U.S. patent application number 09/796725 was filed with the patent office on 2004-01-08 for signal amplification method.
This patent application is currently assigned to Tm Technologies, Inc.. Invention is credited to Benight, Albert S., Faldasz, Brian D., Lane, Michael J..
Application Number | 20040005552 09/796725 |
Document ID | / |
Family ID | 21811080 |
Filed Date | 2004-01-08 |
United States Patent
Application |
20040005552 |
Kind Code |
A1 |
Lane, Michael J. ; et
al. |
January 8, 2004 |
Signal amplification method
Abstract
Methods for detecting the presence or absence of an analyte in a
sample are disclosed. Kits for performing the analysis methods of
the invention are also disclosed.
Inventors: |
Lane, Michael J.;
(Baldwinsville, NY) ; Benight, Albert S.;
(Schaumburg, IL) ; Faldasz, Brian D.; (Maynard,
MA) |
Correspondence
Address: |
MCCARTHY TETRAULT LLP
SUITE 4900, P.O. BOX 48
66 WELLINGTON ST. WEST
TORONTO, ONTARIO
M5K 1E6
CA
|
Assignee: |
Tm Technologies, Inc.
|
Family ID: |
21811080 |
Appl. No.: |
09/796725 |
Filed: |
February 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09796725 |
Feb 27, 2001 |
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09293362 |
Apr 16, 1999 |
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6245513 |
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09293362 |
Apr 16, 1999 |
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08893535 |
Jul 11, 1997 |
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5902724 |
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60022718 |
Jul 12, 1996 |
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Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C12Q 1/6804 20130101;
C12Q 1/682 20130101; C12Q 1/6844 20130101; C12Q 1/682 20130101;
C12Q 1/6804 20130101; G01N 2458/10 20130101; C12Q 1/6844 20130101;
C12Q 2563/179 20130101; C12Q 2525/313 20130101; C12Q 2525/313
20130101; C12Q 2563/131 20130101; C12Q 2525/173 20130101; C12Q
2563/179 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method for detecting the presence or absence of an analyte in
a sample, the method comprising: contacting the sample with a
reagent having a first portion which specifically binds to the
analyte and a second portion comprising a polynucleotide sequence,
such that a complex of the analyte and the reagent is formed;
contacting the complex of the analyte and the reagent with an
amplifying entity having a first polynucleotide sequence and a
second polynucleotide sequence, wherein the first polynucleotide
sequence is complementary to the polynucleotide sequence of the
second portion of the reagent, such that a complex of the analyte,
the reagent, and the amplifying entity is formed; contacting the
complex of the analyte, the reagent, and the amplifying entity with
a plurality of signalling moieties, each of the signalling moieties
comprising a detectable label and a polynucleotide sequence
complementary to the second polynucleotide sequence of the
amplifying entity, to form a detectable complex of the analyte, the
reagent, the amplifying polynucleotide and the signalling moieties;
and detecting the label as indicative of the presence or absence of
analyte in the sample.
2. The method of claim 1, wherein the analyte is a nucleic acid
sequence.
3. The method of claim 2, wherein the reagent first portion is a
nucleic acid sequence which is substantially complementary to the
analyte.
4. The method of claim 1, wherein the analyte is an antibody or
antigen.
5. The method of claim 4, wherein the reagent first portion is an
antibody or antigen which specifically binds with the analyte.
6. The method of claim 1, wherein the amplifying entity first
polynucleotide sequence and second polynucleotide sequence comprise
substantially the same sequence.
7. The method of claim 1, wherein the amplifying entity is a
homopolynucleotide.
8. The method of claim 7, wherein the homopolynucleotide comprises
poly(dA).
9. The method of claim 8, wherein the poly(dA) has a length of at
least about 3000 bases.
10. The method of claim 8, wherein the reagent second portion
comprises poly(dT).
11. The method of claim 8, wherein each of the signalling moieties
comprises poly(dT).
12. The method of claim 1, wherein each of the plurality of
signalling moieties comprises a detectable label selected from the
group consisting of antigens, antibodies, enzymes, radioisotopes,
and fluorescent moieties.
13. The method of claim 1, wherein, prior to the step of contacting
the complex of the analyte, the reagent and the amplifying entity
with the plurality of signalling moieties, the method comprises the
further step of washing the complex of the analyte, the reagent and
the amplifying entity to remove unbound polynucleotide.
14. The method of claim 1, wherein the analyte is immobilized with
an immobilized capture reagent.
15. The method of claim 1, wherein the first polynucleotide
sequence of the amplifying entity and the second polynucleotide
sequence of the amplifying entity comprises the same or
substantially the same sequence.
16. A method for detecting the presence or absence of an analyte in
a sample, the method comprising: contacting the sample with a
reagent having a first portion which specifically binds to the
analyte and a second portion comprising a homopolynucleotide
sequence, such that a complex of the analyte and the reagent is
formed; contacting the complex of the analyte and the reagent with
a homopolynucleotide strand complementary to the homopolynucleotide
sequence of the reagent, such that a complex of the analyte, the
reagent, and the homopolynucleotide is formed; and contacting the
complex of the analyte, the reagent, and the homopolynucleotide
with a plurality of signalling moieties, each of the signalling
moieties comprising a detectable label and a homopolynucleotide
sequence complementary to homopolynucleotide strand, to form a
detectable complex of the analyte, the reagent, the
homopolynucleotide strand and the signalling moieties; and
detecting the label as indicative of the presence or absence of
analyte in the sample.
17. The method of claim 16, wherein the homopolynucleotide strand
is poly(dA) and the reagent second portion and the signalling
moieties comprise poly(dT) or poly(dU).
18. A method for detecting the presence or absence of an analyte in
a sample, the method comprising: contacting the sample with a first
reagent having a first portion which specifically binds to the
analyte and a second portion comprising a polynucleotide sequence,
such that a complex of the analyte and the first reagent is formed;
contacting the complex of the analyte and the first reagent with an
amplifying entity having a first polynucleotide sequence and a
second polynucleotide sequence, wherein the first polynucleotide
sequence is complementary to the polynucleotide sequence of the
second portion of the first reagent, such that a complex of the
analyte, the first reagent, and the amplifying entity is formed;
contacting the complex of the analyte, the first reagent, and the
amplifying entity with a second reagent, the second reagent having
a first portion which includes a polynucleotide sequence
complementary to the second polynucleotide sequence of the
amplifying entity, and a second portion, to form an extendable
complex of the analyte, the first reagent, the amplifying entity
and the second reagent; contacting the extendable complex with an
extension reagent, the extension reagent comprising a first portion
capable of specifically binding to the second portion of the
amplifying entity, and a second portion which comprises a
polynucleotide sequence, such that the extension reagent binds to
the extendable complex to form a complex of the analyte, the first
reagent, the amplifying entity, and the extension reagent; and
contacting the complex of the analyte, the first reagent, the
amplifying entity and the extension reagent with a plurality of
signalling moieties, each of the signalling moieties comprising a
detectable label and a polynucleotide sequence complementary to the
polynucleotide sequence of the extension reagent, to form a
detectable complex of the analyte, the reagent, the amplifying
polynucleotide, the extension reagent and the signalling moieties;
and detecting the label as indicative of the presence or absence of
analyte in the sample.
19. The method of claim 18, wherein the second portion of the
extension reagent comprises a homopolynucleotide.
20. The method of claim 19, wherein the second portion of the
extension reagent comprises poly(dC), and the polynucleotide
sequence of the signalling moieties comprises poly(dG).
21. The method of claim 20, wherein the detectable label is
selected from the group consisting of antigens, antibodies,
enzymes, radioisotopes, and fluorescent moieties.
22. A kit for detecting the presence or absence of an analyte in a
sample, the kit comprising: a container including a reagent having
a first portion which specifically binds to the analyte and a
second portion comprising a polynucleotide sequence; a container
including an amplifying entity having a first polynucleotide
sequence and a second polynucleotide sequence, wherein the first
polynucleotide sequence is complementary to the polynucleotide
sequence of the second portion of the reagent; a container
including a plurality of signalling moieties, each of the
signalling moieties comprising a detectable label and a
polynucleotide sequence complementary to the second polynucleotide
sequence of the amplifying entity; and instructions for detecting
the presence or absence of the analyte in a sample.
