U.S. patent application number 17/349970 was filed with the patent office on 2021-10-21 for multisignal reagents for labeling analytes.
This patent application is currently assigned to Enzo Life Science, Inc.. The applicant listed for this patent is Enzo Life Science, Inc.. Invention is credited to Jack Coleman, Richard Jin, Maciej Szczepanik.
Application Number | 20210325375 17/349970 |
Document ID | / |
Family ID | 1000005683406 |
Filed Date | 2021-10-21 |
United States Patent
Application |
20210325375 |
Kind Code |
A1 |
Coleman; Jack ; et
al. |
October 21, 2021 |
MULTISIGNAL REAGENTS FOR LABELING ANALYTES
Abstract
Provided is a multisignal labeling reagent comprising a first
polymer covalently bound to (a) a reactive group or a first member
of a first binding pair, and (b) more than one digoxigenin
molecule.
Inventors: |
Coleman; Jack; (East
Northport, NY) ; Szczepanik; Maciej; (Mount Sinai,
NY) ; Jin; Richard; (Pennington, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Enzo Life Science, Inc. |
Farmingdale |
NY |
US |
|
|
Assignee: |
Enzo Life Science, Inc.
Farmingdale
NY
|
Family ID: |
1000005683406 |
Appl. No.: |
17/349970 |
Filed: |
June 17, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16202213 |
Nov 28, 2018 |
11073512 |
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17349970 |
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15609360 |
May 31, 2017 |
10184934 |
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16202213 |
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13556425 |
Jul 24, 2012 |
9696298 |
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15609360 |
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13065101 |
Mar 14, 2011 |
9156986 |
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13556425 |
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12399393 |
Mar 6, 2009 |
8394949 |
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13065101 |
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10407818 |
Apr 3, 2003 |
7514551 |
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12399393 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09B 11/08 20130101;
G01N 33/82 20130101; G01N 33/581 20130101; C07H 21/04 20130101;
G01N 33/53 20130101; C07H 21/00 20130101; G01N 33/94 20130101; C09B
11/24 20130101; C12Q 1/6818 20130101 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07H 21/00 20060101 C07H021/00; C07H 21/04 20060101
C07H021/04; C09B 11/08 20060101 C09B011/08; C09B 11/24 20060101
C09B011/24; C12Q 1/6818 20060101 C12Q001/6818; G01N 33/58 20060101
G01N033/58; G01N 33/82 20060101 G01N033/82; G01N 33/94 20060101
G01N033/94 |
Claims
1. A method for providing an oligonucleotide labeled at each of the
5' end and the 3' end of the oligonucleotide, comprising the steps
of: providing an DNA oligonucleotide labeled with a first member of
a first binding pair at its 5' end; adding deoxynucleotides labeled
with a first member of a second binding pair to the 3' end of the
oligonucleotide using terminal transferase.
2. The method of claim 1, wherein the first member of the first
binding pair and the first member of the second binding pair are
the same.
3. The method of claim 1, wherein the first member of the first
binding pair and the first member of the second binding pair are
different.
4. The method of claim 1, wherein the first member of the first
binding pair is streptavidin or avidin.
5. The method of claim 1, therein the first member of the second
binding pair is digoxigenin.
6. The method of claim 4, wherein the first member of the second
binding pair is digoxigenin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 16/202,213 filed Nov. 28, 2018 (now U.S. Pat. No.), which
is a continuation of U.S. patent application Ser. No. 15/609,360
filed May 31, 2017 (now U.S. Pat. No. 10,184,934), which is a
divisional of U.S. patent application Ser. No. 13/556,425 filed
Jul. 24, 2012 (now U.S. Pat. No. 9,696,298), which is a
continuation-in-part of U.S. patent application Ser. No. 13/065,101
filed Mar. 14, 2011 (now U.S. Pat. No. 9,156,986), which is a
continuation-in-part of U.S. patent application Ser. No. 12/399,393
filed Mar. 6, 2009 (now U.S. Pat. No. 8,394,949), which is a
divisional of U.S. application Ser. No. 10/407,818 filed Apr. 3,
2003 (now U.S. Pat. No. 7,514,551), each of which is hereby
incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jun. 16, 2021, is named ENZ-65-CIP2-D1-CON-D1-103-SL.txt and is
1,599 bytes in size.
FIELD OF THE INVENTION
[0003] The present application generally relates to assays for
detecting analytes, e.g., proteins and small molecules (haptens).
More specifically, this application provides reagents for improving
the detection and quantitation of an analytes with assays that
utilize an agent, such as an antibody, that binds to the
hapten.
BACKGROUND OF THE INVENTION
[0004] There is a high demand to determine the presence and
concentration of analytes of interest, for example protein and
small molecule ("hapten") analytes, which can be of environmental
or medical concern. Examples of hapten analytes include fungal or
microbial toxins as a threat to food safety, and drugs, steroids,
hormones, proteins, peptides, lipids, sugars, receptors, nucleic
acids, vitamins, etc., e.g., in mammalian fluid or tissue samples,
to identify an intoxication, control the medication of therapeutic
drugs with a narrow therapeutic window, etc.
[0005] Assays (e.g., immunoassays) utilizing an agent that binds to
an analyte of interest (e.g., an antibody) have been developed for
detecting numerous analyte compounds at very low levels. As is
known in the art, antibodies that bind to small molecule analytes
can be developed by, for example, using phage display techniques,
or by utilizing an immunogen that comprises the analyte, or an
analog of the analyte, covalently conjugated to a carrier protein
or other immunogenic macromolecule. When using hapten immunogens,
antibodies are generally elicited to the portion of the analyte
that is distal to the chemical bond through which the hapten is
conjugated to the carrier, such that the distal portion becomes an
epitope of the carrier-hapten complex.
[0006] Immunoassays for analytes often take the form of competitive
binding assays, where, for example, a labeled analyte competes with
analytes in the sample for binding to antibodies fixed on a solid
matrix. In those assays, the signal from the label decreases with
increasing concentration of analyte in the sample. Examples of such
an assay includes known assays for cAMP and vitamin D (see, e.g.,
Enzo Life Sciences, "cAMP Complete ELISA kit"; GE Life Sciences,
"Cyclic-AMP Assay Kit"; Caymen Chemical Company, "Cyclic AMP EIA
Kit"; PerkinElmer, "AlphaScreen.RTM. cAMP Assay Kit"; and Promega,
"GloSensor.TM. cAMP Assay"). These competitive assays and similar
assays are often improved by improving the signal intensity of the
label.
