U.S. patent application number 14/309695 was filed with the patent office on 2014-12-18 for hydrolase enzyme substrates and uses thereof.
The applicant listed for this patent is General Atomics. Invention is credited to Xiaoru CHEN, Chong-Sheng YUAN.
Application Number | 20140370501 14/309695 |
Document ID | / |
Family ID | 45558418 |
Filed Date | 2014-12-18 |
United States Patent
Application |
20140370501 |
Kind Code |
A1 |
YUAN; Chong-Sheng ; et
al. |
December 18, 2014 |
HYDROLASE ENZYME SUBSTRATES AND USES THEREOF
Abstract
The present invention provides novel methods for determining the
presence or amount of a hydrolytic enzyme in a sample, based on
novel substrates for the enzymes, and also provides compositions
and methods that provide highly sensitive assay methods for such
hydrolytic enzymes.
Inventors: |
YUAN; Chong-Sheng; (San
Diego, CA) ; CHEN; Xiaoru; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
General Atomics |
San Diego |
CA |
US |
|
|
Family ID: |
45558418 |
Appl. No.: |
14/309695 |
Filed: |
June 19, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13352273 |
Jan 17, 2012 |
8795979 |
|
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14309695 |
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61433909 |
Jan 18, 2011 |
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Current U.S.
Class: |
435/6.11 ;
435/18; 435/19; 435/207; 435/21; 435/6.1; 435/6.12; 435/7.92;
536/4.1 |
Current CPC
Class: |
C07H 15/18 20130101;
G01N 2458/00 20130101; G01N 2333/938 20130101; C07H 15/10 20130101;
C12Q 1/34 20130101; C07H 15/203 20130101; G01N 33/573 20130101;
C12Q 1/42 20130101; C12Q 1/44 20130101; C12Q 1/6876 20130101 |
Class at
Publication: |
435/6.11 ;
536/4.1; 435/207; 435/18; 435/19; 435/21; 435/7.92; 435/6.1;
435/6.12 |
International
Class: |
C12Q 1/34 20060101
C12Q001/34; C12Q 1/42 20060101 C12Q001/42; C12Q 1/44 20060101
C12Q001/44; C07H 15/203 20060101 C07H015/203; C07H 15/10 20060101
C07H015/10 |
Claims
1. A hydrolytic enzyme substrate, which is a compound of formula
(I): ##STR00039## wherein: A is an aromatic or heteroaromatic
group, a 1-alkene or a 1-alkyne, each of which is optionally
substituted; each R is independently H or an optionally substituted
C1-C4 alkyl or aryl; n is an integer from 1-4; and X is a group
comprising a substrate moiety, wherein the substrate moiety
comprises a recognition component of a substrate for the hydrolytic
enzyme, and wherein the activity of said hydrolytic enzyme is
capable of hydrolyzing said compound of formula (I) to form
compounds (II) and (III): ##STR00040##
2-10. (canceled)
11. A combination, which comprises: a) a hydrolytic enzyme
substrate of claim 1; and b) a hydrolytic enzyme that is capable of
cleaving said hydrolytic enzyme substrate to produce an aryl
alcohol molecule or unsaturated aliphatic alcohol molecule as a
product of said cleavage reaction catalyzed by said hydrolytic
enzyme, wherein said aryl alcohol molecule or unsaturated aliphatic
alcohol molecule has a structure of said formula (II): ##STR00041##
wherein A, R and n are as defined in claim 1.
12-16. (canceled)
17. A method for assessing activity and/or amount of a hydrolytic
enzyme in a sample, which method comprises: a) contacting a
hydrolytic enzyme substrate of any of the embodiments 1-25 with a
sample containing or suspected of containing a hydrolytic enzyme
with a hydrolytic enzyme substrate having a structure of formula
(I): ##STR00042## under conditions where said hydrolytic enzyme, if
present in said sample, cleaves said substrate to produce an aryl
alcohol molecule or unsaturated aliphatic alcohol molecule having a
structure of formula (II) and a compound having a structure of
formula (III): ##STR00043## wherein A, R, n and X are as defined in
claim 1; and b) assessing the presence and/or amount of said aryl
alcohol molecule or unsaturated aliphatic alcohol molecule to
assess activity and/or amount of said hydrolytic enzyme in said
sample.
18. The method of claim 17, wherein the hydrolytic enzyme is an
esterase, a beta-galactosidase, or a glycosidase.
19. The method of claim 17, wherein the step of assessing the
presence and/or amount of the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule comprises oxidizing the aryl alcohol
molecule or unsaturated aliphatic alcohol molecule with an
oxidizing reagent.
20. The method of claim 19, wherein the presence and/or amount of
the aryl alcohol molecule or unsaturated aliphatic alcohol molecule
is assessed by oxidizing the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule with an aryl alcohol oxidase or an
aliphatic alcohol oxidase in the presence of oxygen to produce
H.sub.2O.sub.2 and assessing the presence and/or amount of the
H.sub.2O.sub.2.
21. The method of claim 17, wherein the presence and/or amount of
the aryl alcohol molecule or unsaturated aliphatic alcohol molecule
is assessed by oxidizing the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule with an aryl alcohol dehydrogenase or an
alcohol dehydrogenase in the presence of NAD.sup.+ or NADP.sup.+ to
produce NADH or NADPH, and assessing the presence and/or amount of
the NAD+, NADP+, NADH or NADPH.
22. The method of claim 17, wherein the presence and/or amount of
the aryl alcohol molecule or unsaturated aliphatic alcohol molecule
is assessed by: a) oxidizing the aryl alcohol molecule or
unsaturated aliphatic alcohol molecule with an aryl alcohol oxidase
or an aliphatic alcohol oxidase in the presence of oxygen to
produce aryl aldehyde molecule or unsaturated aliphatic aldehyde
molecule and H.sub.2O.sub.2; b) reducing the aryl aldehyde molecule
or unsaturated aliphatic aldehyde molecule with an aryl alcohol
dehydrogenase or an alcohol dehydrogenase in the presence of NADH
or NADPH to form a reaction cycle in which the reduced aryl alcohol
molecule or unsaturated aliphatic alcohol molecule is oxidized by
the aryl alcohol oxidase or an aliphatic alcohol oxidase in the
presence of oxygen to produce additional aryl aldehyde molecule or
unsaturated aliphatic aldehyde molecule and H.sub.2O.sub.2; and c)
assessing the presence and/or amount of the H.sub.2O.sub.2, or the
amount of NADH, NADPH, NAD+, or NADP+.
23. The method of claim 17, wherein the hydrolytic enzyme substrate
comprises at least a part of a .beta.-glycosidase substrate
molecule, and the hydrolytic enzyme is a .beta.-glycosidase or
beta-galactosidase.
24. The method of claim 23, wherein the step of assessing the
presence and/or amount of the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule comprises oxidizing the aryl alcohol
molecule or unsaturated aliphatic alcohol molecule with an
oxidizing reagent.
25. The method of claim 23, wherein the presence and/or amount of
the aryl alcohol molecule or unsaturated aliphatic alcohol molecule
is assessed by oxidizing the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule with an aryl alcohol oxidase or an
aliphatic alcohol oxidase in the presence of oxygen to produce
oxidized aryl alcohol molecule or unsaturated aliphatic alcohol
molecule and H.sub.2O.sub.2 and assessing the presence and/or
amount of the H.sub.2O.sub.2.
26. The method of claim 23, wherein the presence and/or amount of
the aryl alcohol molecule or unsaturated aliphatic alcohol molecule
is assessed by oxidizing the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule with an aryl alcohol dehydrogenase or an
alcohol dehydrogenase in the presence of NAD.sup.+ or NADP.sup.+ to
produce NADH or NADPH, and assessing the presence and/or amount of
the NAD+, NADP+, NADH or NADPH.
27. The method of claim 23, wherein the presence and/or amount of
the aryl alcohol molecule or unsaturated aliphatic alcohol molecule
is assessed by: a) oxidizing the aryl alcohol molecule or
unsaturated aliphatic alcohol molecule with an aryl alcohol oxidase
or an aliphatic alcohol oxidase in the presence of oxygen to
produce oxidized aryl alcohol molecule or unsaturated aliphatic
alcohol molecule and H.sub.2O.sub.2; b) reducing the oxidized aryl
alcohol molecule or unsaturated aliphatic alcohol molecule with an
aryl alcohol dehydrogenase or an alcohol dehydrogenase in the
presence of NADH or NADPH to form a reaction cycle in which the
reduced aryl alcohol molecule or unsaturated aliphatic alcohol
molecule is oxidized by the aryl alcohol oxidase or an aliphatic
alcohol oxidase in the presence of oxygen to produce additional
oxidized aryl alcohol molecule or unsaturated aliphatic alcohol
molecule and H.sub.2O.sub.2; and c) assessing the presence and/or
amount of the H.sub.2O.sub.2.
28. The method of claim 17, wherein the hydrolytic enzyme substrate
comprises at least a part of an alkaline phosphatase substrate
molecule, and the hydrolytic enzyme is an alkaline phosphatase.
29. The method of claim 28, wherein the step of assessing the
presence and/or amount of the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule comprises oxidizing the aryl alcohol
molecule or unsaturated aliphatic alcohol molecule with an
oxidizing reagent.
30. The method of claim 28, wherein the presence and/or amount of
the aryl alcohol molecule or unsaturated aliphatic alcohol molecule
is assessed by oxidizing the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule with an aryl alcohol oxidase or an
aliphatic alcohol oxidase in the presence of oxygen to produce
oxidized aryl alcohol molecule or unsaturated aliphatic alcohol
molecule and H.sub.2O.sub.2, and assessing the presence and/or
amount of the H.sub.2O.sub.2.
31. The method of claim 28, wherein the presence and/or amount of
the aryl alcohol molecule or unsaturated aliphatic alcohol molecule
is assessed by oxidizing the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule with an aryl alcohol dehydrogenase or an
alcohol dehydrogenase in the presence of NAD.sup.+ or NADP.sup.+ to
produce NADH or NADPH, and assessing the presence and/or amount of
the NAD+, NADP+, NADH or NADPH.
32. The method of claim 28, wherein the presence and/or amount of
the aryl alcohol molecule or unsaturated aliphatic alcohol molecule
is assessed by a) oxidizing the aryl alcohol molecule or
unsaturated aliphatic alcohol molecule with an aryl alcohol oxidase
or an aliphatic alcohol oxidase in the presence of oxygen to
produce oxidized aryl alcohol molecule or unsaturated aliphatic
alcohol molecule and H.sub.2O.sub.2; b) reducing the oxidized aryl
alcohol molecule or unsaturated aliphatic alcohol molecule with an
aryl alcohol dehydrogenase or an alcohol dehydrogenase in the
presence of NADH or NADPH to form a reaction cycle in which the
reduced aryl alcohol molecule or unsaturated aliphatic alcohol
molecule is oxidized by the aryl alcohol oxidase or an aliphatic
alcohol oxidase in the presence of oxygen to produce additional
oxidized aryl alcohol molecule or unsaturated aliphatic alcohol
molecule and H.sub.2O.sub.2; and c) assessing the presence and/or
amount of the H.sub.2O.sub.2.
33. The method of claim 17, which is conducted as part of an assay,
isolation and/or production of a target.
34. The method of claim 17, which is conducted as part of an
immunoassay, protein sequencing, nucleic acid amplification,
hybridization or sequencing.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/352,273, filed Jan. 17, 2012, now allowed,
which claims the priority benefit of U.S. provisional application
Ser. No. 61/433,909, filed Jan. 18, 2011, the contents of both
applications are incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] This invention generally relates to certain hydrolase enzyme
substrates and uses thereof. In particular, the invention provides
novel compounds that function as substrates for some hydrolytic
enzymes, whereby the hydrolytic enzyme converts the hydrolase
substrate into a hydrolysis product that can be readily detected,
e.g., by enzymatic methods described herein.
BACKGROUND OF THE INVENTION
[0003] Enzymes have been widely utilized as sensitive labels in a
number of biochemical systems, including immunoassays such as ELISA
systems, and nucleic acid assays such as PCR and sequencing
systems. The enzymes are often detected indirectly, based on their
activity, typically based on their transformation of substrate into
product, or of a co-factor between, e.g., oxidized and reduced
states.
[0004] In some implementations, the enzyme to be detected is
attached to a highly specific complexing or binding agent such as
an antibody. When the antibody binds to a target molecule to be
detected, the antibody complex can be detected by observing the
presence of the enzyme label attached to it; the enzyme is readily
detected based on its activity. In other systems, an
oligonucleotide to be expressed is labeled by linking it to a
nucleotide that encodes an enzyme that can function as a label.
When the oligonucleotide is expressed, the protein product that
includes the enzyme label, which facilitates detection, again based
on the activity of the enzyme.
[0005] Detecting the activity of the enzyme provides very efficient
signal amplification. Rather than detecting the often small amount
of enzyme (or target compound) present, one looks for the activity
of the enzyme, i.e., its effect on known substrates that can be
added in relatively large amounts. A single enzyme molecule can
catalyze transformation of a large number of substrate molecules
(e.g., an enzyme may catalyze 10.sup.7 reactions per minute: THE
IMMUNOASSAY HANDBOOK, 3.sup.rd ed. by David Wild, Elsevier Press,
pg. 194 (2005)), so the species actually detected can be a product
formed by the enzyme or the disappearance of a substrate or
co-factor consumed by the enzyme, instead of the enzyme itself.
Thus when observing the enzyme's activity, one detects large
numbers of substrate or product molecules rather than the enzyme
itself, which provides a highly amplified signal.
[0006] A number of such enzyme labels are known: the ones most
commonly used in immunoassays (e.g., ELISA) include horseradish
peroxidase and alkaline phosphatase. Others that have been used
include acetate kinase, firefly luciferase, xanthine oxidase,
beta-D-galactosidase, glucose oxidase, and glucose 6-phosphate
dehydrogenase. Id. at 194-195.
[0007] However, there remains a need for new methods to label
biochemical species to facilitate detection of extremely small
quantities, and novel enzyme labeling systems are thus needed.
There also remains a need for methods to detect trace amounts of
hydrolytic enzymes in other settings where they are not used just
as labels. The present invention provides such methods as well as
compounds and compositions for use in these methods and
conditions.