23. The kit of claim 22, wherein the amplifying entity is a
homopolynucleotide.
24. The kit of claim 23, wherein the homopolynucleotide comprises
poly(dA).
25. The kit of claim 24, wherein the poly(dA) has a length of at
least about 3000 bases.
26. The kit of claim 24, wherein the reagent second portion
comprises poly(dT).
27. The kit of claim 22, further comprising a container of an
analyte-specific capture reagent.
28. The kit of claim 27, wherein the analyte-specific capture
reagent is immobilized on a solid support.
29. A detectable complex for detection of an analyte, the complex
comprising: an analyte; a reagent bound to the analyte, the reagent
having a first portion which specifically binds to the analyte and
a second portion comprising a polynucleotide sequence; an
amplifying entity bound to the reagent, the amplifying entity
having a first polynucleotide sequence and a second polynucleotide
sequence, wherein the first polynucleotide sequence is
complementary to the polynucleotide sequence of the second portion
of the reagent; and a plurality of signalling moieties bound to the
amplifying entity, each of the signalling moieties comprising a
detectable label and a polynucleotide sequence complementary to the
second polynucleotide sequence of the amplifying entity.
30. The detectable complex of claim 29, further comprising an
analyte-specific capture reagent bound to a solid support, the
capture reagent further being bound to the analyte.
31. The detectable complex of claim 30, wherein the analyte is an
antigen.
32. The detectable complex of claim 30, wherein the analyte is a
nucleic acid.
33. An isolated purified single-stranded homopolynucleotide having
a length of at least about 3000 bases.
34. The homopolynucleotide of claim 33, wherein the
homopolynucleotide has a length of at least about 7000 bases.
35. The homopolynucleotide of claim 40, wherein the
homopolynucleotide is selected from the group consisting of
poly(dA), poly(dT), poly(dC), poly(dG), and poly(dU).
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. 119(e) to co-pending U.S. provisional application Serial No.
60/022,718, filed Jul. 12, 1996, the entire content of which is
hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Biochemical assays such as immunoassays (e.g., enzyme-linked
immunosorbent assay (ELISA)) and methods for detecting nucleic acid
sequences in a test sample are well known. In such assays, high
sensitivity is important to ensuring the ability of the assay to
detect low levels of the analyte of interest. Radioactive and
colorimetric methods have often been employed in such assays.
However, achieving high levels of sensitivity has not always been
possible, and methods of increasing the sensitivity of such tests
are desirable.
[0003] One reported method (European Patent Publication EP 128 332)
for detecting analytes, including nucleic acid sequences and
antigens (or antibodies), is the use of a "bridging moiety," which
provides a bridge between an analyte and a "signalling moiety"
which provides a detectable signal. The bridging moiety includes an
analyte-specific region and a signalling moiety-specific region.
This method has the advantage that, by varying the bridging moiety
according to the target analyte, the same signalling moiety can be
employed to detect a variety of analytes. However, the preparation
of the bridging moieties can be rather lengthy and inefficient.
Also, large bridging moieties (such a long polynucleotide
sequences) may be less sensitive at detecting target analyte due to
the presence of large segments which do not bind to the target.
Moreover, this method requires that the analyte-specific region and
a signalling moiety-specific region of the bridging moiety be
different.
[0004] Another publication (U.S. Pat. No. 5,627,030 to Pandian et
al.) describes the use of a primary probe for detecting a target
nucleic acid sequence, and an "amplification probe ", which is a
nucleic acid sequence which includes two regions: a first region
complementary to the primary probe, and a second region which
contains repeated sequences for binding to a plurality of labeled
probes. The amplification probe permits several labeled probes to
bind to each primary probe, amplifying the signal from the binding
of the primary probe to the target molecule in the sample. However,
construction of the amplification probe can be cumbersome.
SUMMARY OF THE INVENTION
[0005] The present invention relates to methods and compositions
for amplifying or amplifying signals in biochemical assays. In
particular, the invention provides methods and compositions useful
for improving sensitivity in biochemical assays involving the
binding of specific binding pairs.
[0006] In one aspect, the invention provides amplifying entities
(also referred to herein as polymeric amplifying moieties (PAMs))
which are capable of binding to both an analyte (e.g., an antigen
or nucleic acid) or analyte-detecting moiety (e.g., an antibody, a
nucleic acid probe, and the like) and to a plurality of signalling
moieties which include a detectable label. By binding to multiple
signalling moieties, the PAM amplifies the signal generated in the
presence of the analyte, thereby detecting the presence or absence
of the analyte in a sample.
[0007] In another aspect, the invention provides a method for
amplifying signals in assay systems. The method includes the steps
of contacting the sample with a reagent having a first portion
which specifically binds to the analyte and a second portion
comprising a polynucleotide sequence, such that a complex of the
analyte and the reagent is formed; contacting the complex of the
analyte and the reagent with an amplifying entity having a first
polynucleotide sequence and a second polynucleotide sequence,
wherein the first polynucleotide sequence is complementary to the
polynucleotide sequence of the second portion of the reagent, such
that a complex of the analyte, the reagent, and the amplifying
entity is formed; contacting the complex of the analyte, the
reagent, and the amplifying entity with a plurality of signalling
moieties, each of the signalling moieties comprising a detectable
label and a polynucleotide sequence complementary to the second
polynucleotide sequence of the amplifying entity, to form a
detectable complex of the analyte, the reagent, the amplifying
polynucleotide and the signalling moieties; and detecting the label
as indicative of the presence or absence of analyte in the
sample.
[0008] The analyte can be a nucleic acid sequence; the reagent
first portion can be a nucleic acid sequence which is substantially
complementary to the analyte; the analyte can be an antibody or
antigen; the reagent first portion can be an antibody or antigen
which specifically binds with the analyte; the amplifying entity
first polynucleotide sequence and second polynucleotide sequence
comprise the same or substantially the same sequence. In certain
embodiments, the amplifying entity is a homopolynucleotide; the
homopolynucleotide can comprise poly(dA); the poly(dA) can have a
length of at least about 3000 bases; the reagent second portion can
comprise poly(dT); each of the signalling moieties comprises
poly(dT); each of the signalling moieties can comprise a detectable
label selected from the group consisting of antigens, antibodies,
enzymes, radioisotopes, and fluorescent moieties. In certain
embodiments, prior to the step of contacting the complex of the
analyte, the reagent and the amplifying entity with the plurality
of signalling moieties, the method comprises the further step of
washing the complex of the analyte, the reagent and the amplifying
entity to remove unbound polynucleotide. In preferred embodiments,
the analyte is immobilized with an immobilized capture reagent.
[0009] In another embodiment, the invention provides a method for
detecting the presence or absence of an analyte in a sample, the
method including the steps of contacting the sample with a reagent
having a first portion which specifically binds to the analyte and
a second portion comprising a homopolynucleotide sequence, such
that a complex of the analyte and the reagent is formed; contacting
the complex of the analyte and the reagent with a
homopolynucleotide strand complementary to the homopolynucleotide
sequence of the reagent, such that a complex of the analyte, the
reagent, and the homopolynucleotide is formed; and contacting the
complex of the analyte, the reagent, and the homopolynucleotide
with a plurality of signalling moieties, each of the signalling
moieties comprising a detectable label and a homopolynucleotide
sequence complementary to homopolynucleotide strand, to form a
detectable complex of the analyte, the reagent, the
homopolynucleotide strand and the signalling moieties; and
detecting the label as indicative of the presence or absence of
analyte in the sample.
[0010] In certain embodiments, the homopolynucleotide strand is
poly(dA) and the reagent second portion and the signalling moieties
comprise poly(dT) or poly(dU).
