U.S. patent application number 12/745140 was filed with the patent office on 2010-12-02 for methods and compositions for signal enhancement using multivalent interactions.
Invention is credited to Robert D. Jenison, Joshua Klonoski.
Application Number | 20100304387 12/745140 |
Document ID | / |
Family ID | 40679211 |
Filed Date | 2010-12-02 |
United States Patent
Application |
20100304387 |
Kind Code |
A1 |
Jenison; Robert D. ; et
al. |
December 2, 2010 |
METHODS AND COMPOSITIONS FOR SIGNAL ENHANCEMENT USING MULTIVALENT
INTERACTIONS
Abstract
Methods and materials are disclosed relating to an improved
method for amplifying a signal in a diagnostic assay for an
analyte, using an amplification polymer that multivalently binds to
one or more non-analyte-specific binding site of the multivalent
bridge conjugate, if present on the solid support.
Inventors: |
Jenison; Robert D.;
(Boulder, CO) ; Klonoski; Joshua; (Longmont,
CO) |
Correspondence
Address: |
BRINKS HOFER GILSON & LIONE/UTAH;UTAH OFFICE
222 South Main Street, Suite 1930
SALT LAKE CITY
UT
84101
US
|
Family ID: |
40679211 |
Appl. No.: |
12/745140 |
Filed: |
November 26, 2008 |
PCT Filed: |
November 26, 2008 |
PCT NO: |
PCT/US08/84990 |
371 Date: |
May 27, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60990755 |
Nov 28, 2007 |
|
|
|
Current U.S.
Class: |
435/5 ; 435/7.9;
530/391.1; 536/112 |
Current CPC
Class: |
G01N 33/54306 20130101;
C12Q 1/6804 20130101; C12Q 2527/137 20130101; C12Q 2527/125
20130101; C12Q 1/682 20130101; C12Q 1/682 20130101; Y10T 436/143333
20150115 |
Class at
Publication: |
435/6 ; 435/7.9;
530/391.1; 536/112 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C12Q 1/68 20060101 C12Q001/68; C07K 16/00 20060101
C07K016/00; C08B 37/02 20060101 C08B037/02 |
Claims
1. A method for detecting the presence or absence of an analyte
bound to a solid support, comprising: (a) Providing a solid support
comprising an analyte-specific capture molecule that has been
contacted with a sample; (b) Applying to the solid support a
multivalent bridge conjugate having an analyte-specific binding
site and a plurality of non-analyte-specific binding sites, wherein
the analyte-specific binding sites of the multivalent bridge
conjugate bind to the analyte, if present on the solid support; (c)
Applying to the solid support an amplification polymer comprising a
plurality of multivalent binding sites having binding specificity
to the non-analyte-specific binding sites of the multivalent
conjugate and a plurality of detection conjugate binding groups,
wherein the amplification polymer binds to one or more
non-analyte-specific binding site of the multivalent bridge
conjugate, if present on the solid support; (d) Applying to the
solid support a plurality of detection conjugates, wherein a
plurality of detection conjugates bind to detection conjugate
binding groups of the amplification polymer, if present on the
solid support; and (e) Washing the solid support following any one
or more of steps (a), (b), or (c) to remove constituents not bound
to the solid support, wherein detection conjugates remaining bound
to the solid support produce a detectable signal indicating the
presence of analyte on the solid support.
2. The method of claim 1, wherein binding of the multivalent bridge
conjugate to the amplification polymer is detected by reacting the
amplification polymer with a detection conjugate comprising a
detectable label.
3. The method of claim 1, wherein binding of the multivalent bridge
conjugate to the amplification polymer is detected by reacting an
enzyme bound to the amplification polymer with a substrate that
produces a product having a detectable label.
4. The method of claim 1, wherein the multivalent bridge conjugate
is selected from the group consisting of antibodies, polyvalent
antibodies, multi-subunit proteins, chimeric proteins,
glycoproteins, allosteric aptamers, and multimeric aptamers.
5. The method of claim 1, wherein the multivalent bridge conjugate
is unmodified streptavidin.
6. The method of claim 1, wherein the multivalent bridge conjugate
is a protein with repeating subunits.
7. The method of claim 6, wherein the protein with repeating
subunits is selected from the group consisting of ferritin and the
hepatitis B surface antigen (HBsAg).
8. The method of claim 1, wherein the multivalent bridge conjugate
is an IgG, IgE or IgM antibody
9. The method of claim 1, wherein the multivalent bridge conjugate
is an oligosaccharide
10. The method of claim 1, wherein the multivalent bridge conjugate
is indirectly bound to the analyte.
11. The method of claim 1, wherein the multivalent bridge conjugate
comprises a plurality of binding sites having substantially
equivalent binding specificity.
12. The method of claim 1, wherein the multivalent bridge conjugate
comprises a plurality of binding sites having substantially
equivalent binding affinity.
13. The method of claim 1, wherein the amplification polymer is
biotin-labeled dextran.
14. The method of claim 1, wherein the amplification polymer is
conformationally flexible.
15. The method of claim 1, wherein the amplification polymer is
soluble in water from 1 fg/ml to 10 mg/mL.
16. The method of claim 1, wherein the amplification polymer is
soluble in 1M monovalent salt from 1 fg/ml to 10 mg/mL.
17. The method of claim 1, wherein the detection conjugate binding
group is selected from a group consisting of biotin, fluorophores,
sugars, nucleotides, and peptides.
18. The method of claim 1, wherein the amplification polymers
comprise from about 5 to about 1000 detection conjugate binding
groups.
19. The method of claim 1, wherein the affinity of the detection
conjugate binding groups for the multivalent detection conjugate is
not altered as a result of the conjugation chemistry used to attach
the capture groups to the amplification polymer.
20. The method of claim 1, wherein the detection conjugate binding
groups are bound to the amplification polymer via linkage groups
having a length ranging from about 14 to about 2000 daltons.
21. The method of claim 1, the detection conjugate binding groups
are bound to the amplification polymer via linkage groups
comprising -CH.sub.2 linkages ranging from 1 to 200 units.
22. The method of claim 1, wherein the analyte is a biological
molecule.
23. The method of claim 1, wherein the multivalent binding sites on
the amplification polymer are present at a density sufficient to
enable two or more separate multivalent binding sites of one
amplification polymer to bind to two or more non-analyte-specific
binding sites of one multivalent bridge conjugate, if present on
the solid support.
24. The method of claim 23, wherein binding of the multivalent
bridge conjugate to the amplification polymer is detected by
reacting the amplification polymer with a detection conjugate
comprising a detectable label.
25. The method of claim 23, wherein binding of the multivalent
bridge conjugate to the amplification polymer is detected by
reacting an enzyme bound to the amplification polymer with a
substrate that produces a product having a detectable label.
26. The method of claim 23, wherein the multivalent bridge
conjugate is selected from the group consisting of antibodies,
polyvalent antibodies, multi-subunit proteins, chimeric proteins,
glycoproteins, allosteric aptamers, and multimeric aptamers.
27. The method of claim 23, wherein the multivalent bridge
conjugate is unmodified streptavidin.
28. The method of claim 23, wherein the multivalent bridge
conjugate is a protein with repeating subunits.
29. The method of claim 28, wherein the protein with repeating
subunits is selected from the group consisting of ferritin and the
hepatitis B surface antigen (HBsAg).
30. The method of claim 23, wherein the multivalent bridge
conjugate is an IgG, IgE or IgM antibody
31. The method of claim 23, wherein the multivalent bridge
conjugate is an oligosaccharide
32. The method of claim 23, wherein the multivalent bridge
conjugate is indirectly bound to the analyte.
33. The method of claim 23, wherein the multivalent bridge
conjugate comprises a plurality of binding sites having
substantially equivalent binding specificity.
34. The method of claim 23, wherein the multivalent bridge
conjugate comprises a plurality of binding sites having
substantially equivalent binding affinity.
35. The method of claim 23, wherein the amplification polymer is
biotin-labeled dextran.
36. The method of claim 23, wherein the amplification polymer is
conformationally flexible.
37. The method of claim 23, wherein the amplification polymer is
soluble in water from 1 fg/ml to 10 mg/mL.
38. The method of claim 23, wherein the amplification polymer is
soluble in 1M monovalent salt from 1 fg/ml to 10 mg/mL.
39. The method of claim 23, wherein the detection conjugate binding
group is selected from a group consisting of biotin, fluorophores,
sugars, nucleotides, and peptides.
40. The method of claim 23, wherein the amplification polymers
comprise from about 5 to about 1000 detection conjugate binding
groups.
41. The method of claim 23, wherein the affinity of the detection
conjugate binding groups for the multivalent detection conjugate is
not altered as a result of the conjugation chemistry used to attach
the capture groups to the amplification polymer.
42. The method of claim 23, wherein the detection conjugate binding
groups are bound to the amplification polymer via linkage groups
having a length ranging from about 14 to about 2000 daltons.
43. The method of claim 23, the detection conjugate binding groups
are bound to the amplification polymer via linkage groups
comprising --CH.sub.2 linkages ranging from 1 to 200 units.
44. The method of claim 23, wherein the analyte is a biological
molecule.
45. A method for detecting the presence or absence of an analyte
bound to a solid support, comprising: (a) Mixing a multivalent
conjugate having an analyte-specific binding site and a plurality
of non-analyte-specific binding sites, with an amplification
polymer comprising a plurality of capture groups that bind
specifically to at least two binding sites of the analyte, to
produce a multivalent conjugate/amplification polymer complex; (b)
Contacting the solid support with the multivalent
conjugate/amplification polymer complex of (a), wherein the
multivalent conjugate/amplification polymer complex binds to
analyte, if present on the solid support; (c) Contacting the solid
support with a plurality of detection conjugates, wherein a
plurality of detection conjugates bind to detection conjugate
binding groups of the amplification polymer, if present on the
solid support; and (d) Detecting multivalent conjugates bound to
both amplification polymer and analyte.
46. A method for detecting the presence or absence of an analyte
bound to a solid support, comprising: (a) Providing an analyte
detection complex having an analyte-specific binding site, wherein
the complex comprises: i. a multivalent bridge conjugate having an
analyte specific binding site and a plurality of
non-analyte-specific binding sites, ii. an amplification polymer
comprising a plurality of capture groups bound to at least two
binding sites of the analyte and a plurality of detection conjugate
binding sites, wherein the amplification polymer binds to one or
more non-analyte-specific binding site of the multivalent bridge
conjugate, if present on the solid support; iii. a plurality of
detection conjugates bound to detection conjugate binding sites of
the amplification polymer; (b) Contacting the analyte detection
conjugate complex of (a) with the solid support, wherein the
complex binds to the analyte, if present on the solid support; and
(c) Detecting analyte detection conjugate complex bound to
analyte.
47. The method of claim 46, wherein the multivalent binding sites
on the amplification polymer are present at a density sufficient to
enable two or more separate multivalent binding sites of one
amplification polymer to bind to two or more non-analyte-specific
binding sites of one multivalent bridge conjugate, if present on
the solid support.
48. A method for preparing an analyte detection complex having an
analyte-specific binding site and improved dissociation properties,
comprising combining: (a) a multivalent bridge conjugate having an
analyte specific binding site and a plurality of
non-analyte-specific binding sites, (b) an amplification polymer
comprising a plurality of capture groups bound to at least two
binding sites of the analyte and a plurality of detection conjugate
binding sites, wherein the amplification polymer binds to one or
more non-analyte-specific binding site of the multivalent bridge
conjugate, if present on the solid support; (c) a plurality of
detection conjugates bound to detection conjugate binding sites of
the amplification polymer.
49. The method of claim 48, wherein the multivalent binding sites
on the amplification polymer are present at a density sufficient to
enable two or more separate multivalent binding sites of one
amplification polymer to bind to two or more non-analyte-specific
binding sites of one multivalent bridge conjugate, if present on
the solid support.
50. The method of claim 49, further comprising the step of
contacting the analyte detection conjugate complex with a solid
support, wherein the complex binds to the analyte, if present on
the solid support.
51. The method of claim 50, further comprising the step of
detecting analyte detection conjugate complex bound to analyte.
52. A method for detecting the presence or absence of a multivalent
analyte bound to a solid support, comprising: (a) Applying to the
solid support an amplification polymer comprising (i) a plurality
of multivalent binding sites having binding specificity to two or
more binding sites of the analyte and (ii) a plurality of detection
conjugate binding groups, wherein the amplification polymer binds
to one or more non-analyte-specific binding site of the multivalent
bridge conjugate, if present on the solid support; (b) Applying to
the solid support a plurality of detection conjugates, wherein a
plurality of detection conjugates bind to detection conjugate
binding groups of the amplification polymer, if present on the
solid support; and (c) Washing the solid support following any one
or more of steps (a), (b), or (c) to remove constituents not bound
to the solid support, wherein detection conjugates remaining bound
to the solid support produce a detectable signal indicating the
presence of analyte on the solid support.
53. The method of claim 52, wherein the multivalent binding sites
on the amplification polymer are present at a density sufficient to
enable each of the two or more multivalent binding sites of one
amplification polymer to bind to one of the two or more multivalent
binding sites of the analyte, if present on the solid support.
54. An amplification polymer comprising a plurality of multivalent
binding sites having binding specificity to a non-analyte-specific
binding sites of the multivalent conjugate and a plurality of
detection conjugate binding groups, wherein the amplification
polymer binds to one or more non-analyte-specific binding site of
the multivalent bridge conjugate, if present on the solid
support.
55. An analyte detection complex comprising (a) a multivalent
bridge conjugate having an analyte specific binding site and a
plurality of non-analyte-specific binding sites conjugated to (b)
an amplification polymer having a plurality of multivalent binding
sites, wherein the multivalent binding sites are present at a
density wherein two or more separate multivalent binding sites of
one amplification polymer are bound to two or more
non-analyte-specific binding sites of one multivalent bridge
conjugate.
56. The analyte detection complex of claim 55, further comprising a
plurality of detection conjugates bound to detection conjugate
binding sites of the amplification polymer.
57. A kit for detecting an analyte in a sample, comprising in
packaged combination, (a) a multivalent bridge conjugate having an
analyte specific binding site and a plurality of
non-analyte-specific binding sites, and (b) an amplification
polymer having a plurality of multivalent binding sites, wherein
the multivalent binding sites are present at a density wherein two
or more separate multivalent binding sites of one amplification
polymer are bound to two or more non-analyte-specific binding sites
of one multivalent bridge conjugate.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Patent Application No. 60/990,755,
filed Nov. 28, 2007, which is incorporated, in its entirety, by
this reference.
TECHNICAL FIELD
[0002] The present invention relates generally to methods and
compositions for amplifying a detectable signal used to detect the
absence or presence of a nucleic acid analyte in a sample.
BACKGROUND
[0003] Many diagnostic assays utilize detectable labels to indicate
binding events that are indicative of the presence or absence of a
target analyte in a sample. Typical target analytes include
proteins, carbohydrates, or nucleic acids. Generally, such
diagnostic assays utilize a target-specific capture molecule that
is immobilized on a solid substrate. A sample is placed on the
solid substrate and the target analyte, if present, binds to a
target-specific capture molecule. The surface-bound target analyte
may then be directly modified by binding, directly or indirectly,
with a detectable label. Alternatively, a second reagent, modified
with a detectable label, may bind to the surface immobilized
target. The label can be detected directly in the case of
radio-labeled or fluorescent labels using devices such as a
phosphor-imager or a fluorescence reader, respectively.
Alternatively, the label may be indirectly detected, for example,
by binding the label with an anti-label/enzyme conjugate that is
subsequently contacted with an enzyme substrate to produce a signal
that can be detected.
[0004] Due to the low frequency of target analytes in some samples,
various methods have been developed to enhance the signal of
diagnostic assays using indirect methods. For example, U.S. Pat.
No. 5,196,306, discloses a method in which a target-specific,
surface-immobilized label is reacted with an amplification polymer
to multiply the number of binding sites for a detectable label
complex, followed by conjugation with an anti-label antibody
conjugate, such as horse radish peroxidase ("HRP") that is then
exposed to a tyramide/label conjugate. The tyramide is activated by
HRP and then reacts with electron rich groups nearby to physically
attach a label molecule.
[0005] Several nucleic acid specific techniques have also been
developed. For example, U.S. Pat. No. 5,124,246 discloses
amplification of a signal by creating branched layers of DNA
hybridization in a target nucleic acid sequence specific manner.
The layers culminate in a branched structure that can hybridize to
hundreds of labels. Other approaches, disclosed in U.S. Pat. No.
6,103,474 and U.S. Pat. No. 6,110,682, amplify a signal by
targeting homopolymeric regions of a target nucleic acid analyte
with multiple-labeled hairpin reporter probes. A method has also
been developed that amplifies biotin-dependent signaling events
(Zhong et al., PNAS (2003) 100:11559-11564). In this approach,
biotinylated probes are immobilized on a surface in a
target-dependent manner, and are then contacted with an
avidin-biotinylated dextran copolymer, resulting in a 50-100 fold
increase in assay sensitivity.