BRIEF SUMMARY OF THE INVENTION
[0008] In one aspect, the present invention provides for substrates
for certain hydrolytic enzymes, compositions containing these
substrates, and methods to use these substrates to determine the
presence of a hydrolytic enzyme that can process the substrates. In
some embodiments, the hydrolytic enzyme may be used as a label for
an assay system such as ELISA, for example, or a nucleic acid
encoding the hydrolytic enzyme may be attached to an
oligonucleotide to be expressed in such a way that expression of
the oligonucleotide produces a polypeptide that includes the
functional hydrolytic enzyme: the compositions and methods
described herein are useful to detect the hydrolytic enzymes in
these and other systems.
[0009] In one aspect, the invention provides for a compound of this
formula, which is a substrate for a hydrolytic enzyme of
interest:
##STR00001##
wherein:
[0010] A is an aromatic or heteroaromatic group, a 1-alkene or a
1-alkyne, each of which is optionally substituted;
[0011] each R is independently H or an optionally substituted C1-C4
alkyl or C6-C10 aryl;
[0012] n is an integer from 1-4;
[0013] and X is a group comprising a substrate moiety, [0014]
wherein the substrate moiety comprises a molecular fragment that is
a substrate for the hydrolytic enzyme, and wherein the activity of
the hydrolytic enzyme is capable of hydrolyzing the compound of
formula (I) to form compounds (II) and (III):
##STR00002##
[0015] In these compounds, A can be an aromatic or heteroaromatic
group, e.g., a 5-6 membered aromatic ring optionally containing up
to three heteroatoms selected from N, O and S as ring members; or a
bicyclic ring system having 8-10 ring members, up to four of which
can be heteroatoms selected from N, O and S. In some embodiments, A
is phenyl or naphthyl. A can be optionally substituted as described
herein, typically with up to three substituents selected from those
described herein as suitable for aryl or heteroaryl groups.
[0016] In alternative embodiments, A is a 1-alkenyl or 1-alkynyl
group, typically containing 2-10 carbon atoms and preferably 2-6
carbon atoms. In these embodiments, A can be substituted with the
groups described herein as suitable for alkyl groups, to the extent
valence permits such substitution. Typically, A is substituted with
up to three substituents in these embodiments.
[0017] In specific embodiments of these compounds, n is 1. Because
A is an aryl, heteroaryl, 1-alkene or 1-alkyne, when n is 1, the
compounds are of the formula:
##STR00003##
[0018] These embodiments are activated toward oxidation of the
hydroxyl group shown, because the A group makes the alcohol in
formula IIB a benzylic, allylic, propargylic, or similarly
activated hydroxyl. These compounds are particularly suitable for
oxidation by aryl alcohol oxidase, alcohol dehydrogenase, and/or
aryl alcohol dehydrogenase enzymes. In some embodiments of these
compounds, R is H, and the product of the oxidation is an aldehyde,
e.g., the product will be a benzaldehyde when A is a phenyl
moiety.
##STR00004##
[0019] These embodiments are also particularly well suited for use
in a cycling system that amplifies the detectable signal and
substantially increases sensitivity. The cycling system requires
the presence of an additional enzyme that oxidizes the initial
product of the hydrolysis reaction (the alcohol IIB shown above) to
form an oxidized product of formula II-ox; and a reducing enzyme to
reduce the oxidized product back to the alcohol IIB:
##STR00005##
[0020] This produces a cycling system that can amplify the
effective signal from the initial hydrolysis of the substrate of
Formula I. The cycling can be conducted using any suitable
reagents, such as the aryl alcohol oxidase and aryl alcohol
dehydrogenase described in Guillen and Evans, Appl. Environmental
Microbiol., 60(8):2811-17 (1994). The cycling system can be used to
detect a small amount or level of a hydrolytic enzyme of interest,
e.g., at millimolar, micromolar, nanomolar, picomolar, femtomolar,
attomolar or even sub-attomole, e.g., zeptomolar or yoctomolar,
level.
[0021] In the present system, the cyclic process increases the
amount of an analyte that can be detected and correlated with
enzymatic activity levels. Rather than detecting only the
hydrolysis product formed by the direct action of the enzyme, which
may already provide good sensitivity, one can detect instead a
by-product from the cycling reaction that is produced in either the
oxidation reaction or a subsequent reaction of the oxidized product
(ketone or aldehyde) as the initial hydrolysis product is cycled
between oxidized and reduced forms. The cycling reaction shown
above can produce large amounts of such by-products, e.g.,
H.sub.2O.sub.2, produced in the oxidation reaction, or NAD+ or
NADP+, produced in the reducing reaction. Additionally or
alternatively, one can monitor disappearance of NADH or NADPH
consumed in the reduction reaction that is involved when the
cycling reaction system is operating. Because the amount of these
detectable species can be much larger than the amount of the
hydrolytic enzyme substrate due to the cycling reaction,
sensitivity can be significantly increased by using such cycling
methods.
[0022] In some embodiments, the oxidation is accomplished by an
aryl alcohol oxidase using O.sub.2 as the oxidant, and producing
H.sub.2O.sub.2 as a by-product. This H.sub.2O.sub.2 can be detected
to determine the presence or amount of hydrolytic enzyme in the
reaction. Methods for determining H.sub.2O.sub.2 in small amounts
are well known in the art. Reagents for measuring H.sub.2O.sub.2
include a peroxidase enzyme, an aminoantipyrine (e.g.,
4-aminoantipyrine (4-AA)), a phenol, and/or an aniline analog. For
example, the Trinder reaction can be used, requiring a phenol or
aniline analog, a peroxidase such as horseradish peroxidase, and
4-AA. This method can be practiced without any need for the
reducing enzyme or the cycling reaction.
[0023] In some embodiments, a reducing enzyme is included in the
test milieu, typically a dehydrogenase (aryl alcohol dehydrogenase,
alcohol dehydrogenase), and a reducing co-factor is included (e.g.,
NADH, NADPH), often in large excess. The co-factor is oxidized to
form NAD+ or NADP+ during the cycling reaction, and the appearance
of this oxidized form (NAD+ or NADP+) or the disappearance of the
reduced form (NADH or NADPH) can be measured by methods well known
in the art. Because the reaction mixture also contains an enzyme
that oxidizes the alcohol of Formula (II) or (IIb), the result can
be a cycling assay system as further described herein.
[0024] The various reagents and/or enzymes, include the hydrolytic
enzymes, alcohol oxidase, aryl alcohol oxidase, alcohol
dehydrogenase, and/or aryl alcohol dehydrogenase, can be provided
and/or used in any suitable forms. In some embodiments, the various
reagents and/or enzymes are provided and/or used in isolated forms.
In other embodiments, the various reagents and/or enzymes are
provided and/or used in a mixture.
[0025] A more detailed description of certain embodiments of the
invention is provided below to illustrate its scope and
operation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1.1 illustrates the reaction of an enzyme substrate of
the invention having a benzyl-substituted galactose, reacting with
a beta-galactosidase to release a benzyl alcohol, which is then
oxidized by aryl alcohol oxidase and oxygen to a benzaldehyde and
hydrogen peroxide.
[0027] FIG. 1.2 illustrates an olefin-containing galactosidic
enzyme substrate that is hydrolyzed by beta-galactosidase to
release an allylic alcohol, followed by oxidation of the allylic
alcohol to an aldehyde and hydrogen peroxide.
[0028] FIG. 2 illustrates a benzylic galactosidic enzyme substrate
that is hydrolyzed by beta-galactosidase to form a benzylic
alcohol, and subsequent oxidation of the benzylic alcohol by an
additional enzyme (aryl alcohol dehydrogenase or alcohol
dehydrogenase). The enzymatic oxidation uses a co-factor, NAD+ or
NADP+, which gets reduced by the oxidation step to form NADH or
NADPH.
[0029] FIG. 3 illustrates use of aryl alcohol oxidase and oxygen to
oxidize the benzylic alcohol produced by a hydrolytic enzyme acting
on a hydrolytic enzyme substrate of the invention to form a
benzaldehyde and hydrogen peroxide, followed by a reduction of the
benzaldehyde back to the benzylic alcohol. The reduction uses NADH
or NADPH and an alcohol dehydrogenase, producing NAD+ or NADP+.
[0030] FIG. 4 illustrates a benzylic phosphate ester as a
hydrolytic enzyme substrate, hydrolyzed by alkaline phosphatase to
produce phosphate and a benzylic alcohol. The benzylic alcohol is
then oxidized by aryl alcohol oxidase and oxygen to produce
benzaldehyde and hydrogen peroxide; hydrogen peroxide formation can
be monitored to measure the rate of the reaction and thus detect
and/or quantify the amount of alkaline phosphatase present.
[0031] FIG. 5 illustrates a benzylic ester acting as a hydrolytic
enzyme substrate for an acetylesterase, releasing a benzylic
alcohol, which is then oxidized by an additional enzyme such as
aryl alcohol oxidase and oxygen. The reaction products include a
benzaldehyde and hydrogen peroxide, which can be measured by
methods known in the art.
[0032] FIG. 6 illustrates a benzylic ester of an alpha-amino acid
acting as a hydrolytic enzyme substrate for an alpha-amino acid
esterase. The enzymatic reaction releases a benzylic alcohol, which
is then oxidized by an additional enzyme such as aryl alcohol
oxidase and oxygen. The reaction products include a benzaldehyde
and hydrogen peroxide, which can be measured by methods known in
the art.
[0033] FIG. 7 illustrates a benzylic ester acting as a hydrolytic
enzyme substrate for a carboxylesterase, releasing a benzylic
alcohol, which is then oxidized by an additional enzyme such as
aryl alcohol oxidase and oxygen. The reaction products include a
benzaldehyde and hydrogen peroxide, which can be measured by
methods known in the art.
[0034] FIG. 8 illustrates a beta-galactosidase substrate as shown
in FIG. 1, and illustrates the option of using a reductive enzyme
(aryl alcohol dehydrogenase or alcohol dehydrogenase) to reduce the
benzaldehyde produced in FIG. 1, providing a cycling enzyme system.
The reduction step regenerates the benzylic alcohol and produces an
oxidized co-factor (NAD+ or NADP+), so the rate of consumption of
reduced co-factor or the rate of formation of the oxidized
co-factor can be monitored to measure the amount of
beta-galactosidase present. Alternatively or in addition, as shown
in FIG. 8, a Trinder reaction can be used to measure the hydrogen
peroxide produced to determine the amount of beta-galactosidase
present.
[0035] FIG. 9 illustrates a benzylic phosphate ester as a
hydrolytic enzyme substrate, reacting with alkaline phosphatase to
produce phosphate and a benzylic alcohol. The benzylic alcohol is
then oxidized by aryl alcohol oxidase and oxygen to produce
benzaldehyde and hydrogen peroxide, as shown in FIG. 4. FIG. 9
further illustrates using a reductive enzyme (aryl alcohol
dehydrogenase or alcohol dehydrogenase) to reduce the benzaldehyde,
providing a cycling enzyme system. The reduction step regenerates
the benzylic alcohol and produces an oxidized co-factor (NAD+ or
NADP+), so the rate of consumption of reduced co-factor or the rate
of formation of the oxidized co-factor can be monitored to measure
the amount of alkaline phosphatase present. Alternatively or in
addition, as shown in FIG. 8, a Trinder reaction can be used to
measure the hydrogen peroxide produced to determine the amount of
alkaline phosphatase present.
[0036] FIG. 10 illustrates detection of aryl alcohol with an
exemplary AAO/AAD cycling system.
[0037] FIG. 11 illustrates detection of alkaline phosphatase with
an exemplary AAO/AAD cycling system.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention provides for substrates for hydrolytic
enzymes that provide for a highly efficient enzyme activity
detection system. The substrates include a recognition moiety that
make them specifically recognizable by the hydrolytic enzyme of
interest, and that can function as part of the substrate for the
hydrolytic enzyme. The recognition moiety is covalently linked to a
molecular fragment that can be cleaved off by the action of the
hydrolytic enzyme. Suitable examples of these substrates are
depicted in FIGS. 1-9.
[0039] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections that follow.
A. DEFINITIONS
[0040] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this invention belongs. All
patents, applications, published applications and other
publications referred to herein are incorporated by reference in
their entireties. If a definition set forth in this section is
contrary to or otherwise inconsistent with a definition set forth
in the patents, applications, published applications and other
publications that are herein incorporated by reference, the
definition set forth in this section prevails over the definition
that is incorporated herein by reference.
[0041] As used herein, "a" or "an" means "at least one" or "one or
more."
[0042] As used herein, "sample" refers to anything which may
contain an analyte for which an analyte assay is desired. The
sample may be a biological sample, such as a biological fluid or a
biological tissue. Examples of biological fluids include urine,
blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal
fluid, tears, mucus, amniotic fluid or the like. Biological tissues
are aggregates of cells, usually of a particular kind together with
their intercellular substance that form one of the structural
materials of a human, animal, plant, bacterial, fungal or viral
structure, including connective, epithelium, muscle and nerve
tissues. Examples of biological tissues also include organs,
tumors, lymph nodes, arteries and individual cell(s).
[0043] As used herein, "blood sample" refers to a whole blood
sample or a plasma or serum fraction derived therefrom. Preferably,
the blood sample refers to a human blood sample such as whole blood
or a plasma or serum fraction derived therefrom. Also preferably,
the blood sample is pre-treated before the assay by removing
substantially all hemoglobin (i.e., red blood cells) in order to
eliminate or significantly reduce the oxidative interference from
the hemoglobin molecules.
[0044] As used herein the term "whole blood" refers to a blood
sample that has not been fractionated and contains both cellular
and fluid components. As used herein, "whole blood" refers to
freshly drawn blood which is tested before it clots, or a
conventionally-drawn blood sample, which may be drawn into a
vacutainer, and which may contain an anticoagulant, such as
lithium-heparin, EDTA, etc., or to which one or more other standard
clinical agents may be added in the course of routine clinical
testing.
[0045] As used herein, the phrase "substantially all hemoglobin has
been removed" refers to a blood sample wherein preferably at least
about 50%, 60% or 70%, more preferably, at least about 80%, 90% or
95%, and most preferably, at least about 96%, 97%, 98%, 99 or 100%
of all hemoglobin-containing red blood cells in the sample have
been removed to eliminate or significantly reduce the oxidative
interference from hemoglobin.
[0046] As used herein, the term "plasma" refers to the fluid,
non-cellular component of the whole blood. Depending on the
separation method used, plasma may be completely free of cellular
components, or may contain various amounts of platelets and/or a
small amount of other cellular components. Because plasma includes
various clotting factors such as fibrinogen, the term "plasma" is
distinguished from "serum" as set forth below.
[0047] As used herein, the term "serum" refers to whole mammalian
serum, such as whole human serum. Further, as used herein, "serum"
refers to blood plasma from which clotting factors (e.g.,
fibrinogen) have been removed.