[0007] The use of non-radioactive labels in biochemistry and
molecular biology has grown exponentially in recent years. Among
the various compounds used as non-radioactive labels, aromatic dyes
that produce a fluorescent or luminescent signal are especially
useful. Notable examples of such compounds include fluorescein,
rhodamine, coumarin and cyanine dyes such as Cy3 and Cy5. Composite
dyes have also been synthesized by fusing two different dyes
together. See, e.g., Lee et al., 1992; and U.S. Pat. Nos. 5,945,526
and 6,008,373.
[0008] Non-radioactive labeling methods have been developed to
attach signal-generating groups onto proteins, nucleic acids and
haptens. This is generally achieved by modifying labels with
chemical groups such that they would be capable of reacting with,
e.g., the amine, thiol, and hydroxyl groups on proteins or haptens.
Examples of reactive groups utilized for this purpose include
activated esters such as N-hydroxysuccinimide esters,
isothiocyanates and other compounds.
[0009] Labeled nucleotides are used for the synthesis of DNA and
RNA probes in many enzymatic methods including terminal transferase
labeling, nick translation, random priming, reverse transcription,
RNA transcription and primer extension. Labeled phosphoramidite
versions of these nucleotides have also been used with automated
synthesizers to prepare labeled oligonucleotides. The resulting
labeled probes are widely used in such standard procedures as
northern blotting, Southern blotting, in situ hybridization, RNase
protection assays, DNA sequencing reactions, DNA and RNA microarray
analysis and chromosome painting.
[0010] There is an extensive literature on chemical modification of
nucleic acids by means of which a signal moiety is directly or
indirectly attached to a nucleic acid. See, e.g., U.S. Pat. Nos.
4,711,955 and 5,241,060, 4,952,685, 5,013,831, 7,166,478 and
7,514,551, and U.S. Patent Publication 2011/0318788.
[0011] The presence and nature of a linker arm may also improve the
signaling characteristics of the labeled target molecule (see,
e.g., U.S. Patent Publication 2011/0218788 and U.S. Pat. No.
7,514,551).
BRIEF SUMMARY OF THE INVENTION
[0012] The invention provided herein is based in part on the
discovery that a multisignal labeling reagent, e.g., as described
in U.S. Patent Publication 2011/0318788 and U.S. Pat. No.
7,514,551, can be advantageously utilized for labeling analytes,
e.g., protein or on small molecule analytes, for example as a
reagent in competitive immunoassays.
[0013] Thus, in some embodiments, a composition comprising an
analyte bound covalently or through a first binding pair to a
polymer is provided. In these embodiments, the analyte is less than
about 2000 MW; the polymer further comprises more than one signal
or first member of a second binding pair; and the analyte is not a
member of the first binding pair or the second binding pair.
[0014] In other embodiments, an assay for an analyte is provided.
The assay comprises: (a) combining a sample suspected of containing
the analyte with a detection reagent and a binding agent that binds
to the analyte, wherein the detection reagent comprises the analyte
or an analyte analog bound covalently or through a first binding
pair to a polymer, said polymer further comprising more than one
signal or first member of a second binding pair, wherein the
analyte or analyte analog is not a member of the first binding pair
or the second binding pair; (b) removing any of the detection
reagent that is not bound to the binding agent; and (c) detecting
the signal or the first member of the second binding pair that is
bound to the binding agent. In these embodiments, the amount of the
signal or the first member of the second binding pair bound to the
binding agent is inversely proportional to the analyte in the
sample.
[0015] Additionally provided is a multisignal labeling reagent
comprising a first polymer covalently bound to (a) a reactive group
or a first member of a first binding pair, and (b) more than one
digoxigenin molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a graph showing the results of an immunoassay for
cAMP utilizing a composition of the present invention.
[0017] FIG. 2 is a graph showing the results of a comparison of
four labeling reagents.
DETAILED DESCRIPTION OF THE INVENTION
[0018] As used herein, the singular forms "a", "an" and "the" are
intended to include the plural forms as well, unless the context
clearly indicates otherwise. Additionally, the use of "or" is
intended to include "and/or", unless the context clearly indicates
otherwise.
[0019] The present invention is based in part on the discovery that
a multisignal labeling reagent, e.g., as described in U.S. Patent
Publication 2011/0318788 and U.S. Pat. No. 7,514,551, can be
advantageously utilized on small molecules, for example as a
reagent in competitive immunoassays.
[0020] Thus, in some embodiments, a composition comprising an
analyte bound covalently or through a first binding pair to a
polymer is provided. In these embodiments, the analyte is less than
about 2000 MW; the polymer further comprises more than one signal
or first member of a second binding pair; and the analyte is not a
member of the first binding pair or the second binding pair.
[0021] In various aspects of these embodiments, the analyte is less
than about 1000 MW, less than about 500 MW, less than about 250 MW,
less than about 200 MW, or less than about 150 MW.
[0022] As used herein, a polymer is an organic molecule comprising
at least 3 repeating monomeric units such as amino acids, sugars,
nucleotides or nucleotide analogs. The polymer of the composition
can be comprised of any form of organic monomer and can be less
than about 10 monomers, less than about 20 monomers, less than
about 50 monomers, less than about 100 monomers, or about 100 or
more monomers.
[0023] Non-limiting examples of polymers made from such monomeric
units include nucleic acids, abasic nucleic acids, peptide nucleic
acids, polypeptides, proteins, oligosaccharides, polysaccharides
and organic polymers. The polymers used in the present invention
may be isolated from biological sources or they may be created
synthetically or in vitro. The polymers may also comprise multiples
of only one particular type of monomeric unit, or they may comprise
different types of monomeric units. For example, a chimeric
oligomer or polymer can be a nucleic acid construct that comprises
both a normal nucleic acid segment and a peptide nucleic acid
segment, a combination of nucleotides and amino acids, or a
combination of a segment of an abasic nucleic acid and a segment
comprising a peptide nucleic acid.
[0024] Additionally, even when the monomeric units of the polymer
are the same type of compound (e.g., all deoxyribonucleotides),
they may be the same or they may be different. For instance, a
nucleic acid polymer may be a homopolymer comprising a reiteration
of a single base or it can be a heteropolymer having varied
nucleotides. A polypeptide polymer may be homopolymeric and
comprise multiples of a single amino acid or it may be
heteropolymeric and comprise different amino acids. The labels in
an oligomeric or polymeric labeling reagent may also be the same or
they may be different. For instance, a labeling reagent that
comprises two different dyes attached at discrete intervals on a
polynucleotide may participate in energy transfer for signal
generation.