[0006] DNA dendrimers have also been used to amplify signals, as
disclosed in U.S. Pat. No. 5,175,270, U.S. Pat. No. 5,487,973, and
U.S. Pat. No. 6,046,038. DNA dendrimers are large cross-linked
structures that can be modified to contain up to several hundred
label groups. These labels groups include biotin, HRP, streptavidin
("SA"), and fluorescent molecules, as disclosed in U.S. Pat. No.
6,072,043; U.S. Pat. No. 6,110,687; and U.S. Pat. No. 6,762,292.
DNA dendrimer can contain mixtures of molecules as well, such as SA
and HRP. The mixture allows for binding of SA to
surface-immobilized biotin, for example. This approach multiplies
the number of HRP molecules at the surface of each biotin molecule
bound and results in amplification of the signal.
[0007] Another technique employs the targeting of homopolymeric
regions of target DNA with multiple-labeled hairpin reporter
probes, as disclosed in U.S. Pat. Nos. 6,103,474; 6,110,682.
[0008] A method was recently disclosed that describes the
amplification of biotin-dependent signaling events (Zhong et al.,
PNAS (2003) 100:11559-11564). Biotinylated probes that were
immobilized onto a surface in a target-dependent manner were
contacted with an avidin-biotinylated dextran copolymer. This was
reported to increase assay sensitivity 50-100 fold increase in
assay sensitivity for detection of biotin DNA probes covalently
immobilized onto a chip surface. However, it has been observed that
this method suffers from some inconsistency and high levels of
non-specific interaction between the avidin-biotinylated dextran
copolymer and the surface immobilized DNA probesresulting in an
improvement in assay sensitivity of only 5-25 fold.
[0009] Existing signal amplification technologies that use multiple
biotin molecules per polymer backbone have two problems: (1) Poor
surface characteristics result in sub-optimal streptavidin binding.
Steric effects as well as surface charge reduce binding affinity.
(2) Poor spacing of biotin molecules also inhibit optimal
interactions between the polymer-conjugated biotin and
streptavidin. Generally only monovalent interactions may occur
between the polymer-conjugated biotin and streptavidin.
[0010] Accordingly, there continues to be a need for improvement in
the sensitivity and accuracy of assays for detecting target nucleic
acid analytes that may be present in samples.
SUMMARY
[0011] The present invention provides improvements in diagnostic
assays for detecting and/or quantitating an analyte (such as a
nucleic acid or a protein) in a sample. The invention provides
improved methods and compositions for amplifying a detectable
signal used to indicate the presence or absence of the analyte in
the sample. The present invention provides reagents and methods for
improving the sensitivity of a signal generated by means of a
plurality of amplification polymers. In particular, the present
invention relates to a method for enhancing the signal of a
detection complex using multivalent interactions to stabilize
and/or decrease the off-rate of binding of one or more intermediate
binding events between the analyte, if present, and the
amplification polymer and detection conjugate.
[0012] In one embodiment of the invention, the amplification
polymer binds to one or more non-analyte-specific binding site of
the multivalent bridge conjugate, if present on the solid support.
One specific embodiment of the invention is a method for detecting
the presence or absence of an analyte bound to a solid support,
comprising: [0013] (a) Providing a solid support comprising an
analyte-specific capture molecule that has been contacted with a
sample; [0014] (b) Applying to the solid support a multivalent
bridge conjugate having an analyte-specific binding site and a
plurality of non-analyte-specific binding sites, wherein the
analyte-specific binding sites of the multivalent bridge conjugate
bind to the analyte, if present on the solid support; [0015] (c)
Applying to the solid support an amplification polymer comprising a
plurality of multivalent binding sites having binding specificity
to the non-analyte-specific binding sites of the multivalent
conjugate and a plurality of detection conjugate binding groups,
wherein the amplification polymer binds to one or more
non-analyte-specific binding site of the multivalent bridge
conjugate, if present on the solid support; [0016] (d) Applying to
the solid support a plurality of detection conjugates, wherein a
plurality of detection conjugates bind to detection conjugate
binding groups of the amplification polymer, if present on the
solid support; and [0017] (e) Washing the solid support following
any one or more of steps (a), (b), or (c) to remove constituents
not bound to the solid support; wherein detection conjugates
remaining bound to the solid support produce a detectable signal
indicating the presence of analyte on the solid support.
[0018] In another embodiment, the multivalent binding sites on the
amplification polymer are present at a density sufficient to enable
two or more separate multivalent binding sites of one amplification
polymer to bind to two or more non-analyte-specific binding sites
of one multivalent bridge conjugate, if present on the solid
support.
[0019] In another aspect, the invention relates to a specific order
of mixing the multivalent conjugate and amplification polymer,
followed by mixing the resulting complex with the sample. This
aspect may include, for example, a method for detecting the
presence or absence of an analyte bound to a solid support,
comprising: [0020] (a) Mixing a multivalent conjugate having an
analyte-specific binding site and a plurality of
non-analyte-specific binding sites, with an amplification polymer
comprising a plurality of capture groups that bind specifically to
at least two binding sites of the analyte, to produce a multivalent
conjugate/amplification polymer complex; [0021] (b) Contacting the
solid support with the multivalent conjugate/amplification polymer
complex of (a), wherein the multivalent conjugate/amplification
polymer complex binds to analyte, if present on the solid support;
[0022] (c) Contacting the solid support with a plurality of
detection conjugates, wherein a plurality of detection conjugates
bind to detection conjugate binding groups of the amplification
polymer, if present on the solid support; and [0023] (d) Detecting
multivalent conjugates bound to both amplification polymer and
analyte.
[0024] In another aspect, the invention relates to a method in
which the multivalent bridge conjugate, amplification polymer and
detection conjugate are premixed, and then mixed with the analyte
(i.e., applied to a solid substrate to which the analyte is bound),
and then detecting the presence, absence or amount that binds to
the analyte. This aspect may include, for example, a method for
detecting the presence or absence of an analyte bound to a solid
support, comprising: [0025] (a) Providing an analyte detection
complex having an analyte-specific binding site, wherein the
complex comprises: [0026] i. a multivalent bridge conjugate having
an analyte specific binding site and a plurality of
non-analyte-specific binding sites, [0027] ii. an amplification
polymer comprising a plurality of capture groups bound to at least
two binding sites of the analyte and a plurality of detection
conjugate binding sites, wherein the amplification polymer binds to
one or more non-analyte-specific binding site of the multivalent
bridge conjugate, if present on the solid support; [0028] iii. a
plurality of detection conjugates bound to detection conjugate
binding sites of the amplification polymer; [0029] (b) Contacting
the analyte detection conjugate complex of (a) with the solid
support, wherein the complex binds to the analyte, if present on
the solid support; and [0030] (c) Detecting analyte detection
conjugate complex bound to analyte.
[0031] In another aspect, the invention relates to a method for
pre-mixing the multivalent bridge conjugate, amplification polymer
and detection conjugate (which may later be bound to the analyte).
This aspect may include, for example, a method for preparing an
analyte detection complex having an analyte-specific binding site
and improved dissociation properties, comprising combining: [0032]
(a) a multivalent bridge conjugate having an analyte specific
binding site and a plurality of non-analyte-specific binding sites,
[0033] (b) an amplification polymer comprising a plurality of
capture groups bound to at least two binding sites of the analyte
and a plurality of detection conjugate binding sites, wherein the
amplification polymer binds to one or more non-analyte-specific
binding site of the multivalent bridge conjugate, if present on the
solid support; [0034] (c) a plurality of detection conjugates bound
to detection conjugate binding sites of the amplification
polymer.
[0035] In yet another aspect, the invention relates to a method
used to detect the presence or absence or amount of an analyte
which itself has multiple binding sites. This aspect of the
invention may include, for example, a method for detecting the
presence or absence of a multivalent analyte bound to a solid
support, comprising: [0036] (a) Applying to the solid support an
amplification polymer comprising (i) a plurality of multivalent
binding sites having binding specificity to two or more binding
sites of the analyte and (ii) a plurality of detection conjugate
binding groups, wherein the amplification polymer binds to one or
more non-analyte-specific binding site of the multivalent bridge
conjugate, if present on the solid support; [0037] (b) Applying to
the solid support a plurality of detection conjugates, wherein a
plurality of detection conjugates bind to detection conjugate
binding groups of the amplification polymer, if present on the
solid support; and [0038] (c) Washing the solid support following
any one or more of steps (a), (b), or (c) to remove constituents
not bound to the solid support, wherein detection conjugates
remaining bound to the solid support produce a detectable signal
indicating the presence of analyte on the solid support.
[0039] In yet another aspect, the invention relates to a
composition comprising a multivalent bridge conjugate,
amplification polymer and detection conjugate (which may be bound
to the analyte). This aspect of the invention may include, for
example, an analyte detection complex having an analyte-specific
binding site, wherein the complex comprises: [0040] (a) a
multivalent bridge conjugate having an analyte specific binding
site and a plurality of non-analyte-specific binding sites, [0041]
(b) an amplification polymer comprising a plurality of capture
groups bound to at least two binding sites of the analyte and a
plurality of detection conjugate binding sites, wherein the
multivalent binding sites on the amplification polymer are present
at a density sufficient to enable each of the two or more
multivalent binding sites of one amplification polymer to bind to
one of the two or more multivalent binding sites of the analyte, if
present on the solid support; [0042] (c) a plurality of detection
conjugates bound to detection conjugate binding sites of the
amplification polymer.
[0043] In another aspect, the methods and compositions of the
invention may further comprise the step of combining the
amplification complex or label with one or more solvating compounds
in order to increase the number of amplification polymers that form
a complex with the detectable labels.
DETAILED DESCRIPTION
[0044] Units, prefixes, and symbols may be denoted in their SI
accepted form. Numeric ranges recited herein are inclusive of the
numbers defining the range and include and are supportive of each
integer within the defined range. Unless otherwise noted, the terms
"a" or "an" are to be construed as meaning "at least one of." The
section headings used herein are for organizational purposes only
and are not to be construed as limiting the subject matter
described. All documents, or portions of documents, cited in this
application, including but not limited to patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety for any purpose, and
are understood to represent methods and materials generally known
to those skilled in the art.
[0045] As utilized in the present disclosure, the following terms,
unless otherwise indicated, shall be understood to have the
following meanings
Definition of Terms
[0046] "Acetylating compound" means a compound that reacts with
amine groups on the amplification polymer under high salt
conditions to acetylate the amine groups not bound with a
detectable label complex.
[0047] "Amplification polymer" means a polymeric compound that
specifically binds, either directly or indirectly, to a target
nucleic acid analyte and has a plurality of other binding sites
that bind to the detection conjugate, which multiplies the number
of detectable labels that can be bound to each amplification
polymer associated with a given target nucleic acid analyte. In
some embodiments of the invention the amplification polymer may
comprise a polymer having a plurality of reactive amine groups to
which biotin molecules can be covalently attached. The biotin
molecules, when bound to the amplification polymer, can then be
used as a binding substrate for a detectable label complex that
generates a detectable signal. Because each biotin molecule
generates an independent signal, there are multiple signals
generated relative to a single analyte to which the polymer binds,
thereby amplifying the signal of each analyte. The amplification
polymer may be, for example, a dextran copolymer. The amplification
polymer has a plurality of binding sites, which may include one or
more binding sites having different binding specificity to one or
more different binding sites of the multivalent binding bridge
complex. Preferably, the amplification polymer will also be
conformationally flexible, to allow for optimal movement and
placement of biotins for a multivalent interactions. The
amplification polymer will also permit appropriate spacing and
density of biotin molecules on the backbone.
[0048] "Analyte" means a molecule, macromolecule, or compound that
is the target of an assay. Although "analyte" is often used in the
singular in this application, it should be understood that most
samples consist of millions or billions of the identical analyte.
Examples of analytes include, but are not limited to, proteins or
polypeptide molecules, polynucleotide molecules, organic or
inorganic compounds, DNA, polymorphisms of DNA, and RNA. An analyte
will generally have one or more unique binding sites (or epitopes)
to which other molecules may bind. In particular embodiments
disclosed herein, the analyte may be bound to a solid support via
an analyte-specific capture molecule that is directly or indirectly
bound to the solid support, leaving alternative binding sites
available for binding to the multivalent binding conjugate.
[0049] "Analyte-specific" means that a compound binds specifically,
though not necessarily exclusively, to the analyte in a sample.
[0050] "Binds" means the formation of an attractive force between
two molecules, which includes ionic bonds, covalent bonds, polar
covalent bonds, or noncovalent bonds.
[0051] "Capture molecule" means a label comprising a functional
binding group that binds covalently or non-covalently to the
analyte, and further comprising a second functional binding group
that binds covalently or non-covalently to an amplification polymer
or secondary amplification polymer functional binding group.
[0052] "Conjugate" or "complex" means one or more molecules
covalently or non-covalently coupled together.
[0053] "Detectable label" means a chemical compound that can be
either directly or indirectly detected by visual or instrumental
means. A detectable label may consist of a molecule that itself
produces a signal that can be detected, such as a fluorescent,
chemiluminescent or radioactive signal. Alternatively, the
signaling label may comprise a molecule that requires reaction with
another molecule to generate a signal that can be detected.
Detectable labels also include compounds that can be detected
visually, for example, colored dyes.
[0054] "Detection conjugates" and "detectable label complex" means
one or more molecules associated together that enable visual or
instrumental detection of a detectable label. May be single
compound with a detectable label, May be complex of compounds with
a detectable label. In other embodiments, the detectable label
complex may comprise molecules that react with other molecules to
produce other products that can be detected. For example, the
detectable label complex may comprise an enzyme that is reacted
with a substrate to produce reaction products having a detectable
label, or detectable property.
[0055] "Label" means, in its generic sense, a molecule or binding
site of a molecule that is capable of binding either covalently or
non-covalently to other molecules, and being used itself as a
binding substrate for another molecule or as a signal for
detection. Labels often have different chemical functional groups
that react with other chemical functional groups on other
molecules. A label can also have multiple functions, for example a
capture label could also be a signaling label. Labels may be, for
example, an enzyme, antibody, or protein. Labels may also be
detectable labels that are used to generate a signal that can be
detected for purposes of indicating the presence or absence of an
analyte of interest in a sample.
[0056] "Multivalent bridge conjugate" means a compound or complex
of compounds having an analyte-specific binding site and one or
more non-analyte-specific binding sites. The analyte-specific
binding site of the multivalent bridge conjugate binds directly or
indirectly to an analyte, for example, that is bound to a solid
support. The non-analyte specific binding sites of the multivalent
bridge conjugate bind to the binding sites of the amplification
polymer. The multivalent binding sites may all have equivalent
binding specificity to the same substrate, or they may bind to
different substrates. By way of example, a multivalent bridge
conjugate may comprise a streptavidin molecule having four
equivalent binding sites that bind to biotin, one of which is used
to link directly or indirectly to the analyte, and one or more of
which is used to link to an amplification polymer. In other
embodiments, the multivalent bridge conjugate may include one or
more compounds that bind to the signaling groups of the
amplification polymer.
[0057] "Signal" means a property or characteristic of a detectable
label that permits it to be visually or instrumentally detected
and/or distinguished. Typical signals include fluorescent signals,
dyes, radioactive signals, etc.
[0058] "Solid support" means a substrate to which an analyte, if
present in a sample, binds.
[0059] "Specifically binds" and "having binding specificity" means
that a compound binds specifically, though not necessarily
exclusively, to the analyte in a sample.
[0060] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA techniques, and oligonucleotide
synthesis which are within the skill of the art. The foregoing
techniques and procedures are generally performed according to
conventional methods well known to one skilled in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification. See e.g.,
Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic
Acid Hybridization (B. D. Hames & S. J. Higgins, eds., 1984); A
Practical Guide to Molecular Cloning (B. Perbal, 1984); and a
series, Methods in Enzymology (Academic Press, Inc.), the contents
of all of which are incorporated herein by reference. Enzymatic
reactions and purification techniques are performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The nomenclatures utilized in
connection with, and the laboratory procedures and techniques of
biochemistry, analytical chemistry, synthetic organic chemistry,
and medicinal and pharmaceutical chemistry described herein are
those well known and commonly used in the art. Standard techniques
are used for chemical syntheses, chemical analyses, pharmaceutical
preparation, formulation, and delivery, and diagnosis of
patients.
[0061] It is to be understood that the foregoing descriptions of
embodiments of the present invention are exemplary and explanatory
only, are not restrictive of the invention, as claimed, and merely
illustrate various embodiments of the invention. It will be
appreciated that other particular embodiments consistent with the
principles described in the specification but not expressly
disclosed may fall within the scope of the claims. Various aspects
and embodiments of the methods and compositions of the invention
are described in further detail in the following subsections.
[0062] In order to achieve highly sensitive assay performance, high
affinity binding interactions between bio-molecules is of critical
importance. High affinity binding is particularly important for low
amounts or densities of target molecules; higher affinity reagents
are capable of binding to lower concentrations of target analyte. A
method to further enhance the affinity of a bio-molecule is to
increase its valency so that more than one interaction is possible
per molecule. Multivalent interactions have a strong impact on
bio-molecular interactions in multiple ways. First, a higher local
concentration of bio-molecule will increase association kinetics.