[0048] As used herein, the term "fluid" refers to any composition
that can flow. Fluids thus encompass compositions that are in the
form of semi-solids, pastes, solutions, aqueous mixtures, gels,
lotions, creams and other such compositions.
[0049] As used herein, the term "disease" or "disorder" refers to a
pathological condition in an organism resulting from, e.g.,
infection or genetic defect, and characterized by identifiable
symptoms.
[0050] As used herein, "contacting" means bringing two or more
components together. "Contacting" can be achieved by mixing all the
components in a fluid or semi-fluid mixture. "Contacting" can also
be achieved when one or more components are brought into contact
with one or more other components on a solid surface such as a
solid tissue section or a substrate.
[0051] As used herein, the term "chromogenic substrate" refers to a
chemical composition that can participate in a particular enzymatic
reaction as either a donor or an acceptor for the reaction and that
changes color during the reaction. For example, myeloperoxidase
converts hydrogen peroxide to water by borrowing two hydrogen atoms
from a donor molecule. When the donor molecule is a chromogenic
substrate, the oxidation of the chromogenic substrate causes the
substrate to change to a detectable color. For example,
3,3',5,5'-tetramethylbenzidine (TMB) is colorless in the reduced
state but blue in the oxidized state or yellow in the diamine
state.
[0052] As used herein, the term "non-chromogenic co-substrate"
refers to a chemical composition that participates in the same
enzymatic reaction as the chromogenic substrate but does not change
color during the reaction. In the example above, hydrogen peroxide
is a non-chromogenic co-substrate because both water and hydrogen
peroxide are colorless.
[0053] As used herein, the term "comparing" generally means
examining in order to note similarities or differences between two
or more values. Preferably, "comparing" refers to quantitative
comparisons such as, for example, subtracting one value from
another, calculating a ratio of two values, calculating a
percentage of one value with respect to another, or combining these
types of calculations to produce a single number. As used herein,
"comparing" further refers to comparisons made by a human,
comparisons made by a computer or other processor, and comparisons
made by a human in combination with a computer or other
processor.
[0054] As used herein, the terms "alkyl," "alkenyl" and "alkynyl"
unless otherwise indicated include straight-chain, branched-chain
and cyclic monovalent hydrocarbyl radicals, and combinations of
these, which contain only C and H when they are unsubstituted.
Examples include methyl, ethyl, isobutyl, cyclohexyl,
cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. The total
number of carbon atoms in each such group is sometimes described
herein, e.g., when the group can contain up to ten carbon atoms it
can be represented as 1-10C or as C1-C10 or C1-10. When heteroatoms
(e.g., N, O and S typically) are allowed to replace carbon atoms as
in heteroalkyl groups, for example, the numbers describing the
group, though still written as e.g. C1-C6, represent the sum of the
number of carbon atoms in the group plus the number of such
heteroatoms that are included as replacements for carbon atoms in
the ring or chain being described.
[0055] Typically, the alkyl, alkenyl and alkynyl substituents of
the invention contain 1-10C (alkyl) or 2-10C (alkenyl or alkynyl).
Preferably they contain 1-8C (alkyl) or 2-8C (alkenyl or alkynyl).
Sometimes they contain 1-4C (alkyl) or 2-4C (alkenyl or alkynyl). A
single group can include more than one type of multiple bond, or
more than one multiple bond; such groups are included within the
definition of the term "alkenyl" when they contain at least one
carbon-carbon double bond, and are included within the term
"alkynyl" when they contain at least one carbon-carbon triple
bond.
[0056] The terms "1-alkene" and "1-alkyne" as used herein refer to
an alkene or alkyne respectively that is attached to a base
molecule being described through a carbon atom that is double or
triple bonded to an adjacent carbon atom of the alkene or alkyne
moiety.
[0057] Alkyl, alkenyl and alkynyl groups are often substituted to
the extent that such substitution makes sense chemically. Typical
substituents include, but are not limited to, halo, .dbd.O,
.dbd.N--CN, .dbd.N--OR, .dbd.NR, OR, NR.sub.2, SR, SO.sub.2R,
SO.sub.2NR.sub.2, NRSO.sub.2R, NRCONR.sub.2, NRCOOR, NRCOR, CN,
COOR, CONR.sub.2, OOCR, COR, and NO.sub.2, wherein each R is
independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C3-C8
heterocyclyl, C4-C10 heterocyclyclalkyl, C1-C8 acyl, C2-C8
heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl,
C2-C8 heteroalkynyl, C6-C10 aryl, or C5-C10 heteroaryl, and each R
is optionally substituted with halo, .dbd.O, .dbd.N--CN,
.dbd.N--OR', .dbd.NR', OR', NR'.sub.2, SR', SO.sub.2R',
SO.sub.2NR'.sub.2, NR'SO.sub.2R', NR'CONR'.sub.2, NR'COOR',
NR'COR', CN, COOR', CONR'.sub.2, OOCR', COR', and NO.sub.2, wherein
each R' is independently H, or an unsubstituted group selected from
C1-C8 alkyl, C2-C8 heteroalkyl, C3-C8 heterocyclyl, C4-C10
heterocyclyclalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl and
C5-C10 heteroaryl. Alkyl, alkenyl and alkynyl groups can also be
substituted by C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10
heteroaryl, each of which can be substituted by the substituents
that are appropriate for the particular group. Where a substituent
group contains two R or R' groups on the same or adjacent atoms
(e.g., --NR.sub.2, or --NR--C(O)R), the two R or R' groups can
optionally be taken together with the atoms in the substituent
group to which they are attached to form a ring having 5-8 ring
members, which can be substituted as allowed for the R or R'
itself, and can contain an additional heteroatom (N, O or S) as a
ring member.
[0058] "Heteroalkyl", "heteroalkenyl", and "heteroalkynyl" and the
like are defined similarly to the corresponding hydrocarbyl (alkyl,
alkenyl and alkynyl) groups, but the `hetero` terms refer to groups
that contain 1-3 O, S or N heteroatoms or combinations thereof
within the backbone residue; thus at least one carbon atom of a
corresponding alkyl, alkenyl, or alkynyl group is replaced by one
of the specified heteroatoms to form a heteroalkyl, heteroalkenyl,
or heteroalkynyl group. The typical and preferred sizes for
heteroforms of alkyl, alkenyl and alkynyl groups are generally the
same as for the corresponding hydrocarbyl groups, and the
substituents that may be present on the heteroforms are the same as
those described above for the hydrocarbyl groups. For reasons of
chemical stability, it is also understood that, unless otherwise
specified, such groups do not include more than two contiguous
heteroatoms except where an oxo group is present on N or S as in a
nitro or sulfonyl group.
[0059] While "alkyl" as used herein includes cycloalkyl and
cycloalkylalkyl groups, the term "cycloalkyl" may be used herein to
specifically describe a carbocyclic non-aromatic group that is
connected via a ring carbon atom, and "cycloalkylalkyl" may be used
to describe a carbocyclic non-aromatic group that is connected to
the molecule through an alkyl linker. Similarly, "heterocyclyl" (or
the equivalent term `heterocycloalkyl`) may be used to describe a
non-aromatic cyclic group that contains at least one heteroatom as
a ring member and that is connected to the molecule via a ring
atom, which may be C or N; and "heterocyclylalkyl" may be used to
describe such a group that is connected to another molecule through
a linker. The sizes and substituents that are suitable for the
cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl
groups are the same as those described above for alkyl groups. As
used herein, these terms also include rings that contain a double
bond or two, as long as the ring is not aromatic.
[0060] As used herein, "acyl" encompasses groups comprising an
alkyl, alkenyl, alkynyl, aryl or arylalkyl radical attached at one
of the two available valence positions of a carbonyl carbon atom,
and heteroacyl refers to the corresponding groups wherein at least
one carbon other than the carbonyl carbon has been replaced by a
heteroatom chosen from N, O and S. Thus heteroacyl includes, for
example, --C(.dbd.O)OR and --C(.dbd.O)NR.sub.2 as well as
--C(.dbd.O)-heteroaryl.
[0061] Acyl and heteroacyl groups are bonded to any group or
molecule to which they are attached through the open valence of the
carbonyl carbon atom. Typically, they are C1-C8 acyl groups, which
include formyl, acetyl, pivaloyl, and benzoyl, and C2-C8 heteroacyl
groups, which include methoxyacetyl, ethoxycarbonyl, and
4-pyridinoyl. The hydrocarbyl groups, aryl groups, and heteroforms
of such groups that comprise an acyl or heteroacyl group can be
substituted with the substituents described herein as generally
suitable substituents for each of the corresponding component of
the acyl or heteroacyl group.
[0062] "Aromatic" moiety or "aryl" moiety refers to a monocyclic or
fused bicyclic moiety having the well-known characteristics of
aromaticity; examples include phenyl and naphthyl. Similarly,
"heteroaromatic" and "heteroaryl" refer to C5-C6 monocyclic or
C8-C10 fused bicyclic ring systems which contain as ring members
one or more heteroatoms selected from O, S and N. The inclusion of
a heteroatom permits aromaticity in 5-membered rings as well as
6-membered rings. Typical heteroaromatic systems include monocyclic
C5-C6 aromatic groups such as pyridyl, pyrimidyl, pyrazinyl,
thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, and
imidazolyl and the fused bicyclic moieties formed by fusing one of
these monocyclic groups with a phenyl ring or with any of the
heteroaromatic monocyclic groups to form a C8-C10 bicyclic group
such as indolyl, benzimidazolyl, indazolyl, benzotriazolyl,
isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl,
pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolinyl, and the
like. Any monocyclic or fused ring bicyclic system which has the
characteristics of aromaticity in terms of electron distribution
throughout the ring system is included in this definition. It also
includes bicyclic groups where at least the ring which is directly
attached to the remainder of the molecule has the characteristics
of aromaticity. Typically, the ring systems contain 5-12 ring
member atoms. Preferably the monocyclic heteroaryls contain 5 or 6
ring members, and the bicyclic heteroaryls contain 8, 9 or 10 ring
members.
[0063] Aryl and heteroaryl moieties may be substituted with a
variety of substituents including C1-C8 alkyl, C2-C8 alkenyl, C2-C8
alkynyl, C3-C8 cycloalkyl, C5-C12 aryl, C1-C8 acyl, and heteroforms
of these, each of which can itself be further substituted; other
substituents for aryl and heteroaryl moieties include halo, OR,
NR.sub.2, SR, SO.sub.2R, SO.sub.2NR.sub.2, NRSO.sub.2R,
NRCONR.sub.2, NRCOOR, NRCOR, CN, COOR, CONR.sub.2, OOCR, COR, and
NO.sub.2, wherein each R is independently H, C1-C8 alkyl, C2-C8
heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl,
C2-C8 heteroalkynyl, C3-C8 heterocyclyl, C4-C10 heterocyclyclalkyl,
C6-C10 aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12
heteroarylalkyl, and each R is optionally substituted as described
above for alkyl groups. Where a substituent group contains two R or
R' groups on the same or adjacent atoms (e.g., --NR2, or
--NR--C(O)R), the two R or R' groups can optionally be taken
together with the atoms in the substituent group to which they are
attached to form a ring having 5-8 ring members, which can be
substituted as allowed for the R or R' itself, and can contain an
additional heteroatom (N, O or S) as a ring member. The substituent
groups on an aryl or heteroaryl group may of course be further
substituted with the groups described herein as suitable for each
type of such substituents or for each component of the substituent.
Thus, for example, an arylalkyl substituent may be substituted on
the aryl portion with substituents described herein as typical for
aryl groups, and it may be further substituted on the alkyl portion
with substituents described herein as typical or suitable for alkyl
groups.
[0064] Similarly, "arylalkyl" and "heteroarylalkyl" refer to
aromatic and heteroaromatic ring systems which are bonded to their
attachment point through a linking group such as an alkylene,
including substituted or unsubstituted, saturated or unsaturated,
cyclic or acyclic linkers. Typically the linker is C1-C8 alkyl or a
hetero form thereof. These linkers may also include a carbonyl
group, thus making them able to provide substituents as an acyl or
heteroacyl moiety. An aryl or heteroaryl ring in an arylalkyl or
heteroarylalkyl group may be substituted with the same substituents
described above for aryl groups. Preferably, an arylalkyl group
includes a phenyl ring optionally substituted with the groups
defined above for aryl groups and a C1-C4 alkylene that is
unsubstituted or is substituted with one or two C1-C4 alkyl groups
or heteroalkyl groups, where the alkyl or heteroalkyl groups can
optionally cyclize to form a ring such as cyclopropane, dioxolane,
or oxacyclopentane. Similarly, a heteroarylalkyl group preferably
includes a C5-C6 monocyclic heteroaryl group that is optionally
substituted with the groups described above as substituents typical
on aryl groups and a C1-C4 alkylene that is unsubstituted or is
substituted with one or two C1-C4 alkyl groups or heteroalkyl
groups, or it includes an optionally substituted phenyl ring or
C5-C6 monocyclic heteroaryl and a C1-C4 heteroalkylene that is
unsubstituted or is substituted with one or two C1-C4 alkyl or
heteroalkyl groups, where the alkyl or heteroalkyl groups can
optionally cyclize to form a ring such as cyclopropane, dioxolane,
or oxacyclopentane.
[0065] Where an arylalkyl or heteroarylalkyl group is described as
optionally substituted, the substituents may be on either the alkyl
or heteroalkyl portion or on the aryl or heteroaryl portion of the
group. The substituents optionally present on the alkyl or
heteroalkyl portion are the same as those described above for alkyl
groups generally; the substituents optionally present on the aryl
or heteroaryl portion are the same as those described above for
aryl groups generally.
[0066] "Arylalkyl" groups as used herein are hydrocarbyl groups if
they are unsubstituted, and are described by the total number of
carbon atoms in the ring and alkylene or similar linker. Thus a
benzyl group is a C7-arylalkyl group, and phenylethyl is a
C8-arylalkyl.
[0067] "Heteroarylalkyl" as described above refers to a moiety
comprising an aryl group that is attached through a linking group,
and differs from "arylalkyl" in that at least one ring atom of the
aryl moiety or one atom in the linking group is a heteroatom
selected from N, O and S. The heteroarylalkyl groups are described
herein according to the total number of atoms in the ring and
linker combined, and they include aryl groups linked through a
heteroalkyl linker; heteroaryl groups linked through a hydrocarbyl
linker such as an alkylene; and heteroaryl groups linked through a
heteroalkyl linker. Thus, for example, C7-heteroarylalkyl would
include pyridylmethyl, phenoxy, and N-pyrrolylmethoxy.