[0011] In still another aspect, the invention provides a method for
detecting the presence or absence of an analyte in a sample, the
method comprising the steps of contacting the sample with a first
reagent having a first portion which specifically binds to the
analyte and a second portion comprising a polynucleotide sequence,
such that a complex of the analyte and the first reagent is formed;
contacting the complex of the analyte and the first reagent with an
amplifying entity having a first polynucleotide sequence and a
second polynucleotide sequence, wherein the first polynucleotide
sequence is complementary to the polynucleotide sequence of the
second portion of the first reagent, such that a complex of the
analyte, the first reagent, and the amplifying entity is formed;
contacting the complex of the analyte, the first reagent, and the
amplifying entity with a second reagent, the second reagent having
a first portion which includes a polynucleotide sequence
complementary to the second polynucleotide sequence of the
amplifying entity, and a second portion, to form an extendable
complex of the analyte, the first reagent, the amplifying entity
and the second reagent; contacting the extendable complex with an
extension reagent, the extension reagent comprising a first portion
capable of specifically binding to the second portion of the
amplifying entity, and a second portion which comprises a
polynucleotide sequence, such that the extension reagent binds to
the extendable complex to form a complex of the analyte, the first
reagent, the amplifying entity, and the extension reagent; and
contacting the complex of the analyte, the first reagent, the
amplifying entity and the extension reagent with a plurality of
signalling moieties, each of the signalling moieties comprising a
detectable label and a polynucleotide sequence complementary to the
polynucleotide sequence of the extension reagent, to form a
detectable complex of the analyte, the reagent, the amplifying
polynucleotide, the extension reagent and the signalling moieties;
and detecting the label as indicative of the presence or absence of
analyte in the sample.
[0012] In certain embodiments, the second portion of the extension
reagent comprises a homopolynucleotide; the second portion of the
extension reagent comprises poly(dC), and the polynucleotide
sequence of the signalling moieties comprises poly(dG); the
detectable label is selected from the group consisting of antigens,
antibodies, enzymes, radioisotopes, and fluorescent moieties.
[0013] In another aspect, the invention provides a kit for
detecting the presence or absence of an analyte in a sample. The
kit includes a container including a reagent having a first portion
which specifically binds to the analyte and a second portion
comprising a polynucleotide sequence; a container including an
amplifying entity having a first polynucleotide sequence and a
second polynucleotide sequence, wherein the first polynucleotide
sequence is complementary to the polynucleotide sequence of the
second portion of the reagent; a container including a plurality of
signalling moieties, each of the signalling moieties comprising a
detectable label and a polynucleotide sequence complementary to the
second polynucleotide sequence of the amplifying entity; and
instructions for detecting the presence or absence of the analyte
in a sample.
[0014] In preferred embodiments, the amplifying entity is a
homopolynucleotide; the homopolynucleotide can comprise poly(dA);
the poly(dA) can have a length of at least about 3000 bases; the
reagent second portion can comprise poly(dT). In certain
embodiments, the kit further includes a container of an
analyte-specific capture reagent; analyte-specific capture reagent
can be immobilized on a solid support.
[0015] In another aspect, the invention provides a detectable
complex for detection of an analyte, the complex comprising a
reagent bound to an analyte, the reagent having a first portion
which specifically binds to the analyte and a second portion
comprising a polynucleotide sequence; an amplifying entity bound to
the reagent, the amplifying entity having a first polynucleotide
sequence and a second polynucleotide sequence, wherein the first
polynucleotide sequence is complementary to the polynucleotide
sequence of the second portion of the reagent; and a plurality of
signalling moieties bound to the amplifying entity, each of the
signalling moieties comprising a detectable label and a
polynucleotide sequence complementary to the second polynucleotide
sequence of the amplifying entity. The detectable complex can
further include an analyte-specific capture reagent bound to a
solid support, the capture reagent further being bound to the
analyte. In preferred embodiments, the analyte can be an antigen or
nucleic acid.
[0016] In still another aspect, the invention provides an isolated
purified single-stranded homopolynucleotide having a length of at
least about 3000 bases, more preferably a length of at least about
7000 bases. In preferred embodiments, the homopolynucleotide is
selected from the group consisting of poly(dA), poly(dT), poly(dC),
poly(dG), and poly(dU).
[0017] In the methods and compositions of the invention, the PAM
can bind to the analyte-detecting moiety and to a plurality of
signalling moieties. The present invention is based, at least in
part, on the discovery that the portions of the PAM which recognize
the analyte-detecting moiety and the signalling moieties can be the
same. This finding permits simplified synthesis and use of the
PAMs, thus reducing the time and cost required to provide suitable
assay systems. Furthermore, the simplified PAMs of the invention
can be constructed to permit the binding of greater numbers of
signalling moieties than heretofore contemplated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 depicts a generalized scheme for detection of an
analyte according to the methods of the invention.
[0019] FIG. 2 depicts another embodiment of a detection method
according to the invention.
[0020] FIG. 3 shows another embodiment of a detection method
according to the invention in which a second amplified is
employed.
[0021] FIG. 4 is a bar graph comparing a detection method of the
invention to direct detection of a nucleic acid sequence.
[0022] FIG. 5 depicts an ELISA with signal amplification according
to the invention.
[0023] FIG. 6 graphically depicts the results an HIV p24 assay
according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention provides methods and compositions for
amplification of signals in binding assays.
[0025] The methods and compositions of the invention are useful in
assays in which an analyte, which is a member of a specific binding
pair, is detected by binding of the other member of the
specific-binding pair. Specific binding pairs are known in the art
and include pairs such as antibody-antigen, hormone-receptor,
binding ligand-substrate, lectin-sugar, enzyme-inhibitor, and the
like. The term "detecting" as used herein, can include
determination of the presence or absence of an analyte in a sample,
and/or quantitation of the amount of analyte in the sample.
[0026] The amplifying entity (or polymeric amplifying moiety (PAM))
of the invention can be any moiety capable of binding to (i) an
analyte or analyte-binding moiety and (ii) a plurality of
signalling moieties. For example, in a preferred embodiment, a PAM
is a polynucleotide, more preferably a homopolynucleotide,
preferably poly(A) or, more preferably, poly(dA). Poly(dA) can bind
to a complementary sequence of an analyte or analyte-binding
moiety, and to a complementary sequence of a plurality of
signalling moieties. For example, poly(dA) can bind to a sequence
comprising poly(dT), poly(dU), or a sequence comprising a polymer
of dT and dU. In other embodiments, the PAM can be a
polysaccharide, a polypeptide, or other polymer capable of binding
to an analyte or analyte-binding moiety and to multiple signalling
moieties. A PAM can also be a hybrid molecule, e.g., a
protein-nucleic acid conjugate, and the like.
[0027] The signalling moiety can be any molecular entity which can
bind to the amplifying entity and comprises a portion that can
generate a signal, or which can bind to or interact with a moiety
that can generate a signal. Suitable signalling moieties can be
prepared according to methods known in the art, and can be prepared
to bind to a pre-selected amplifying entity. The signalling moiety
can comprise a detectable label. The label, if present, can be,
e.g., a radionuclide, an enzyme (such as alkaline phosphatase (AP)
or horse radish peroxidase (HRP)), an antibody, an antigen (such as
FITC), a member of a specific binding pair (such as
biotin/streptavidin), a fluorescent moiety, a dye, and the like. In
an exemplary embodiment, if the amplifying entity is poly(dA), a
suitable signalling moiety would be labelled poly(dT), e.g.,
FITC-dT.sub.15 (FITC-fluorescein isothiocyanate). In this
illustrative embodiment, the poly(dT) portion of the signalling
moiety can bind to an amplifying entity such as poly(dA), while the
FITC portion of the signalling moiety can bind to, e.g., an
anti-FITC/alkaline phosphatase conjugate. The anti-FITC/alkaline
phosphatase conjugate can generate a colorimetric signal by
reaction with an appropriate substrate for the enzyme, as is
conventional in the art.
[0028] A plurality of signalling moieties are used to bind to each
PAM; the signalling moieties can be the same or may be different.
For example, if the PAM is poly(dA) a plurality of, e.g.,
FITC-dT.sub.15 moieties can be used to bind to each strand of
poly(dA), and the signal detected by use of an anti-FITC-alkaline
phosphatase conjugate, which can react with a substrate to produce
a colored product. Alternatively, a FITC-dT.sub.15 signalling
moiety could be used together with a .sup.32P-dT.sub.15 moiety.
Both signalling moieties could then bind to the poly(dA) strand to
provide both colorimetric and radioactive detection modes.