Second, the effective dissociation rate is slowed as well by
allowing for rapid rebinding to occur. The net effect is an
improved apparent binding constant.
[0063] In one specific embodiment of the above method,
multiple-labeled biotin polymers will be prepared of sufficient
density and size to permit a multivalent interaction with
streptavidin; more than one biotin will bind per streptavidin for
optimal signal amplification. This multivalent binding is a
requirement for optimal signal enhancement because the biotinylated
polymer will have a very slow off rate, allowing for optimal assay
performance. Key factors include: [0064] (1) Appropriate spacing
and density of biotin on the backbone to provide steric ability of
multivalent binding (i.e., binding groups on one molecule are
spaced so as to match the spacing of two binding epitopes of
another molecule); [0065] (2) Adequate polymer solubility and
flexibility to allow for moving biotins into place for a
multivalent interaction; and [0066] (3) The total number of biotin
molecules present (on a properly configured polymer, more biotins
should correlate with greater signal amplification).
[0067] This approach is a novel embodiment of the principle of
multivalent interactions. It has been reported that multivalent
interactions lead to better affinity constants and therefore,
better binding (Yoshitani and Takasaki Anal. Biochem (2000) 277:
127-134). For example, a multivalent drug used to inhibit
interactions between two molecules leads to drugs with better
potency than a monovalent drug. The use of a multivalent
interaction to maximize signal amplification of an amplification
polymer is a novel approach to signal enhancement.
[0068] The embodiments of the invention presented herein
demonstrate the principle of multivalent binding to create a highly
efficient signal amplification method, termed AMPED. The system
utilizes two multivalent molecules to amplify signal; the first
multivalent molecule binds to a cognate label on immobilized target
molecules. This interaction serves as a "bridge" to a second
multivalent molecule (amplification polymer) by also binding to
labels on the amplification polymer. There are multiple labels on
the amplification polymer available to bind to the bridge molecule.
The binding of the bridge to the amplification polymer may be one
or more interactions, and combined with the interaction with the
target label, multivalent binding occurs. The labels on the
amplification polymer may now be detected either directly or
indirectly by treatment with anti-label/enzyme conjugates that are
subsequently reacted with an enzyme substrate to create a signal.
The multivalent interactions improve signal amplification in
multiple ways: slowed off-rate and improved limits of detection.
Slowed off-rate may be accomplished by placing multiple labels on
the amplification polymer, rebinding may occur. This slows the
effective off-rate, leaving more amplification polymer present to
be detected. Limits of detection may be improved by creating a
higher affinity interaction with target molecules on the target,
lower concentration of target will be bound, improving assay
sensitivity.
[0069] The present invention relates to improved methods and
compositions for detection of analytes. Improved methods and
reagents are disclosed for detection, quantification, and
characterization of analytes, such as proteins, carbohydrates,
nucleic acids, or other molecules, in a sample. Clinically useful
diagnostic methods must be capable of detecting and/or quantifying
the presence of an analyte of interest that is present in extremely
small quantities in a complex mixture containing similar species.
Methods for such diagnostic assays have previously used detectable
labels, such as radiolabeling, radiobioassay and immunoassay
techniques. For example, immunological reagents have been used
extensively for detecting and/or quantitating a broad spectrum of
molecular species such as proteins, lipids, carbohydrates,
steroids, nucleic acids, drugs, carcinogens, antibiotics, inorganic
salts etc. Polyvalent and monoclonal antibodies are very important
diagnostic tools in most areas of clinical medicine today.
[0070] The methods and compositions of the present invention
improve upon methods of the prior art by amplifying the signal
generated by an analyte-specific detection complex. In particular
embodiments, the improved methods disclosed herein increase the
number of amplification polymers available for reaction with a
detectable label. In many diagnostic assays, a capture molecule
that can recognize specific regions of a target analyte interest is
bound to a solid surface and used to capture and immobilize the
target analyte on the solid surface. The target analyte bound to
the solid surface may then be directly or indirectly modified with
a detectable label. The target analyte may be directly labeled with
a detectable label, such as a radio-label or fluorescent label.
Alternatively, the target analyte may be indirectly labeled, for
example, using an anti-label/enzyme conjugate which is then
contacted with an enzyme substrate to produce a signal that can be
detected. A significant advantage of indirect detection is that
intermediate molecules can be conjugated to the target analyte to
amplify the number of signals per target analyte bound to the
capture molecule. The present invention provides improved methods
for signal amplification that reduce interference caused by
non-specific binding of amplification polymers.
[0071] In a particular embodiment, the present invention relates to
methods for detecting an analyte, such as a DNA polymorphism, in a
sample. An analyte-specific capture label is conjugated to an
amplification complex, which comprises a plurality of amplification
polymers that are on a polymer or other macromolecule.
Amplification polymers are also known in the art as "binding
sites." Each polymer or macromolecule has a plurality of
amplification groups, such as amine groups or other functional
binding groups. A detectable label complex is bound to one of the
amplification polymers, which can then be detected. Amplification
polymer complexes may be, such as biotin, will bind to
substantially all of the available amine groups on the polymer or
macromolecule. The detectable label will then either directly or
indirectly produce a signal that can be detected. In accordance
with the methods of the present invention, the unbound
amplification polymers are bound with a capping compound to reduce
non-specific binding of the amplification complex. The detectable
labels are then bound to the to which an analyte is conjugated are
separated from the detection labels to which an analyte is not
conjugated. If the detectable labels are detected, then the
presence of the analyte is inferred. In one embodiment of the
invention the unbound amplification polymers are amine groups on a
polymer. The capping compound could be an acylating compound that
interacts with the amino group and converts it into an acetyl
group, which is more stable and less likely to nonspecifically bind
to other molecules in the assay.
[0072] The methods of the invention provide a novel approach to
amplification of a detectable signal conjugated to an analyte, such
as a nucleic acid analyte. In particular, the invention provides
methods for amplification of a detectable signal conjugated to an
analyte in high salt concentrations typically used in nucleic acid
diagnostic assays. In some embodiments of the invention, the amine
groups of the amplification polymer are reacted with an acetylating
compound in a salt solution under conditions of ionic strength
greater than about 0.5M, to produce an amide group. The capping of
the amine group by acetylation provides a neutrally charged, water
soluble complex. Salt solutions compatible with nucleic acid
detection are well-known to those in the art. In some embodiments,
the salt solution comprises a salt that is monovalent. In other
embodiments, the salt is selected from the group consisting of NaCl
and LiCl.
[0073] In particular embodiments, the improved methods comprise the
step of reacting the amplification polymers with a capping compound
that specifically binds the amplification polymers with greater
affinity than the detectable label complex. In other embodiments,
the improved methods further comprise the step of combining the
amplification polymer with a solvating compound.
Solid Supports
[0074] In some embodiments, the methods of the present invention
may be practiced by first capturing an analyte of interest on a
solid support. In such embodiments, a capture molecule that
specifically or selectively binds the analyte of interest is first
attached to a solid support. The present invention can also be
practiced with or without a solid support. Without a solid support,
for example, a capture label binds to the analyte and an
electrophoretic separator can be used to separate bound analyte
from unbound analyte. However, use of a solid support, such as a
chip, may be more cost-effective and accurate.
[0075] Solid supports include any material that can be used to
immobilize an analyte-specific capture label for use in diagnostic
tests and in separation procedures. Natural or synthesized
materials, which have or have not been modified chemically, can be
used as the solid support, in particular polysaccharides such as
cellulose-based materials, for example paper, cellulose derivatives
such as cellulose acetate and nitrocellulose, dextran; polymers
such as vinyl polychlorides, polyethylenes, polystyrenes,
polyacrylates, polyamides, or copolymers based on aromatic vinyl
monomers, alkyl esters of alpha-beta unsaturated acids, esters of
unsaturated carboxylic acids, vinylidene chloride, dienes or
compounds exhibiting nitrile functions (acrylonitrile); polymers of
vinyl chloride and propylene; polymers of vinyl chloride and vinyl
acetate; copolymers based on styrenes or substituted derivatives of
styrene; natural fibers such as cotton and synthetic fibers such as
nylon; inorganic materials such as silica, glass, ceramic and
quartz; latexes, that is, an aqueous colloid dispersion of any
polymer insoluble in water; magnetic particles; metallic
derivatives. The solid support according to the invention can be,
in the forms which are customarily suitable, for example, in the
form of a chip, microchip, microtitration plate, a sheet, a cone, a
tube, a well, beads, particles or the like. The choice of a support
material can be made, in each particular case, on the basis of
simple routine experiments.
[0076] Methods are also known in the art for binding to a solid
support an oligonucleotide probe for use in detecting specific
nucleic acid sequences in a target nucleic acid. For example,
oligonucleotides may be immobilized to a solid support by covalent
attachment. See, e.g., PCT patent publication Nos. WO 89/10977 and
89/11548. See Chee et al., U.S. Pat. No. 5,837,832. See Strategies
for Attaching Oligonucleotides to Solid Supports, Eric J. Devor and
Mark A. Behlke, Integrated DNA Technologies (2005). The present
invention can be used with all of the above methods.
Multivalent Bridge Conjugate
[0077] The present invention provides a method for enhancing a
signal in a detection assay by using multivalent interactions
between an analyte and a detectable label. Although multivalent
interactions have been previously used in other applications, the
present invention provides a novel application of multivalent
interactions to enhance the signal in a detection assay.
[0078] In one aspect the invention provides a multivalent bridge
conjugate which comprises a complex of one or more molecules, one
of which specifically binds to an analyte of interest. One or more
of the molecules in the complex further serves as a substrate for
the binding of a detectable label complex comprising an
amplification molecule having a plurality of multivalent binding
sites that specifically bind to the non-analyte-specific binding
sites of the multivalent conjugate and a plurality of detection
conjugate binding groups to which is bound a molecule having a
detectable label or that can be used to produce a detectable label.
In the methods of the invention, the multivalent bridge conjugate
may be either directly bound or indirectly bound to the
analyte.
[0079] In some embodiments, the amplification polymer binds to one
or more non-analyte-specific binding site of the multivalent bridge
conjugate, if present on the solid support. In other embodiments,
the multivalent binding sites on the amplification polymer are
present at a density sufficient to enable two or more separate
multivalent binding sites of one amplification polymer to bind to
two or more non-analyte-specific binding sites of one multivalent
bridge conjugate, if present on the solid support.
[0080] In one aspect, the present invention provides novel
complexes for amplifying a signal in a diagnostic assay for a
nucleic acid analyte. The complexes of the invention may, for
example, comprise (i) an amplification polymer bound to an analyte,
such as a nucleic acid, (ii) wherein the amplification polymer
comprises a plurality of binding sites that bind to a detectable
label complex. The binding sites may comprise, for example, amine
groups to which a biotin molecule (linked to a detectable label or
molecules capable of producing a detectable label) can bind. The
amine groups may also be capped to prevent non-specific binding of
other molecules. For example, the amine groups may be capped by
reacting them with an acetylating compound to produce non-reactive
amide groups. The analyte-specification detection complex will, in
some preferred embodiments, be neutrally charged and water
soluble.
[0081] The multivalent bridge conjugate comprises a plurality of
binding sites having substantially equivalent binding specificity
or binding affinity. The multivalent bridge conjugate may be, for
example, an antibody, a polyvalent antibody, a multi-subunit
protein, a chimeric protein, a glycoprotein, an allosteric aptamer,
or a multimeric aptamer. In particular embodiments, the multivalent
bridge conjugate may be a unmodified streptavidin molecule. In
other embodiments, the multivalent bridge conjugate may be a
protein with repeating subunits. Proteins having repeating subunits
include, for example, ferritin and the hepatitis B surface antigen
(HBsAg). The multivalent bridge conjugate may also comprise an IgG,
IgE or IgM antibody. The multivalent bridge conjugate may also be
an oligosaccharide.
Analytes
[0082] In accordance with the methods of the invention, an analyte
of interest is bound to a detection complex that specifically binds
to the analyte. Appropriate analytes include any substance for
which there exists an analyte-specific binding molecule that can be
chemically conjugated to other compounds typically used in chemical
or biological assays. The analyte-specific detection complex may
comprise may be a protein or polypeptide molecule, a carbohydrate,
a polynucleotide molecule, or an organic or inorganic compound. For
example, the capture label may be an antibody, a lectin, a DNA
repressor protein, a stereospecific receptor-protein, a high
affinity enzyme, a sequence specific polynucleotide binding
protein, avidin, streptavidin, a hormone or a complementary
polynucleotide sequence. Target molecules may be any inorganic or
organic species that is capable of producing an affinity with a
detecting agent. Other examples of analytes that have been
disclosed in the prior art are: proteins, lipids, carbohydrates,
phospholipids, fats, nucleotides, nucleosides, nucleoside bases,
polynucleotides, polypeptides, cancerogenic agents, drugs,
antibiotics, pharmaceutical agents, controlled substances,
polymers, silicones, organometallic compounds, heavy metals,
metal-protein complexes, toxic inorganic salts, and other agents or
compounds produced by or having an effect upon a biological
organism or material derived from such molecules. The present
invention could be used with any of the examples from the prior
art.
Detection Complex
[0083] In some embodiments, binding of the multivalent bridge
conjugate to the amplification polymer is detected by reacting the
amplification polymer with a detection conjugate comprising a
detectable label. In other embodiments, multivalent bridge
conjugate to the amplification polymer is detected by reacting an
enzyme bound to the amplification polymer with a substrate that
produces a product having a detectable label.
[0084] The detection complex may include an analyte-specific
molecule, such as an antibody, a lectin, a DNA repressor protein, a
stereospecific receptor-protein, a high affinity enzyme, a sequence
specific polynucleotide binding protein, avidin, streptavidin, a
hormone, a complementary polynucleotide sequence, or some other
molecule.
[0085] Capture molecules may include, for example, proteins (such
as receptor molecules or ligands that bind to a specific cognate
molecule), oligonucleotides that specifically hybridize to a
complementary polynucleotide sequence, or any other molecule known
to bind to a cognate molecule with a high degree of specificity.
Methods for attaching capture molecules to a solid support are
well-known to those skilled in the art, and can be readily
selected, as appropriate. See, e.g., Strategies for Attaching
Oligonucleotides to Solid Supports, Eric J. Devor and Mark A.
Behlke, Integrated DNA Technologies (2005).
[0086] An especially preferred method for detection of target
molecules is based upon the foregoing preferred arrangement, but
includes a second bridging component. The complex, i.e., avidin or
streptavidin-(biotin ligand)-visualization polymer, is used to
complex with a biotin labeled second antibody. The second antibody
is a general reagent for the first antibody detecting agent which
in turn is specific for the target. The first antibody is incubated
with the target to form an antigen-antibody conjugate. Then the
second antibody is incubated with this conjugate. Following the
second incubation, the amplification molecule is added which binds
to the second antibody and enables detection.
[0087] Yet another method, according to the invention, also
utilizes the indirect complexing ligand arrangement. In this
arrangement, the detecting agent is a complementary polynucleotide
sequence and the target is the corresponding native polynucleotide
sequence which will hybridize with the complementary sequence. The
detecting agent and the visualization polymer are labeled with a
biotin or iminobiotin group. A complex of avidin or
streptavidin-(biotin ligand)-amplification molecule is formed. The
labeled polynucleotide detecting agent is added to the complex
biological mixture containing the native polynucleotide sequence to
be detected. Hybridization is allowed to take place, then the
complex is added which binds to the hybridized and labeled
polynucleotide detecting agent and which provides
visualization.
[0088] Multivalent conjugates can be synthesized as described, for
example, by Ooya et al (J. Controlled Release (2002) 80: 219-228)
in different applications relating to a model system for drug
targeting and receptor mediated drug delivery. Ooya et al. describe
binding of biotin-polyotaxane polymer binding to streptavidin,
using a modified polymers containing 11, 35, or 78 biotins per
backbone. These different conjugates were tested for association
and dissociation kinetics using surface plasmon resonance (SPR) and
found no difference in the association rate but a strong effect on
dissociation kinetics. The polymer with 78 biotins had an off rate
that was 7.5 and 30 times slower than the 35 biotin and 11 biotin
polymer, respectively, leading to improved affinity constants with
the 78 biotin backbone. The authors hypothesized that this result
was due to multivalent (more than one biotin per streptavidin)
interactions with greater numbers of biotin present per polymer. To
test this idea, the authors attempted to fit the dissociation rate
data to a pseudo first order kinetic equation, and discovered a
flattening of the dissociation curve with increasing numbers of
biotin. This effect was due partly to avidity--higher local
concentration of biotin leads to improved dissociation constants.
They also identified another unexpected factor--rebinding had a
strong effect on the off rate, not due solely to the avidity
provided by multivalency and high local concentration of biotin,
but also due to concurrent multiple biotin/streptavidin
interactions on the same streptavidin molecule. By conjugating the
biotins to the polyotaxane backbone with adequate spacing, the 78
biotin/backbone was able to bind more than one biotin per
streptavidin, whereas the 11 and 35 biotin/backbone could not.