[0068] "Alkylene" as used herein refers to a divalent hydrocarbyl
group; because it is divalent, it can link two other groups
together. Typically it refers to --(CH.sub.2).sub.n-- where n is
1-8 and preferably n is 1-4, though where specified, an alkylene
can also be substituted by other groups, and can be of other
lengths, and the open valences need not be at opposite ends of a
chain. Thus --CH(Me)- and --C(Me).sub.2- may also be referred to as
alkylenes, as can a cyclic group such as cyclopropan-1,1-diyl.
Where an alkylene group is substituted, the substituents include
those typically present on alkyl groups as described herein.
[0069] In general, any alkyl, alkenyl, alkynyl, alkylene, acyl, or
aryl or arylalkyl group or any heteroform of one of these groups
that is contained in a substituent may itself optionally be
substituted by additional substituents. The nature of these
substituents is similar to those recited with regard to the primary
substituents themselves if the substituents are not otherwise
described. Thus, where an embodiment of, for example, R.sup.7 is
optionally substituted alkyl, this alkyl may optionally be
substituted by the remaining substituents listed as embodiments for
R.sup.7 where this makes chemical sense, and where this does not
undermine the size limit provided for the alkyl per se; e.g., alkyl
substituted by alkyl or by alkenyl would simply extend the upper
limit of carbon atoms for these embodiments, and is not included.
However, alkyl substituted by aryl, amino, alkoxy, .dbd.O, and the
like would be included within the scope of the invention, and the
atoms of these substituent groups are not counted in the number
used to describe the alkyl, alkenyl, etc. group that is being
described. Where no number of substituents is specified, each such
alkyl, alkenyl, alkynyl, acyl, or aryl group may be substituted
with a number of substituents according to its available valences;
in particular, any of these groups may be substituted with fluorine
atoms at any or all of its available valences, for example.
[0070] "Heteroform" as used herein refers to a derivative of a
group such as an alkyl, aryl, or acyl, wherein at least one carbon
atom of the designated carbocyclic group has been replaced by a
heteroatom selected from N, O and S. Thus the heteroforms of alkyl,
alkenyl, alkynyl, acyl, aryl, and arylalkyl are heteroalkyl,
heteroalkenyl, heteroalkynyl, heteroacyl, heteroaryl, and
heteroarylalkyl, respectively. It is understood that no more than
two N, O or S atoms are ordinarily connected sequentially, except
where an oxo group is attached to N or S to form a nitro or
sulfonyl group.
[0071] "Optionally substituted" as used herein indicates that the
particular group or groups being described may have no non-hydrogen
substituents, or the group or groups may have one or more
non-hydrogen substituents. If not otherwise specified, the total
number of such substituents that may be present is equal to the
number of H atoms present on the unsubstituted form of the group
being described. Where an optional substituent is attached via a
double bond, such as a carbonyl oxygen (.dbd.O), the group takes up
two available valences, so the total number of substituents that
may be included is reduced according to the number of available
valences.
[0072] "Halo", as used herein includes fluoro, chloro, bromo and
iodo. Fluoro and chloro are often preferred.
[0073] "Amino" as used herein refers to NH.sub.2, but where an
amino is described as "substituted" or "optionally substituted",
the term includes NR'R'' wherein each R' and R'' is independently
H, or is an alkyl, alkenyl, alkynyl, acyl, aryl, or arylalkyl group
or a heteroform of one of these groups, and each of the alkyl,
alkenyl, alkynyl, acyl, aryl, or arylalkyl groups or heteroforms of
one of these groups is optionally substituted with the substituents
described herein as suitable for the corresponding group. The term
also includes forms wherein R' and R'' are linked together to form
a 3-8 membered ring which may be saturated, unsaturated or aromatic
and which contains 1-3 heteroatoms independently selected from N, O
and S as ring members, and which is optionally substituted with the
substituents described as suitable for alkyl groups or, if NR'R''
is an aromatic group, it is optionally substituted with the
substituents described as typical for heteroaryl groups.
[0074] "Amino acid" as used herein refers to an amino substituted
carboxylic acid compound; typical examples are the 20 common
alpha-amino acids, as well as analogs thereof having the amine in
the beta or gamma position relative to the carboxylic acid.
"Alpha-amino acid" as used herein refers to an amino acid of the
formula HO.sub.2C--CH(NH.sub.2)--R.sup.a, where R.sup.a is an
optionally substituted C1-C6 alkyl group, or optionally substituted
aryl or arylalkyl group, or optionally substituted heteroaryl or
heteroarylalkyl group. Specific examples include glycine, alanine,
valine, leucine, isoleucine, serine, threonine, aspartic acid,
glutamic acid, glutamine, phenylalanine, tyrosine, tryptophan,
lysine, histidine, methionine, cysteine, arginine, asparagine, and
proline.
[0075] "Saccharide" as used herein refers to a carbohydrate moiety
containing one or more sugars, typically in a branched or
unbranched chain of sugars. Saccharides are typically of the
formula (CH.sub.2O).sub.n, where n is an integer, such as 1-1000 or
3-50, or 5-25. Typical examples are glucose, sucrose, starches, and
cellulose. These saccharides can be monosaccharides, disaccharides,
or polysaccharides; the term `oligosaccharides` is used to describe
saccharides that consist of about 3 to 25 sugar groups, usually in
an unbranched chain.
[0076] "A recognition component of a substrate for a hydrolytic
enzyme" or a "recognition moiety" refers to a portion of an enzyme
substrate molecule that is sufficiently similar to a portion of a
natural substrate for the hydrolytic enzyme to cause the hydrolytic
enzyme to bind to the enzyme substrate molecule.
[0077] As used herein, an "esterase" refers to an enzyme that
splits esters into an acid and an alcohol in a chemical reaction
with water called hydrolysis. A wide range of different esterases
exist that differ in their substrate specificity, their protein
structure, and their biological function. Exemplary esterases
include acetylesterases, thiolester hydrolases, phosphoric
monoester hydrolases (or phosphomonoesterases), phosphodiesterases,
triphosphoric monoester hydrolases, sulfuric ester hydrolases
(sulfatases), diphosphoric monoester hydrolases, phosphoric
triester hydrolases, exonucleases (deoxyribonucleases and
ribonucleases), and endonucleases (deoxyribonucleases and
ribonucleases). It is intended to encompass esterase with
conservative amino acid substitutions or functional fragments that
do not substantially alter its activity.
[0078] As used herein, a "glycoside hydrolase (also called
glycosidase)" refers to an enzyme that catalyzes the hydrolysis of
the glycosidic linkage to release smaller sugars. Glycoside
hydrolases are typically classified into EC 3.2.1 as enzymes
catalyzing the hydrolysis of O- or S-glycosides. Glycoside
hydrolases can also be classified according to the stereochemical
outcome of the hydrolysis reaction: thus they can be classified as
either retaining or inverting enzymes. Glycoside hydrolases can
also be classified as exo or endo acting, dependent upon whether
they act at the (usually non-reducing) end or in the middle,
respectively, of an oligo/polysaccharide chain. Glycoside
hydrolases may also be classified by sequence or structure based
methods. Exemplary glycoside hydrolases include
.beta.-galactosidase (also called beta-gal or (.beta.-gal),
glucosidase, xylannase, lactase, amylase, chitinase, sucrase,
maltase, neuraminidase, invertase, hyaluronidase and lysozyme. It
is intended to encompass glycoside hydrolase with conservative
amino acid substitutions or functional fragments that do not
substantially alter its activity.
[0079] As used herein, a "phosphatase" refers to an enzyme that
removes a phosphate group from its substrate by hydrolysing
phosphoric acid monoesters into a phosphate ion and a molecule with
a free hydroxyl group. Phosphatases can be subdivided based upon
their substrate specificity, such as tyrosine specific
phosphatases, serine/threonine specific phosphatases, dual
specificity phosphatases, histidine specific phosphatases and lipid
phosphatases. Exemplary phosphatases include alkaline phosphatases
(ALP, ALKP). It is intended to encompass phosphatase with
conservative amino acid substitutions or functional fragments that
do not substantially alter its activity.
[0080] As used herein, an "alcohol oxidase" refers to an enzyme
that catalyzes the following chemical reaction:
a primary alcohol+O.sub.2an aldehyde+H.sub.2O.sub.2
The systematic name of this enzyme class is alcohol:oxygen
oxidoreductase. This enzyme is also called ethanol oxidase. It is
intended to encompass alcohol oxidase with conservative amino acid
substitutions or functional fragments that do not substantially
alter its activity.
[0081] As used herein, an "aryl alcohol oxidase" refers to an
enzyme that catalyzes the following chemical reaction:
an aromatic primary alcohol+O.sub.2an aromatic
aldehyde+H.sub.2O.sub.2
The systematic name of this enzyme class is aryl-alcohol:oxygen
oxidoreductase. Other names in common use include veratryl alcohol
oxidase, and arom. alcohol oxidase. It is intended to encompass
aryl alcohol oxidase with conservative amino acid substitutions or
functional fragments that do not substantially alter its
activity.
[0082] As used herein, an "aryl alcohol dehydrogenase" refers to an
enzyme that catalyzes the following chemical reaction:
an aromatic alcohol+NAD.sup.+an aromatic aldehyde+NADH+H.sup.+
The systematic name of this enzyme class is aryl-alcohol:NAD+
oxidoreductase. Other names in common use or examples include
p-hydroxybenzyl alcohol dehydrogenase, benzyl alcohol
dehydrogenase, and coniferyl alcohol dehydrogenase. It is intended
to encompass aryl alcohol dehydrogenase with conservative amino
acid substitutions or functional fragments that do not
substantially alter its activity.
[0083] As used herein, an "alcohol dehydrogenase (ADH)" refers to a
group of dehydrogenase enzymes that occur in many organisms and
facilitate the interconversion between alcohols and aldehydes or
ketones with the reduction of NAD.sup.+ to NADH. It is intended to
encompass alcohol dehydrogenase with conservative amino acid
substitutions or functional fragments that do not substantially
alter its activity.
[0084] As used herein the term "assessing" is intended to include
quantitative and qualitative determination in the sense of
obtaining an absolute value for the amount or concentration of the
analyte present in the sample, and also of obtaining an index,
ratio, percentage, visual or other value indicative of the level of
analyte in the sample. Assessment may be direct or indirect and the
chemical species actually detected need not of course be the
analyte itself but may for example be a derivative thereof or some
further substance.
[0085] As used herein, a "binding reagent (or binder)" refers to
any substance that binds to target or analyte with desired affinity
and/or specificity. Non-limiting examples of the binding reagent
include cells, cellular organelles, viruses, particles,
microparticles, molecules, or an aggregate or complex thereof, or
an aggregate or complex of molecules.
[0086] As used herein, "antibody" includes not only intact
polyclonal or monoclonal antibodies, but also fragments thereof
(such as Fab, Fab', F(ab').sub.2, Fv), single chain (ScFv), a
diabody, a multi-specific antibody formed from antibody fragments,
mutants thereof, fusion proteins comprising an antibody portion,
and any other modified configuration of the immunoglobulin molecule
that comprises an antigen recognition site of the required
specificity. An antibody includes an antibody of any class, such as
IgG, IgA, or IgM (or sub-class thereof), and the antibody need not
be of any particular class.
[0087] As used herein, the term "specifically binds" refers to the
specificity of a binding reagent, e.g., an antibody, such that it
preferentially binds to a defined analyte or target. Recognition by
a binding reagent or an antibody of a particular analyte or target
in the presence of other potential targets is one characteristic of
such binding. In some embodiments, a binding reagent that
specifically binds to an analyte avoids binding to other
interfering moiety or moieties in the sample to be tested.
[0088] As used herein the term "avoids binding" refers to the
specificity of particular binding reagents, e.g., antibodies or
antibody fragments. Binding reagents, antibodies or antibody
fragments that avoid binding to a particular moiety generally
contain a specificity such that a large percentage of the
particular moiety would not be bound by such binding reagents,
antibodies or antibody fragments. This percentage generally lies
within the acceptable cross reactivity percentage with interfering
moieties of assays utilizing the binding reagents or antibodies
directed to detecting a specific target. Frequently, the binding
reagents, antibodies or antibody fragments of the present
disclosure avoid binding greater than about 90% of an interfering
moiety, although higher percentages are clearly contemplated and
preferred. For example, binding reagents, antibodies or antibody
fragments of the present disclosure avoid binding about 91%, about
92%, about 93%, about 94%, about 95%, about 96%, about 97%, about
98%, about 99%, and about 99% or more of an interfering moiety.
Less occasionally, binding reagents, antibodies or antibody
fragments of the present disclosure avoid binding greater than
about 70%, or greater than about 75%, or greater than about 80%, or
greater than about 85% of an interfering moiety.
[0089] As used herein, "mammal" refers to any of the mammalian
class of species. Frequently, the term "mammal," as used herein,
refers to humans, human subjects or human patients.
[0090] As used herein, the term "subject" is not limited to a
specific species or sample type. For example, the term "subject"
may refer to a patient, and frequently a human patient. However,
this term is not limited to humans and thus encompasses a variety
of mammalian species.
[0091] As used herein, "stringency" of nucleic acid hybridization
reactions is readily determinable by one of ordinary skill in the
art, and generally is an empirical calculation dependent upon probe
length, washing temperature, and salt concentration. In general,
longer probes require higher temperatures for proper annealing,
while shorter probes need lower temperatures. Hybridization
generally depends on the ability of denatured nucleic acid
sequences to reanneal when complementary strands are present in an
environment below their melting temperature. The higher the degree
of desired homology between the probe and hybridizable sequence,
the higher the relative temperature that can be used. As a result,
it follows that higher relative temperatures would tend to make the
reaction conditions more stringent, while lower temperatures less
so. For additional details and explanation of stringency of
hybridization reactions, see Current Protocols in Molecular Biology
(Ausubel et al. eds., Wiley Interscience Publishers, 1995);
Molecular Cloning: A Laboratory Manual (J. Sambrook, E. Fritsch, T.
Maniatis eds., Cold Spring Harbor Laboratory Press, 2d ed. 1989);
Wood et al., Proc. Natl. Acad. Sci. USA, 82:1585-1588 (1985).
[0092] As used herein the term "isolated" refers to material
removed from its original environment, and is altered from its
natural state. For example, an isolated polypeptide could be
coupled to a carrier, and still be "isolated" because that
polypeptide is not in its original environment.