[0029] A preferred PAM is a single-stranded polynucleotide sequence
which includes at least two non-overlapping occurrences of a
nucleic acid sequence (each occurrence is referred to herein as a"
subsequence"). A polynucleotide having at least two non-overlapping
occurrences of a nucleic acid sequence is referred to herein as a
"repeating polynucleotide" (RP). It will be appreciated that a
subsequence can itself include repeated units or can be a
homopolynucleotide sequence. An RP can include DNA, RNA, mixtures
of DNA and RNA, or nucleic acid analogs or congeners including
phosphorothioates and peptide nucleic acid (PNA). An RP can have
two occurrences of a nucleic acid sequence (the subsequence), but
more preferably includes at least 3, 5, 10, 15, 20, 30, 50, 100,
200, 300, 500, 700, 1000, 2000, 5000, or 10,000 non-overlapping
occurrences of a subsequence. In preferred embodiments, an RP has a
total length of at least about 30 bases, more preferably at least
about 50 bases, more preferably at least about 100 bases, more
preferably at least about 500 bases, more preferably at least about
1000 bases, and most preferably at least about 5000 bases. In
certain embodiments, an RP has a length less than about 10,000
bases, less than about 5000 bases, less than about 1000 bases, or
less than about 500 bases.
[0030] Complementary polynucleotide sequences should be long enough
to permit stable hybridization under stringency conditions
associated with washing, detection, and the like, e.g., binding of
a subsequence to its complement should not be substantially
disrupted by normal washing conditions and the like. For example,
the polynucleotide sequence of the reagent second portion should be
long enough to stably hybridize to the first polynucleotide
sequence of the amplification entity under washing conditions, and
the polynucleotide sequence of the signalling moiety which is
complementary to the amplifying entity second polynucleotide
sequence should be long enough to stably hybridize to the second
polynucleotide sequence of the amplification entity. Thus, in
preferred embodiments, a subsequence is at least about 6 bases in
length, more preferably at least about 10 bases in length, at least
about 15 bases in length, at least about 20 bases in length, at
least about 25 bases in length, at least about 30 bases in length,
at least about 50 bases in length, or at least about 100 bases in
length. In a preferred embodiment, an amplifying entity is a linear
(not circular) polynucleotide sequence.
[0031] An RP can include "contiguous" subsequences (in which two or
more subsequences are contiguous with each other), "non-contiguous"
subsequences (in which at least one base intervenes between
subsequences), or a combination of contiguous and non-contiguous
subsequences. For example, if a subsequence (which is at least six
bases in length) is represented as X, an RP including the
subsequence could be represented as follows: X-Y-X-X-Z-X, in which
Y and Z are bases or sequences which do not include subsequence X.
This exemplary RP includes both contiguous subsequences
(illustrated as X-X) and non-contiguous subsequences (such as
X-Z-X, in which the sequence portion Z separates two subsequences).
In certain embodiments, contiguous subsequences are preferred,
because there are fewer extraneous bases in the RP. In a
particularly preferred embodiment, the RP comprises contiguous
subsequences, with substantially no non-contiguous subsequences or
non-subsequence regions.
[0032] Subsequences can be selected according to factors such as
the ease of preparation of the RP, ease of preparation of a
complement to the subsequence (for example, a labelled signalling
moiety or probe), and the like. An RP preferably does not include
self-complementary regions which could form secondary structures
such as loops; such secondary structure formation could interfere
with the ability of the RP to hybridize to the analyte or to the
signalling moiety. It will be understood that an RP can include
more than one sequence which is repeated (i.e., can include more
than one type of subsequence). Thus, an RP could have the structure
X-Y-X-Y-X-Y, in which X and Y are different nucleic acid
subsequences.
[0033] It will be understood from the discussion herein that a
preferred amplifying entity includes a first polynucleotide
sequence complementary to a polynucleotide portion of an
analyte-specific reagent, and a second polynucleotide sequence
which is complementary to a polynucleotide portion of a signalling
moiety. In a particularly preferred embodiment, the first and
second polynucleotide sequences of the amplifying entity comprise
the same or substantially the same sequence. In a preferred
embodiment, each of the first and second polynucleotide sequences
of the amplifying entity have a length of at least about 6 bases,
more preferably at least about 12 bases, 15 bases, 20 bases, 25
bases, or 35 bases. This result can be obtained by using a
homopolynucleotide as the amplifying entity. A homopolynucleotide
can be viewed as including two portions, C and D, both including
the same homopolynucleotide sequence. Thus, a particularly
preferred amplifying moiety is a homopolynucleotide sequence, i.e.,
a nucleotide sequence composed of a single nucleotide base N; a
homopolynucleotide having n bases is designated N.sub.n, in which n
is an integer in the range of 6 to 10,000, inclusive. It will be
appreciated that a homopolynucleotide of length 100 includes 2
subsequences of length 50, 4 subsequences of length 25, etc. Other
homopolynucleotide sequences will similarly include
homopolynucleotide subsequences. Thus, a homopolynucleotide
provides an efficient amplifying entity, due to the availability of
multiple subsequences (for bonding to a plurality of signalling
moieties) without extraneous bases which do not bind to signalling
moiety or analyte.
[0034] As described in detail below, an amplifying entity which
comprises a homopolynucleotide can be obtained commercially and/or
readily prepared in the laboratory. Preferred amplifying entities
include dA.sub.n, dT.sub.n, dC.sub.n, dG.sub.n, dU.sub.n in which
dA, dT, dC, dG and dU represent deoxyadenosine, deoxythymidine,
deoxycytosine, deoxyguanine, and deoxyuracil, respectively, and n
is an integer greater than 100, preferably greater than 500, more
preferably at least about 1000, more preferably at least about
3000, more preferably at least about 5000, more preferably at least
about 7000, and still more preferably greater than 9000.
[0035] According to the present invention, the PAM (e.g., an
amplifying entity such as a homopolynucleotide) and the signalling
moiety are selected such that at least two signalling moieties can
bind to each strand of PAM, e.g., after washing to remove i) PAM
which is not complexed to an analyte molecule (directly or through
a analyte-specific reagent) and/or ii) unbound signalling moieties.
In preferred embodiments, the number of signalling moieties bound
to each strand of amplifying entity is at least about 5, more
preferably at least about 10, more preferably at least about 20,
50, 100, 200, 300, 500, 1000 or 2000.
[0036] Without wishing to be bound by theory, it is believed that
the ability of a nucleic acid to bind to its complementary sequence
can be affected by the size of the nucleic acid strand. It is
further believed that such effects can reduce the sensitivity of
conventional nucleic acid probes which include long segments not
complementary to the target (analyte) sequence. It is believed that
such non-complementary sequences can decrease the ability of a
probe to sensitively and selectively bind to its target. The
present invention provides amplification systems in which the
ability of an analyte-binding reagent to bind to an analyte is not
significantly impaired by the PAM. Thus, the methods of the
invention retain high analyte specificity and sensitivity while
providing significant amplification of a generated signal.
[0037] I. Methods
[0038] In one aspect, the invention provides a method for detecting
the presence or absence of an analyte in a sample. The analyte can
be, e.g., a nucleic acid sequence (such as a DNA sequence or RNA
sequence) or a member of a specific binding pair such as
antibody/antigen, hormone/receptor, and the like. The sample can
be, inter alia, a biological sample such as a tissue biopsy or a
sample of a biological fluid such as blood, urine, semen, saliva,
and the like.
[0039] In general, the methods of the invention include the steps
of contacting the sample with a reagent having a first portion
which specifically binds to the analyte and a second portion
comprising a polynucleotide sequence, such that a complex of the
analyte and the reagent is formed; contacting the complex of the
analyte and the reagent with an amplifying entity having a first
polynucleotide sequence and a second polynucleotide sequence,
wherein the first polynucleotide sequence is complementary to the
polynucleotide sequence of the second portion of the reagent, such
that a complex of the analyte, the reagent, and the amplifying
entity is formed; contacting the complex of the analyte, the
reagent, and the amplifying entity with a plurality of signalling
moieties, each of the signalling moieties comprising a detectable
label and a polynucleotide sequence complementary to the second
polynucleotide sequence of the amplifying entity, to form a
detectable complex of the analyte, the reagent, the amplifying
polynucleotide and the signalling moieties; and detecting the label
as indicative of the presence or absence of analyte in the sample.