[0089] In addition, Wands et al. (PNAS (1981) 78:1214-1218)
describe the effect of multivalent binding for detection of
hepatitis B. The binding affinity of various antibodies that bind
to the surface antigen, HBsAg (a protein with repeating epitopes)
was measured. An IgM antibody, which is pentameric, had the highest
affinity by greater than 10-fold compared with IgG antibodies and
had the fastest on rate. Also, this IgM clone (5D3) was the most
sensitive antibody for showing a positive hemagglutination results,
which suggested multivalent interactions. In a sandwich ELISA
format the authors further discovered that IgM-IgM pairing gave the
best limits of sensitivity (100 pg/mL of HBsAg). When IgM (on
surface)-IgG (secondary in solution) pair was used, the sensitivity
was worse (5 ng/mL), but worked at higher HBsAg concentrations
(>10 ng/mL). The authors observed that the IgG was binding to a
low density epitope on HBsAg, so that as the concentration of HBsAg
was lowered, and IgG binding was limited due to lack of multivalent
interactions.
[0090] By extension to multivalent binding, the apparent affinity
constant is slowed by a multiplicative factor of the two binding
events. Further, it has been observed that the binding of a second
biotin leads to a conformation change in streptavidin that
effectively locks the biotins into place (Sano and Cantor J B C
(1990), 265:3369-3373).
[0091] The amplification polymer may further comprise a dextran
copolymer having a plurality of oligosaccharide chains. For
example, Yoshitani and Takasaki (Analytical Biochemistry (2000)
277:127-134) describe the conjugation of 30 to 180 oligosaccharide
chains on a dextran backbone (AsFet-oligo-Dex), which may be used
in conjunction with an amplification polymer and a detectable label
complex, as described herein.
[0092] In other embodiments, multivalent interactions may be
facilitated with the use of antibodies having multivalent binding
specificity, as described by Zuckier et al (Cancer Res (2000)
60:7008-7013).
[0093] In preferred aspects of the invention, the detection
conjugate binding group may be biotin, fluorophores, sugars,
nucleotides, and peptides. The amplification polymers comprise from
about 5 to about 1000 detection conjugate binding groups. In
preferred embodiments, the amplification polymers comprise from
about 20 to 500 detection conjugate binding groups. In more
preferred embodiments, the amplification polymers comprise from
about 50 to about 200 detection conjugate binding groups.
[0094] Suitable detection conjugate binding groups include those
that do not alter the affinity of the detection conjugate binding
groups for the multivalent detection conjugate as a result of the
conjugation chemistry used to attach the capture groups to the
amplification polymer.
[0095] In some embodiments, the detection conjugate binding groups
are bound to the amplification polymer via linkage groups having a
length ranging from about 14 to about 2000 daltons. The detection
conjugate binding groups are bound to the amplification polymer via
linkage groups comprising, for example, --CH.sub.2 linkages ranging
from 1 to 200 units
Amplification Molecules
[0096] The methods of the invention further contemplate the use of
an amplification molecule conjugated to the target analyte of
interest. The amplification molecule comprises, for example, a
plurality of amplification polymers. Amplification polymers perform
the function of providing, for each analyte, multiple bindings
sites for a detectable label. Because each analyte is conjugated to
multiple binding sites to which a detectable label can be bound,
rather than just one binding site for each analyte, the signal
associated with each analyte is multiplied or amplified.
Amplification polymers are typically in the form of
macro-molecules, such as polymers, that have multiple binding
groups to which other molecules or complexes can bind and be used
as a binding substrate for a detectable label or some other
signal.
[0097] For example, amplification polymers may be comprised of a
biotinylated biomolecule such as an enzyme or protein. Numerous
biotinylated biomolecules are known and available to those skilled
in the art. Nonlimiting examples of biotinylated biomolecules
include biotinylated lectins, antibodies, mitogens, DNA, RNA, tRNA,
rRNA fragments, nucleosomes, membranes, membrane proteins,
glycoproteins, synthetic peptides.
[0098] The polymer or other macromolecule used in the amplification
complex can come in many different forms. For example, in the prior
art, the reactive chemical groups or backbone moieties of polymer
subunits have been used to link the detectable label to the polymer
or other macromolecule. For example, if the unit was a protein and
was found to contain a dipeptide side chain ending with cysteine,
the mercaptan group of the cysteine was cross-linked to cysteine of
another similar protein by reaction with bis
(N-butylenylmaleimide). The groups and moieties identified may
include amine groups, mercaptan groups, carboxyl groups, hydroxyl
groups, sugar groups, carbohydrate groups, ester groups, lipid
groups, and amide bonds, labile carbon-carbon bonds and
carbon-hydrogen bonds. Other measurements such as the relation of
derivatization and site activity, relation of pH and site activity
and type of site reaction produced in the case of an enzyme will
help determine a priority for the functional groups based upon the
probability of their presence within the vicinity of the active
site. A typical ranking of priority would be: 1) an epsilon or
primary amine group, 2) a sugar group, 3) a carboxyl group, 4) a
mercaptan group, 5) a hydroxyl group, and 6) a lipid group. If
derivatization of amine groups such as those of lysine residues
produces a derivatized product devoid of site activity, then the
foregoing priority will change and the amine group will be last.
The present invention could be used with each of the preceding
functional groups.
[0099] Tagged natural or synthetic polypeptide, polyol, polyolefin
or carbohydrate have had amplification polymers which are
substantially less sensitive to the chemical group/backbone moiety
bonding arrangement. The fluorescent group, dye, luminescent group,
radioactive group or electron dense group which acts as the tag
typically have not been subject to variations in activity when
adjacent chemical groups or backbone moieties are directly bonded
or indirectly linked with coupling agent. Moreover, the prior art
has shown that if the tag is to be converted to an active group
after the polymer-analyte conjugation is made, then the position of
the chemical group or backbone moiety linkage should not interfere
with the conversion. Each of these teachings can be applied to the
present invention.
[0100] The amplification complex will be conjugated to multiple
detectable labels, either directly interbonded or cross-linked by a
coupling agent. The structural and functional character of the
polymer will be similar to that of the monomer units. The number of
units per polymer will depend upon the extent of coupling, the
stability of the resulting polymer, the reactivity of the chemical
groups or backbone moieties relative to the polymer chain length
and the position of the groups or moieties along the unit
backbone.
[0101] Generally, the number of units incorporated into the polymer
may vary from as few as two to thousands per polymer. Higher
multiples have been possible when the polymer chain length is not
of an order which will render the polymer extremely insoluble in
aqueous solution or will be extremely susceptible toward mechanical
cleavage. The present invention can be used in a similar
fashion.
[0102] The polymer may be linear or it may be branched. There may
be single or multiple coupling between two adjacent units. Coupling
may occur at any point along the unit chain so that adjacent units
may lie end to end, or may partially or fully overlap. As a result,
the three dimensional structure of the polymer may have all of
these features. It may be linear, but more typically, it will be a
combination of linear and branching units. Partial overlap will
typically occur and multiple coupling will also be present.
[0103] The accessibility of the chemical groups or backbone
moieties has also been shown to affect polymer length. If they are
buried within the unit structure, steric inhibition will tend to
hinder coupling of a high number of units. This effect may be
compensated by use of coupling agents having a chain length greater
than about ten carbons in length. Coupling readily accessible
groups or moieties with agents which will hold apart the units of
the polymer has at times proved advantageous. This has allowed for
the facile approach of substrate or reactant and has prevented
adverse interaction among the units of the polymer. Typically,
agents having a carbon chain length of from about 4 to about 20
carbons have been preferred. The present invention contemplates
being used in conjunction with all of the aforementioned methods in
the prior art.
[0104] The coupling agent linking units together generally is
derived from a bifunctional or multifunctional organic
cross-linking reagent. In this context, the term coupling agent has
indicated the group in its coupled form with a chemical group or
backbone moiety. The term cross-linking reagent has been used to
indicate the chemical form of the agent before it is reacted with a
chemical group or backbone moiety.
[0105] The choice of the coupling agent/cross-linking reagent has
depended upon the choice of the reactive chemical group or backbone
moiety to be coupled and the agent chain length which would avoid
intraunit interference within the polymer. See "Reagents For
Organic Synthesis", L. Fiezer, M. Fiezer, Vol. 1-8, Wiley &
Son; "Cross Linking Reagents" (1980 Ed.), Pierce Biochemical
Reagent Catalog, Pierce Chemical Co., Rockford Ill. and references
therein, or "Advanced Organic Chemistry" J. March, McGraw Hill
(1968).
[0106] The amplification molecules and complexes of the present
invention detect and chemically amplify the presence of minute
quantities of inorganic or organic target molecules which may be
found in biological material. Generally, the detection is based
upon interaction between the polymer, its complex and the target
molecule to be detected. The polymer is carried in a complex
carrying arrangement which can bind with specific target molecules
and exclude others. Quantitative determination of the target is
made by measuring the amount of polymer present in the association
formed between the target molecule and carrying arrangement. Signal
amplification is provided by the multiple units in the polymer in
each association.
[0107] The units of the polymer are an important feature providing
visualization of the target carrying arrangement association. The
units can contain visualization tags or can react with a substrate
which can be utilized as a means for quantitative measurement. This
measurement may be accomplished by production of a readily
identifiable substrate product or production of a spectroscopic
signal, as well as other, similar types of nondestructive
quantitative analytic methods for measurement. Preferably, the
visualization will be based upon the production of color,
fluorescence, luminescence, radioactivity, high electron density as
well as other forms of spectroscopic measurements.
[0108] When the units are enzymes they can generate products which
are capable of producing such spectroscopic measurement. For
example, they may catalyze reaction of substrates to produce
colored, fluorescent, luminescent, electron dense or radioactive
products.
[0109] Alternatively, the tagged units may be directly utilized as
tools for spectroscopic measurement. For example, the natural or
synthetic polypeptides, polyols, polyolefins or carbohydrates may
be tagged with chemical groups which have coloration, fluorescent,
luminescent, electron dense or radioactive properties. These may
then be used for spectroscopic measurement.
[0110] Enzymes and tagged polypeptides, polyols, polyolefins or
carbohydrates possessing the foregoing properties are well-known as
means for spectroscopic quantification. When placed in an
appropriate spectrometer, the enzymatic substrate or tag will cause
a spectrographic change which will indicate the quantity of target
present. This process is commonly referred to as visualization and
the spectral change is termed the signal produced by the
visualization group (the substrate or tag).
[0111] The quantity of target to be detected usually will be minute
and if the signal from the complex-target association were produced
on an equivalent basis, it also would be extremely weak. However,
the carrying arrangement and its visualization polymers chemically
amplify the signal so that minute quantities of target will produce
a strong, readily determined signal. Amplification is achieved by
the polymer because it comprises multiple visualization units. The
signal provided by each unit is maintained by the polymer.
Consequently, its signal is the sum of the signals of its units. In
addition, the carrying arrangement may contain multiple numbers of
polymers. Although it is not necessary, this multiple arrangement
is preferred since it provides further amplification.
[0112] The visualization polymer of the invention comprises
multiple visualization units monomer directly bonded together or
indirectly linked together by a coupling agent bonded to chemical
groups or backbone moieties of the units. Each unit also possesses
a site or sites which provide the visualization signal. That is it
may be a site for enzymatic action or a site to which a
visualization tag or tags are attached. The visualization signal
activity of the polymer depends upon production of a signal by each
unit. Accordingly, the visualization site or sites should be
substantially preserved in its or their original form so that the
site activity is not substantially decreased. It follows that
chemical modification of the units should be conducted in a manner
which does not substantially affect the site or sites.
[0113] To this end, the direct bonding or coupling agent linkage
should join chemical groups or backbone moieties of the units which
are at least one atom and in some embodiments at least 3 to 5 atoms
away from the visualization site or sites. Also, the choice of
chemical groups or backbone moieties for direct bonding or linking
with coupling agent should be limited to those which are not
present within the site or which are not necessary for site
conformation and three dimensional configuration. This choice will
be more important for enzyme proteins than for tagged natural or
synthetic polypeptides polyols, polyolefins or carbohydrates;
however, interference with the production of tag fluorescence,
luminescence, coloration, radioactivity or high electron density
should also be avoided.
[0114] Generally, these site preservation requirements may be met
in several ways. If the types of biochemical substructures or
chemical residues making up the monomer structure are known, then
one which is not part of the visualization site may be chosen as
the structure containing the reactive chemical groups or backbone
moieties for coupling. Usually, however, a semi-empiric method will
be used for choice of the appropriate reactive chemical groups or
backbone moieties.
[0115] According to the substructure/residue method, the chemical
construction of the units will be investigated. The unit backbone
substituted groups and functional structures such as sugar groups,
lipids, oligomer side chains and the like which are not necessary
for visualization site action will be identified. Typically, this
would be determined by removal modification or modification of such
substructures and study of the activity of the resulting product.
Chemical groups or backbone moieties present primarily within these
substructures may then be used for direct bonding or indirect
linking with the coupling agent. For example, the sugar groups of a
glycoprotein which are not necessary for enzymatic activity can be
oxidized to dialdehyde groups and reacted with a hydrazine coupling
agent to form the visualization polymer.
[0116] If the chemical sequence of the unit, such as the amino acid
sequence of a protein, can be determined, this may also be utilized
to guide direct bonding or indirect linking Analysis of the
sequence for the active site as well as the three dimensional
configuration will show which unit structural subunits are not
essential to functioning of the site and/or not present within it.
The reactive chemical groups or backbone moieties of these subunits
may be used for bonding or linking with the coupling agent. For
example, if the unit is a protein and it is found to contain a
dipeptide side chain ending with cysteine, the mercaptan group of
the cysteine may be cross-linked to cysteine of another similar
protein by reaction with bis (N-butylenylmaleimide).
[0117] According to the semi-empiric method, the reactive chemical
groups and backbone moieties of the unit can be determined by
appropriate spectrographic and chemical analysis. These include
techniques such as NMR, IR, chemical derivatization,
electrophoresis, osmometry, amino acid analysis, elemental
analysis, mass spectrometry and the like. The groups and moieties
identified may include amine groups, mercaptan groups, carboxyl
groups, hydroxyl groups, sugar groups, carbohydrate groups, ester
groups, lipid groups, and amide bonds, labile carbon-carbon bonds
and carbon-hydrogen bonds the like [JO needs to clarify this part
based on RJ's disclosure] . Other measurements such as the relation
of derivatization and site activity, relation of pH and site
activity and type of site reaction produced in the case of an
enzyme will help determine a priority for the functional groups
based upon the probability of their presence within the vicinity of
the active site. A typical priority will be 1. an epsilon or
primary amine group, 2. sugar group, 3. carboxyl group, 4.
mercaptan group, 5. hydroxyl group, 6. lipid group. If
derivatization of amine groups such as those of lysine residues
produces a derivatized product devoid of site activity, then the
foregoing priority will change and the amine group will be
last.
[0118] Under usual emperic procedures, several versions of polymer
will be prepared using a selection of several of the reactive
chemical groups or backbone moieties. The activities of the several
versions are then tested and the one selected of which has the
highest activity. Typically, the selection of chemical groups or
backbone moieties will encompass three or four types which are
least likely to affect the activity of the visualization site. Each
type of reactive chemical group or backbone moiety may eventually
be tried if results with the first few are unsatisfactory. Emperic
examination of each version of polymer will allow identification of
the one with the highest activity.
[0119] The units having visualization sites which are very
sensitive to the chemical group/backbone moiety bonding arrangement
are enzymes. The catalytic site typically will have a conformation
closely fitting the substrate and chemical modification which
disturbs the three dimensional configuration of the catalytic site
may adversely affect the activity of the polymer. Following the
foregoing procedures, enzyme site activity can be preserved.
Furthermore, the enzyme catalytic site may be protected during
bonding or linking by reversibly binding it with substrate.
[0120] The units may be any enzyme which will react with an
appropriate substrate to produce a colored, fluorescent,
luminescent, electron dense or radioactive product. Also, the
enzyme may react with a colored, fluorescent or luminescent
substrate and quench it. The production or quenching of color,
fluorescence or luminescence may result from direct enzyme
catalysis or the enzyme may produce an intermediate which enters
into a chain of reactions to produce or quench color fluorescence
or luminescence.
[0121] If an electron dense or radioactive substrate is to be used,
the enzyme will act to immobilize it. This may be accomplished by
rendering the substrate insoluble, chemically reactive toward the
enzyme or otherwise generating an immobilizing physical
characteristic. With this type of visualization polymer, the
quantity of radioactivity immobilized by the enzymatic reaction or
an electron microscopy determination of the quantity of electron
dense material present will allow analysis of the minute quantity
of target. Examples of such enzymes include peroxidase, alkaline or
acidic phosphatase, galactosidase, glucose oxidase, NADPase,
luciferase, carboxypeptidase and the like.