[0093] As used herein, "test substance (or candidate compound)"
refers to a chemically defined compound (e.g., organic molecules,
inorganic molecules, organic/inorganic molecules, proteins,
peptides, nucleic acids, oligonucleotides, lipids, polysaccharides,
saccharides, or hybrids among these molecules such as
glycoproteins, etc.) or mixtures of compounds (e.g., a library of
test compounds, natural extracts or culture supernatants, etc.)
whose effect on a target is determined by the disclosed and/or
claimed methods herein.
[0094] As used herein, high-throughput screening (HTS) refers to
processes that test a large number of samples, such as samples of
diverse chemical structures against disease targets to identify
"hits" (see, e.g., Broach, et al., High throughput screening for
drug discovery, Nature, 384:14-16 (1996); Janzen, et al., High
throughput screening as a discovery tool in the pharmaceutical
industry, Lab Robotics Automation: 8261-265 (1996); Fernandes, P.
B., Letter from the society president, J. Biomol. Screening, 2:1
(1997); Burbaum, et al., New technologies for high-throughput
screening, Curr. Opin. Chem. Biol., 1:72-78 (1997)). HTS operations
are highly automated and computerized to handle sample preparation,
assay procedures and the subsequent processing of large volumes of
data.
[0095] As used herein, "plant" refers to any of various
photosynthetic, eucaryotic multi-cellular organisms of the kingdom
Plantae, characteristically producing embryos, containing
chloroplasts, having cellulose cell walls and lacking
locomotion.
[0096] As used herein, "animal" refers to a multi-cellular organism
of the kingdom of Animalia, characterized by a capacity for
locomotion, nonphotosynthetic metabolism, pronounced response to
stimuli, restricted growth and fixed bodily structure. Non-limiting
examples of animals include birds such as chickens, vertebrates
such fish and mammals such as mice, rats, rabbits, cats, dogs,
pigs, cows, ox, sheep, goats, horses, monkeys and other non-human
primates.
[0097] As used herein, "bacteria" refers to small prokaryotic
organisms (linear dimensions of around 1 micron) with
non-compartmentalized circular DNA and ribosomes of about 70S.
Bacteria protein synthesis differs from that of eukaryotes. Many
anti-bacterial antibiotics interfere with bacteria proteins
synthesis but do not affect the infected host.
[0098] As used herein, "eubacteria" refers to a major subdivision
of the bacteria except the archaebacteria. Most Gram-positive
bacteria, cyanobacteria, mycoplasmas, enterobacteria, pseudomonas
and chloroplasts are eubacteria. The cytoplasmic membrane of
eubacteria contains ester-linked lipids; there is peptidoglycan in
the cell wall (if present); and no introns have been discovered in
eubacteria.
[0099] As used herein, "archaebacteria" refers to a major
subdivision of the bacteria except the eubacteria. There are three
main orders of archaebacteria: extreme halophiles, methanogens and
sulphur-dependent extreme thermophiles. Archaebacteria differs from
eubacteria in ribosomal structure, the possession (in some case) of
introns, and other features including membrane composition.
[0100] As used herein, "virus" refers to an obligate intracellular
parasite of living but non-cellular nature, consisting of DNA or
RNA and a protein coat. Viruses range in diameter from about 20 to
about 300 nm Class I viruses (Baltimore classification) have a
double-stranded DNA as their genome; Class II viruses have a
single-stranded DNA as their genome; Class III viruses have a
double-stranded RNA as their genome; Class IV viruses have a
positive single-stranded RNA as their genome, the genome itself
acting as mRNA; Class V viruses have a negative single-stranded RNA
as their genome used as a template for mRNA synthesis; and Class VI
viruses have a positive single-stranded RNA genome but with a DNA
intermediate not only in replication but also in mRNA synthesis.
The majority of viruses are recognized by the diseases they cause
in plants, animals and prokaryotes. Viruses of prokaryotes are
known as bacteriophages.
[0101] As used herein, "fungus" refers to a division of eucaryotic
organisms that grow in irregular masses, without roots, stems, or
leaves, and are devoid of chlorophyll or other pigments capable of
photosynthesis. Each organism (thallus) is unicellular to
filamentous, and possesses branched somatic structures (hyphae)
surrounded by cell walls containing glucan or chitin or both, and
containing true nuclei.
[0102] As used herein, NAD refers to nicotinamide adenine
dinucleotide or a suitable derivative such as acetyl-NAD.sup.+ or
thio-NAD.sup.+. NADH refers to the reduced form of NAD.sup.+ and a
suitable derivative such as acetyl-NADH or thio-NADH. NADP.sup.+
refers to nicotinamide adenine dinucleotide phosphate or a suitable
derivative such as acetyl-NADP.sup.+ or thio-NADP.sup.+. NADPH
refers to the reduced form of NADP.sup.+ and a suitable derivative
such as acetyl-NADPH or thio-NADPH.
B. HYDROLYTIC ENZYMES
[0103] Enzymes are catalytic proteins that act on substrate(s) to
yield product(s). hydrolytic enzymes or hydrolases are enzymes that
catalyze the hydrolysis of a chemical bond by addition of water.
For example, an enzyme that catalyzes the following reaction is a
hydrolase: A-B+H.sub.2O.fwdarw.A-OH+B--H. In enzymology, hydrolases
are typically classified as EC 3 in the EC number classification of
enzymes. It is intended to encompass hydrolase with conservative
amino acid substitutions or functional fragments that do not
substantially alter its activity.
[0104] Hydrolases can be further classified into several subclasses
based upon the bonds that they act upon: for example, EC3.1.: ester
bonds (esterases, nuclease, phosphodiesterase, lipase,
phosphatase); EC3.2.: sugars (glycoside hydrolases); EC 3.3.: ether
bonds; EC 3.5.: carbon-nitrogen bonds, other than peptide bonds;
EC. 3.6.: acid anhydrides (acid anhydride hydrolases, including
helicases and GTPase); EC 3.7.: carbon-carbon bonds; EC 3.8.:
halide bonds; EC 3.9.: phosphorous-nitrogen bond; EC 3.10:
sulfur-nitrogen bonds; EC 3.11: carbon-phosphorous bonds; EC 3.12:
sulfur-sulfur bonds; EC 3.13: carbon-sulfur bonds
[0105] The methods described herein can be used with any suitable
hydrolytic enzyme, i.e., any enzyme that splits a substrate
molecule into two products, one of which is a hydroxylated organic
molecule, while adding a molecule of water. Glycosidases,
esterases, lipases, nucleases, and phosphatases are typical
examples. The glycosidases hydrolyze a glycosylated alcohol to
produce a sugar and the free alcohol. Esterases hydrolyze an ester
to produce a carboxylic acid and a free alcohol. Phosphatases
typically hydrolyze a phosphate ester to produce phosphate (or
diphosphate or triphospate) and an alcohol.
[0106] Some glycosidases of special interest include
beta-D-galactosidase Some esterases of special interest include
alpha-amino acid esterases, carboxylesterases, acetylesterases, and
the like. Some phosphatases of special interest include alkaline
phosphatase, which can be easily conjugated to a carrier or
antibody, phosphodiesterases, and the like. Bovine alkaline
phosphatase is one suitable example, which is well known for use in
ELISA assays.
C. HYDROLYTIC ENZYME SUBSTRATES
[0107] The hydrolytic enzyme substrates of the invention generally
comprise an alcohol that is linked by a cleavable linkage to a
recognition moiety that is recognized by the hydrolytic enzyme as
part of its substrate. The recognition moiety renders the
hydrolytic enzyme substrate specific for the chosen hydrolytic
enzyme, e.g., the recognition moiety makes the substrate
susceptible to transformation by the chosen hydrolytic enzyme and
not subject to transformation at comparable rates by other enzymes
that may be present in a typical system.
[0108] Examples of recognition moieties for some specific
hydrolytic enzymes are shown in FIGS. 1-9. The galactosyl ring of
the substrate shown in FIG. 1.1 is a recognition moiety that makes
the benzylic substrate specifically recognizable by and hyrolyzable
by beta-D-galactosidase. Similarly, the phosphate of the substrate
in FIG. 4 makes that hydrolytic enzyme substrate specifically
susceptible to hydrolytic action of alkaline phosphatase; the
acetyl ester of the substrate in FIG. 5 provides a substrate
selectively recognized by acetylesterase; and the alpha-amino acid
ester of the substrate in FIG. 6 provides a substrate that is
selectively hydrolyzed by alpha-amino acid esterase activity.
[0109] Typically, the hydrolytic enzyme substrates are not a
substrate for the additional enzyme or enzymes used in the assay
methods described herein (aryl alcohol oxidase, alcohol
dehydrogenase, aryl alcohol dehydrogenase, etc.); only after the
substrate is hydrolyzed by a suitable hydrolytic enzyme does it
serve as a substrate for the oxidizing and/or reducing enzymes that
are used in the assay systems described herein.
[0110] The hydrolytic enzyme substrates of the invention include
compounds of Formula (I):
##STR00006##
wherein:
[0111] A is an aromatic or heteroaromatic group, a 1-alkene or a
1-alkyne, each of which is optionally substituted;
[0112] each R is independently H or an optionally substituted C1-C4
alkyl or aryl;
[0113] n is an integer from 1-4;
[0114] and X is a group comprising a substrate moiety, [0115]
wherein the substrate moiety comprises a recognition component of a
substrate for the hydrolytic enzyme, and wherein the activity of
the hydrolytic enzyme is capable of hydrolyzing the compound of
formula (I) to form a detectable product of Formula II:
##STR00007##
[0116] In some embodiments, the reaction of the hydrolytic enzyme
with the hydrolytic enzyme substrate of Formula (I) produces a
compound of formula (II) and a by-product of Formula (III):
##STR00008##
[0117] `X` in these compounds includes a recognition moiety for the
particular hydrolytic enzyme of interest. Examples of the
by-products of Formula (III) can be seen in FIGS. 1-9. These
include galactose, phosphate, carboxylic acids, amino acids, and
the like.
[0118] In some embodiments of the hydrolytic enzyme substrates of
the invention, A is an optionally substituted aromatic or
heteroaromatic group. Suitable aromatic groups include phenyl and
naphthyl. Suitable heteroaromatic groups include pyridyl,
pyrimidinyl, triazinyl, indolyl, imidazolyl, benzimidazolyl,
pyrazolyl, benzopyrazolyl, quinolinyl, isoquinolinyl, thienyl,
furanyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, and
isothiazolyl, as examples. These aryl and heteroaryl groups can be
substituted as further described herein, or they can be
unsubstituted.
[0119] In other embodiments of the hydrolytic enzyme substrates of
the invention, A is a 1-alkene or a 1-alkyne. In some embodiments,
it is a 1-alkene of the formula (IV):
##STR00009##
[0120] wherein the wavy line indicates the point of attachment of A
to --[CH(R)].sub.n--O--X in Formula (I), and each G, G' and G'' is
independently H or an optionally substituted group selected from
the group consisting of C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl,
C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, aryl, and heteroaryl. In
certain of these embodiments, A is a group of Formula (IVb):
##STR00010##
[0121] where G, G' and G'' are defined as for Formula (IV). An
example of this type of compound is shown as HDEGP in the
examples.
[0122] In other embodiments of the hydrolytic enzyme substrate, A
is a 1-alkyne of the formula (V):
##STR00011##
[0123] wherein the wavy line indicates the point of attachment of A
to --[CH(R)].sub.n--O--X in Formula (I), and G is H or an
optionally substituted member selected from the group consisting of
C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C3-C8
heterocycloalkyl, aryl, and heteroaryl. In some such embodiments, G
can be optionally substituted phenyl.
[0124] In any of the foregoing embodiments, R can be H, Methyl or
phenyl, for example. In preferred embodiments, R is H, so the
product of Formula (II) is a primary alcohol. Where n is 1, this
product becomes a benzylic alcohol when A is phenyl, an allylic
alcohol when A is a 1-alkene, or a propargylic alcohol when A is a
1-alkyne. Preferred embodiments include compounds wherein R is H
and n is 1.
[0125] In some embodiments of the hydrolytic enzyme substrates of
the invention, X comprises a saccharide, e.g., a monosaccharide or
a disaccharide. In some embodiments, X is a D-galactosyl ring or
another D-sugar such as glucose, allose, mannose, xylose, gulose,
talose, altrose, idose, ribose, arabinose, lyxose, and the like. In
some embodiments, the hydrolytic enzyme substrate is a compound of
Formula (VIa) or (VIb):
##STR00012##
[0126] wherein R.sup.2 is H or --CH.sub.2OQ, and each Q is
independently H or a monosaccharide, disaccharide or
oligosaccharide, and A, R and n are as defined for Formula (I). In
some embodiments, each Q is H; in some embodiments, n is 1; R can
be H, and in some embodiments, R.sup.2 is --CH.sub.2OH or H.
[0127] In other embodiments, the hydrolytic enzyme substrate is an
ester of formula (VII):
##STR00013##
[0128] wherein R.sup.3 is H or an optionally substituted aryl,
heteroaryl, C1-C8 alkyl, C3-C8 cycloalkyl, or C3-C8 heterocyclyl
group,
[0129] and A, R and n are as defined for Formula (I).
[0130] R.sup.3 in these compounds can vary widely, provided it acts
as a recognition moiety for the hydrolytic enzyme of interest. In
specific embodiments, R.sup.3 is selected from the group consisting
of Me, Et, and phenyl, or it is an amino acid radical such that
HO.sub.2C--R.sup.3 is an alpha-amino acid. Thus --R.sup.3 can be a
group of the formula --CH(NH.sub.2)--R.sup.aa, where R.sup.aa is
the side chain of one of the 20 commonly recognized essential amino
acids. In some of these embodiments, n is 1.
[0131] In other embodiments, the hydrolytic enzyme substrate can be
a compound of formula (VIII):
##STR00014##
[0132] wherein Z is N, S, S.dbd.O, P, or P--OH, and R.sup.4 is O,
hydroxy, C1-C4 alkoxy, C1-C4 alkyl, or aryl. These can be, for
example, phosphatase substrates, e.g., compounds of Formula I,
wherein X comprises a phosphate group, so that Z in Formula (VII)
is P--OH. Examples of these would be a compound of the formula
(IX):
##STR00015##
or a salt thereof.
[0133] In the foregoing hydrolytic enzyme substrates, n can be 1;
and wherever A can be an aryl group, it can be an optionally
substituted phenyl group. In these embodiments, the phenyl group
can be unsubstituted, or it can be substituted with 1-3 groups
selected from halo, hydroxy, CN, NO.sub.2, COOR', CONR'.sub.2,
NR'.sub.2, OR', optionally substituted C1-4 alkyl, SR', SO.sub.2R',
or SO.sub.2NR'.sub.2, wherein each R' is independently H or
optionally substituted C1-4 alkyl, and two R' on the same or
adjacent atoms can be taken together to form an optionally
substituted C3-C8 heterocyclic ring.