This embodiment is illustrated in FIG. 1. As shown in FIG. 1, an
analyte 10 (depicted as a nucleic acid in FIG. 1, which can be
immobilized with a capture reagent 12 (such as a complementary
probe) bound to a solid support 14) is contacted with reagent 20,
which includes an analyte-binding first portion 22 and a second
portion 24 which includes a polynucleotide sequence complementary
to a first polynucleotide sequence of an amplifying entity 30. The
amplifying entity includes a first polynucleotide sequence which is
complementary to the polynucleotide sequence of the second portion
of the reagent, and a second polynucleotide sequence. A plurality
of signalling moieties 34 (including detectable label portion 36)
and a polynucleotide sequence complementary to the second
polynucleotide sequence of the amplifying entity 30 to provide a
signal which indicates the presence of analyte 10 in the
sample.
[0040] The reagent is selected to have a first portion capable of
binding selectively to the analyte. If the analyte is a nucleic
acid, this selective binding portion can be, e.g., a nucleic acid
sequence complementary to at least a portion of the analyte; a
nucleic acid binding protein which selectively binds to the analyte
sequence; an antibody which selectively binds to the analyte
nucleic acid sequence; and the like. If the analyte is, e.g., an
antigen, the first portion of the reagent can be an antibody which
binds to the antigen. If the analyte is an antibody, the first
portion of the reagent can be an antigen to which the antibody
binds, or the first portion of the reagent can be an anti-analyte
antibody. If the analyte is a hormone, the first portion of the
reagent can be a receptor for the hormone. Other examples of
selective binding portions of the reagent will be apparent to the
ordinarily skilled artisan.
[0041] The selective binding first portion of the reagent is
covalently bonded to a second portion which is includes a
polynucleotide sequence complementary to a polynucleotide sequence
of an amplifying entity. The second portion can be readily prepared
to be complementary to an amplifying entity, e.g., by convention
chemical or biochemical nucleotide synthesis. Methods for
covalently linking an first portion of a reagent with a second
portion of the reagent are also routine to one of ordinary skill in
the art. For example, a protein (such as an antibody) can be linked
to a nucleic acid through use of a bifunctional linking reagent
(see, e.g., Example 3, infra). Similarly, a nucleic acid (which is
complementary to a nucleic acid analyte) can be covalently bonded
to a nucleic acid which is complementary to the amplifying entity
by conventional chemical or biochemical methods. For example, an
analyte-specific probe (A) can first be synthesized, a
polynucleotide sequence (B) complementary to the amplifying entity
can then be synthesized, and then the A portion ligated to the B
portion (e.g., by chemical synthesis or ligation with an enzyme
such as ligase) to form the reagent. Alternatively, the reagent
could be synthesized as a single strand, e.g., using an automated
nucleotide synthesizer. It will be appreciated that the reagent can
have a plurality of portions which are complementary to the
amplifying entity (i.e., can be represented by the formula
A-(B).sub.n, in which A is the first portion, B is a polynucleotide
sequence complementary to the amplifying entity, and n is an
integer greater than one). For example, the antibody-nucleic acid
conjugate reagents prepared in Example 3, infra, may have more than
one nucleic acid "tail" per antibody portion. Such a reagent can
provide additional signal enhancement by binding to a plurality of
amplifying entities, thus providing additional binding sites for
signalling moieties.
[0042] The analyte-binding reagent will in general be contacted
with the analyte under conditions such that an analyte:reagent
complex can form. Thus, for example, appropriate conditions of
stringency will be employed to ensure hybridization to a nucleic
acid analyte, and conditions suitable for binding of an antibody to
an antigen will be employed where the analyte is an antigen. If
desired, the analyte:reagent complex can be washed to remove
impurities, such as impurities and other components of the sample,
extra reagents, and the like. If a washing step is employed, care
should be taken to ensure that the conditions of the wash do not
cause substantial dissociation of the analyte:reagent complex.
[0043] In certain embodiments, it is preferred that the analyte be
immobilized to a solid support to facilitate the washing process.
Thus, for example, a nucleic acid analyte can be immobilized on a
nitrocellulose filter, or can be immobilized by hybridization to an
immobilized capture moiety, such as the hairpin capture moieties
described in PCT Publication No. PCT/US96/13546. A hairpin capture
moiety can be selected so that the capture moiety binds to (is
complementary to) a region of the target nucleic acid sequence
which is contiguous with the region of the target sequence to which
the analyte-specific reagent is bound (where the reagent first is a
target-complementary sequence). This arrangement has the advantage
that the binding of the reagent to the hairpin-bound target will
result in a base-stacking interaction between the hairpin and the
first portion polynucleotide sequence of the reagent, which can
provide additional mismatch discrimination. If the analyte is an
antigen, it can be immobilized by methods known in the art. For
example, an immobilized antibody (bound to a solid support) which
binds to the antigen can be employed to immobilize the antigen.
[0044] Once the analyte:reagent complex is formed (and, optionally,
washed to remove impurities), the analyte:reagent complex is
contacted with an amplifying entity such that an
analyte:reagent:amplifying entity complex is formed. As described
above, the contacting step should occur under such conditions, and
for sufficient time, to ensure that an analyte:reagent:amplifying
entity complex is formed, i.e., the appropriate conditions of
stringency will be employed to ensure hybridization of the
analyte:reagent complex to the amplifying entity. Once the
analyte:reagent:amplifying entity complex has formed, it is
preferably washed to remove impurities, excess reagents, and the
like, as described above.
[0045] The method includes the further step of contacting the
analyte:reagent:amplifying entity complex with a plurality of
detectably-labeled RP-complementary signalling moieties to form a
detectable complex, such that the presence or absence of analyte in
the sample is detected. The term "detectable complex" refers to a
complex of an analyte, a reagent as described above, an amplifying
entity, and a detectably-labelled signalling moiety. The signalling
moiety includes a polynucleotide sequence which is complementary to
the second polynucleotide sequence of the amplifying entity. A
detectably-labelled moiety can be detected either directly or
indirectly. For example, a radioisotope is a detectable label,
which can be detected, e.g., by scintillation counting or with
X-ray film, and the like. Other detectable labels include, e.g.,
the labels described above. A "plurality" of signalling moieties
can include more than one occurrence of a single signalling moiety,
or can include multiple different signalling moieties. For example,
an amplifying entity having the structure X-Y-X-Y-X-Y (in which X
and Y are different sequences) could be contacted with a plurality
of signalling moieties of the formula X.sub.c*, in which X.sub.c
represents a sequence complementary to X, and * represents a
detectable label; or the RP could be contacted with a plurality of
signalling moieties of the formula Y.sub.c*, in which Y.sub.c
represents a sequence complementary to Y, and * represents a
detectable label; or the RP could be contacted with a plurality of
signalling moieties including a mixture of X.sub.c* and
Y.sub.c*.
[0046] As described herein, the detectable complex (if such has
formed due to the presence of analyte in the sample) can be
detected by detection of the label on the signalling moieties. In a
preferred embodiment, the detectable complex is washed to remove
impurities, unbound signalling moieties, and the like, to reduce
background noise in the detection process.
[0047] In a preferred embodiment, the amplifying entity is
poly(dA), and each of the signalling moieties comprises poly(dT) or
poly(dU), preferably dT.sub.m or dU.sub.m, in which m is an integer
in the range of 15 to 35, more preferably about 25. In this
embodiment, the analyte-binding reagent second portion preferably
includes a poly(dT) "tail" to which the poly(dA) amplifying entity
can bind.
[0048] It will be appreciated by the skilled artisan that the
presence of multiple signalling moieties bound to the
analyte:reagent:amplifying entity complex (due to multiple
signalling-moiety binding sites of the amplifying entity) can
provide an amplified signal compared to methods in which only a
single signal moiety (or detectable label) is bound to each analyte
molecule. Thus, the methods of the invention provide amplified
signal compared to many conventional methods. In preferred
embodiments, the signal, or the signal-to-noise ratio, is enhanced
by at least about a factor of two, more preferably by a factor of
at least about 5, 10, 20, 50, 100, 200, 500, or 1000. In preferred
embodiments, the methods of the invention provide methods which are
at least 2, 5, 10, 50, 100, 500, 1000, or 5000-fold more sensitive
than non-amplified assays using the same detectable label.