[0122] The units may also be natural or synthetic polypeptides,
polyols, polyolefins or carbohydrates which are tagged. These may
be based upon a polyamide backbone, a polyether backbone, a
polyvinyl backbone, or poly (sugar) backbone. For the polyamide,
the amino acid or diamine compound and dicarboxylic acid compound
used to make the backbone may be nonfunctional, i.e., composed of a
methylene unit chain ending in the appropriate functional groups,
or it may be substituted with groups which would provide side chain
functionality. Examples would include glycine, alanine, serine,
lysine, aspartic acid and the like as amino acids. Examples of
diacids and diamines include arylene or alkylene dicarboxylic acid
having at least 6 carbons in the arylene group or 1 to 20 carbons
in the alkylene group, and arylene or alkylene diamines having at
least 6 carbons in the arylene group and 1 to 20 carbons in the
alkylene group. Examples will include poly(3-aminopropionic acid),
polyglycine poly(glycyl-lysine), poly(N-(aminohexyl)alipic amide),
poly(N-(aminobutyl)terephthalamide) and the like.
[0123] For the polyethers, epoxides and/or oxacyclic compounds with
or without hydroxyl substitution can be used as backbone building
blocks. Acidic condensation will couple the oxide compounds. Also,
the polyols may have a poly(vinyl) backbone with hydroxylic
substitution. These may be formed by vinyl/free radical
polymerization of alkyl alcohol, butene diol and the like.
[0124] For the polyvinyls, vinyl compounds with or without chemical
group substitution may be used as backbone building blocks.
Vinyl/free radical polymerization of such compounds as acrylamide,
acrylic acid, maleic acid, alkyl sulfide, acrylonitrile, methyl
acrylate, hydroxyethyl acrylate, alkenyl amine, acrolein, etc. will
produce the polyolefin monomers.
[0125] For the poly(sugar), glycosidic linking through hemi-ketal
condensation of simple sugar building blocks can be used as the
carbohydrate backbone formation process. Carbohydrates such as
methoxy cellulose, poly(glucose) starch, dextran, polymaltose,
amylose, etc. are examples.
[0126] The chemical tags include the known, colored, fluorescent,
luminescent, radioactive and electron dense probes which will
chemically bond with substituents present in a natural or synthetic
polypeptides polyols, polyolefins and carbohydrates. These include
probes with carboxylic acid derivative substituents, sulfonic acid
substituents, imino ester substituents, maleimide substituents,
aldehyde substituents, azide substituents and amine substituents
which will react with the appropriate functional group of the unit
as outlined in Scheme I and Table 1. The probes will be
monofunctional rather than difunctional so that they may react only
once with a unit chemical group or backbone moiety. Examples of
color tags include azido indigo dye, and congo red with sulfonyl
chloride substitution. Examples of fluorescent tags include
fluorescein with an azido or sulfonyl chloride reactive
substituent, 3-azido-(2,7)-naphthalene disulfonate and rhodamine.
Examples of radioactive tags include wood reagent (methyl
p-hydroxybenzimidate) HCl which can be iodinated, and
p-iodobenzenesulfonyl chloride. Examples of electron dense tags
include collodial gold, colloidal silver, ferritin, metal binding
proteins and reactive lead salts.
[0127] Isolation and purification of the visualization polymer of
the invention may be accomplished by known techniques used for
polymer isolations. These include dialyzation, lyophilization,
chromatography, electrophoresis, centrifugation, precipitation by
electrolyte adjustment or solvent lipophilicity and the like.
[0128] The carrying arrangement of visualization polymer and
detecting agent may be direct or indirect. The direct carrying
arrangement will have the detecting agent covalently bonded to the
visualization polymer by a bifunctional or multifunctional
cross-linking reagent. Generally, the bonding will follow Scheme I
and method given for linking the visualization units of the
polymer. These methods are generally known; for example see K.
Peters, et. al., Ann Rev. Biochem., 46, 523-551 (1977); F. Wold,
"Methods In Enzymology XXV", pp 623-651 (1972) or M. Das, et al.,
Ann Rev. Biophys. Bioeng., 8 165-193 (1979). As with the
visualization polymer, covalent linkage with chemical groups or
backbone moieties of the detecting agent should take place in a
region of the agent which will not interfere with its ability to
detect the target. This may be determined by any of the methods
given above, especially the emperic method.
[0129] The indirect carrying arrangement may be of two types. In
the first, the detecting agent may be multivalent and have an
affinity for the visualization polymer as well as the target. For
example, it may be accomplished by employing a multivalent antibody
which cross-reacts with the units of the visualization polymer and
by utilizing the appropriate amount of antibody and polymer so that
at least one of the affinity sites of the antibody remains open.
The visualization polymer may also be bonded to a ligand which
complexes with a multivalent detecting agent. This will accomplish
the same kind of carrying arrangement.
[0130] In the second type of indirect carrying arrangement, there
will be an intermediate ligand binding compound interspersed
between the detecting agent and the visualization polymer. It will
display a high affinity for specific ligands and will include an
antibody, lectin, avidin, streptavidin, a DNA repressor protein, a
high affinity enzyme, a sequence specific polynucleotide binding
protein or a complementary polynucleotide sequence. The agent and
polymer will be correspondingly labeled with the appropriate
ligand. The ligand may be joined to the detecting agent and polymer
through a linker similar to a bi or multifunctional cross-linking
reagent. Also, the ligand may be substituted for a reactive group
of the bi or multifunctional cross-linking reagent.
[0131] Alternatively, the ligand may be covalently bonded directly
to the detecting agent and polymer. That is, the ligand may be
bonded to a chemical group of the polymer and detecting agent which
may include an amine group, mercaptan group, carboxylic acid group,
hydroxy group, aldehyde group or a C--H group. The procedures and
reagents for the appropriate reaction will be chosen depending upon
the kind of reactive group present on the ligand.
[0132] Methods for the preparation of the carrying arrangements and
complexes of the invention follow the well known procedures given
in the foregoing background. Examples include use of ligands such
as biotin, iminobiotin, polynucleotide sequences, enzyme
substrates, sugars, haptenes such as 2,4-dinitrophenol,
2,4-dinitrophenylalkylcarboxylic acid having from 1 to 20 carbons
in the alkyl group, and carboxylic acid derivatives thereof Other
examples of haptenes include 2,4-dinitrophenylalkylamine having
from 1 to 20 carbons in the alkyl, phenylarsenate, inistol and
trinetrobenzene.
[0133] An example of this type of carrying arrangement and complex
is based upon use of a complementary strand of polynucleotide as a
detecting agent for a specific native polynucleotide sequence.
Avidin or streptavidin is used as the ligand binding compound and a
functionalized biotin or imino biotin derivative is used as the
ligand. Bonding the biotin or imino biotin to the visualization
polymer and polynucleotide detecting agent may be accomplished
directly or through use of a linker group. These methods are known
in the art; see Langer et al., Proc. Nat'l. Acad. Sci. U.S.A., 78,
6633-7 (1981); and follow the methods given for Scheme I except
that one end of the bifunctional cross-linking reagent will have
been reacted with biotin or iminobiotin. Accordingly, the complex
includes avidin or streptavidin-(biotin or iminobiotin
ligand)-visualization polymer. The carrying arrangement in addition
includes the biotin or imino biotin labeled polynucleotide
detecting agent.
[0134] The method of the invention utilizing this example can be
practiced as follows. An isolated double strand of native
polynucleotide to be detected, such as viral DNA, is broken or
nicked with a DNAase at random points along each strand. Labeled
nucleotide monomers are then translated into the nicks using a
polymerase enzyme and the other associated strand as a template.
Alternatively, the complementary strands can be directly labeled
with biotin label. The labeled complementary pair of polynucleotide
strands are then denatured and mixed with a denatured mixture of
unknown native polynucleotides, suspected as containing the
polynucleotide to be detected. If it is present, hybridization will
occur and the labeled double strand may be visualized with the
polymer complex.
[0135] A second example of a complex is derived from the methods
given in the Background for PAP or ABC complex methods or according
to Langer et al., supra. In this example, avidin or streptavidin is
used as the intermediate ligand, an antibody, lectin, or a sequence
specific polynucleotide binding protein is used as the detecting
agent and a biotin or imino biotin compound is used as the ligand
complexing the visualization polymer and detecting agent with
avidin or streptavidin.
[0136] In either of these two examples, the biotin or imino biotin
compound may be directly coupled with amine or hydroxy groups of
the polymer and agent through the use of amide bond or ester bond
forming coupling reagents respectively. It may also be coupled
through a linker group such as that described above. The linker
group is similar to the bifunctional cross-linking reagent except
that one of the two reactive groups will be an amine or
acylhydrazide group which is coupled with biotin or
iminobiotin.
[0137] The visualization polymer of the present invention may be
used to detect minute quantities of target molecules. These
molecules may be found in biological material such as tissue and
fluid as well as in artificial or synthetic systems. Examples
include blood, lymph, urine, feces, organ tissue such as lung,
liver, skin, kidney and the like, microorganisms, plant tissue,
cultured cells, hybrid cells, cells with recombinant DNA, synthetic
mixtures of polypeptides, immobilized enzyme systems, synthesized
DNA and other biological material.
[0138] The target molecules may constitute any inorganic or organic
species which is capable of producing an affinity with a detecting
agent. Preferred targets will be found in the foregoing biological
material and systems. Examples include proteins, lipids,
carbohydrates, phospholipids, fats, nucleotides, nucleosides,
nucleoside bases, polynucleotides, polypeptides, cancerogenic
agents, drugs, antibiotics, pharmaceutical agents, controlled
substances, polymers, silicones, organometallic compounds, heavy
metals, metal-protein complexes, toxic inorganic salts, and other
agents or compounds produced by or having an effect upon a
biological organism or material derived therefrom.
[0139] Generally, the procedures for combination and, incubation of
the detecting agents and targets are well known. They follow
methods used for affinity and immumodiagnostics assays; see for
example L. A. Sternbeyer, "Immunohistochemistry" cited above. For
example, combination of metered amounts of agent and target in
buffered aqueous solution followed by incubation at temperatures
from ambient to about 37.degree. C. for periods such as 5 minutes
to 18 hr. will cause conjugation. Addition of the visualization
polymer or its complex under similar conditions will then provide
visualization. Finally, if the agent is bonded to the visualization
polymer, similar techniques can be followed.
[0140] Use of the visualization polymer for the foregoing detection
purposes has advantage since it allows detection of extremely
minute quantities of target molecules. It may be employed in
medical diagnostic laboratory as an analytical technique for
identification of biological products in fluids and tissues which
are indicative of a disease state. These would include for example,
abnormal amounts of growth hormone, the presence of human
gonadotropin indicating cancer, detection of viral invasion,
quantification of hormone and regulatory enzyme levels. Also, it
may be employed to perform normal fluid and tissue chemistry
analyses and may be employed in the biochemical research laboratory
as a tool for identification of biochemical substances.
[0141] The visualization polymer may be used in synthetic protein
or polynucleotide work to identify synthesized, semisynthetic or
native proteins and synthesized, recombinant or native
polynucleotides. Applications will be found in the course of
preparative or bulk work to produce useful proteins such as
insulin, interferon, ACTH, gonadotropin, oxytocin, pituitary
hormone, LH, FSH and the like by such techniques as recombinant DNA
or hybridomas.
[0142] The carrying arrangement of detecting agent and
visualization polymer complex will be the form for use to perform
the foregoing analyses. Since the polymer will provide multiple
signals from the carrying arrangement association with the target,
chemical amplification will result. In the preferred form of the
carrying arrangement wherein a complex of polymer and ligand
binding compound is employed, the signal amplification by the
polymer will be further increased by multivalent liganding of
multiple numbers of polymer to each molecule of detecting agent.
Accordingly, in the preferred embodiments employing an antibody or
complementary polynucleotide sequence detecting agent, biotin or
immobiotin labels, on the agent and polymer, and an avidin or
streptavidin, detection of femtomole (10-15) quantitites can be
achieved. This will also depend in part upon employing a sensitive
visualization unit system and the appropriate carbon chain linker
lengths for both the biotin labels and the coupling agent of the
polymer. An example would be use of the enzymes alkaline
phosphatase or horseradish peroxidase coupled as visualization
polymer by epsilon amino group bonding with an active diacyl
derivative of suberic acid, and use of biotin labels with carbon
chain linkers of from 6 to 14 carbon in length.
[0143] The polymer, complex and carrying arrangement of the
invention may be formulated as an integral part of a solid or
liquid detection system and kit. Colorimetric, fluorescent,
luminescent and radioactive systems may be prepared in this manner.
Such systems and kits would include the detecting components, i.e.,
the polymer, its complex with a ligand, a ligand binding compound,
and the detecting agent as well as the appropriate chemicals,
reagents and solutions in metered amounts and standardized
concentrations also. For example, if enzymatic action with a
substrate to produce a colored product is to be the visualization
procedure employing the polymer, the system and kit will contain
the chemicals, substrate and reagents necessary for performing this
analysis. These materials will be present as metered quantities so
that the light absorption produced by the colored product may be
used in conjunction with a standard Beer's Law mathematical formula
to determine the concentration of target detected. Usually, a
standard reaction of polymer with substrate will be employed as a
control and reference, although standard graphs of absorption
relative to concentration may also be utilized.
[0144] Fluorimetric, lumimetric and radiometric analyses may be
performed in a similar fashion. The intensity of fluorescence,
luminescence or radioactivity produced by the polymer in the
carrying arrangement associated with the target will be measured by
the appropriate electronic machine. Necessary reagents and
chemicals will also be present. Metered amounts of components will
be employed so that the intensity value may be correlated with the
quantity of target using a standard Beer's Law mathematical
formula.
[0145] In these systems, a concentration of detecting
agent-visualization polymer complex will be used in the test
solution which is sufficient to associate with all the target to be
detected. Preferably, the concentration will provide an excess
amount. The target may be grossly separated from other material by
sedimentation, by centrifugation, or otherwise separated by such
techniques as high pressure liquid chromatography, gel permeation
chromatography, electrophoresis, precipitation, thin layer
chromatography, paper chromatography or similar techniques.
However, this is not necessary for the purposes of this invention.
The signal producing reaction will be initiated by forming the
target-detecting agent conjugate followed by forming the
visualization polymer-detecting agent associative arrangement and
measuring the visualization signal from this arrangement.
Comparison of the signal intensity with a standard graph will yield
the quantity of target. Other techniques such as conjugate-complex
exchange, which are known in the field of immunoanalysis, may also
be used.
[0146] With all of the foregoing liquid and solid analysis methods,
qualitative detection may also be made. Since this object will be
determination of the presence of the target to be detected rather
than quantity, standardization need not be used. The qualitative
techniques will generally follow the methods for the foregoing
quantitative techniques.
Aptamers
[0147] Aptamers have some advantages over antibodies, which may not
be able to detect low concentrations of analyte if the binding
affinity between an antibody and an analyte are too low. Aptamers
have been developed to bind specifically to target molecules for
purposes of identifying the molecules for disease analysis. PCT
application number WO 99-07724, by Nextar Pharmaceuticals, Inc.,
authored by Heilig and Gold, "Nucleic Acid Ligands for Blood-Brain
and Cerebrospinal Fluid-Blood Barriers by Tissue SELEX," published
Feb. 18, 1999, discloses use of the SELEX system of obtaining a
nucleic acid that has a sequence capable of binding a target
protein with high affinity and specificity, in this case for
components of cerebrospinal fluid and the blood-brain barrier.
Aptamers have been developed for a variety of different types of
target materials. See also, for example, PCT application number WO
95/07364, by Nexagen, Inc., authored by Gold et al., "Nucleic Acid
Ligands and Improved Methods for Producing the Same," published
Mar. 16, 1995; and PCT application number WO 91/19813, by
University of Colorado Foundation, authored by Gold and Tuerk,
"Nucleic Acid Ligands," published Dec. 26, 1991. The foregoing
publications and the references cited therein are hereby
incorporated herein by reference. Aptamers and similar structures
of the prior art may also be used in conjunction with the present
invention.
Conjugation of Compounds
[0148] The capture molecule may be directly conjugated with the
amplification complex via direct covalent or non-covalent bonding
with the polymer, or indirect bonding through an intermediate
covalent or non-covalent binding group. The capture label may also
be conjugated to the polymer or other macromolecule through an
intermediate ligand binding complex. In a direct binding
arrangement, the capture label acts as a ligand binding compound
also and the corresponding ligand is bound to the amplification
polymer. In an indirect binding arrangement, a first ligand is
bound to the agent, a second ligand is bound to the polymer and
they are sandwiched with a ligand binding compound such that the
first and second ligands function as bridges that form a complex
with the compound.
[0149] Methods for conjugating the amplification complex are well
known in the prior art. Examples include use of ligands such as
biotin, iminobiotin, polynucleotide sequences, enzyme substrates,
sugars, haptenes such as 2,4-dinitrophenol,
2,4-dinitrophenylalkylcarboxylic acid having from 1 to 20 carbons
in the alkyl group, and carboxylic acid derivatives thereof. Other
examples of haptenes include 2,4-dinitrophenylalkylamine having
from 1 to 20 carbons in the alkyl, phenylarsenate, inistol and
trinitrobenzene. All of the foregoing examples can be used with the
present invention.
[0150] Complementary strands of a polynucleotide have been used as
a detecting agent for a specific native polynucleotide sequence.