[0025] The polymers in these compositions may comprise a single
chain structure linking the monomeric units together or they may
comprise more than one chain. For example, branched,
double-stranded and triple-stranded nucleic acids may all find use
with present invention. Additionally, the polymer comprising the
signals or the first members of a second binding pair can be
hybridized to the polymer that is bound to the analyte. Such
multi-chain structures may provide useful properties. For example,
a double-stranded nucleic acid is more rigid than a single stranded
nucleic acid. The use of a double-stranded structure may allow
better control over the distribution or spacing of labeled moieties
where proximity or lack of proximity may be desirable. As is known,
efficient signal generation by means of energy transfer depends
upon a close proximity of donor and acceptor moieties and as such,
establishment of a proximity between these moieties can be
beneficial. Additionally, if a single dye species is being used as
signal generators, a close proximity of some dye molecules can lead
to a self-quenching phenomenon, and spreading out the locations of
the dyes could thus be beneficial. The use of more than one chain
may also convey other useful properties such as increasing the
amount of signal generated or increasing the charge number.
Multiple chains may also endow the system with flexibility of use.
For example, a first nucleic acid strand may comprise a reactive
group and a second nucleic acid strand with complementary sequences
can comprise signal groups. By complementary base pairing between
these strands, a complex can be formed that comprises a reactive
group and signaling groups. See, e.g., FIG. 1 of U.S. Pat. No.
7,514,551. For example, they may comprise termini or extended
chains with extended multiple charged groups. Other groups that may
offer useful additional properties may also find use with the
present invention.
[0026] In some embodiments, the polymer is an oligopeptide. The
oligopeptide polymer can be more than about 100 amino acids or
amino acid analogs, or less than about 100 amino acids or amino
acid analogs, e.g., less than about 90, 80, 70, 60, 50, 40, 30, 20,
10 or 5 amino acids or amino acid analogs. In other embodiments,
the polymer is a nucleic acid. Such a polymer can comprise any
known nucleotide or nucleotide analog known in the art. The nucleic
acid polymer can be more than about 100 nucleotides or nucleotide
analogs, or less than about 100 nucleotides or nucleotides analogs,
e.g., less than about 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10 or
5 nucleotides or nucleotide analogs. The nucleic acid polymers can
be synthesized by any means known in the art, for example as
described in U.S. Patent Publication 2011/0318788 and U.S. Pat. No.
7,514,551.
[0027] In the invention compositions, the polymer can be bound to
the analyte either covalently or through a first binding pair.
Where the analyte is covalently bound to the polymer, the analyte
and polymer can be joined by any method known in the art for the
particular analyte and polymer. See, e.g., Example 1. As is known
in the art, the analyte (or the polymer) can be modified to
comprise a reactive group that reacts with a moiety on the polymer
(or analyte). Where an immunoassay is already available for the
analyte, the methods utilized to conjugate the analyte to the
carrier protein to prepare the immunogen can generally also be
utilized to conjugate the analyte to the polymer. Examples of
reactive groups include but are not limited to active esters,
groups capable of forming a carbon-carbon bonds and groups capable
of forming bonds with O, N or S. Specific examples of such groups
are isothiocyanate, isocyanate, monochlorotriazine,
dichlorotriazine, mono- or di-halogen substituted pyridine, mono-
or di-halogen substituted diazine, maleimide, aziridine, sulfonyl
halogen substituted diazine, maleimide, aziridine, sulfonyl halide,
acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide
ester, imido ester, hydrazine, azidonitrophenyl, azide,
3-(2-pyridyl dithio)-proprionamide, glyoxal, aldehyde,
carbon-carbon double bonds, mercury salts, and any group capable of
reacting with carbon-carbon double bonds, amines, hydroxyl groups,
sulfhydryl groups and halogens.
[0028] Where the analyte is bound to the polymer through a first
binding pair, the analyte is conjugated to one member of the first
binding pair and the polymer is conjugated to the other member of
the first binding pair. Non-limiting examples of binding pairs are
a sugar-lectin, an antigen-antibody, a ligand-ligand receptor, a
hormone-hormone receptor, an enzyme-substrate, a biotin-avidin, and
a biotin-streptavidin. Methods for conjugating haptens and polymers
to various members of binding pairs are well-known in the art. For
the purposes of the invention compositions, a DNA supramolecular
binding molecule or two DNA supramolecular binding molecules
covalently joined by a linker group, as described in U.S. Patent
Publication 2011/0318788, is considered a member of a binding pair,
where the nucleic acid to which the DNA supramolecular binding
molecule(s) bind is considered both the polymer and the other
member of the binding pair.
[0029] As used herein, a "linker" or "linker arm" is a chemical
moiety that separates one component of the composition (e.g., the
polymer, hapten, binding pair member or signal) from another
component. Thus, a linker may be used for example to separate the
hapten from the first binding pair, the hapten from the polymer,
the polymer from the first binding pair, the polymer from the
second binding pair, the polymer from the signal, or the second
binding pair from the signal.
[0030] A linker comprises a chain of atoms of any length that may
be comprised of carbon, nitrogen, oxygen, sulfur in any combination
and any other possible atom. The connecting chain can be saturated,
unsaturated or can contain aromatic rings and the linking chain can
be flexible or rigid. The connecting chain can further comprise any
of the rigid units disclosed, e.g., in U.S. Patent Publication
2005/0137388.
[0031] The linker in these compounds can be rigid or flexible.
Rigid linkers have been utilized with dimer intercalators. See,
e.g., Glover et al. (2003). However, flexible linkers do not
require the precise design required of rigid linkers, where the
linker must precisely separate and orient the DNA supramolecular
binding molecules to properly insert into the nucleic acid.
[0032] In some embodiments, the linker comprises an unsubstituted
C.sub.1-C.sub.20 straight-chain, branched or cyclic alkyl, alkenyl
or alkynyl group, a substituted C.sub.1-C.sub.10 straight-chain,
branched or cyclic alkyl, alkenyl or alkynyl group wherein one or
more C, CH or CH.sub.2 groups are substituted with an O atom, N
atom, S atom, NH group, CO group or OCO group, or an unsubstituted
or substituted aromatic group. In more specific embodiments, the
linker is (CH.sub.2)1-10-NH--(CH.sub.2)1-10-. In still more
specific embodiments, the linker is
--(CH.sub.2).sub.1-5--NH--(CH.sub.2).sub.1-5--. One useful linker
within these embodiments is
--(CH.sub.2).sub.3--NH--(CH.sub.2).sub.4-(spermidine--see Examples
20-22 of U.S. Patent Publication 2011/0318788).