[0049] It will further be appreciated that the signal can be
amplified even further by providing additional binding sites for
signalling moieties. Such a result can be obtained by "extending"
the (first) amplifying entity with additional (second, third, etc.)
amplifying entities which provide a greater number of binding sites
for signalling moieties. For example, as shown in FIG. 2, the
analyte:reagent:(first)amp- lifying entity complex 40 (in which the
analyte 10 is illustrated as an antigen, which is recognized by
antibody first portion 22 of the reagent 20) can be contacted with
an extension reagent 31 which includes a first portion 32 which
includes a polynucleotide sequence complementary to the (first)
amplifying entity, but also includes a second portion 33 which
comprises a polynucleotide portion to which a plurality of
signalling moieties can bind. The second portion 33 of the
extension reagent may include the same polynucleotide sequence as
the second portion of the amplifying entity, or, preferably, can be
different. Binding of the extension reagent 31 to the complex 40
results in formation of an "extended" complex. The "extended"
complex can then be contacted with a plurality of signalling
moieties 34, which include a detectable label and a polynucleotide
sequence complementary to the second portion of the extension
reagent 31, to detect the analyte. An example of reagent 31
suitable for use with a poly(dA) amplifying entity 30 is a
poly(dT)-poly(dC) strand (prepared as described in Example 1,
infra); the poly(dT) portion binds to the poly(dA) strand, while
the poly(dC) portion serves as a second RP to which a plurality of
signalling moieties, such as (dG).sub.14-FITC can bind.
[0050] Additional binding sites for signalling moieties can be
provided as shown in FIG. 3, in which the analyte 10 is depicted as
an antigen which is immobilized on solid support 14 by a capture
reagent 12 (e.g., a bound antibody). The steps of contacting the
analyte with an analyte-complementary reagent to form an
analyte:reagent complex, and contacting the analyte:reagent complex
with a first amplifying entity to form an
analyte:reagent:first-amplifying-entity complex, can be performed,
e.g., as described hereinabove. In FIG. 3, the
analyte:reagent:first-amplifying-entity 40 is contacted with a
bifunctional reagent 50. The bifunctional reagent 50 includes a
first portion which includes a polynucleotide sequence
complementary to the second polynucleotide sequence of the first
amplifying entity, and a second portion 52 which can be
specifically recognized and bound by an extension reagent 60. An
example of second portion 52 is a member of a specific-binding
pair, e.g., as described herein. A preferred portion 52 is biotin.
Extension reagent 60 includes a first portion 62 (e.g.,
streptavidin) which is capable of binding to portion 52 of the
bifunctional reagent 50, and a second portion 64 which comprises a
polynucleotide sequence to which a plurality of signalling moieties
can bind. A plurality of signalling moieties 34 can bind to the
second portion 64 as described herein to provide a signal which
indicates the presence of the analyte 10 in the sample.
[0051] Thus, in another embodiment, the invention provides a method
for detecting the presence or absence of an analyte in a sample,
including the steps of contacting the sample with a first reagent
having a first portion which specifically binds to the analyte and
a second portion comprising a polynucleotide sequence, such that a
complex of the analyte and the first reagent is formed; contacting
the complex of the analyte and the first reagent with an amplifying
entity having a first polynucleotide sequence and a second
polynucleotide sequence, wherein the first polynucleotide sequence
is complementary to the polynucleotide sequence of the second
portion of the first reagent, such that a complex of the analyte,
the first reagent, and the amplifying entity is formed; contacting
the complex of the analyte, the first reagent, and the amplifying
entity with a second reagent, the second reagent having a first
portion which includes a polynucleotide sequence complementary to
the second polynucleotide sequence of the amplifying entity, and a
second portion, to form an extendable complex of the analyte, the
first reagent, the amplifying entity and the second reagent;
contacting the extendable complex with an extension reagent, the
extension reagent comprising a first portion capable of
specifically binding to the second portion of the amplifying
entity, and a second portion which comprises a polynucleotide
sequence, such that the extension reagent binds to the extendable
complex to form a complex of the analyte, the first reagent, the
amplifying entity, and the extension reagent; and contacting the
complex of the analyte, the first reagent, the amplifying entity
and the extension reagent with a plurality of signalling moieties,
each of the signalling moieties comprising a detectable label and a
polynucleotide sequence complementary to the polynucleotide
sequence of the extension reagent, to form a detectable complex of
the analyte, the reagent, the amplifying polynucleotide, the
extension reagent and the signalling moieties; and detecting the
label as indicative of the presence or absence of analyte in the
sample.
[0052] In a preferred embodiment, the second portion of the
bifunctional reagent comprises biotin, and the extension reagent
first portion comprises streptavidin. In certain preferred
embodiments, the first amplifying entity and the second amplifying
entity do not include the same subsequence, to avoid non-specific
binding. Thus, in an embodiment in which the first amplifying
entity is poly(dA), the extension reagent second portion preferably
comprises a different homopolynucleotide, preferably poly(dC). In
an embodiment in which the second amplifying entity is poly(dC),
each of the detectably-labeled signalling moieties preferably
comprises poly(dG).
[0053] It will be appreciated that by appropriate selection of
bifunctional reagents and additional amplifying entities, complexes
which include several "layers" of amplifying entities can be
formed, multiplying the number of signalling moieties which can
bind to the complex and increasing the signal. However, it will be
understood that as the number of amplifying entities is increased,
the background "noise" generally increases.
[0054] II. Kits
[0055] In another aspect, the invention provides kits for the
detection of an analyte in the sample.
[0056] In one embodiment, a kit comprises a container including a
reagent having a first portion which specifically binds to the
analyte and a second portion comprising a polynucleotide sequence;
a container including an amplifying entity having a first
polynucleotide sequence and a second polynucleotide sequence,
wherein the first polynucleotide sequence is complementary to the
polynucleotide sequence of the second portion of the reagent; a
container including a plurality of signalling moieties, each of the
signalling moieties comprising a detectable label and a
polynucleotide sequence complementary to the second polynucleotide
sequence of the amplifying entity; and instructions for detecting
the presence or absence of the analyte in a sample. In a preferred
embodiment, the amplifying entity is a homopolynucleotide, more
preferably poly(dA), which preferably has a length of at least
about 3000 bases, more preferably at least about 5000 bases, more
preferably at least about 7000 bases, and still more preferably at
least about 9000 bases.
[0057] In a preferred embodiment, the kit further comprises a
container of an analyte-specific capture moiety. The
analyte-specific capture moiety can be immobilized on a surface of,
e.g., a container (e.g., a surface of the container of the
analyte-specific capture moiety), which can optionally be a
reaction vessel such as a 96-well plate. Alternatively, the
analyte-specific capture moiety can be immobilized on the surface
of a particle such as a bead, e.g., a magnetic microbead.
[0058] III. Compositions
[0059] In another aspect, the invention provides compounds and
detectable complexes useful for detecting the presence or absence
of an analyte in a sample.
[0060] In one embodiment, the invention provides the detectable
complexes described hereinabove. In one embodiment, the invention
provides a complex which includes a reagent which can bind to an
analyte, the reagent having a first portion which specifically
binds to the analyte and a second portion comprising a
polynucleotide sequence; an amplifying entity bound to the reagent,
the amplifying entity having a first polynucleotide sequence and a
second polynucleotide sequence, wherein the first polynucleotide
sequence is complementary to the polynucleotide sequence of the
second portion of the reagent; and a plurality of signalling
moieties bound to the amplifying entity, each of the signalling
moieties comprising a detectable label and a polynucleotide
sequence complementary to the second polynucleotide sequence of the
amplifying entity. The complex can be formed in the presence or
absence of analyte; if formed in the absence of the analyte, the
complex can be contacted with a sample suspected of containing the
analyte to detect the presence or absence of the analyte. In
certain embodiments, the complex includes at least about 50
signalling moieties, more preferably at least about 100 signalling
moieties, at least about 200 signalling moieties, or at least about
1000 signalling moieties. In certain embodiments, the complex
includes the analyte, and the reagent first portion is bound to the
analyte.