Avidin or streptavidin is used as the ligand binding compound and a
functionalized biotin or imino biotin derivative is used as the
ligand. Bonding the biotin or imino biotin to the amplification
complex may be accomplished directly or through use of a linker
group. These methods are known in the art. See Langer et al., Proc.
Nat'l. Acad. Sci. U.S.A., 78, 6633-7 (1981). Accordingly, the
amplification complex may be composed of an avidin or
streptavidin-(biotin or iminobiotin ligand)-polymer.
[0151] A biotin or imino biotin compound may be directly coupled
with amine or hydroxy groups of the polymer and agent through the
use of amide bond or ester bond forming coupling reagents,
respectively. It may also be coupled through a linker group such as
that described above. The linker group is similar to the
bifunctional cross-linking reagent. The present invention could be
used with biotin or imino biotin compounds.
Biotinylated Molecules
[0152] Examples of the detectable label include, but are not
limited to, biotin or any derivatized form or analog thereof, or
any molecule having an affinity for avidin including monomeric
avidin, streptavidin, or any protein having biotin-binding
properties including recombinant forms of any of the above. It
should be noted that streptavidin has four binding sites for
biotin; thus many examples in the prior art include a
biotin-streptavidin-biotin complex. Patents and literature are
replete with the various biotin compounds including various
spacers, linking groups and the like, for use in the present
applications. Nonlimiting examples can be found in M. D. Savage, et
al. (1992), Pierce Chemical Co., Avidin-Biotin Chemistry: A
Handbook; DE 3629194, U.S. Pat. Nos. 5,180,828, 4,709,037 and
5,252,743, 4,798,795, 4,794,082, WO 85/05638 incorporated herein by
reference. For a basic reference on using biotin and horseradish
peroxidase signals, see Adams, J. Histochem. Cytochem. 1992
October; 40:1457-63. The prior art discloses a modification of the
Adam's protocol wherein biotin amplification was applied to early
gene screening and also to enhance the metal portion of
diaminobenzidines used in an immunoperoxidase method. Berghorn, et
al., J. Histochem. Cytochem., 1994 December; 42: 1635-42. The same
or similar method could be used with the present invention.
Amplification Polymers
[0153] The presence of a target analyte of interest may be
visualized by binding to the target analyte an amplification
molecule that amplifies the number of binding sites per target
analyte. As used herein, the term "amplification polymer" is used
to refer to the binding sites of an amplification molecule. An
amplification molecule may comprise, for example, a polymer having
multiple binding sites covalently linked together by polymerization
or non-covalently coupled together. The amplification molecule
binds to the analyte or an intermediate molecule via a binding site
on the amplification molecule. Each unit of the polymer is coupled
to at least one signal label, and the units are linked in a manner
which preserves the intrinsic activity of the binding sites or
amplification polymers of the units. An amplification unit can
generate or produce color, fluorescence, luminescence, localization
of radioactivity or localization of electron dense material. The
units may be selected from an enzyme or a tagged natural or
synthetic polypeptide, a tagged polyol, tagged polyolefin, or a
tagged carbohydrate. Thus, each amplification molecule that binds
to an analyte provides multiple additional binding sites (or
"amplification polymers") to which a detectable label can be bound,
thereby providing amplification of the number of signaling events
per target molecule bound to the solid substrate.
[0154] The units may be directly linked by polymerization or
indirectly linked by a coupling agent. Direct polymerization or
agent coupling bonds chemical groups or unit backbone moieties of
adjacent units. The chemical groups or backbone moieties utilized
for each unit of polymer will be independently selected from an
amine group, an oxidized form of a 1,2-diol group, a carboxy group,
a mercaptan group, a hydroxy group or a carbon-hydrogen bond. For
example, oxidative enzymes such as horseradish peroxidase can be
used to polymerize monomer units by oxidative cross-linking.
[0155] Alternatively, a coupling agent may be used, which may be
derived from a bifunctional or multifunctional organic
cross-linking reagent, bonds with the appropriate chemical group or
backbone moiety of the units. In this context the term "coupling
agent" denotes the linkage group after bonding and the term
cross-linking reagent denotes the linkage compound before
bonding.
[0156] Bound amplification polymers refers to those functional
groups on the polymer that bind to the signal labels. Unbound
amplification polymers on the amplification polymer, such as a
dextran polymer, refers to those functional binding groups that
could bind to the signal labels, such as biotin, but remain unbound
because under experimental conditions, chemical reactions almost
never go completely to completion.
[0157] In some embodiments, at least some of the plurality of
amplification compounds are not bound to a detectable label
complex, and the plurality of amplification polymers not bound to a
detectable label are reacted with the capping compound.
[0158] In some embodiments, the amplification polymer is selected
from the group consisting of multi-valent proteins, dimerized
proteins, dimerized antibodies, multimerized proteins, multimerized
antibodies, and allosteric aptamers.
[0159] The amplification polymers may be comprised of functional
binding groups selected from the group consisting of amines,
carboxylates, sulfhydryls, arginines, maleimides, or aldehydes. For
example, the amplification polymer may be selected from the group
consisting of the following polymers: dextran, acrylic acid, poly
(acrylamide-co-acrylic acid), poly-L-lysine, poly-L-aspartic acid,
poly-benzyl-L-glutamate, poly-benzyl-L-aspartate, poly (Arg,Trp),
poly (Lys,Phe), polymaleimide and poly-L-glutamic acid. In
particular embodiments, the amplification polymer is a dextran
polymer, an acrylic acid polymer, or a poly-L-lysine polymer. In a
particular embodiment, the amplification polymer is biotin-labeled
dextran.
[0160] In other embodiments, the molecular weight of the
amplification polymer ranges from between about 6,000 to about
1,000,000, or alternatively from between about 70,000 to about
500,000.
[0161] The amplification polymer is preferably conformationally
flexible. The amplification polymer is also preferably soluble in
water from 1 fg/ml to 10 mg/mL. Preferably, the amplification
polymer is also soluble in 1M monovalent salt from 1 fg/ml to 10
mg/mL.
[0162] In order to indicate the presence or absence of a target
molecule, the amplification polymer is conjugated to a target
analyte, which may, for example, be bound to a solid substrate.
Capping Amine Groups
[0163] The methods of the present invention may also employ
techniques used for amplifying a signal in a diagnostic assay for a
nucleic acid, comprising the steps of:
[0164] (a) providing an amplification polymer bound to a nucleic
acid analyte, wherein the amplification polymer comprises a
plurality of reactive amine groups;
[0165] (b) binding amine groups on the amplification polymer with a
detectable label complex; and
[0166] (c) reacting under an acetylating compound with amine groups
not bound with a detectable label complex.
[0167] (d) performing test in a salt solution having an ionic
strength greater than about 0.5M.
The salt may be monovalent. In some embodiments, the salt may be
selected from the group consisting of NaCl and LiCl.
[0168] In other embodiments, the capping compounds use in the
methods of the invention will not displace the detectable label
complex. In some embodiments, the capping compound may be a
stronger base than the functional binding groups to which it binds.
For example, the capping compound may be an amine-reactive
compound, such as a compound that converts the functional binding
groups into amides or imides. By way of example, the capping
compound may be an acetylating reagent. Amine-reactive compounds
may include compounds from one or more of the following chemical
classes: N-hydroxysuccinimidyl (NHS) esters, imidoesters, aryl
halides, acyl halides, isocyanates, isothiocyanates, nitrophenyl
esters, carbonyls, carboxylates, and acid anhydrides. Particular
amine-reactive compounds may include, for example, any one or more
compounds selected from the group consisting of NHS acetate,
disuccinimidyl suberate (DSS),
succinimidyl-3-(tri-N-butylstannyl)benzoate, methyl
N-succinimidyladipate (MSA), mono(latosylamido)
mono(succinimidyl)suberate, acetic anhydride, aryl chlorides, acyl
chlorides, 2,4-dinitrofluorobenzene (DFNB), sulfonyl halides,
aldehydes, 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide (EDC)
based activation chemistries, maleic anhydride, succinic anhydride,
acetyl chlorides, benzoyl chlorides, propionyl chlorides, butyryl
chlorides, and penylethanoyl chlorides. The capping compound may
also be selected from non-acetylating agents, such as
diazoacetates, imidoesters, carbodimides, maleimides,
.quadrature.-haloacetyls, aryl halides, dicarbonyl compounds,
sulfhydryls, and hydrazides. By way of example, specific
non-acetylating compounds may be selected from the group consisting
of, for example, N-ethylmaleimide,
N-.quadrature.-maleimidopropionic acid,
N-.quadrature.-maleimidocaprioic acid, iodoacetic acid,
N-[iodoethyl](trifluoroacetamide), 3,4-difluoronitrobenzene (DFNB),
sulfonyl halide, (ammonium 4-chloro-7-sulfobenzo-furazan)-chloride
(SBF-chloride), glyoxal, phenyglyoxal, 2,3-butanedione,
1,2-cyclohexanedione, 2-mercaptoethanol, dithiothreitol (DTT)
followed by sulfhydryl chemistries, (2,4,6-trinitrobenzene sulfonic
acid (TNBSA), and 2-mercaptoethanol. The capping compound may also
contain a detectable label.
[0169] The finding that capping the unbound functional groups on
polymers could lead to an increase in signal was unexpected.
Unreacted amine groups are known to bind non-specifically to
surfaces or molecules in the assay, and this non-specific
interaction interferes with the amplification of the signal.
Initially, it was believed that this problem was due to charge
interactions with the amine NH2+ ions. Ordinarily, such charge
interactions can be reduced by increasing the salt concentration so
that there are more negatively charged Cl-- ions in solution.
However, it was observed that the higher salt concentration did not
decrease non-specific binding. Reactive amine groups were then
capped, which resulted in amplification of the signal by
100-fold.
[0170] Reactive amine groups may be capped using various chemical
processes. For example, an iminoester salt reagent may be reacted
with an amine chemical group to produces an amidine coupling agent
linkage. The reagent may be generated from the acidic alcoholysis
of the corresponding nitrile. The amidine formation reaction may be
conducted in aqueous or polar organic solvent under mild
conditions. The methods and procedures are known. See, e.g.,
Lockhart, et. al., Can. J. Biochem., 53, 861-867 (1975) and Pierce
Biochemical Reagent Catalog and references therein, supra.
[0171] Amine groups may also be reacted with an aldehyde reagent to
form a bis Schiff base (imine) in a condensation reaction. Examples
include glutaraldehyde and other tissue fixing reagents. Conditions
include use of polar organic solvent and mild temperatures.
[0172] In another embodiment found in the prior art, an aldehyde
chemical group is reacted with amine groups and amine derivative
reagents to form imine and imine derivative compounds. These
reagents and reactions included primary amine reagents and reagents
which react to form a Schiff base (imine). In the prior art other
embodiments included substituted hydrazine reagents, which react to
form substituted hydrazones, and acyl hydrazide reagents, which
also react to form acyl hydrazones. The present invention
contemplates similar applications.
[0173] Other capping chemistry options include maleic anhydride,
acetic anhydride, succinic anhydride, N-maleimide derivatives, aryl
halides, alkyl halides, aldehyde, ketone derivatives, and
chemistries that create carboxylates. Other substituent groups may
be useful for capping functional groups.
[0174] The following list contains other chemical classes and
examples of chemicals from those classes that in theory could be
used to cap unbound functional groups: carboxylate reactive
chemistries, such as diazoacetate, imidoesters, carbodimides;
sulfhydryl reactive chemistries, such as maleimides
(N-ethylmaleimide, N-beta-maleimidopropionic acid,
N-epsilon-maleimidocaprioic acid), alpha-haloacetyls (iodoacetic
acid, N-[iodoethyl]trifluoroacetamide), aryl halides (DFNB,
sulfonyl halide, SBF-Chloride); arginine reactive chemistries, such
as dicarbonyl compounds (glyoxal, phenyglyoxal, 2,3-butanedione,
1,2-cyclohexanedione); maleimide reactive chemistries, such as
sulfhydryl, e.g. 2-mercaptoethanol, DTT followed by sulfhydryl
chemistries; fluorescent protecting groups; fluorescent protecting
groups, such as sulfhydryls (SBF-chloride), amine (TNBSA); aldehyde
reactive chemistries, and hydrazides.
[0175] Amine-reactive compounds and compounds that are not
amine-reactive may also be used, for example, acetylating reagents,
such as NHS-acetate and acetic anhydride. Aldehydes, sulfhydryls,
and carboxylates may also be used to cap polymers with free
reactive groups other than amine groups. Some chemistries can react
with multiple groups. For example free-SH and free-NH2 can show
similar reactivities depending on the pH of the solution.
Amine-Reactive Compounds
[0176] If there are free, reactive amine groups on the polymer or
other macromolecule, they could first be protected with a removable
protecting group such as a Schiff base, i.e., condensation of the
amine groups with an aromatic aldehyde such as
p-methoxybenzaldehyde or benzaldehyde which could be removed with
dilute hydrogen chloride in acetone. Other known amine protecting
groups may also be used. These include dinitrofluorobenzene,
t-butoxy groups and organosilanes.
[0177] After protection, esterification can be conducted using an
activated acid reagent. Unit residues that have esterified in this
fashion have included amino acid residues of serine, threonine,
hydroxylysine, tyrosine, thyroxine, hydroxyproline, carbohydrate,
starch, lipid and olefinic residues with hydroxyl substitutions,
including hexoses, pentoses, dextrans, amyloses, glycerols, fatty
acid derivatives, methylhydroxymethacrylate, hydroxymethyl acrylate
and similar compounds. The present invention could be used with all
of the foregoing examples.
Detectable Label
[0178] The methods and compositions disclosed herein contemplate
the use of a detectable label that is conjugated directly or
indirectly to the analyte of interest. Detectable labels may be
selected from the group consisting of biotin, fluorochromes,
di-nitro-phenol, and digoxigenin. The detectable label may be
structurally integrated with a complex that is conjugated to the
analyte of interest, or may be a product of the complex. The
detectable label complex may comprise, for example, biotin
molecules to which are conjugated streptavidin and other molecules
capable of being used to generate a detectable signal.
[0179] Preferred detection methods and preferred amplification
molecule include polymers having multiple units of an enzyme or
multiple units of a natural or synthetic polypeptide or polyolefin
chemically bonded to a tag selected from a fluorescent group, a
dye, a luminescent group or an electron dense group. Preferred
enzymes include alkaline phosphatase, peroxidase, galactosidase,
glucose oxidase, acid phosphatase and luciferase. Preferred
polypeptides include polyamides of dicarboxylic acids and diamine,
polyamides, oligomers and copolymers of alpha amino acids such as
glycine, lysine, aspartic acid, cysteine, ornithine and the like.
Polyolefins include polyacylamide, polyacrylic acid, polymaleic
acid, poly(hydroxyethylacrylic ester) and the like. These
polypeptides and polyolefins will be tagged with such groups as
fluorescein, rhodamine, a diazo dye, colloidal gold, luciferin,
radioactive iodine and the like.
[0180] The detectable labels may be directly utilized as tools for
spectroscopic measurement. For example, the natural or synthetic
polypeptides, polyols, polyolefins or carbohydrates may be tagged
with chemical groups which have coloration, fluorescent,
luminescent, electron dense or radioactive properties. These may
then be used for spectroscopic measurement.
[0181] The detectable labels of the units can be sites of
biological activity. For example, sites for enzymatic action will
provide visualization when reacted with an appropriate substrate.
In this manner, the visualization sites can be utilized to generate
soluble or insoluble bodies of color, fluorescence, luminescence,
radioactivity or high electron density which can be measured and
correlated with the quantity of target molecules detected.
[0182] The sites may also be created chemically. Combining a
natural or synthetic polypeptide, polyol, polyolefin or
carbohydrate with a visualization tag selected from a fluorescent
chemical group, a dye, a radioactive group, a photon emitter (a
luminescent group) or an electron dense moiety will produce monomer
units which can be visualized.
[0183] The detectable label will be present at a ratio greater than
one unit of detectable label per target analyte. In some
embodiments, the detectable label may comprise an enzyme, which may
be conjugated to a polymer, such that the number of enzyme
molecules conjugated to each polymer molecule is, for instance, 1
to 200, 2 to 50, 2 to 25, or some other ratio. In some embodiments
the secondary amplification polymer may be a gold particle, a
radioactive isotope, or a color label, e.g. a low molecular weight
fluorophore, and the number of detectable labels conjugated to each
polymer molecule is, for instance and not by way of limitation, 1
to 500 or 2 to 200. In some embodiments the detectable label may
comprise a protein fluorophore. The detectable label and may be
detected by numerous methods including reflectance, transmittance,
light scatter, optical rotation, and fluorescence or combinations
hereof in the case of optical labels or by film, scintillation
counting, or phosphorimaging in the case of radioactive labels. See
e.g., Larsson, 1998, Immunocytochemistry: Theory and Practice, (CRC
Press, Boca Raton, Fla.); Methods in Molecular Biology, vol. 80,
1998, John D. Pound (ed.) (Humana Press, Totowa, N. J.). In some
embodiments more than one type of detectable label or more than one
detectable label may be employed. The present invention
contemplates using all of the aforementioned embodiments.