[0134] In additional embodiments of the hydrolytic enzyme
substrates, A can be a group of the formula (X):
##STR00016##
[0135] wherein the wavy line indicates the point of attachment of A
to --[CH(R)].sub.n--O--X in Formula (I), and each G is
independently H or an optionally substituted group selected from
the group consisting of C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8
alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl,
aryl, and heteroaryl.
[0136] In any of the foregoing embodiments, the optional
substituents for alkyl, alkenyl alkynyl, and heterocyclic groups
can be as set forth below; or they can be selected from halo, oxo,
CN, NO.sub.2, COOR'', CONR''.sub.2, NR''.sub.2, OR'', optionally
substituted C1-4 alkyl, SR', SO.sub.2R'', or SO.sub.2NR''.sub.2,
wherein each R'' is independently H or C1-4 alkyl. Likewise the
optional substituents for aryl and heteroaryl groups can be as
described in the definitions herein, or they can be selected from
halo, CN, NO.sub.2, COOR'', CONR''.sub.2, NR''.sub.2, OR'',
optionally substituted C1-4 alkyl, SR', SO.sub.2R'', or
SO.sub.2NR''.sub.2, wherein each R'' is independently H or C1-4
alkyl.
[0137] In preferred embodiments of any of the foregoing hydrolytic
enzyme substrates, R is H; and n is 1.
[0138] Preferred hydrolytic enzymes for the substrates of the
invention include glycosidases; esterases; beta-D-galactosidase;
alpha-amino acid esterases; and phosphatases. Suitable esterases
can be a carboxylesterase, an acetyl esterase or an alpha-amino
acid esterase.
D. HYDROLYTIC ENZYME SUBSTRATE COMPOSITIONS
[0139] The hydrolytic enzyme substrates of the invention can be
used in combination with at least one additional enzyme, besides
the hydrolytic enzyme that hydrolyzes the substrate. The additional
enzyme is one that promotes efficient detection of the initial
product of the hydrolytic enzyme activity, the compound of Formula
(II), by converting it into another species. Therefore compositions
that include the hydrolytic enzyme substrates described above and
at least one additional enzyme are useful as components of an assay
system for detecting and/or quantifying the presence of the
hydrolytic enzymes of interest. Similarly, combinations of the
hydrolytic enzyme substrate with other materials that are required
in some embodiments of the methods described herein are also useful
for these assays and are also an aspect of the invention.
[0140] Thus in another aspect, the invention provides compositions
that comprise any of the hydrolytic enzyme substrates described
herein in combination with an additional enzyme as described
herein, or enzyme co-factor that can be used to detect or quantify
the products of hydrolysis of the hydrolytic enzyme substrate by
the hydrolytic enzyme, or a reagent for detection of the product of
hydrolysis of the hydrolytic enzyme substrate.
[0141] Thus in some embodiments, the invention provides a
composition that comprises a combination of a hydrolytic enzyme
substrate as described above plus at least one of the
following:
[0142] the hydrolytic enzyme that recognizes and hydrolyzes the
hydrolytic enzyme substrate, which enzyme may be present as a
conjugate with a recognition element such as an antibody, or it may
be present as a polynucleotide sequence that will be expressed as a
functional enzyme before detection;
[0143] an additional enzyme that can transform the product of
Formula (II) that is produced by action of the hydrolytic enzyme on
the hydrolytic enzyme substrates of the invention into a new
chemical species, often by an oxidation reaction (e.g., an aryl
alcohol oxidase, alcohol dehydrogenase, or aryl alcohol
dehydrogenase;
[0144] a co-factor utilized by the additional enzyme that can help
transform the product of Formula (II) that is produced by action of
the hydrolytic enzyme on the hydrolytic enzyme substrates of the
invention, e.g., NAD+ or NADP+ that can promote oxidation of the
compound of Formula II to a carbonyl compound of Formula
A-C(.dbd.O)--R (II-ox); and a reagent for detecting a by-product
formed by the additional enzyme when it transforms the product of
Formula (II) into another species, e.g., a reagent for detection of
hydrogen peroxide formed upon oxidation of an aryl alcohol of
Formula IIB into a carbonyl compound of Formula A-C(.dbd.O)--R
(II-ox); and/or an enzyme or co-factor that can help transform the
carbonyl compound formed by oxidation of the compound of Formula
(II) into another species that facilitates detection, e.g., a
reducing enzyme as described herein that converts the carbonyl
compound back into an alcohol of Formula (II), or a co-factor for
such a reducing enzyme.
[0145] In some embodiments, the hydrolytic enzyme is not included
in these reagent combinations, because for example, the hydrolytic
enzyme may be directly linked to the target to be detected. The
combination may be prepared to be contacted with a separate sample
containing the hydrolytic enzyme. For example, the hydrolytic
enzyme can be part of a fusion protein produced from a nucleic acid
that encodes a target of interest and also encodes the hydrolytic
enzyme. Alternatively, the hydrolytic enzyme may be included in the
reagent combinations, and reaction mixtures contacted with the
sample will often contain the hydrolytic enzyme, which may be
linked or conjugated to a specific recognition moiety such as an
antibody directed to the target of interest.
[0146] In some embodiments, the invention provides a combination of
the hydrolytic enzyme substrate of any of the embodiments described
above, and the corresponding hydrolytic enzyme for that particular
substrate, i.e., a hydrolytic enzyme that is capable of cleaving
the particular hydrolytic enzyme substrate to produce a detectable
product of Formula (II). In some embodiments, an aryl alcohol
molecule or unsaturated aliphatic alcohol molecule is the product
of that hydrolytic cleavage reaction, and the aryl alcohol molecule
or unsaturated aliphatic alcohol molecule has a structure of said
formula (II):
##STR00017##
[0147] wherein A, R and n are as defined for Formula (I).
[0148] In some of these embodiments, the hydrolytic enzyme is an
esterase, a phosphatase, or a glycosidase. In particular
embodiments, the hydrolytic enzyme is selected from the group
consisting of an acetylesterase, an amino acid esterase, a
carboxylesterase, a nuclease, a phosphodiesterase, a lipase and a
phosphatase. For example, the hydrolytic enzyme can be alkaline
phosphatase.
[0149] In some specific embodiments, the hydrolytic enzyme can be
an alkaline phosphatase, an .alpha.-amino acid esterase, a
galactosidase or a .beta.-glycosidase.
[0150] In some of these embodiments, the combination further
comprises an oxidizing reagent that is capable of oxidizing the
aryl alcohol molecule or unsaturated aliphatic alcohol molecule
produced by the cleavage reaction catalyzed by the hydrolytic
enzyme. In some such embodiments, the oxidizing reagent is an aryl
alcohol oxidase or an alcohol oxidase that is capable of oxidizing
the aryl alcohol molecule or unsaturated aliphatic alcohol molecule
in the presence of oxygen to produce an aryl aldehyde molecule or
unsaturated aliphatic aldehyde molecule (assuming R is H) and
H.sub.2O.sub.2.
[0151] Where an oxidizing reagent is used and H.sub.2O.sub.2 can be
produced as a by-product of the oxidation reaction, in some
embodiments, the combination also comprises a reagent for detecting
and/or measuring the H.sub.2O.sub.2. Suitable reagents are well
known in the art, including those used in Trinder reactions. Thus
suitable reagents include a peroxidase such as horseradish
peroxidase; a phenol such as phenol; an antipyrine such as
4-aminoantipyrine (4-AA), and/or an aniline analog. Some suitable
aniline analogs known for use in these modified Trinder's reactions
include ADOS, ADPS, ALPS, DAPS, DAOS, TOOS, MAOS, and MAPS. See,
e.g., U.S. Pat. No. 5,156,955 for some suitable Trinder reaction
components that can be used in the methods of the invention.
[0152] In some embodiments of these combinations, the oxidizing
reagent that oxidizes the compound of Formula (II) to a carbonyl
compound (aldehydes when R is H; ketone when R is not H) is an aryl
alcohol dehydrogenase or an alcohol dehydrogenase that is capable
of oxidizing the aryl alcohol molecule or unsaturated aliphatic
alcohol molecule in the presence of NAD or NADP.sup.+ to produce a
carbonyl compound and NADH or NADPH. In these embodiments, the
combination optionally further comprises NAD.sup.+ or NADP.sup.+,
which acts as a co-factor to promote the oxidation reaction, and is
transformed by the oxidation reaction into NADH or NADPH.
Optionally in these embodiments, the combination further comprises
a reagent for measuring the NADH or NADPH formed by this oxidation
reaction.
[0153] The combination compositions of the invention include
combinations of the hydrolytic enzyme substrate with NADH or NADPH
and/or an aryl alcohol dehydrogenase or an alcohol dehydrogenase
that is capable of reducing the aryl aldehyde molecule or
unsaturated aliphatic aldehyde molecule (compounds of Formula
II-ox, where R is H) in the presence of NADH or NADPH. These
combinations optionally also include an oxidizing enzyme that can
oxidize the initially formed hydrolysis product of Formula II into
a carbonyl compound (e.g., Formula II-ox) as described above.
Preferably, the oxidizing enzyme is different from the enzymes for
reducing the aryl aldehydes or unsaturated aliphatic aldehydes, and
preferably the different enzymes do not share the same co-factors.
In one preferred embodiment, the oxidizing enzyme is an alcohol
oxidase or aryl alcohol oxidase that uses O.sub.2 to promote
oxidation of the compound of Formula (II), and the reducing enzyme
is a dehydrogenase that uses NADH or NADPH to reduce the aldehyde
back to an alcohol of Formula II.
[0154] Where such complimentary oxidizing and reducing enzymes are
employed, a cycling reaction system is formed (see, e.g., FIG. 3),
whereby signal amplification can be achieved to greatly increase
the sensitivity of the assay system. The cycling assay system can
be monitored by measuring hydrogen peroxide formation, by measuring
consumption of NADH or NADPH in the reduction reaction, and/or by
measuring formation of NAD+ or NADP+ in the reduction reaction. In
some embodiments of these compositions, the combination further
comprises a reagent for measuring the H.sub.2O.sub.2. Suitable
reagents for measuring H.sub.2O.sub.2 can include at least one of a
peroxidase, an antipyrine, a phenol such as 2-chlorophenol,
2,4-dichlorophenol, 4-chlorophenol, 2,6-dichlorophenol, and/or an
aniline analog such as DMA, TOOS, TOPS, ADOS, ALOS, ADPS, ALPS,
DAPS, DAOS, HDAPS, HDAOS, MAOS, MAPS, or EMAE.
[0155] In some embodiments of the foregoing combinations, the
hydrolytic enzyme substrate comprises at least a part of a
.beta.-glycosidase substrate molecule, and the hydrolytic enzyme is
a .beta.-glycosidase. In others, the hydrolytic enzyme substrate
comprises a phosphate ester, and the hydrolytic enzyme is alkaline
phosphatase. In other embodiments, the hydrolytic enzyme substrate
comprises a beta-galactosidic group and the hydrolytic enzyme is
beta-galactosidase. In other embodiments, the hydrolytic enzyme
substrate is an ester, and the hydrolytic enzyme is an alpha-amino
acid esterase, a carboxylesterase, or an acetylesterase.
[0156] In some of these compositions, the combination comprises any
of the hydrolytic enzyme substrates described herein and at least
one of the following: [0157] a) an aryl alcohol oxidase or an
aliphatic alcohol oxidase that is capable of oxidizing the aryl
alcohol molecule or unsaturated aliphatic alcohol molecule in the
presence of oxygen to produce an aryl aldehyde molecule or
unsaturated aliphatic aldehyde molecule and H.sub.2O.sub.2; and/or
[0158] b) NADH or NADPH; and/or [0159] c) an aryl alcohol
dehydrogenase or an alcohol dehydrogenase that is capable of
reducing the aryl aldehyde molecule or unsaturated aliphatic
aldehyde molecule in the presence of NADH or NADPH; and/or [0160]
d) a reagent for measuring H.sub.2O.sub.2.
[0161] In another aspect, the invention provides kits for
determining and/or quantifying the amount of a hydrolytic enzyme,
which include any of the compounds described herein as suitable
hydrolytic enzyme substrates for purposes of the invention and
optionally any of the combination compositions described above. In
some embodiments, any one of the combinations set forth above is
provided in the form of a kit. The kit can include the components
of the combination packaged separately, or it can include mixtures
of the components of an embodiment of the combinations premixed in
a single container, where the components are compatible for mixing.
The kit may further comprise one or more standards useful for
calibration of the assay system, and instructions for performing an
assay with the hydrolytic enzyme substrate or combination
composition.
[0162] Any of the foregoing combinations can be comprised in an
assay, isolation and/or production system for a target to be
produced or detected. The target is an analyte to be detected or
quantified, or a product to be produced and detected or quantified,
and the combinations and kits described above can include a moiety
that is specific for the target, such as a PCR primer or an
antibody. In some embodiments, the target is an inorganic molecule,
an organic molecule and/or a complex thereof. In some embodiments,
the target is an organic molecule selected from the group
consisting of an amino acid, a peptide, a protein, a nucleoside, a
nucleotide, an oligonucleotide, a nucleic acid, a vitamin, a
monosaccharide, an oligosaccharide, a carbohydrate, a lipid and a
complex thereof.
[0163] Any suitable alcohol oxidase can be used in the present
combinations. For example, the alcohol oxidase disclosed and/or
claimed U.S. Pat. Nos. 7,160,708, 5,166,329, 4,956,290, 4,729,956
and 4,619,898 can be used. In another example, the alcohol oxidase
disclosed in Janssen and Ruelius, Biochim. Biophys. Acta., 151(2):
330-42 (1968), and Suye, Curr. Microbiol., 34(6): 374-7 (1997) can
be used.
[0164] Any suitable aryl alcohol oxidase can be used in the present
combinations. For example, the aryl alcohol oxidase disclosed
and/or claimed in U.S. Pat. Nos. 3,183,235, 3,290,326 and
6,835,212, and U.S. patent application US2009/053780 A1 can be
used. In another example, the aryl alcohol oxidase disclosed in
Farmer et al., Biochem. J. 74:257-62 (1960) and Guillen and Evans,
Applied and Environmental Microbiology, 60(8):2811-2817 (1994) can
be used.