[0061] In another aspect, the invention provides
homopolynucleotides useful a amplification entities. In one
embodiment, the invention provides an isolated purified
single-stranded homopolynucleotide having a length of at least
about 3000 bases. In preferred embodiments, the homopolynucleotide
has a length of at least about 7000 bases. In certain embodiments,
the homopolynucleotide is selected from the group consisting of
poly(dA), poly(dT), poly(dC), poly(dG), and poly(dU), most
preferably poly(dA).
[0062] The following examples are intended to illustrate, but not
to limit, the methods and compositions of the invention.
EXAMPLE 1
[0063] Long poly(dA) was prepared by a modification of a published
procedure (Methods in Molecular Biology, Vol. 16, Ch. 7, pp. 95-105
(1993)). Terminal deoxyribonucleotidyl sransferase (TDT, EC
2.7.7.31)) was used to extend poly(dA) purchased from Sigma
Chemical Co. (St. Louis, Mo.). The literature procedure was
modified by, inter alia, addition of excess TDT to improve the
reaction time and/or yield of lengthened poly(dA).
[0064] A poly(dA) solution was provided by dissolving poly(dA) in
deionized water to a concentration which provided 12.5 A.sub.260
units/ml. A solution of dATP in deionized water (concentration of
100 mM) was also prepared and frozen until use.
[0065] In a 1.5 ml microcentrifuge tube, the following reagents
were combined:
[0066] 100 ul poly(dA)
[0067] 200 ul 5.times. buffer (500 mM cacodylate pH 6.8, 5 mM
CoCl.sub.2, 0.5 mM DTT)
[0068] 20 ul dATP solution
[0069] 660 ul deinonized water
[0070] 750 units TDT
[0071] The reaction mixture was incubated overnight at 37.degree.
C.
[0072] The reaction mixture was purified with a buffer exchange
column, eluted with deionized water, and further purified (e.g., to
remove remaining excess ATP) by ultrafiltration with a Centricon
100 centifuge filter (Amicon, Beverly Mass.). The above extension
procedure could be repeated to further extend the poly(dA).
[0073] An agarose gel analysis showed that the poly(dA) before
lengthening had an average length of about 1600-1650 bases. After
one cycle of lengthening, the average length of the poly(dA) strand
was about 5000 bases; after two cycles, about 7000 bases; after
three cycles, about 9000 bases. Further cycles of lengthening
appeared to provide only minimal increases in strand length.
[0074] A similar procedure for lengthening poly(dC) was performed
to provide longer poly(dC) sequences. To provide a hybrid reagent,
a poly(dT) strand was successfully extended with dC bases in an
analogous reaction.
EXAMPLE 2
[0075] The ability of poly(dA) to bind to a signalling moiety was
tested using FITC-T.sub.20 and FITC-T.sub.40 conjugates.
Spectrophotometric analysis (260 nm) of the binding of the
signalling moiety to poly(dA), in the absence of other components,
showed that commercially-available poly(dA) (Sigma), with a length
of about 1650 bases per strand, bound about 282 FITC-T.sub.20
moieties or about 93 FITC-T.sub.40 moieties per strand of poly(dA)
at room temperature. This is in excess of the maximum number of
signalling moieties which could bind to the poly(dA) in a linear
manner; the reason for the discrepancy is not fully understood.
[0076] Binding of signalling moieties to a sample of lengthened
poly(dA) with an average length of about 8000 bases per strand
(prepared as in Example 1) was performed as described above. The
results indicated that about 1100 FITC-T.sub.20 moieties or about
484 FITC-T.sub.40 moieties bound per strand of poly(dA) at room
temperature. Again, this is in excess of the maximum number of
signalling moieties which could bind in a linear manner to the
poly(dA).
[0077] It can be seen that, as expected, the lengthened poly(dA)
prepared in Example 1 provides a greater number of binding sites
for signalling moieties than does commercially-available
poly(dA).
EXAMPLE 3
[0078] An analyte-specific reagent for detection of human IgG was
prepared as described below. The reagent included an anti-IgG
portion and a poly(dT) "tail".
[0079] Goat anti-human IgG (available from Sigma Chemical Co., St.
Louis. Mo.) in 5 mM EDTA was reduced with 2-mercaptoethylamine
hydrochloride (MEA, Pierce, Rockford, Ill.) in buffer A (100 mM
sodium phosphate, 5 mM EDTA, pH 6.0) to cleave the disulfide bond
between the F(ab) fragments and provide a free sulfhydryl group.
When reaction was complete (incubation was at 37.degree. C. for 90
minutes), the mixture was diluted with sterile buffer B (20 mM
sodium phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.4) and purified on a
Bio-Rad Econo-Pac 10DG column, eluting with Buffer B. Fractions
were collected and assayed for protein with a BCA assay (BCA
Protein Assay Reagent kit, Pierce) being careful to distinguish
false positives due to the reducing reagent (MEA). The
protein-containing fractions were pooled and the yield was
calculated. An assay for determination of free sulfhydryl groups
(Ellman's reagent) indicated that each antibody fragment may have
several sulfhydryl groups. (However, some of these free sulfhydryl
groups may not react with the modified DNA in the derivatization
step, infra.)
[0080] 3'-terminal amine-modified (dT).sub.35 was obtained from
Oligos Inc., and treated with
sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexan- e-1-carboxylate
(Sulfo-SMCC, Pierce, 25 mole equiv.) in sterile PBS (20 mM sodium
phosphate, 150 mM NaCl, pH 7.2) to derivative the (dT).sub.35 amino
group. The reaction was typically incubated for 60 minutes at room
temperature or for 30 minutes at 37.degree. C. The derivatized
(dT).sub.35 was purified (on a Bio-Rad Econ-Pac column eluting with
Buffer B). Fractions containing modified DNA were detected by
measuring the UV absorbance at 260 nm. The derivatized DNA was then
conjugated to the cleaved F(ab) fragments prepared from anti-human
IgG (molar ration of modified DNA to protein was 10:1) by
incubation for at least 2 hours at 4.degree. C. (or overnight). The
conjugate was purified with a Centricon 60 centrifuge filter
(Amicon) to provide the analyte-specific reagent.
[0081] Other protein conjugates were derivatized with a (dT).sub.35
tail according to this procedure with only minor changes. Thus,
analyte-specific reagents were prepared from polyclonal antibodies
(goat anti-human IgG, goat anti-mouse IgG, and sheep anti-rabbit
IgG), monoclonal antibodies (mouse monoclonal anti-FITC, mouse
monoclonal antibiotin), and other proteins (streptavidin and
alkaline phosphatase (AP)).
EXAMPLE 4
[0082] The following protocol was used for this example:
[0083] 1 pmol of the capture hairpin was incubated with the
appropriate target in 100 microliters of reaction buffer (100 mM
Tris-HCl, 1M NaCl, 0.08% Triton-X-100, pH 8) per well. The reaction
was heated to 95.degree. C. for 5 minutes and then cooled to room
temperature. The reaction mixture was loaded into high-capacity
streptavidin-coated microtiter plates (available from, e.g.,
Boehringer Mannheim) and incubated 45 minutes. The wells were
washed 6.times. with reaction buffer.
[0084] For direct detection, the protocol described at "Signal
generation," below, was then followed. For amplified reactions, a
secondary probe molecule (a T.sub.20-tailed polynucleotide
complementary to a target sequence) was suspended in reaction
buffer at a concentration of 100 pmol per 100 microliters of
reaction buffer. The target mixture was loaded into wells and
incubated for 45 minutes. A poly(dA) solution (250 ng/100
microliters reaction buffer) was then added (100 microliters) to
each well and incubated for 45 minutes. The wells were washed
6.times. with reaction buffer.
[0085] A solution of signal moiety (FITC-T.sub.15-FITC) was
dissolved in reaction buffer (4 pmol signal/00 microliters buffer).
The signal solution was loaded into wells, incubated 45 minutes,
and then washed 6.times. with reaction buffer, followed by signal
generation.