[0184] Isolation and purification of the detectable labels that are
conjugated to the analyte may be accomplished by any one of various
techniques used for polymer isolation known to those skilled in the
art, including dialyzation, lyophilization, chromatography,
electrophoresis, centrifugation, precipitation by electrolyte
adjustment or solvent lipophilicity and the like.
Detection
[0185] A variety of procedures are available to visualize specific
antigen-antibody interactions fluorimetrically or colorimetrically.
Since the utility of immunodiagnostic procedures often depends upon
the sensitivity and the specificity with which the target antigen
or molecule can be detected, new methods for increasing these
detection parameters are highly desirable. A detailed discussion of
the advantages and disadvantages of immunologic methods can be
found in any standard textbook on immunocytochemistry. See, for
example, L. A. Sternberger, "Immunohistochemistry," 2nd Ed., John
Wiley and Sons, New York, 1979.
[0186] Detecting the presence of a detectable label often requires
that the detection label be conjugated to some type of label that
produces a signal. Producing the detectable signal may be performed
using any of the methods in the prior art. For example, chemical
tags include the known, colored, fluorescent, luminescent,
radioactive, and electron dense probes which will chemically bond
with substituents present in a natural or synthetic polypeptide or
carbohydrate. These include probes with carboxylic acid derivative
substituents, sulfonic acid substituents, imino ester substituents,
maleimide substituents, aldehyde substituents, azide substituents
and amine substituents which will react with the appropriate
functional group. Probes may be monofunctional rather than
bifunctional so that they may react only once with a unit chemical
group or backbone moiety. Examples of color tags include azido
indigo dye, and congo red with sulfonyl chloride substitution.
Examples of fluorescent tags include fluorescein with an azido or
sulfonyl chloride reactive substituent, 3-azido-(2,7)-naphthalene
disulfonate and rhodamine. Examples of radioactive tags include
wood reagent (methyl p-hydroxybenzimidate) HCl which can be
iodinated, and p-iodobenzenesulfonyl chloride. Examples of electron
dense tags include colloidal gold, colloidal silver, ferritin,
metal binding proteins and reactive lead salts. The present
invention is contemplates using the foregoing methods
[0187] Immunologic detection methods can utilize direct or indirect
visualization techniques for measurement of the formed immune
complex. In general, these methods visually indicate the presence
of the complex through use of an entity coupled to the complex
which produces a detectable, quantifiable signal such as color,
fluorescence, radioactivity, enzymatic action and the like. The
greater the signal intensity present per complex, the better will
be the sensitivity for the presence of a minute quantity of target
molecule. Enzymes and tagged polypeptides, polyols, polyolefins or
carbohydrates are well-known as means for spectroscopic
quantification. When placed in an appropriate spectrometer, the
enzymatic substrate or tag will cause a spectrographic change which
will indicate the quantity of target present.
[0188] Of the various methods available in the art, the simplest
and least sensitive is direct immunofluorescence. In this method, a
primary antibody (or specific ligand-binding protein) is chemically
linked to a fluorochrome, such as rhodamine or fluorescein which
functions as the signal entity. Indirect immunofluorescence
methods, in which a primary antibody is used unmodified and it, in
turn, is detected with a fluorescently-labeled secondary antibody,
generally will increase the detection sensitivity. An additional
three to five-fold enhancement in sensitivity has been reported
using a "haptene-antibody sandwich" technique. See Cammisuli, et
al., J. Immunol., 117,1695 (1976); Wallace, et al., J. Immunol
Methods, 25, 283 (1979). According to this technique, ten to
fifteen molecules of a small haptene determinant such as
2,4-dinitrophenol are chemically coupled to each primary antibody
molecule. Then, by use of a fluorescently-labeled second antibody
which complexes with the haptene molecules, rather than with the
primary antibody itself, more of the secondary visualization
protein can be bound per antigen site, thus further increasing the
sensitivity.
[0189] Secondary antibodies have been coupled to monomeric
horseradish peroxidase and used the catalytic activity of
peroxidase enzyme to reveal either the site, or the amount, of
antigen in the test sample. See Nakane, et. al., J. Histochem.
Cytochem., 22, 1084 (1974); Wilson, et. al. "Immunofluorescence and
Related Staining Techniques", W. Knapp, H. Holuban and G. Wick,
Eds. Elsevier/North-Holland Biomedical Press, 215. Similar
enzymatic assays have been developed with intestinal or bacterial
alkaline phosphatase conjugated secondary antibodies. See Avrameas,
Immunochemistry, 6, 43, (1969); Mason, et. al., J. Clin. Path., 31,
454 (1978).
[0190] The enzymatic signal of this method can occur in at least
two ways. Enzymatic conversion of a soluble enzyme substrate into
an insoluble, colored product permitted the direct localization of
the antigen by direct macroscopic visualization, light microscopic
examination, or by using other types of apparatus.
[0191] Alternatively, colorless substrates were enzymatically
converted into soluble colored products which were used to
quantitate antigen concentrations by direct colorimetric analysis.
The latter method is the basis of the Enzyme-Linked Immuno-Sorbent
Assay (ELISA), which has been widely used in clinical laboratories
around the world. See Sternberger, Immunohistochemistry, 2d ed.,
John Wiley and Sons, N.Y. (1979); Engvall, et. al., Immunochem., 8,
871 (1972); Engvall, et al., J. Immunol., 109, 129 (1972); Guesdon,
et. al., J. Histochem. and Cytochem., 27, 1131 (1979); Voller et.
al., "The Enzyme Linked Immuno Sorbent Assay (ELISA)", Dynatech
Laboratories Inc., Alexandria (1979). These enzyme-based detection
methods are generally more sensitive than direct or indirect
immunofluorescence methods since the high turnover of substrate by
the enzyme continuously accumulates a measurable product over long
periods of time.
[0192] To further increase the sensitivity of immunoenzyme assays,
a three stage peroxidase-antiperoxidase (PAP) assay method has been
used. See Sternberger, et. al. J. Histochem, Cytochem. 18, 315
(1970). Following the addition of a primary antibody and a
secondary antibody, which acts as a bridge between the primary
antibody and antiperoxidase antibody, a peroxidase-antiperoxidase
antibody complex (PAP complex) is added to the sample prior to the
development of the enzymatic reaction. Since the PAP complex
contains two immunoglobulins (antiperoxidase antibodies) and three
active peroxidase molecules, the net effect is to provide more
enzyme at the antigen site with which to amplify the detection
signal. Although quite useful, the PAP detection system has
limitations. The secondary "bridge" antibody has to be used at
saturating levels to ensure optimal binding of the PAP complex.
Furthermore, the antiperoxidase and the primary antibody should be
of the same, or an immunologically cross-reacting, species so that
the secondary antibody will bridge to both. Although the present
invention contemplates the use of the foregoing, the present
invention also contemplates the use of biotin and streptavidin/avid
analogs.
[0193] Specific interaction between biotin, a small water soluble
vitamin, and avidin, a 68 kDa glycoprotein from egg white, can be
exploited to develop antigen or ligand detection systems. See Bayer
and Wilchek in Voller, et. al., "The Enzyme Linked Immuno Sorbent
Assay (ELISA)", Dynatech Laboratories Inc., Alexandria (1979).
Biotin may be covalently conjugated to amino, carboxyl, thiol and
hydroxyl groups present in proteins, glycoproteins,
polysaccharides, steroids and glycolipids using well established
chemical reactions. See Guesdon, et. al., J. Histochem. and
Cytochem., 27, 1131 (1979); Sternberger, et. al., J. Histochem.
Cytochem., 18, 315 (1970); Bayer, et. al., Methods Biochem. Anal.,
26, 1, (1980); Bayer, et. al., J. Histochem. Cytochem., 24, 933
(1976); Heitzmann, et. al., Proc. Natl. Acad. Sci. USA, 71, 3537
(1974). Biotin may also be introduced into other macromolecules,
such as DNA, RNA and co-enzymes, by enzymatic methods that utilize
biotin-labeled nucleotide precursors. See Langer, et al., Proc.
Natl. Acad. Sci. USA, 78, 6633 (1981). Similarly, avidin may be
coupled to a host of molecular species by standard chemical
reactions. See Sternberger, Immunohistochemistry, 2nd Edition, John
Wiley and Sons, N.Y. (1979); Nakane, et. al., J. Histochem.
Cytochem., 22, 1084 (1974); Guesdon, et. al., Histochem. and
Cytochem., 27, 1131 (1979); Bayer et. al., Methods Biochem. Anal.,
26, 1, (1980). This allows for great flexibility in designing
detection systems for use in immunology, immunopathology and
molecular biology.
[0194] Avidin-biotinylated horseradish peroxidase complex (ABC) has
also been used for antigen detection. Hsu, et. al., Amer. J. Clin.
Path., 75, 734 (1981); Hsu, et al., J. Histochem. Cytochem., 29,
577 (1981). In a three-step procedure, the primary antibody
incubation is followed by an incubation period with a
biotin-labeled secondary antibody and then with the ABC complex,
formed by preincubating avidin with a titrated amount of
biotinylated peroxidase. Since avidin has four biotin-binding sites
per molecule, at least three peroxidase enzymes can be added to
avidin without interfering with its ability to interact with the
biotinylated secondary antibody. The ABC detection procedure was
reported to be 4-8 times more sensitive in detecting antigens in
tissues than either the immunoperoxidase or the PAP detection
systems. The ABC method is four-fold more sensitive for antigen
detection using an ELISA system than either the immunoperoxidase or
the PAP techniques. Madri, et. al., Lab. Invest., 48, 98
(1983).
[0195] The sensitivity for the ABC method, however, is limited.
Typically, only 30 to 100 pg of a target molecule can be detected.
This is significantly higher than the upper limit required for
detection of a single molecule per cell. Limits for other less
sensitive methods are even higher. Accordingly others have
developed visualization methods which substantially improve
sensitivity over that provided by known visualization
techniques.
Non-Ionic Polymers
[0196] The methods of the invention further comprise the step of
reacting the amplification polymer and detection complex in the
presence of a high molecular weight non-ionic polymer. The
non-ionic polymers are useful in increasing the detection
sensitivity of the assay by reducing background noise from
non-specific binding between amplification complexes, detectable
labels nucleic acids, etc. Useful non-ionic polymers include, for
example, a dextran sulfate, an amino dextran, a polyvinyl
pyrollidone (PVP), a polyvinyl sulfate (PVS), a polyethylene glycol
(PEG), a carboxymethyl cellulose, a hyaluronic acid or a
polyacrylic acid (PAA), or co-polymers such as poly(acrylic
acid-co-maleic acid). Non-ionic polymers are obtainable in
differing degrees of polymerisation, i.e. with different molecular
weights. For the present invention, high molecular weight non-ionic
polymers are preferred, the upper limit of the molecular weight
depending upon the molecular weight at which the polymer is no
longer sufficiently soluble to be effective according to the
present invention. For use in the process according to the present
invention, polyethylene glycol has a molecular weight of from about
6 kD to about 300 kD, with a molecular weight of about 40 kD being
particularly useful. Polyvinylpyrrolidone is also useful as
non-ionic polymer, having a molecular weight of at least about 40
kD, and up to about 100-750 kD. Dextran may be used which has
molecular weight of about 200 kD, and up to about 500-1,000 kD. The
concentration of the non-ionic polymer may be, for example, from
about 0.5 to about 3% by weight, and can be present as powder,
lyophilisate or solution.
Kits
[0197] In another aspect, the invention provides kits for
amplifying a detectable signal. The kits of the present invention
may include (i) a capture molecule that specifically binds the
nucleic acid analyte; (ii) an amplification polymer adapted to be
conjugated to the nucleic acid analyte, wherein the amplification
polymer comprises a plurality of amine groups; (iii) a conjugation
compound capable of conjugating the amplification polymer to the
nucleic acid analyte; (iv) an acetylating compound capable of
reaction with amine groups on the amplification polymer to create
amide groups; and (v) a detectable label complex.
[0198] In another aspect, the invention provides kits for detecting
an analyte in a sample, comprising in packaged combination, (a) a
multivalent bridge conjugate having an analyte specific binding
site and a plurality of non-analyte-specific binding sites, and (b)
an amplification polymer having a plurality of multivalent binding
sites, wherein the multivalent binding sites are present at a
density wherein two or more separate multivalent binding sites of
one amplification polymer are bound to two or more
non-analyte-specific binding sites of one multivalent bridge
conjugate.
Conjugated Complexes
[0199] The present invention also provides novel conjugated
complexes as intermediates in the methods of the invention. The
conjugated complexes may, for example, be prepared in advance of
testing procedures for inclusion in a kit.
[0200] Such conjugated complexes may comprise (a) a multivalent
bridge conjugate having an analyte specific binding site and a
plurality of non-analyte-specific binding sites conjugated to (b)
an amplification polymer having a plurality of multivalent binding
sites, wherein the multivalent binding sites are present at a
density wherein two or more separate multivalent binding sites of
one amplification polymer are bound to two or more
non-analyte-specific binding sites of one multivalent bridge
conjugate.
[0201] The invention also provides, for use in conjugating in a
complex, novel amplification polymers comprising a plurality of
multivalent binding sites having binding specificity to a
non-analyte-specific binding sites of the multivalent conjugate and
a plurality of detection conjugate binding groups, wherein the
amplification polymer binds to one or more non-analyte-specific
binding site of the multivalent bridge conjugate, if present on the
solid support.
[0202] The invention is illustrated by the following examples.
These examples are not limiting and other similar procedures as
shown by the examples will be readily apparent to those skilled in
the art. All measurements are provided in the metric system unless
otherwise noted.
Examples
Example 1
[0203] The following three chips were used: Biotin Chip dil #1,
Biotin Chip dil #2, and the MRSA Chip. Chips were purchased from
Inverness Medical--Biostar Inc. Surfaces were coated with 5 ug/mL
of poly (Lys-Phe) in 1.times. PBS, 2M NaCl pH 6 overnight. Surfaces
were washed with water and then coated with 10 uM SFB in 0.1M
Borate buffer pH 8.5 for 2 hours at room temperature. Chips were
again washed with water, dried with a stream of nitrogen, and
stored in a dry box purged with nitrogen and protected from
light.
[0204] Biotin Chip dil #1 contained four 120 nL spots of
biotinylated probe. 5'-Hydrazide-A18 probes with 3'-biotinTEG were
diluted with 5'-Hydrazide, un-modified A18 probe to a constant
final concentration of 100 nM in 0.1M Sodium Phosphate pH 7.8, 10%
glycerol. The spots were immobilized to the chip's surface using a
non-contact printer, and arranged in a vertical line with the
lowest concentration at the top. Each spot corresponded to 110 pM,
330 pM, 1 nM, or 3 nM of biotinylated probe.
[0205] Biotin Chip dil #2 contained five 1000 nL spots of
biotinylated probe that were arranged in an "X" pattern on a chip.
5'-Hydrazide-A18 probes with 3'-biotinTEG were diluted with
5'-Hydrazide, un-modified A18 probe to a constant final
concentration of 100 nM in 0.1M Sodium Phosphate pH 7.8, 10%
glycerol. The control spot of 100 nM un-labeled A18 was located in
the center of the chip. Starting with the highest concentration in
the upper left corner and then proceeding from the left to the
right, the four remaining spots represented the concentrations of
300 pM, 60 pM, 12 pM, and 2.4 pM of biotinylated probe.
[0206] The MRSA Chip consisted of a chip with two columns of four
spots arranged vertically. The left column are fiducial spots of
dried latex particles to orient the viewer. The test spots
contained probes that specifically recognize sequences in:
[0207] mec A gene to identify methicillin-resistance,
[0208] fem B gene for specific recognition of S. aureus
[0209] tuf gene for recognition of the Staphylococcu genus
[0210] A control probe to ensure the chemistry was performed
properly.
Example 2
Testing Procedures for Polymer Detection and Standard Detection
Chips
A) Biotin Chips
[0211] Polymer Enhanced Detection. 125 uL of Streptavidin was
applied to the biotin chip at a concentration of 1 .mu.g/mL in
1.times. Hyb buffer (i.e., 5.times.SSC, 0.1% SDS, and 0.1%
Blockaid.TM.) and incubated at room temperature for five minutes.
The chip was then washed four times with wash A (i.e.,
0.1.times.SSC and 0.1% SDS) and wash B (i.e., 0.1.times.SSC). The
sample was then incubated with 125 uL of 1 .mu.g/mL of the biotin
polymer diluted into 1.times.Hyb buffer for five minutes at room
temperature. The chip was washed four times with wash B. Poly
(horse radish peroxidase)-Streptavidin ("pHSA") was diluted to 1
.mu.g/ml in 1.times. Hyb buffer and 125 uL was added to the chip,
for 10 minutes incubation at room temperature. The chip was washed
6 times with wash B, then each chip was incubated with
tetramethylbenzidine (TMB) for 10 minutes, washed with water,
dried, and analyzed.