[0165] Any suitable aryl alcohol dehydrogenase can be used in the
present combinations. For example, the aryl alcohol dehydrogenase
disclosed and/or claimed in U.S. Pat. Nos. 4,020,070, 5,182,209,
6,262,295, 7,750,135, and U.S. patent application US2009/017510 A1,
US2009/186900 A1, US2006/074060 A1, and JP2147956 A can be used. In
another example, the aryl alcohol dehydrogenase disclosed in Suhara
et al., Arch. Biochem. Biophys., 130(1): 422-9 (1969), and Yamanaka
and Minoshima, Agric. Biol. Chem., 48:1161-1171 (1984) can be
used.
[0166] Any suitable alcohol dehydrogenase can be used in the
present combinations. For example, the alcohol dehydrogenase
disclosed and/or claimed in U.S. Pat. Nos. 7,750,135, 7,354,751,
6,552,249, 6,432,688, 6,255,092, 6,225,099, 5,908,924, 5,855,881,
5,695,973, 5,445,943, 5,385,833, 5,344,777, 5,162,516, 5,162,203,
4,241,184, 4,131,727, and 4,111,751 can be used. In another
example, the alcohol dehydrogenase disclosed in Yakushi and
Matsushita, Appl Microbiol Biotechnol., 86(5):1257-65 (2010) and
Yin, Alcohol Alcohol Suppl., 2:113-9 (1994) can be used.
[0167] The combinations of the invention can be embodied in a
system such as a system for immunoassay, protein sequencing,
nucleic acid amplification, hybridization or sequencing. Exemplary
immunoassays include sandwich or competitive assay, enzyme-linked
immunosorbent assay (ELISA), immunoblotting, immunoprecipitation,
immunostaining, lateral flow immunoassay, u-capture assay,
inhibition assay and avidity assay. Exemplary nucleic acid
sequencing technologies include DNA sequencing technology using a
hydrolytic enzyme, e.g., an alkaline phosphatase, to generate a
signal readout. See e.g., Patel and Nash, Biotechniques,
18(2):328-33 (1995).
[0168] The combinations of the invention can be used in any
suitable assay formats or configurations. In some embodiments, the
combinations of the invention can be used in heterogeneous assay
formats. In other embodiments, the combinations of the invention
can be used in homogeneous assay formats. Exemplary homogeneous
assay formats include cloned enzyme donor immunoassay (CEDIA),
multiplied immunoassay techniques (EMIT), apoenzyme reactivation
immunoassay (ARIA), cofactor-labeled immunoassay and
inhibitor-labeled immunoassay. See e.g., U.S. Pat. Nos. 4,708,929,
5,120,653, 5,244,785, and 5,362,625, WO 96/41172 A1, and Jenkins,
J. Immunol. Meth., 150:91-97 (1992).
E. METHODS OF USING THE HYDROLYTIC ENZYME SUBSTRATES
[0169] In another aspect, the invention provides for methods for
using the hydrolytic enzyme substrates and/or combinations
described above to detect the presence or amount of the hydrolytic
enzyme in a sample, which in some embodiments is used to detect the
presence or amount of a target molecule in the sample. The target
molecule may be conjugated with the hydrolytic enzyme where the
enzyme functions as a label, for example; or the hydrolytic enzyme
may be attached or conjugated to a binding moiety that is specific
for the target molecule, e.g., an antibody that specifically
recognizes and binds to the target molecule, or to a complex of the
target molecule with another moiety such as another enzyme as part
of a sandwich assay. In some embodiments, the hydrolytic enzyme
itself may be the species to be detected or quantified.
[0170] The hydrolytic enzyme will typically be in a sample, which
can be any suitable composition. Frequently it will be a solution
or suspension, primarily aqueous, and containing suitable buffering
agents to maintain an appropriate pH for the hydrolytic enzyme to
function. Selection of suitable temperature, pH, and concentration
and other parameters are within the ordinary level of skill for a
given hydrolytic enzyme.
[0171] In some embodiments, the invention provides a method for
assessing activity and/or amount of a hydrolytic enzyme in a
sample, which method comprises:
[0172] a) contacting a sample containing or suspected of containing
a hydrolytic enzyme with a hydrolytic enzyme substrate having a
structure of formula (I):
##STR00018##
[0173] under conditions where said hydrolytic enzyme, if present in
said sample, cleaves said substrate to produce an aryl alcohol
molecule or unsaturated aliphatic alcohol molecule having a
structure of formula (II) and a compound having a structure of
formula (III):
##STR00019##
[0174] wherein A, R, n and X are as defined above for Formula (I);
and
[0175] b) assessing the presence and/or amount of said aryl alcohol
molecule or unsaturated aliphatic alcohol molecule to assess
activity and/or amount of said hydrolytic enzyme in said
sample.
[0176] The amount or presence of the aryl alcohol or unsaturated
aliphatic alcohol can be assessed directly or indirectly, by any
convenient method. In some embodiments, the presence or amount of
the alcohol is detected by converting it into a carbonyl compound
as described herein, typically by an enzymatic oxidation. The
oxidation can be accomplished with various enzymes as described
herein (e.g., aryl alcohol oxidase; alcohol dehydrogenase; aryl
alcohol dehydrogenase). The hydrolytic enzyme substrate can be any
of those described above, provided it is selected to be compatible
with the hydrolytic enzyme to be detected and thus contains a
recognition moiety specific for that hydrolytic enzyme, and is
capable of being hydrolyzed by that hydrolytic enzyme.
[0177] The method is thus an assay that involves contacting the
sample with the hydrolytic enzyme substrate, either alone or in any
of the combinations described above. The conditions for the assay
are selected such that the hydrolytic enzyme, if present, will
hydrolyze the hydrolytic enzyme substrate to produce products as
discussed above. Typically, an excess amount of the hydrolytic
enzyme substrate will be included relative to the amount of enzyme
likely to be present, so the amount of product can exceed the
amount of enzyme, thus enhancing the effective signal strength and
making assay more sensitive, and giving a substantially linear rate
of product formation.
[0178] It will be understood that the assays described herein can
be quantitative or qualitative. Qualitatively, one can detect the
product formed to verify that the hydrolytic enzyme is present,
often by convenient color-change or spectrophotometric assays that
confirm some product has formed. Where a quantitative result is
desired, it will frequently be necessary to test one or more
standards in order to interpret the data from the assay, which will
typically reflect the rate of formation of the products rather than
directly describing the amount of enzyme. It is thus often
necessary to test with the assay, in addition to the test sample,
at least one and optionally more than one standard sample having a
known amount of the hydrolytic enzyme in order to determine the
amount of hydrolytic enzyme present from the data on rate of
product formation. Such calibration methods are well known to those
of ordinary skill in the art.
[0179] In some embodiments of these methods, the hydrolytic enzyme
is an esterase, or a glycosidase. In some embodiments, the
hydrolytic enzyme is an esterase selected from the group consisting
of an acetylesterase, an amino acid esterase, and a
carboxylesterase; in some embodiments, the enzyme is a nuclease, a
phosphodiesterase, a lipase or a phosphatase. In some particular
embodiments, the hydrolytic enzyme is an alkaline phosphatase. In
other particular embodiments, the hydrolytic enzyme is an
.alpha.-amino acid esterase; or a .beta.-glycosidase.
[0180] In some embodiments of these methods, the step of assessing
the presence and/or amount of the aryl alcohol molecule or
unsaturated aliphatic alcohol molecule comprises oxidizing the aryl
alcohol molecule or unsaturated aliphatic alcohol molecule with an
oxidizing reagent. Where R is H, the oxidation product is an
aldehyde; where R is alkyl or aryl, the oxidation product is a
ketone. In some preferred embodiments, n is 1 and R is H, so the
product of the oxidation is an aldehydes of formula A-CHO, where A
is as described for formula (I).
[0181] In some of these embodiments, the presence and/or amount of
the aryl alcohol molecule or unsaturated aliphatic alcohol molecule
is assessed by oxidizing the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule with an aryl alcohol oxidase or an
aliphatic alcohol oxidase in the presence of oxygen to produce
H.sub.2O.sub.2 and assessing the presence and/or amount of the
H.sub.2O.sub.2. Methods for assessing the presence or amount of
hydrogen peroxide are well known in the art, and include variations
of the Trinder reaction. In some such embodiments, a reagent
comprising at least one of a peroxidase, a phenol, an antipyrine,
and/or an aniline analog is used to detect the presence and/or
amount of the H.sub.2O.sub.2. Suitable reagents are discussed, for
example, in U.S. Pat. No. 5,156,955.
[0182] In alternative methods, the presence and/or amount of the
aryl alcohol molecule or unsaturated aliphatic alcohol molecule is
assessed by oxidizing the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule with an aryl alcohol dehydrogenase or an
alcohol dehydrogenase in the presence of NAD.sup.+ or NADP.sup.+ to
produce NADH or NADPH, and assessing the presence and/or amount of
the NAD+, NADP+, NADH or NADPH. Methods to monitor reactions using
NAD+/NADH or NADP+/NADPH are well known in the art, and are
conveniently applied to these assay methods.
[0183] In some embodiments, the presence and/or amount of the aryl
alcohol molecule or unsaturated aliphatic alcohol molecule is
assessed by:
[0184] a) oxidizing the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule with an aryl alcohol oxidase or an
aliphatic alcohol oxidase in the presence of oxygen to produce aryl
aldehyde molecule or unsaturated aliphatic aldehyde molecule and
H.sub.2O.sub.2;
[0185] b) reducing the aryl aldehyde molecule or unsaturated
aliphatic aldehyde molecule with an aryl alcohol dehydrogenase or
an alcohol dehydrogenase in the presence of NADH or NADPH to form a
reaction cycle in which the reduced aryl alcohol molecule or
unsaturated aliphatic alcohol molecule is oxidized by the aryl
alcohol oxidase or an aliphatic alcohol oxidase in the presence of
oxygen to produce additional aryl aldehyde molecule or unsaturated
aliphatic aldehyde molecule and H.sub.2O.sub.2; and c) assessing
the presence and/or amount of the H.sub.2O.sub.2, or the amount of
NADH, NADPH, NAD+, or NADP+.
[0186] In these embodiments, two different enzymes are used, one to
oxidize alcohol to aldehydes, and another to reduce aldehydes back
to alcohol. The different enzymes use different co-factors and
produce different by-products; and the combination of the two
different enzymes operating simultaneously provides a cycling assay
system that results in effective amplification of the signal from
the initial hydrolysis of the hydrolytic enzyme substrate. The
amount of by-products formed from the cycling oxidation/reduction
combination can far exceed the amount of hydrolytic enzyme
substrate used. This system thus introduces two amplification
steps, the initial amplification from amount of enzyme to the
amount of hydrolyzed substrate; and the further amplification
provided by cycling the hydrolyzed substrate molecule between
oxidized and reduced states.
[0187] The amount or presence of H.sub.2O.sub.2 can be measured by
known methods such as the Trinder reactions as discussed above, as
can the amounts of the co-factors present. It is also possible in
these methods to monitor both hydrogen peroxide formation and the
rate of the NAD+/NADH or NADP+/NADH formation.
[0188] In some embodiments of these methods, the hydrolytic enzyme
substrate comprises at least a part of (a recognition moiety of) a
.beta.-glycosidase substrate molecule, and the hydrolytic enzyme is
a .beta.-glycosidase.
[0189] In other embodiments, the hydrolytic enzyme substrate
comprises at least a part of an alkaline phosphatase substrate
molecule (e.g., an optionally substituted benzyl phosphate), and
the hydrolytic enzyme is an alkaline phosphatase.
[0190] The methods described herein can be used as an analytical
assay to detect the presence of a target or as part of an isolation
method for a target; or as part of a process for production of a
target molecule. The target molecule can be organic or inorganic or
a complex, as described above. In some embodiments, the target is
an organic molecule selected from the group consisting of an amino
acid, a peptide, a protein, a nucleoside, a nucleotide, an
oligonucleotide, a nucleic acid, a vitamin, a monosaccharide, an
oligosaccharide, a carbohydrate, a lipid and a complex thereof.
[0191] In some embodiments, the methods are utilized in an
immunoassay, protein sequencing, nucleic acid amplification,
hybridization or sequencing. In some embodiments, the methods are
used in an RNA or DNA sequencing system. In these embodiments,
alkaline phosphatase is a preferred hydrolytic enzyme.
[0192] Any suitable alcohol oxidase can be used in the present
methods. For example, the alcohol oxidase disclosed and/or claimed
U.S. Pat. Nos. 7,160,708, 5,166,329, 4,956,290, 4,729,956 and
4,619,898 can be used. In another example, the alcohol oxidase
disclosed in Janssen and Ruelius, Biochim. Biophys. Acta., 151(2):
330-42 (1968), and Suye, Curr. Microbiol., 34(6): 374-7 (1997) can
be used.
[0193] Any suitable aryl alcohol oxidase can be used in the present
methods. For example, the aryl alcohol oxidase disclosed and/or
claimed in U.S. Pat. Nos. 3,183,235, 3,290,326 and 6,835,212, and
U.S. patent application US2009/053780 A1 can be used. In another
example, the aryl alcohol oxidase disclosed in Farmer et al.,
Biochem. J. 74:257-62 (1960) and Guillen and Evans, Applied and
Environmental Microbiology, 60(8):2811-2817 (1994) can be used.
[0194] Any suitable aryl alcohol dehydrogenase can be used in the
present methods. For example, the aryl alcohol dehydrogenase
disclosed and/or claimed in U.S. Pat. Nos. 4,020,070, 5,182,209,
6,262,295, 7,750,135, and U.S. patent application US2009/017510 A1,
US2009/186900 A1, US2006/074060 A1, and JP2147956 A can be used. In
another example, the aryl alcohol dehydrogenase disclosed in Suhara
et al., Arch. Biochem. Biophys., 130(1): 422-9 (1969), and Yamanaka
and Minoshima, Agric. Biol. Chem., 48:1161-1171 (1984) can be
used.
[0195] Any suitable alcohol dehydrogenase can be used in the
present methods. For example, the alcohol dehydrogenase disclosed
and/or claimed in U.S. Pat. Nos. 7,750,135, 7,354,751, 6,552,249,
6,432,688, 6,255,092, 6,225,099, 5,908,924, 5,855,881, 5,695,973,
5,445,943, 5,385,833, 5,344,777, 5,162,516, 5,162,203, 4,241,184,
4,131,727, and 4,111,751 can be used. In another example, the
alcohol dehydrogenase disclosed in Yakushi and Matsushita, Appl
Microbiol Biotechnol., 86(5):1257-65 (2010) and Yin, Alcohol
Alcohol Suppl., 2:113-9 (1994) can be used.