[0033] Linkers can be covalently attached to any of the
substituents of the instant compositions by any means known in the
art, e.g., through a reactive group as discussed above.
[0034] In some embodiments, the polymer is covalently bound to the
signal moieties either directly or through a linker. Non-limiting
examples of signals useful for these compositions are fluorescent
dyes, colored dyes, radioactive molecules, chemiluminescent
molecules and enzymes. Where the signal is an enzyme, the enzyme is
capable of modifying a substrate to create a detectable signal.
Non-limiting examples of such enzymes include alkaline phosphatase,
horseradish peroxidase and luciferase.
[0035] In other embodiments, the polymer is covalently bound to
more than one first member of a second binding pair, e.g.,
digoxigenin, fluorescein, biotin or dinitrophenol. In these
embodiments, the first member of the second binding pair can be
made into a detectable signal by adding a signal molecule that is
bound to the second member of the second binding pair. Thus, a
polymer bound to a hapten (e.g., cAMP--see Examples) that further
comprises, e.g., more than one biotin moiety can be utilized in an
assay where the biotin moieties are detected by adding
streptavidin-alkaline phosphatase then an alkaline phosphatase
substrate. See Example 3 below. In some of these embodiments, the
composition further comprises more than one signal covalently bound
to a second member of the second binding pair, wherein each
signal-second member of the second binding pair is noncovalently
bound to each first member of the second binding pair.
[0036] The compositions of these embodiments may also contain
additional alkyl, aryl and/or polar or charged groups on any
component of the composition, e.g., the hapten, the polymer, a
binding pair member, a linker, or the signal. The polar or charged
groups may include but are not limited to halogen, substituted or
unsubstituted alkyl or aryl groups, saturated or unsaturated alkyl
groups, alkoxy, phenoxy, amino, amido, and carboxyl groups, polar
groups such as nitrates, sulfonates, sulfhydryl groups, nitrites,
carboxylic acids, phosphates or any other such group or
substituent.
[0037] The analytes for these compositions can be any hapten that
can be manipulated as described above. Useful analytes include
mammalian cellular metabolites, metabolites of microorganisms,
medications, illicit drugs, vitamins, environmental pollutants,
pesticides, and toxins. Specific non-limiting examples include
cAMP, cGMP, 8-bromoadenosine 3',5'-cyclic monophosphate,
cholesterol, a hydroxycholesterol, vitamin D, 25-hydroxy vitamin D,
vitamin B12, vitamin E, vitamin B1, vitamin B6, ascorbic acid,
retinol, biotin, folate, legumin, salbutamol, melamine,
sulfaquinoxaline, an inositol phosphate, a phosphatidyl inositol
phosphate, prednisone, pregnenolone, dexamethasone, triamcinolone,
fludrocortisone, dihydrotachysterol, oxandrolone, testosterone,
dihydrotestosterone, nandrolone, norethindrone, medroxyprogesterone
acetate, progesterone, a glucocorticoid, aldosterone, estrogen,
oxytocin, androstanediol glucuronide, bilirubin, warfarin, an
estrogen, methotrexate, tobramycin, acetaminophen, encainide,
fluoxetine, gentamycin, an aminoglycoside, amikacin, coenzyme Q10,
theophylline, phenytoin, cimetidine, disulfiram, trazodone,
ethanol, halothane, phenylbutazone, azapropazone, ibuprofen,
amiodarone, imipramine, miconazole, metronidazole, nifedipine,
chloramphenicol, trimethoprim, a sulfonamide, rifampin, cisplatin,
vinblastine, bleomycin, oxacillin, nitrofurantoin, phenobarbital,
primidone, carbamazepine, metoclopramide, cholestyramine,
colestipol, neomycin, sulfasalazine, digoxin, indomethacin,
diltiazem, erythromycin, tetracycline, itraconazole, nicardipine,
triamterene, spironolactone, chlorpromazine, a cyclosporin,
nortriptyline, ethosuximide, imipramine, codeine, lorazepam,
topiramate, disopyramide, levetiracetam, clobazam, oxcarbazepine,
fructosamine, caffeine, methaqualone, meprobamate, fluphenazine, a
barbiturate, a phenothiazine, serotonin, valproic acid, digitoxin,
maprotiline, lidocaine, mexiletine, primidone, risperidone,
OH-rispiridone, a porphyrin, haloperidol, flecainide, tocainide,
acetazolamide, a sulfonamide, verapamil, a metanephrine, an
oxidized nucleotide, a mycotoxin, tetrahydrocannabinol, a
cannabinoid, cocaine, LSD, an amphetamine, a barbiturate, heroin,
methadone, nicotine, cotinine, a benzodiazepine, bis(2-ethylhexyl)
phthalate, bisphenol A, hydralazine, atrazine, an organochloride
insecticide, an organophosphate insecticide, or a carbamate
insecticide. In some embodiments, the analyte is a steroid hormone,
a glucocorticoid, oxytocin, cAMP, cGMP, hydroxycholesterol, vitamin
D or 25-hydroxy vitamin D. In these compositions, the analyte could
also be an analog of a compound of interest, e.g., any of the above
compounds, particularly where the analog is able to compete with
sample analyte for binding sites to a binding agent (e.g., an
antibody), thus making the analog-polymer composition useful for
competitive assays for the analyte (see further discussion
below).
[0038] It is noted that certain analytes known to be members of a
binding pair can also usefully be utilized as analytes in the
invention compositions. An example is digoxigenin as an analyte,
e.g., where the polymer is attached to the digoxigenin analyte
through a biotin-streptavidin binding pair. In such a case, the
digoxigenin is considered an analyte and not a member of a binding
pair.
[0039] The above-described compositions, as well as multisignal
labeling reagents bound to larger analytes such as proteins (for
example as described in U.S. Patent Publication 2011/0318788 and
U.S. Pat. No. 7,514,551) are particularly useful in assays for
identification or quantitation of the analyte in a sample. The
assays utilize a binding agent to the analyte, e.g., an antibody or
analyte-binding fragment thereof.
[0040] Thus, an assay for an analyte is provided. The assay
comprises: (a) combining a sample suspected of containing the
analyte with a detection reagent and a binding agent that binds to
the analyte, wherein the detection reagent comprises the analyte or
an analyte analog bound covalently or through a first binding pair
to a polymer, said polymer further comprising more than one signal
or first member of a second binding pair, wherein the analyte or
analyte analog is not a member of the first binding pair or the
second binding pair; and (b) detecting the signal or the first
member of the second binding pair that is bound to the binding
agent. In these embodiments, the amount of the signal or the first
member of the second binding pair bound to the binding agent is
inversely proportional to the analyte in the sample.