[0212] Standard ELISA Detection. An anti-biotin horse radish
peroxidase (anti-biotin/HRP) conjugate was diluted to 1 .mu.g/mL in
1.times. Hyb buffer. 125 uL of the diluted anti-biotin/HRP was
added to the chip, and incubated at room temperature for 10
minutes. The chip was then washed 6 times with wash B. Finally, TMB
was added to each chip. After 10 minutes of incubation, the chip
was washed with water, dried, and analyzed.
B) MRSA Chip
[0213] Target sequences from the femA gene in Staphylococcus aureus
were mixed in water with 20 nM each of two biotinylated detector
probe sequences. Ten .mu.L aliquots of the samples were heated to
95.degree. C. for 3 minutes and then diluted into 90 .mu.L of
1.times. Hyb buffer (i.e., 5.times. SSC, 0.1% SDS, and 0.5%
Blockaid.TM.) that had already been pre-warmed on the surface of
the chip. The samples were incubated at 53.degree. C. for 30
minutes and then washed with 4 washes each of wash A
(0.1.times.SSC, 0.1% SDS) and wash B (0.1.times.SSC).
[0214] Polymer Detection. For polymer enhanced detection,
streptavidin was diluted to 1 .mu.L/mL in 1.times. Hyb buffer and
125 uL was incubated on the chip for 5 minutes. The chips were
washed 4 times with wash B. Next, a biotin dextran polymer was
diluted to 1 .mu.L/mL in 1.times. Hyb buffer and 125 uL was
incubated on the chip at room temperature for 10 minutes. The chip
was washed 6 times with wash B. Mouse monoclonal anti-biotin/HRP
was diluted to 1 .mu.g/mL in 1.times. Hyb and 125 uL was incubated
on the chip for 10 minutes. Finally, each chip was incubated with
125 uL of TMB for 10 minutes, washed with water, dried, and
analyzed.
[0215] Standard Detection. Mouse monoclonal anti-biotin/HRP was
diluted to 1 .mu.g/mL in 1.times. Hyb and 125 uL was incubated on
the chip for 10 minutes. The chip was washed six times with wash B.
Then, 125 uL of a precipitable formulation of the substrate TMB was
added to each chip and incubated at room temperature for 10
minutes.
Example 3
[0216] The following example describes a basic method for forming a
capped enhanced detection system (cEDS) by acetylating biotinylated
dextran polymers.
[0217] Methods. A stock of 2 mg/ml of 500 kDa amino dextran
(Molecular Probes; P/N D7144) and 5 mg/ml of 70 kDa amino dextran
(Molecular Probes; P/N D1862) was prepared in water. NHS-LC-biotin
(Pierce, P/N 21335) was dissolved to a concentration of 10 mM (5.56
mg/ml) in water immediately before use. Varying volumes of the
dextran polymer were diluted into 0.1 M borate buffer, pH 8.5.
Varying volumes of the NHS-LC-biotin stock were combined with said
dextran polymer solutions. The reactions were incubated on a shaker
for 3 hours at room temperature. Immediately before it was used,
NHS-sulfo-acetate (Pierce, P/N 26777) was dissolved in water to
form the concentration of 30 mM. An equal volume of diluted
NHS-sulfo-acetate was added to an equal volume of NHS-LC-biotin for
each reaction. The reactions were incubated with shaking for 3
hours at room temperature. The samples were purified on a PD-10
(Pharmacia) chromatography column.
Example 4
[0218] The following method describes use of hydrazone chemistry to
conjugate hydrazide-biotin to an aldehyde-modified polymer.
[0219] 5 mg of aldehyde dextran polymer (70 kDa, Pierce) was
dissolved in water. 5 mg of biotin hydrazide (Pierce) was dissolved
in 450 uL DMSO. 32 mg sodiumcyanoborohydride was dissolved in 0.5
mL PBS. 200 uL of the aldehyde dextran was mixed with 30-100 uL of
biotin hydrazide (If reduction of bond is sought also add 200 uL
sodium borohydride). PBS was added to bring the volume of the
solution to 800 uL. The solution was then reacted overnight at room
temperature with agitation. The solution was then purified over
PD-10 column.
Example 5
[0220] This example describes a method for determining the extent
of biotinylation for various enhanced detection system molecules.
Levels of biotinylation were determined with a
([2-(4'-hydroxyazobenzene)]benzoic acid) ("HABA") kit from Pierce.
The HABA formed a HABA-avidin complex, and the biotin in the sample
displaced the HABA, causing a change in absorbance when measured at
500 nM. Since the change in absorbance was directly proportional to
the amount of biotin, this assay was used to determine the extent
of biotinylation per molecule.
[0221] Methods. The HABA-avidin mixture was equilibrated to room
temperature. The spectrophotometer was blanked with 800 .mu.l of
PBS, pH 7.2. 100 .mu.l of ddH.sub.2O was, first, added to the
HABA-avidin microtube and, second, pipetted into a cuvette
containing PBS buffer. The absorbance at 500 nM (A500) for the
HABA-avidin and PBS mixture was recorded as the absorption level
for HABA-avididn. 100 .mu.l of biotinylated HRP was added as a
positive control to the HABA-avidin cuvette mix and recorded at
A500 of HRP+. Steps 1-4 were repeated for each of the biotinylated
samples. 100 .mu.l of biotinylated sample was added to the
HABA-avidin sample; the sample's level of absorption was then
recorded. Absorbance is equal to or above 0.3 at steps 5 and 8, if
not dilute sample and the A500 dilution was determined. At steps 5
and 8, if the absorption level was below 0.3 absorbance units, then
the biotinylated sample was diluted, retested, and A500 was
recorded.
[0222] The following chart shows the level of biotinylation that
was obtained for various types of EDS.
TABLE-US-00001 TABLE 1 mol Biotins Available EDS Type biotin/amino
Backbone Type Present Biotin Sites 3x 70 kDa 3 70 kDa Dextran ~18
18 1/3x 500 kDa 0.3 500 kDa Dextran 34 85 1x 500 kDa 1 500 kDa
Dextran 65 85 3x 500 kDa 3 500 kDa Dextran ~85 85 3x Chromalink 3
500 kDa Dextran 77.1 85 4% Acrylate Acrylate 27 650 10% Acrylate
Acrylate 72 650 Molecular Probes NA 500 kDa Dextran 79 85
Example 6
Effect of Various EDS Formulations on Assay Sensitivity
[0223] A dilution series ranging from high concentration to low
concentration (specifically, 1 pM, 100 fM, 33 fM, 11 fM, 3.75 fM,
1.25 fM, control) was tested on a chip containing model target DNA
sequences from the femA gene in methicillin-resistant strains of
Staphylococcus aureus ("MRSA"). A standard detection assay, which
used an anti-biotin antibody conjugated to a horse radish
peroxidase, was compared to a biotin polymer assay in 1.times.
Hyb.
[0224] The standard assay on a thin film biosensor produced a
visible signal at 1 pM but did not produce a visible signal at 100
fM. The LLOD for the standard detection approach was approximately
300 fM. The data for the 500 kD biotin polymer was a solid signal
at 30 fM, a faint signal at 3.75 aM, and an even fainter signal at
1.25 fM. Therefore, the 500 kD biotin polymer improved the
detection limit to a concentration within the range of 1.25 fM to
3.75 fM, which was an improvement of 80-240 fold in LLOD. The chart
below outlines the performance for each of the EDS types tested as
described:
TABLE-US-00002 TABLE 2 Fold- enhancement over EDS Type Chip tested
standard detection 3x 70 kDa MRSA 20-120 fold 1/3x 500 kDa Biotin
chip dil#2 ~25 fold 1x 500 kDa Biotin chip dil#2 ~40 fold 3x 500
kDa MRSA, biotin ~80 fold 3x 500 kDa chip dil#1 80-240 fold 3x
Chromalink Biotin chip dil#1 80-240 fold 4% Acrylate Biotin chip
dil#1 5-40 fold 10% Acrylate Biotin chip dil#1 5-40 fold Molecular
Probes Biotin chip dil#1 3-10 fold Molecular Probes MRSA ND
[0225] The use of smaller polymers resulted in less than 2-4 fold
intensity of detectable signal, as compared to the 500 kDa polymer.
However, in theory the 70 kDa cEDS could work just as well since
the 70 kDa cEDS could pack more densely and may have faster binding
kinetics than the 500 kDa cEDS
[0226] The effect of the number of biotin molecules conjugated per
EDS was tested. The data showed that increasing the number of
biotins from 34 to 65 then to 85 per backbone was roughly
correlated with an improvement in LLOD. However, a second
experiment that compared increasing number of biotins in the
acrylate EDS polymers did not show the same correlation. The
experimenters concluded that the type of polymer was important
factor for obtaining an optimal signal enhancement. An experiment
was performed comparing 3.times.500 kD dextran polymer
(approximately 85 biotins) with the 10% acrylate (approximately 71
biotins). Even though the number of biotins was approximately the
same, the dextran polymer was over 10 times as strong.
[0227] In theory, one could also use biotinylation reagents with
appropriate linker length to prevent interference between the
biotin on the polymer and strepavidin. Also, some linkers may
provide for better performance based on properties such as
flexibility or solubility. Several examples are NHS-LC-biotin
(Pierce), NHS-LC-LC-biotin (Pierce), NHS-Chromalink (Solulink),
NHS-PEGn-biotin (Nektar), and NHS-DNA probes.
[0228] The following experiment was designed to test the effect of
linker type on biotinylation. Biotin polymers were created with
NHS-LC-biotin (.about.85 biotins/polymer) and NHS-Chromalink (77
biotins/polymer) with approximately the same number of
biotins/polymer. The chromalink conjugated polymers were compared
to NHS-LC-biotin in Biotin chip dil #1. The data suggested that the
polymers have appreciably the same activity for signal enhancement
on the biotin chip and that the chemistry options with respect to
linker type are numerous.
[0229] The cEDS reagent was compared to the 500 kDa biotin polymer
from Molecular Probes on the Biotin chip dil #1. Under the normal
test conditions, cEDS were at least 25-fold better than the biotin
polymers made by Molecular Probes. The signal was apparent in all
four dilutions with the polymer but it was very weak by the 3rd
dilution with the Molecular Probes polymer in hybridization buffer.
Therefore, the GBS polymer, is at least 25-fold more sensitive than
the Molecular Probe. (Both polymers had the same molecular weight
and approximately the same number of biotins/polymers. GBS had 85
biotins/polymer and MP polymer had 79 biotins/polymer.
[0230] The cEDS and biotin dextran amplification polymer were
compared on an MRSA chip. In this experiment, femA target sequences
were tested as described. The data showed the cEDS can be clearly
detected down to 1.25-3.75 fM, whereas the MP biotin dextran has
significantly non-specific interactions with the chip surface,
making detection of the specific probe untenable.
Example 7
Comparison of Capped (cEDS) and Uncapped (EDS) Biotin Dextran
Polymers
[0231] In one experiment, the acylation of the remaining amino
groups, with NHS-acetate, on the polymer after modification with
NHS biotin, and subsequent purification on a size exclusion column
improved signal enhancement. Signal enhancement, which was
reproducible, was improved by at least 10-fold. Different fractions
of 500 kDa biotin dextran polymer were tested on a Biotin chip dil
#2, using the following methodology: a. biotinylated polymer
untreated, b. biotinylated polymer acetylated with NHS-acetate, c.
acetylated, biotinylated polymer passed over a PD10 column to
remove excess NHS-acetate. The unacetylated gel and the acetylated
gel showed spots of similar intensity at 12 pM, 300 pM, and 60 pM
concentration of biotinylated probe. The results of the gel showed
that simply acetylating the polymer had no effect on LLOD. After
the acetylated polymer was passed over the PD10 column, signal was
also detected at the 2.4 pM spot, which was approximately a ten to
twenty fold improvement.
[0232] The acylation further served to mitigate nonspecific
interactions of the polymers with the surface. In a test of the
same series of polymers described on Biotin chip dil #2, the
unacetylated fraction of the biotin polymer the results appeared
sporadically, which is typical of non-specific binding. The
sporadic results were most likely caused by the remaining unblocked
amino groups on the dextran polymer as they interacted
non-specifically with the surface of the chip. The results of the
unacetylated gel showed spotting at the 300 pM and the 60 pM spots.
The results of the acetylated gel show an additional faint spot at
12 pM ant 2.4 pM. The results of the PD10 purification were similar
to the acylated results, except that the intensity of the spotting
was increased at the 12 pM concentration. This observation likely
accounted for the observation that the amplification polymer cannot
be used with the assay enhancer PVP. The amplification polymer
contains 147 free lysines that can contribute to non-specific
interactions. In the following experiment, polymers were tested as
described on Biotin chip dil #1. The results of the experiment were
that PVP enhanced the performance of the GBS polymer .about.3-fold,
whereas it created a surface passivation with the amplification
polymer. Amplification polymers having greater solubility in
buffers at basic pH were found to perform better.
[0233] The samples that contained GBS amplification polymer,
biotin, dextran, and hybridization buffer gave off a medium-level
signal. Exchanging the buffer for buffer 2% PVP resulted in an
increase of signal at all 4 spots. The sample that contained the
commercially obtained amplification (Molecular Probes) polymer,
biotin, dextran, and hybridization buffer only produced signal at 1
nM and 3 nM, with a very faint signal at 330 pM.
Example 9
[0234] The addition of large water soluble polymers such as
polyvinyl pyrollidone (PVP) and polyethylene glycol (PEG) enhance
signaled an additional 2-4 fold when used in conjunction with cEDS.
This was not likely due to general improvement in detection of
surface-immobilized biotin because direct detection of the primary
biotin with anti-biotin/HRP and TMB is not effected. Overall
enhancements for detecting surface-immobilized biotin were improved
to 160 to 480-fold.
[0235] The addition of 0.5% PVP to a femA gene detection assay
buffer enhanced signal. A dilution series of model target sequences
from the femA gene in Staphylococcus aureus was tested on the MRSA
chip to determine the effect of cEDS on the LLOD. The PVP was added
to the 1.times. Hyb buffer used to dilute cEDS and the polyHRP/SA.
The results were that polymers alone improved the LLOD by 80 to 240
fold. Addition of 0.5% PVP and polymer improved the LLOD to 160 to
480 fold.
[0236] The addition of a non-ionic polymer compound during the cEDS
incubation step improved signal enhancement by .about.3-fold
compared to cEDS incubated without the compound. Other non-ionic
polymer compounds may enhance signal also. Addition of 2% polyvinyl
pyrrolidone (PVP) 40 kD, or 1-2% polyethylene glycol (PEG) 8 kD to
the 1.times. Hyb buffer for the cEDS and polyHRP/SA incubations
improved signal enhancement by an additional 3-fold above that of
cEDS alone.
[0237] Several other non-ionic polymer compounds may also work to
enhance the performance of the cEDS system. The following table
summarizes data from the testing of various polymeric buffer
additives on Biotin chip dil #1. Polyvinyl-X polymers with
X=pyrollidone, sulfate, or carboxylate worked roughly equivalently
at >40 kDa molecular weight. Polyethylene glycol in the range of
8-40 kDa were close in efficacy to 40kDa polyvinyl pyrrolidone.
Larger molecular weight dextran sulfate and low percentages of
carboxy methyl cellulose also had a measurable effect on enhancing
cEDS performance. Interestingly, alcohol and stearate side groups
on polyvinyl backbones did not enhance the performance of the cEDS
reagents.
TABLE-US-00003 TABLE 3 Molecular Fold- Buffer Additive Weight
Useful Range Enhance EDS Dextran sulfate 5 kD 0.5-3% None Dextran
sulfate 12 kD 0.5-2% None Dextran sulfate 500 kD 0.5-2% 2-4* Amino
dextran 70 kD 0.5-3% 2-4 Polyvinyl pyrollidone 10 kD 0.5-3% None
Polyvinyl pyrollidone 40 kD 0.5-2% 2-4* Polyvinyl sulfate 170 kD
0.5-1% 2-4* Polyvinyl stearate 90 kD 0.5-3% None Polyvinyl alcohol
40 kD 0.1-1% None Polyethylene glycol 8 kD 2-4 Polyethylene glycol
40 kD 0.5-2% 2-10* Carboxymethyl cellulose 0.05-0.36% 2-4*
Polyacrylic acid 100 kD .sup. 0.5% 2-4* Polyacrylic acid 250 kD
.sup. 0.5% 2-4* *Indicates that significant surface passivation
occurs at higher concentrations
[0238] Additional experiments indicated that addition of PVP had no
general effect on assay performance. The first sample contained
1.times. Hyb buffer was used to dilute the anti-biotin/HRP complex,
and the second sample was the same as the first sample (except that
it also contained PVP) was compared with the same sample and
compared with no added PVP in testing on the Biotin chip dil #2. No
signal enhancement was observed with the addition of up to 4% PVP
in the general assay, indicating that the effect is specific to
enhanced cEDS reagent performance.
[0239] It is to be understood that the foregoing descriptions of
embodiments of the present invention are exemplary and explanatory
only, are not restrictive of the invention, as claimed, and merely
illustrate various embodiments of the invention. It will be
appreciated that other particular embodiments consistent with the
principles described in the specification but not expressly
disclosed may fall within the scope of the claims.
* * * * *