[0086] Signal Generation:
[0087] The anti-FITC/alkaline phosphatase conjugate was diluted
6000-fold in Tris-buffered saline (150 mM NaCl, 20 mM Tris, pH 8),
and 100 microliters was loaded into each well and incubated for 45
minutes. The wells were washed 6.times. with {fraction
(1/10)}-strength reaction buffer. The alkaline phosphatase
substrate was added, the color developed, and the results were read
by a plate reader.
[0088] In this example, a synthetic target molecule was employed as
analyte. The target was custom synthesized by and purchased from
Oligo Therapeutics (Wilsonville, Oreg.), and had the following
sequence:
1 5'-FITC-AAC AAG CGG CTA GGA GTT CCG GAG TAT GGA TCG GCA GAG GAG
CC-3'
[0089] The presence of the FITC moiety did not prevent the analyte
from binding to the capture hairpin probe or to the detector
moiety. The analyte was captured by an immobilized hairpin probe
having the following sequence:
2 5'-CTAGT CGACG TGGTC CTTU.sub.BT TGGAC CACGT CGACT AG GGCTC CTCTG
CCGAT CCATA-3
[0090] in which U.sub.B indicates a biotinylated uracil through
which the hairpin was immobilized to a streptavidin-coated plate.
The secondary probe had the following sequence:
[0091] 5'-CTG CGG AAC TCC TAG CCG CTT GTT TTTTT TTTTTT TTTTT-3"
[0092] FIG. 4 shows a comparison of direct and amplified DNA
detection. In FIG. 4, the gray bars represent direct detection of
DNA, while the black bars represent detection according to the
method of the invention The inset shows an expanded scale for the
direct detection data. The vertical axis is in units corresponding
to the change in absorbance (OD) per unit time (seconds); this
slope was more readily determined with our plate reader than a
fixed OD reading.
[0093] According to FIG. 4, the amplified DNA detection system
provided more signal than did the direct detection scheme (however,
the amplified system also had greater noise, as seen by the larger
signal when no analyte was present (columns labelled "nil")). The
amplified system detected analyte at levels as low as 104 pmol
(signal greater than baseline noise level). In contrast, direct
detection was unable to detect levels of analyte below 10.sup.-2
pmol (signal level not above background).
EXAMPLE 5
[0094] A model antibody-based amplification system was prepared and
compared to a direct detection scheme.
[0095] Microtiter plates were coated with human IgG (Sigma) at
concentrations from 50 ng to 50 fg per well. Control wells
(labelled "nil" in FIG. 5) had no IgG. The presence of IgG on the
plates was then detected with either an anti-IgG/alkaline
phosphatase (AP) conjugate (Sigma) with calorimetric detection, or
with the anti-IgG/poly(dT) conjugate prepared in Example 3, supra,
with amplified detection.
[0096] The results are shown in FIG. 5. The test bars (Prep #1,
Prep #2 and Prep #3) represent the results of assays using three
different preparations of the anti-IgG/poly(dT) reagent described
in Example 3. The black bar represents assays using a conventional
(Sigma) anti-IgG/alkaline phosphatase (AP) reagent for signal
generation. The different panels were obtained using the
concentration of anti-IgG reagent (either anti-IgG/poly(dl) or
anti-IgG/AP) indicated in the inset caption. The vertical axis is
change in optical density (OD) per second. The horizontal line
corresponds to the signal level generated by direct detection in
the control sample.
[0097] FIG. 5 shows the three preparations of anti-IgG/poly(dT)
reagent generally gave similar results, although Prep #3 generated
the largest signal. The amplified detection method provided greater
signal in this model system than the direct detection at high
concentrations of immobilized IgG. At anti-IgG concentration of 25
ng/well, the amplified reaction had a detection limit similar to
direct detection, while at anti-IgG concentrations of 250 or 2500
ng/well, the amplified detection system was similar to or more
sensitive than direct detection of IgG.
EXAMPLE 6
[0098] A comparison of direct detection with two amplifications
according to the present invention was performed, using biotin as a
sample analyte.
[0099] Nunc Maxisorp sample plates (Nunc, Inc.) were coated with
BSA-biotin (Sigma) to provide a biotin-coated surface. The
concentration of BSA-biotin was varied in 10-fold increments to
provide plates having varying amounts of BSA-biotin analyte
immobilized on the surface; the lowest amount loaded was 0.01 pg,
up to a maximum of 1000 pg. Control plates had no biotin analyte
immobilized on the surface.
[0100] "Direct" detection was performed using a streptavidin-horse
radish peroxidase (HRP) conjugate (available from, e.g., Sigma) to
bind to immobilized biotin; colorimetric detection of bound HRP was
performed according to a standard protocol.
[0101] Amplification (referred to in FIG. 6 as "lx amplification")
was accomplished by contacting the plate with streptavidin (0.05
pmol), followed by washing and addition of a biotin-dT.sub.30
conjugate (purchased from Oligos) (1 pmol) to bind to the bound
streptavidin. Lengthened poly(dA) (produced as in Example 1) was
then added (5 ng), followed by a FITC-T.sub.20-FITC signalling
moiety (available from Oligos). The presence of signalling moiety
was detected with an anti-FITC/biotin conjugate (Sigma) (0.25
pmol), followed by streptavidin-HRP conjugate with colorimetric
detection of HRP as above. (An alternate detection scheme used
biotin-T.sub.30 conjugate signalling moieties to hybridize to the
poly(dA), followed by addition of streptavidin-HRP conjugate to
detect the signalling moieties.)
[0102] Amplification with an extension reagent (referred to in FIG.
6 as "2.times. amplification") was performed by contacting the
plate sequentially with streptavidin, biotin-T.sub.30 conjugate and
poly(dA) as described above. Biotin-T.sub.30 conjugate was then
added as an extension reagent (to bind to the poly(dA)). This
complex was then extended by addition of streptavidin (0.05 pmol)
to bind to the biotinylated extension reagent, followed by
biotin-T.sub.30 conjugate, then poly(dA) (5 ng). FITC-T.sub.20-FITC
signalling moiety was then added and detected as described
above.
[0103] The results of the assays are shown in FIG. 6. "Direct"
detection is labeled as "Control" in FIG. 6; plates with no biotin
analyte are labeled "Nil". It can be seen that both amplification
methods provided greater signal intensities than the direct
detection method; the extended amplification scheme generally
provided the greatest signal. However, the amplification methods
did result in greater "noise" than direct detection, as seen by
comparison of the "nil" (no analyte) assay results. Despite the
increased noise, the amplification methods provided greater
signal-to-noise ratios at lower concentrations. The signal with
direct detection dropped to background levels below 100 pg loading
of analyte, while both amplification methods provided signal above
background at 1 pg analyte loading. The amplification methods thus
appear to provide a 10-100 fold increase in detection sensitivity
in this system.
[0104] An alternative form of extension amplification, using a
(dT).sub.20-poly(dC) strand (about 500-700 dC bases were tailed
onto the dT.sub.20) as both extension reagent and second
amplification entity, and (dG).sub.14-FITC as signalling moieties,
was found to provide generally similar results.
EXAMPLE 7
[0105] Human immunodeficiency virus (HIV) infection can be
diagnosed with an antibody-based reaction system based on the
presence of the p24 antigen of HIV in blood. In this example, a
"direct" detection system was compared with an amplified detection
system.
[0106] Wells of a reaction plate were coated with
commercially-available anti-p24 antibody (available in a kit from
Dupont, Boston, Mass.). Samples containing varying levels of p24
antibody were added to the wells (a control well had no antigen),
followed by addition of a biotinylated polyclonal anti-p24 antibody
(Dupont, Boston, Mass.). The "direct" detection of p24 antigen was
performed by addition of streptavidin-HRP conjugate and
colorimetric detection with an HRP substrate (OPD, provided with
the Dupont kit). Amplified detection was performed by addition of
streptavidin-T.sub.35 conjugate, followed by poly(dA), then
biotin-T.sub.30 conjugate, and finally streptavidin-HRP conjugate,
with colorimetric detection as above.
[0107] The results showed that the amplified detection system was
able to detect the presence of p24 at levels at least 10-fold lower
than the direct detection system.
[0108] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the following claims.
[0109] The contents of all references and patent applications cited
herein are hereby incorporated by reference.
* * * * *