[0041] The antibodies useful for these assays can be
immunoglobulins of any vertebrate species, e.g., rabbit, goat,
mouse, sheep, chicken, etc. and can be polyclonal or monoclonal.
They can include the Fc region or they can be Fab or Fab2 fragments
or otherwise engineered or manipulated to exclude all or part of
the Fc region. Additionally, they can be from any source, e.g.,
from the serum of an animal injected with an immunogen such as any
of the immunogens described above, or they can be from culture or
ascites as is known in the art of hybridoma technology.
Alternatively, they can be from recombinant sources, e.g., as
described in Winter et al., 1994, or Charlton and Porter, 2002.
[0042] Any immunoassay known in the art as useful for analyte
detection can be utilized for the instant assays. The relevant
immunoassays generally utilize a competitive format, i.e., where
the analyte in the sample competes with a labeled analyte or
labeled hapten or hapten analog ("detection reagent") for
anti-analyte antibody binding sites such that less detection
reagent is bound when there is more hapten in the sample. Thus, in
these competitive assays, an increasing amount of analyte in the
sample results in less detection reagent bound to the solid phase,
and consequently less signal. In various competitive assays, the
sample can be added with the detection reagent to compete directly
for antibody binding sites, or the sample and detection reagent can
be added sequentially such that the detection reagent simply binds
where the sample hapten is not bound.
[0043] The competitive assay for these embodiments can be
homogeneous, where the signal detection step is performed without
removing detection reagent that is not bound to the reagent.
Nonlimiting examples include assays that utilize a change in FRET
from a fluorescent signal when the detection reagent is bound to
the binding agent. Alternatively, the assay can be heterogeneous,
where detection reagent that is not bound to the binding agent is
removed, so that the only signal is from bound detection
reagent.
[0044] In some embodiments, the immunoassay is a direct competitive
assay, utilized where the detection reagent comprises the analyte
bound to the polymer comprising the signal, or an indirect
competitive immunoassay, where the detection reagent comprises a
first member of a second binding pair, e.g., a second antibody (for
example an anti-digoxigenin antibody), biotin, or streptavidin. In
the latter case, the amount of bound detection reagent is
determined by adding the second member of the second binding pair
that is bound to a signal (e.g., a digoxigenin-fluorescent dye
complex, a biotin-alkaline phosphatase complex, a
streptavidin-luciferase complex, etc.).
[0045] The immunoassays provided herein can take any format known
in the art. In some embodiments, analyte antibodies are bound to
the solid phase, either directly or indirectly, the latter being
where the solid phase is coated with an anti-antibody (for example
goat antibodies that bind to rabbit IgG antibodies [goat
anti-rabbit IgG]) and the analyte antibodies are bound to the
anti-antibody. The anti-antibodies are also known as "second
antibodies." In these assays, the sample and detection reagent is
added to the solid phase to compete with antibody binding sites on
the coated solid phase. After washing, the signal is generated,
which measures the amount of detection reagent that is bound to the
solid phase. Numerous particular assays with this configuration can
be devised without undue experimentation.
[0046] Numerous specific immunoassay formats are known that could
be utilized with analyte-multisignal labeling reagents to determine
an analyte in a sample. See, e.g., U.S. Patent Publication
2012/0115169, which provides a description of various relevant
immunoassays. As described therein, the assay is performed in a
liquid phase or on a solid phase, e.g., on a bead or a microplate,
for example a 96 well microtiter plate. Nonlimiting examples of
immunoassays useful in these methods are a radioimmunoassay, a
Luminex.RTM. assay (see, e.g., Wong et al., 2008), a microarray
assay, a fluorescence polarization immunoassay (see, e.g., U.S.
Pat. No. 4,585,862), an immunoassay comprising a Forster resonance
energy transfer (FRET) signaling system (see, e.g., Blomberg et
al., 1999; Mayilo et al., 2009), a scintillation proximity assay, a
fluorescence polarization assay, a homogeneous time-resolved
fluorescence assay, an amplified luminescence assay
("ALPHAScreen"), an enzyme complementation assay, an
electrochemiluminescence assay, and an enzyme immunoassay (a.k.a.
enzyme linked immunosorbent assay [ELISA]). As is well known in the
art, in ELISA, an enzyme combined with a substrate that becomes
colored upon reaction with the enzyme provides the signal to
quantify the antigen in the sample. See, e.g., O'Beirne and Cooper,
1979.
[0047] In various embodiments of these assays, the binding agent is
an antibody or analyte-binding fragment thereof. In additional
embodiments, the binding agent is bound to a solid phase.
[0048] In some embodiments, the analyte is less than about 2000 MW.
In these embodiments, the detection reagent can be any of the
analyte-polymer compositions described above. In some embodiments,
the detection reagent comprises an analog to the analyte of
interest bound to the polymer. Such a configuration may be favored
to provide for more convenient production of the detection reagent,
where the analyte analog provides for easier chemistry for
conjugation to the polymer or first binding pair member.
Utilization of an analyte analog in the detection reagent can also
provide for more favorable conditions for binding to the binding
agent, for example where a detection reagent with an analyte analog
competes with sample analyte for antibody binding sites more
favorably (e.g., providing better sensitivity or less background)
than a detection reagent that utilizes the analyte.
[0049] In some aspects of these embodiments, the polymer comprises
(a) more than one signal or (b) more than one first member of a
second binding pair and a signal covalently bound to a second
member of the second binding pair. In these aspects, each
signal-second member of the second binding pair is noncovalently
bound to each first member of the second binding pair. In
alternative aspects, the signal covalently bound to a second member
of the second binding pair is added after the combining step or the
removing step.
[0050] With any of the assays described above, the signal can be
any detectable label known in the art, for example a fluorescent
dye, a colored dye, a radioactive molecule, a chemiluminescent
molecule or an enzymes. Examples of useful enzymes in these
embodiments are horseradish peroxidase, alkaline phosphatase and
luciferase.
