
replication of the amplification target circle,
[0142] wherein replication of the amplification target circle
results in the formation of tandem sequence DNA.
[0143] As used herein, the term "open circle probe" (or OCP) refers
to a single stranded oligonucleotide comprising terminal segments
complementary to an oligonucleotide primer sequence. When contacted
with a primer, the OCP hybridizes to the primer to form an open
circle in which the 5'- and 3'- ends are adjacent. Treatment with a
ligase or other chemical entity then joins the 3'- and 5'-
nucleotides of the OCP to form a complete circle (i.e., an
amplification target circle or ATC) which now provides a template
for rolling circle amplification starting from the 3'- end of the
primer. Of course, to produce the open circle probe the terminal
segments of said probe must be complementary to contiguous segments
of the RCA primer sequence. Such probes, primers and methods are
well known in the literature (see: Lizardi, U.S. Pat. No.
5,854,033, issued 29 Dec. 1998, the disclosure of which is hereby
incorporated by reference in its entirety). In general, any type of
rolling circle amplification (RCA) can be used to detect and
quantitate the immunoglobulin complexes formed through the process
of the invention and are in no way limiting of the invention
disclosed herein.
[0144] In a preferred embodiment thereof, the present invention
further relates to a process for detecting proteins, the method
comprising the steps of:
[0145] (a) mixing or otherwise contacting a blocked antibody
composition with a target sample comprising a target molecule
wherein a target sequence is coupled to the blocked antibody
composition, wherein the blocked antibody composition binds to the
target molecule,
[0146] wherein the target molecule is a protein,
[0147] (b) mixing or otherwise contacting an open circle probe with
the target sample, to produce an OCP-target sample mixture, and
incubating the OCP-target sample mixture under conditions that
promote hybridization between the open circle probe and the target
sequence in the OCP-target sample mixture,
[0148] (c) mixing or otherwise contacting a ligase with the
OCP-target sample mixture, to produce a ligation mixture, and
incubating the ligation mixture under conditions that promote
ligation of the open circle probe to form an amplification target
circle,
[0149] (d) mixing or otherwise contacting a rolling circle
replication primer with the ligation mixture, to produce a
primer-ATC mixture, and incubating the primer-ATC mixture under
conditions that promote hybridization between the amplification
target circle and the rolling circle replication primer in the
primer-ATC ATC mixture, and
[0150] (e) mixing or otherwise contacting a DNA polymerase with the
primer-ATC mixture, to produce a polymerase-ATC mixture, and
incubating the polymerase-ATC mixture under conditions that promote
replication of the amplification target circle,
[0151] wherein replication of the amplification target circle
results in the formation of tandem sequence DNA.
[0152] DNA polymerases useful in the rolling circle replication
step of RCA must perform rolling circle replication of primed
single-stranded circles (or each strand of a duplex substrate).
Such polymerases are referred to herein as rolling circle DNA
polymerases. For rolling circle replication, it is preferred that a
DNA polymerase be capable of displacing the strand complementary to
the template strand, termed strand displacement, and lack a 5' to
3' exonuclease activity. Strand displacement is necessary to result
in synthesis of multiple tandem copies of the ATC. A 5' to 3'
exonuclease activity, if present, might result in the destruction
of the synthesized strand. It is also preferred that DNA
polymerases for use in the disclosed method are highly processive.
The suitability of a DNA polymerase for use in the disclosed method
can be readily determined by assessing its ability to carry out
rolling circle replication. Preferred rolling circle DNA
polymerases are bacteriophage .phi.29 DNA polymerase (U.S. Pat.
Nos. 5,198,543 and 5,001,050 to Blanco et al.), phage M2 DNA
polymerase (Matsumoto et al., Gene 84:247 (1989)), phage PRD1 DNA
polymerase (Jung et al., Proc. Natl. Acad. Sci. USA 84:8287 (1987),
and Zhu and lto, Biochim. Biophys. Acta. 1219:267-276 (1994)),
VENT.RTM. DNA polymerase (Kong et al., J. Biol. Chem. 268:1965-1975
(1993)), Klenow fragment of DNA polymerase I (Jacobsen et al., Eur.
J. Biochem. 45:623-627 (1974)), T5 DNA polymerase (Chatterjee et
al., Gene 97:13-19 (1991)), and T4 DNA polymerase holoenzyme
(Kaboord and Benkovic, Curr. Biol. 5:149-157 (1995)). .phi.-29 DNA
polymerase is most preferred. Equally preferred polymerases include
T7 native polymerase, Bacillus stearothermophilus (Bst) DNA
polymerase, Thermoanaerobacter thermohydrosulfuricus (Tts) DNA
polymerase (U.S. Pat. No. 5,744,312), and the DNA polymerases of
Thermus aquaticus, Thermus flavus or Thermus thermophilus. Equally
preferred are the .phi.29-type DNA polymerases, which are chosen
from the DNA polymerases of phages: .phi.29, Cp-1, PRD1, .phi.15,
.phi.21, PZE, PZA, Nf, M2Y, B103, SF5, GA-1, Cp-5, Cp-7, PR4, PR5,
PR722, and L17. In a specific embodiment, the DNA polymerase is
bacteriophage .phi.29 DNA polymerase wherein the multiple primers
are resistant to exonuclease activity and the target DNA is high
molecular weight linear DNA.
[0153] For carrying out a ligation step many suitable ligases are
known, such as T4 DNA ligase, E.coli DNA ligase, Taq DNA ligase,
Tth DNA ligase, Thermus scotoductus DNA ligase, Rhodothermus
marinus DNA ligase, Ampligase.TM., Bst ligase, T4 RNA ligase and
cappases.
[0154] References for protein arrays:
[0155] 1. Mendoza, L. G., McQuary, P., Mongan, A., R. Gangadharan,
R., Brignac, S. & Eggers, M. High-throughput microarray-based
enzyme-linked immunoabsorbant assay (ELISA). Biotechniques 27,
778-788 (1999).
[0156] 2. Haab, B. B., Dunham, M. J. & Brown P. O. Protein
microarrays for highly parallel detection and quantitation of
specific proteins and antibodies in complex solutions. Genome
Biology 2, 1-13 (2001)
[0157] 3. Schweitzer, B., Wiltshire, S., Lambert, J., O'Malley, S.,
Kukanskis, K., Zhu, Z., Kingsmore, S. F., Lizardi, P. M. &
Ward, D. C. Immunoassays with rolling circle DNA amplification: a
versatile platform for ultrasensitive antigen detection. Proc.
Natl. Acad. Sci. (USA) 97, 10113-10119 (2000).
[0158] 4. MacBeath, G. &. Schreiber, S. L Printing proteins as
microarrays for high-thoughput function determination. Science 289,
1760-1763 (2000).
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