[0051] These assays can be used to detect any analyte that has a
cognate binding agent or to which a binding agent (e.g., an
antibody) can be synthesized. Useful analytes include mammalian
cellular metabolites, metabolites of microorganisms, medications,
illicit drugs, vitamins, environmental pollutants, pesticides, and
toxins. Specific non-limiting examples include cAMP, cGMP,
8-bromoadenosine 3',5'-cyclic monophosphate, cholesterol, a
hydroxycholesterol, vitamin D, 25-hydroxy vitamin D, vitamin B12,
vitamin E, vitamin B1, vitamin B6, ascorbic acid, retinol, biotin,
folate, legumin, salbutamol, melamine, sulfaquinoxaline, an
inositol phosphate, a phosphatidyl inositol phosphate, prednisone,
pregnenolone, dexamethasone, triamcinolone, fludrocortisone,
dihydrotachysterol, oxandrolone, testosterone, dihydrotestosterone,
nandrolone, norethindrone, medroxyprogesterone acetate,
progesterone, a glucocorticoid, aldosterone, estrogen, oxytocin,
androstanediol glucuronide, bilirubin, warfarin, an estrogen,
methotrexate, tobramycin, acetaminophen, encainide, fluoxetine,
gentamycin, an aminoglycoside, amikacin, coenzyme Q10,
theophylline, phenytoin, cimetidine, disulfiram, trazodone,
ethanol, halothane, phenylbutazone, azapropazone, ibuprofen,
amiodarone, imipramine, miconazole, metronidazole, nifedipine,
chloramphenicol, trimethoprim, a sulfonamide, rifampin, cisplatin,
vinblastine, bleomycin, oxacillin, nitrofurantoin, phenobarbital,
primidone, carbamazepine, metoclopramide, cholestyramine,
colestipol, neomycin, sulfasalazine, digoxin, indomethacin,
diltiazem, erythromycin, tetracycline, itraconazole, nicardipine,
triamterene, spironolactone, chlorpromazine, a cyclosporin,
nortriptyline, ethosuximide, imipramine, codeine, lorazepam,
topiramate, disopyramide, levetiracetam, clobazam, oxcarbazepine,
fructosamine, caffeine, methaqualone, meprobamate, fluphenazine, a
barbiturate, a phenothiazine, serotonin, valproic acid, digitoxin,
maprotiline, lidocaine, mexiletine, primidone, risperidone,
OH-rispiridone, a porphyrin, haloperidol, flecainide, tocainide,
acetazolamide, a sulfonamide, verapamil, a metanephrine, an
oxidized nucleotide, a mycotoxin, tetrahydrocannabinol, a
cannabinoid, cocaine, LSD, an amphetamine, a barbiturate, heroin,
methadone, nicotine, cotinine, a benzodiazepine, bis(2-ethylhexyl)
phthalate, bisphenol A, hydralazine, atrazine, an organochloride
insecticide, an organophosphate insecticide, or a carbamate
insecticide. In some embodiments, the analyte is a steroid hormone,
a glucocorticoid, oxytocin, cAMP, cGMP, hydroxycholesterol, vitamin
D or 25-hydroxy vitamin D.
[0052] The inventors have also discovered that multisignal labeling
reagents, e.g., as described in U.S. Patent Publication
2011/0318788 and U.S. Pat. No. 7,514,551, are particularly
effective when digoxigenin/anti-digoxigenin is used as a binding
pair to join the polymer with the signal. See Examples 4-6.
[0053] Thus, in some embodiments, a multisignal labeling reagent is
provided. The reagent comprises a first polymer covalently bound to
(a) a reactive group or a first member of a first binding pair, and
(b) more than one digoxigenin molecule. Here, polymers, reactive
groups and binding pairs are as described above.
[0054] In various embodiments, the digoxigenin molecules on the
polymer are used as a binding pair member for joining a signal
molecule, where the other member of the binding pair is an
anti-digoxigenin antibody that is further bound to at least one
signal molecule. Signal molecules are described above, and could
be, e.g., a fluorescent dye, a radioactive molecule or an enzyme.
In various embodiments, each signal comprises an enzyme that is
alkaline phosphatase, horseradish peroxidase or luciferase.
[0055] The multisignal labeling reagent of these embodiments can
further comprise a linker moiety covalently linking (a) the first
polymer to the reactive group or first member of the first binding
pair and/or (b) the first polymer to the more than one digoxigenin
molecules. Linker groups here are as described above.
[0056] In some embodiments, the first polymer is further bound to a
second polymer, wherein the second polymer further comprises a
signal molecule or a first member of a second binding pair. In most
constructs, this configuration would cause a further amplification
of the signal, e.g., for use where high sensitivity is desired. In
some of these embodiments, the first polymer is covalently bound to
the second polymer. Alternatively, the first polymer is bound to
the second polymer through a third binding pair.
[0057] In various aspects of these multisignal labeling reagents,
the reagent is bound to a target molecule through the reactive
group or the first binding pair. The target molecule is not
narrowly limited to any class of compound. Examples include
haptens, proteins, oligopeptides, nucleic acids, nucleic acid
analogs, oligosaccharides, polysaccharides, lipids and organic
polymers.
[0058] Preferred embodiments are described in the following
examples. Other embodiments within the scope of the claims herein
will be apparent to one skilled in the art from consideration of
the specification or practice of the invention as disclosed herein.
It is intended that the specification, together with the examples,
be considered exemplary only, with the scope and spirit of the
invention being indicated by the claims, which follow the
examples.
Example 1. Attaching an Oligonucleotide to cAMP
(a) Synthesis of 2'-O-Succinyl-cAMP-NHS Ester
[0059] 2'-O-succinyl-cAMP (3.0 mg, 6.9 .mu.mol) was dissolved in
650 .mu.l of anhydrous, amine-free DMSO and N-hydroxysuccinimide
(11.5 mg, 100 .mu.mol) was added. To the above clear solution,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDCI) (13.4 mg, 70
.mu.mol) dissolved in 150 .mu.l of DMSO was added and the reaction
mixture was incubated at room temperature overnight. The reaction
progress was followed by TLC analysis (silica, isopropyl
alcohol:NH.sub.4OH:H.sub.2O 6:3:1). The crude product was used as
DMSO solution in the following step (b).
(b) Addition of Oligo to 2'-O-Succinyl-cAMP-NHS ester
[0060] To a solution of 5amCap 22-mer (20 nmole, 5'-amino-C6-TTG
CTG AGG TCA TGG ATC GAG A-3') (SEQ ID NO:1) in Modification Buffer
(100 mM sodium phosphate, 150 mM NaCl, pH 8.0), 45 molar
equivalents of 2'-O-succinyl-cAMP-NHS ester in DMSO was added. The
reaction mixture was incubated at room temperature for 18 h and
after diluting with nuclease free water to decrease the amount of
DMSO to 5% of the total, the mixture was purified on a 3k Amicon
diafiltration device. The cAMP-oligomer bioconjugate (.about.8
nmole) was identified and analyzed by HPLC analysis (Zorbax Oligo
column, phosphate:acetonitrile gradient).
Example 2. Adding Poly Biotin to cAMP-Oligo
[0061] The cAMP-oligo described in Example 1 (0.6 nmole) was mixed
with 0.9 nmole of Targext99 (5'-TAT ATT ATA TTA TAT TAT ATT ATA TTA
TAT TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT
ATC TCG ATC CAT GAC CTC AGC-3') (SEQ ID NO:2). The mixture
contained 43 nmoles biotin-16-dUTP and 76 nmoles each of dATP, dCTP
and dGTP in 10 mM Tris-HCl, pH 7.9, 10 mM MgCl.sub.2, 1 mM DTT and
50 mM NaCl. T4 DNA polymerase exo minus (13 units) (Lucigen,
Middleton, Wis.) was added, and the mixture was incubated at
37.degree. C. for 90 min. The reaction was stopped with the
addition of EDTA to a final concentration of 12.5 mM. The resulting
poly-biotin cAMP was separated from free nucleotides using a
NucAway (Ambion, Foster City, Calif.) size exclusion spin column
equilibrated with phosphate buffered saline (PBS).
Example 3. Using cAMP Poly-Biotin to Quantify cAMP in a Sample
[0062] cAMP poly-biotin was used instead of cAMP-alkaline
phosphatase conjugate to quantify low concentrations of cAMP in
samples, in a competitive ELISA assay. Samples (100 .mu.l), serial
diluted standards, or a no cAMP control were added to microtiter
wells coated with goat-anti-rabbit antibody (Enzo Life Sciences).
50 .mu.l of cAMP poly-biotin (10 pmol/ml) were added to the wells
followed by an addition of 50 .mu.l antibody solution containing
rabbit polyclonal antibody to cAMP (Enzo Life Sciences). Controls
lacking rabbit anti-cAMP antibody were included. The plate was
sealed and mixed briefly on a plate shaker (.about.500 rpm) for 5 s
and incubated at 4.degree. C. for greater than 12 hr. The contents
of each well was emptied and washed three times, each with 400
.mu.l wash buffer. After washing, 200 .mu.l of
streptavidin-alkaline phosphatase (2 ng/ml) (Thermo Scientific) was
added to each well and incubated for 15 min on a plate shaker as
above. The contents of the plate were emptied and wells washed 4
times with wash buffer as before. Alkaline Phosphatase Yellow
(pNPP) Liquid Substrate (Sigma) (100 .mu.l) was then added to each
well and the plate incubated at room temperature for 1 hour without
shaking. Stop solution consisting of 2 N NaOH (50 .mu.l) was added
to each well and the optical density of each well was read at 405
nm using a plate reader.
[0063] The sensitivity of the assay, defined as the concentration
of cAMP measured at 2 standard deviations from the mean of 16
control wells without added cAMP was determined to be between
0.03-0.06 pmol/ml, a 5-10 fold increase over current assays using a
cAMP-alkaline phosphatase conjugate. A sample result is shown in
FIG. 1.
Example 4. Labeling a Biotinylated Oligonucleotide with Multiple
Digoxigenin Molecules
[0064] The oligonucleotide 5'-biotin-triethylene glycol
(TEG)-TTGCTGAGGTCATGGATCGAGA-3' (SEQ ID NO:1) was extended with
terminal deoxynucleotidyl transferase as follows. The
oligonucleotide (160 pmoles) was mixed with 25 nmoles dATP, 5 (or
10) nmoles digoxigenin-labeled dUTP (Roche Diagnostics,
Indianapolis, Ind.), 1 mM cobalt chloride, 1.times. TdT reaction
buffer (ENZO Life Sciences, Farmingdale, N.Y.) and 40 units of
terminal deoxynucleotidyl transferase in a total volume of 25
.mu.l. This was incubated at 30.degree. C. for 2 h. The reaction
was stopped by the addition of EDTA to 12.75 mM. The extended oligo
was purified using a Nucaway spin column (Ambion, Austin, Tex.),
following the manufacturer's instructions. Oligo dT.sub.21 (SEQ ID
NO:3) (800 pmoles) was added to bind to the poly dA tracts created
using terminal transferase. A complex of streptavidin with the
biotinylated digoxigenin labeled oligonucleotide was created by
mixing 150 pmoles of oligo with 136 pmoles streptavidin while
vortexing.
Example 5. Labeling an Oligonucleotide Conjugated to Streptavidin
with Multiple Digoxigenin Moieties
[0065] The oligonucleotide 5'-aminoC6-TTGCTGAGGTCATGGATCGAGA-3'
(SEQ ID NO:1) was attached to streptavidin as described in Example
16 of U.S. Patent Publication 2011/0318788. The streptavidin
oligonucleotide was extended with terminal transferase as follows.
The streptavidin oligonucleotide (180 pmoles) was mixed with 25
nmoles dATP, 5 (or 10) nmoles digoxigenin labeled dUTP (Roche
Diagnostics, Indianapolis, Ind.), 1 mM cobalt chloride, 1.times.TdT
reaction buffer (ENZO Life Sciences, Farmingdale, N.Y.) and 40
units of terminal deoxynucleotidyl transferase in a total volume of
25 .mu.l. This was incubated at 30.degree. C. for a total of 2
hours. The reaction was stopped by the addition of EDTA to 12.75
mM. The extended oligo streptavidin was purified using a Nucaway
spin column (Ambion, Austin, Tex.), following the manufacturer's
instructions. 800 pmoles of oligo dT.sub.21 (SEQ ID NO:3) was added
to bind to the poly dA tracts created using terminal
transferase.
Example 6. Labeling an Oligonucleotide Conjugated to Streptavidin
Using DNA Polymerase and a Template
[0066] The oligonucleotide 5'-aminoC6-TTGCTGAGGTCATGGATCGAGA-3'
(SEQ ID NO:1) was attached to streptavidin as described in Example
16 of U.S. Patent Publication 2011/0318788. The streptavidin
oligonucleotide was extended using a template oligonucleotide as
follows. A second oligo (5'-ACTTCTACTT CTACTTCTAC TTCTACTTCT
ACTTCTACTT CTACTTCTAC TCTTACTCTT ACTCTTCATT GGTCATCTCG ATCCATGACC
TCAGC-3') (SEQ ID NO:4) (MWG Operon, Huntsville, Ala.) was
hybridized to the oligo streptavidin. The streptavidin oligo
construct (113 pmoles) was incubated with 338 pmoles of template
oligo in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl.sub.2, 1 mM
dithiothreitol, pH 7.9, 6.1 nMol S4FB-dUTP, 21 nMol each of dATP,
dCTP and dGTP and 5.4 units of T4 DNA polymerase exo.sup.-
(Lucigen, Middleton, Wis.) in a total volume of 30 .mu.l, at
37.degree. C. for 90 min. The extension reaction was stopped with 1
.mu.l of 500 mM EDTA, and the unincorporated nucleotides are
removed using NucAway spin columns (Applied Biosystems/Ambion,
Austin, Tex.) as described by the manufacturer.
Example 7. Binding and Detecting Poly-Digoxigenin Labeled
Streptavidin
[0067] The streptavidin-poly-digoxigenin from Example 5 and a
streptavidin-alkaline phosphatase complex were tested by binding to
biotin attached to a 96-well microplate. Individual wells in the
microplate were coated with varying concentrations of
biotinylated-bovine serum albumin (BSA). Biotinylated BSA was
produced by mixing 0.75 .mu.mol BSA (fraction V, Sigma-Aldrich, St.
Louis, Mo.) with 3.75 .mu.mol ENZOTIN (NHS ester of biotin, ENZO
Life Sciences, Farmingdale, N.Y.) in 50 mM sodium tetraborate, pH
8.5 at 22.degree. C. for 4 hours. Unreacted biotin was removed
using a Zeba spin column (Thermo-Pierce, Rockford, Ill.) as
described by the manufacturer. The biotinylated-BSA (250 pg, 83.3
pg, 27.8 pg and 0 pg) was added to separate wells of black Maxisorb
(Nunc, Roskilde, Denmark) 96-well microplate, diluted in PBS. After
12 hours at 4.degree. C., the excess biotin BSA was washed out of
the plate using 3 washes of 0.05% Tween20.RTM. in PBS for 5 minutes
each. Blocking buffer (Thermo-Pierce, Rockford, Ill.) containing
0.05% Tween20.RTM. and 50 .mu.g/ml single-stranded salmon sperm DNA
was added to each well and the plate was incubated at 22.degree. C.
for 15 minutes with shaking. The blocking buffer was then removed,
and the streptavidin-poly digoxigenin or commercial streptavidin
alkaline phosphatase (Life Technologies, Carlsbad, Calif.) was then
added to each well (100 .mu.l, 4 nM) in the same blocking buffer
and incubated with shaking for 60 minutes at 22.degree. C. After 60
minutes, the streptavidin complexes were removed from the
digoxigenin wells, and those wells were washed once with 200 .mu.l
PBS with 0.05% Tween20.RTM.. After removal of the wash, 100 .mu.l
of alkaline phosphatase labeled digoxigenin antibody (Roche,
Indianapolis, Ind.) diluted 750 fold in blocking buffer was added
to those wells. Incubation was continued at 22.degree. C. for 60
minutes. All liquid was removed, and each well was washed 3 times
with 0.05% Tween20.RTM. in PBS, for 5 min each. CDP-Star (Life
Technologies, Carlsbad, Calif.) (100 .mu.l) was added to each well,
and the chemiluminescence produced was quantified using a BioTek
SynergyMX (Winooski, Vt.) plate reader. The results are shown in
FIG. 2.
[0068] All of the complexes with digoxigenin improved the signal
compared to commercial alkaline phosphatase-labeled streptavidin.
The oligonucleotide was more efficiently extended with terminal
transferase if it was not attached to streptavidin first. Extension
with T4 DNA polymerase on the streptavidin-oligonucleotide complex
appears to be efficient.
REFERENCES
[0069] Blomberg et al., 1999, Clin. Chem. 45:855-61. [0070] Caymen
Chemical Company, "Cyclic AMP EIA Kit" [0071] Chabardes et al.,
1980, J Clin Invest 65:439-448. [0072] Charlton and Porter, 2002,
Meth. Mol. Biol. 178:159-71. [0073] Enzo Life Sciences, "cAMP
Complete ELISA Kit" [0074] GE Life Sciences, "Cyclic-AMP Assay
Kit." [0075] Glover et al., 2003, J. Am. Chem. Soc. 125:9918-9919.
[0076] Grill and Cerasi, 1974, J Biol Chem 249:4196-4201. [0077]
Haynes, 1958, J Biol Chem 233:1220-1222. [0078] Lee et al., (1992)
Nucl. Acids Res. 20:2471-2488. [0079] Lipkin et al., 1959, Journal
of the American Chemical Society 81: 6198-6203. [0080] Mayilo et
al., 2009, Analytica Chimica Acta 646:119-22. [0081] O'Beirne and
Cooper, 1979, J. Histochem. Cytochem. 27:1148-62. [0082]
PerkinElmer, "AlphaScreen.RTM. cAMP Assay Kit" [0083] Promega,
"GloSensor.TM. cAMP Assay" [0084] Szentivanyi, 1968, Journal of
Allergy 42: 203-232. [0085] Wang et al., 2011, Assay Drug Dev
Technol 9: 522-531. [0086] Winter et al., 1994, Ann. Rev. Immunol.
12:433-55. [0087] Wong et al., 2008, Cancer Epidemiol. Biomarkers
17:3450-6. [0088] U.S. Pat. No. 4,081,525. [0089] U.S. Pat. No.
4,585,862. [0090] U.S. Pat. No. 5,945,526. [0091] U.S. Pat. No.
6,008,373. [0092] U.S. Pat. No. 7,514,551. [0093] U.S. Pat. No.
4,711,955. [0094] U.S. Pat. No. 5,241,060. [0095] U.S. Pat. No.
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[0101] In view of the above, it will be seen that several
objectives of the invention are achieved and other advantages
attained.
[0102] As various changes could be made in the above methods and
compositions without departing from the scope of the invention, it
is intended that all matter contained in the above description and
shown in the accompanying drawings shall be interpreted as
illustrative and not in a limiting sense.
[0103] All references cited in this specification are hereby
incorporated by reference. The discussion of the references herein
is intended merely to summarize the assertions made by the authors
and no admission is made that any reference constitutes prior art.
Applicants reserve the right to challenge the accuracy and
pertinence of the cited references.
Sequence CWU 1
1
4122DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1ttgctgaggt catggatcga ga
22299DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2tatattatat tatattatat tatattatat
tatattatat tatattatat tatattatat 60tatattatat tatattatat ctcgatccat
gacctcagc 99321DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 3tttttttttt tttttttttt t
21495DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 4acttctactt ctacttctac ttctacttct
acttctactt ctacttctac tcttactctt 60actcttcatt ggtcatctcg atccatgacc
tcagc 95
* * * * *