[0196] The present methods can be used in any suitable assays such
as immunoassay, protein sequencing, nucleic acid amplification,
hybridization or sequencing. Exemplary immunoassays include
sandwich or competitive assay, enzyme-linked immunosorbent assay
(ELISA), immunoblotting, immunoprecipitation, immunostaining,
lateral flow immunoassay, u-capture assay, inhibition assay and
avidity assay. Exemplary nucleic acid sequencing technologies
include DNA sequencing technology using a hydrolytic enzyme, e.g.,
an alkaline phosphatase, to generate a signal readout. See e.g.,
Patel and Nash, Biotechniques, 18(2):328-33 (1995).
[0197] The present methods can be used in any suitable assay
formats or configurations. In some embodiments, the present methods
can be used in heterogeneous assay formats. In other embodiments,
the present methods can be used in homogeneous assay formats.
Exemplary homogeneous assay formats include cloned enzyme donor
immunoassay (CEDIA), multiplied immunoassay techniques (EMIT),
apoenzyme reactivation immunoassay (ARIA), cofactor-labeled
immunoassay and inhibitor-labeled immunoassay. See e.g., U.S. Pat.
Nos. 4,708,929, 5,120,653, 5,244,785, and 5,362,625, WO 96/41172
A1, and Jenkins, J. Immunol. Meth., 150:91-97 (1992).
[0198] The present combinations and/or methods can be used to
detect an analyte in any suitable sample liquid. In some
embodiments, the liquid sample can be body fluid sample, such as a
whole blood, a serum, a plasma, a urine sample or an oral fluid.
Such body fluid sample can be sued directly or can be processed,
e.g., enriched, purified, or diluted, before use. In other
embodiments, the liquid sample can be a liquid extract, suspension
or solution derived from a solid or semi-solid biological material
such as a phage, a virus, a bacterial cell, an eukaryotic cell, a
fugal cell, a mammalian cell, a cultured cell, a cellular or
subcellular structure, cell aggregates, tissue or organs. In
specific embodiments, the sample liquid is obtained or derived from
a mammalian or human source. In still other embodiments, the liquid
sample is a sample derived from a biological, a forensics, a food,
a biowarfare, or an environmental source. In other embodiments, the
sample liquid is a clinical sample, e.g., a human or animal
clinical sample. In still other embodiments, the sample liquid is a
man-made sample, e.g., a standard sample for quality control or
calibration purposes.
[0199] The present combinations and/or methods can be used to
detect the presence, absence and/or amount of an analyte in any
suitable sample liquid. In some embodiments, the present test
devices are used to detect the presence or absence of an analyte in
any suitable sample liquid, i.e., to provide a yes or no answer. In
other embodiments, the present test devices are used to quantify or
semi-quantify the amount of an analyte in a liquid sample.
[0200] The combinations and/or methods can be used to detect the
presence, absence and/or amount of a single analyte in any suitable
sample liquid. Alternatively, the present test devices can be used
to detect the presence, absence and/or amount of multiple analytes
in a liquid sample. In still other embodiments, the present test
devices can be used to quantify or semi-quantify the amounts of the
multiple analytes in the liquid sample.
[0201] The combinations and/or methods can be used to detect the
presence, absence and/or amount of any suitable analyte in a sample
liquid. Exemplary analytes include inorganic molecules, organic
molecules or complexes thereof. Exemplary organic molecules can be
an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an
oligonucleotide, a nucleic acid, e.g., a DNA or RNA molecule or a
hybrid thereof, a vitamin, a monosaccharide, an oligosaccharide, a
carbohydrate, a lipid and a complex thereof. In some embodiments,
the analyte is a cell, a virus or a molecule. In other embodiments,
the analyte is a disease or disorder marker, an antigen of an
infectious organism, an antibody to an infectious organism,
etc.
[0202] The combinations and/or methods can be used for any suitable
purpose. For example, present combinations and/or methods can be
used for clinical diagnosis, prognosis, risk assessment and
prediction, stratification and treatment monitoring and adjustment.
In another example, present combinations and/or methods can be used
for various research purposes, such as basic research, drug
candidate screening, animal studies, and clinical trials. In still
another example, present combinations and/or methods can be used in
tests for standard setting, quality control, illegal drug
screening, food safety, environmental safety, industrial safety,
and pollution, etc. The present combinations and/or methods can be
used in any suitable settings, such as tests in the labs, clinics,
hospitals, physician's offices, homes, natural environments and
battle fields.
F. EXEMPLARY EMBODIMENTS
[0203] Hydrolytic enzymes are widely used in bioassays for research
and clinical diagnostics. Two of the most commonly used hydrolytic
enzymes in bioassays are beta-galactosidase and alkaline
phosphatase. Beta-galactosidase is often used as the enzyme for the
CEDIA (cloned enzyme donor immunoassay) platform (See, e.g., U.S.
Pat. Nos. 4,708,929, 5,120,653, 5,244,785, and 5,362,625, and WO
96/41172 A1) which is widely used in many assays such as clinical
diagnostics for detecting various analytes including hormones,
vitamins, therapeutic drugs and tests for drug-of-abuse.
[0204] In the CEDIA assay, two fragments of beta-galactosidase (EC
3.2. 1. 23), typically prepared by recombinant DNA technology, are
used. The larger fragment is referred as enzyme acceptor or EA, and
the smaller fragment is referred as enzyme donor or ED. Both
fragments are enzymatically inactive when they are separated. When
these fragments are mixed in solution, they spontaneously assemble
into a fully active enzyme like a native beta-galactosidase. In
many assays including clinical diagnostics, homogeneous assays are
desirable because they save time, save reagents, and are easy to
automate. Homogeneous assays allow a simple "mix and read" process,
without requiring lengthy, time consuming wash steps to remove
unbound constituents.
[0205] CEDIA assay is a homogenous assay that meets some of the
desired clinical testing requirements. The CEDIA homogenous assay
platform operates by controlling the spontaneous assembly of the EA
and ED through target-binder, e.g., an antigen-antibody, reaction.
In some embodiments, an analyte or biomarker can be covalently
attached to the ED in a way such that there is no interference in
forming active beta-galactosidase enzyme when the ED conjugate is
mixed with EA. Adding to the system a binder or an antibody to the
analyte or the biomarker will inhibit the spontaneous assembly of
enzyme. Placing this system in competition for an analyte in a
sample, e.g., a patient's serum, will create active enzyme in
direct proportion to the amount of free unknown analyte or
biomarker in the sample. The amount of enzyme created is monitored
through the hydrolysis of an appropriate enzyme substrate such as
o-nitrophenyl-beta-D-galactopyranoside or chlorophenol red
-beta-D-galactopyranoside. However, these substrates have
limitations in their extinction coefficients, and are not suitable
to make the CEDIA assay a highly sensitive assay system similar to
the chemiluminescent based heterogenous immunoassay.
[0206] In some embodiments, to improve the CEIDA system, we have
designed a series of new substrates for beta-galactosidase. One
distinctive characteristic of the new substrates is the linkage of
an aryl alcohol molecule through its hydroxyl group to a
beta-D-galactopyranoside. Hydrolysis of these substrates by
beta-galactosidase generates free aryl alcohol molecules which are
oxidized to aryl aldehyde by aryl alcohol oxidase with the
concomitant formation of a hydrogen peroxide (H.sub.2O.sub.2). Aryl
aldehyde is then reduced back to aryl alcohol by an enzyme aryl
alcohol dehydrogenase or alcohol dehydrogenase. This oxidation and
reduction reactions form an enzyme cycling with an accumulation of
a reaction by-product H.sub.2O.sub.2 which is exponentially
amplified in each reaction cycle.
[0207] Any suitable aryl alcohol oxidase and aryl alcohol
dehydrogenase can be used. For example, the aryl alcohol oxidase
and aryl alcohol dehydrogenase from a fungus, e.g., Pleurotus
eryngii, can be used (Guillen and Evans, Applied and Environmental
Microbiology, 60(8):2811-2817 (1994)). Use of the novel substrate
for beta-galactosidase in the presently described assays allows for
a coupled enzyme cycling reaction which significantly amplifies the
reaction signal for detection, and thus improving the assay
sensitivity of the CEDIA homogenous platform.
[0208] As another example, alkaline phosphatase is an enzyme that
is widely used as a reporting enzyme in various assays including
immunoassays such as ELISA, and DNA sequencing. The new substrates
described herein include aryl alcohol analogs linking the hydroxyl
group with a phosphate group (H.sub.2PO.sub.4). Hydrolysis of these
substrates by an alkaline phosphatase will generate free aryl
alcohol molecules which will serve as substrate for aryl alcohol
oxidase and can be coupled to the aryl alcohol
oxidase/dehydrogenase based enzyme cycling system for signal
amplification. This will give a more sensitive detection for
immunoassays and DNA sequencing.
[0209] The following enumerated embodiments represent certain
aspects of the invention: [0210] 1. A hydrolytic enzyme substrate,
which is a compound of formula (I):
##STR00020##
[0210] wherein:
[0211] A is an aromatic or heteroaromatic group, a 1-alkene or a
1-alkyne, each of which is optionally substituted;
[0212] each R is independently H or an optionally substituted C1-C4
alkyl or aryl;
[0213] n is an integer from 1-4;
[0214] and X is a group comprising a substrate moiety, [0215]
wherein the substrate moiety comprises a recognition component of a
substrate for the hydrolytic enzyme, and wherein the activity of
said hydrolytic enzyme is capable of hydrolyzing said compound of
formula (I) to form compounds (II) and (III):
##STR00021##
[0216] 2. The hydrolytic enzyme substrate of embodiment 1, wherein
A is an optionally substituted aromatic or heteroaromatic
group.
[0217] 3. The hydrolytic enzyme substrate of embodiment 2, wherein
A is optionally substituted phenyl or naphthyl.
[0218] 4. The hydrolytic enzyme substrate of claim 1, wherein A is
a 1-alkene of the formula (IV):
##STR00022##
[0219] wherein the wavy line indicates the point of attachment of A
to --[CH(R)].sub.n--O--X in Formula (I), and each G, G' and G'' is
independently H or an optionally substituted group selected from
the group consisting of C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl,
C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, aryl, and heteroaryl.
[0220] 5. The hydrolytic enzyme substrate of embodiment 1, wherein
A is a 1-alkyne of the formula (V):
##STR00023##
[0221] wherein the wavy line indicates the point of attachment of A
to --[CH(R)].sub.n--O--X in Formula (I), and G is H or an
optionally substituted member selected from the group consisting of
C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C3-C8
heterocycloalkyl, aryl, and heteroaryl.
[0222] 6. The hydrolytic enzyme substrate of embodiment 1, wherein
R is H, Me or phenyl.
[0223] 7. The hydrolytic enzyme substrate of embodiment 1, wherein
X comprises a saccharide.
[0224] 8. The hydrolytic enzyme substrate of embodiment 7, wherein
the compound is of the formula (VIa) or (VIb):
##STR00024## [0225] wherein R.sup.2 is H or --CH.sub.2OQ, and each
Q is independently H or a monosaccharide, disaccharide or
oligosaccharide, and A, R and n are as defined in claim 1.
[0226] 9. The hydrolytic enzyme substrate of embodiment 1, wherein
the compound is an ester of formula (VII):
##STR00025##
[0227] wherein R.sup.3 is H or an optionally substituted aryl,
heteroaryl, C1-C8 alkyl, C3-C8 cycloalkyl, or C3-C8 heterocyclyl
group, and A, R and n are as defined in claim 1.
[0228] 10. The hydrolytic enzyme substrate of embodiment 9, wherein
R.sup.3 is selected from the group consisting of Me, Et, and
phenyl, or wherein HO.sub.2C--R.sup.3 is an alpha-amino acid.
[0229] 11. The hydrolytic enzyme substrate of embodiment 1, wherein
the compound is of the formula (VIII):
##STR00026##
[0230] wherein Z is N, S, S.dbd.O, P, or P--OH, and R.sup.4 is O,
hydroxy, C1-C4 alkoxy, C1-C4 alkyl, or aryl.
[0231] 12. The hydrolytic enzyme substrate of embodiment 1, wherein
X comprises a phosphate group.
[0232] 13. The hydrolytic enzyme substrate of embodiment 12,
wherein the compound is of the formula (IX):
##STR00027##
[0233] or a salt thereof.
[0234] 14. The hydrolytic enzyme substrate of any of embodiments
1-3 or 6-13, wherein A is an optionally substituted phenyl
group.
[0235] 15. The hydrolytic enzyme substrate of embodiment 14,
wherein the phenyl group is unsubstituted, or is substituted with
1-3 groups selected from halo, hydroxy, CN, NO.sub.2, COOR',
CONR'.sub.2, NR'.sub.2, OR', optionally substituted C1-4 alkyl,
SR', SO.sub.2R', or SO.sub.2NR'.sub.2,
[0236] wherein each R' is independently H or optionally substituted
C1-4 alkyl, and two R' on the same or adjacent atoms can be taken
together to form an optionally substituted C3-C8 heterocyclic
ring.
[0237] 16. The hydrolytic enzyme substrate of any of the preceding
embodiments, wherein optional substituents for alkyl and
heterocyclic groups are selected from halo, oxo, CN, NO.sub.2,
COOR'', CONR''.sub.2, NR''.sub.2, OR'', optionally substituted C1-4
alkyl, SR', SO.sub.2R'', or SO.sub.2NR''.sub.2, wherein each R'' is
independently H or C1-4 alkyl.
[0238] 17. The hydrolytic enzyme substrate of embodiment 1, wherein
A is a group of the formula (X):
##STR00028##
[0239] wherein the wavy line indicates the point of attachment of A
to --[CH(R)].sub.n--O--X in Formula (I),
[0240] and each G is independently H or an optionally substituted
group selected from the group consisting of C1-C8 alkyl, C2-C8
alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl,
aryl, and heteroaryl.
[0241] 18. The hydrolytic enzyme substrate of any of the preceding
embodiments, wherein R is H.
[0242] 19. The hydrolytic enzyme substrate of any of the preceding
embodiments, wherein n is 1.
[0243] 20. The hydrolytic enzyme substrate of embodiment 1, wherein
X comprises a substrate moiety for a glycosidase.
[0244] 21. The hydrolytic enzyme substrate of embodiment 20,
wherein the glycosidase is a beta-galactosidase.
[0245] 22. The hydrolytic enzyme substrate of embodiment 1, wherein
X comprises a substrate moiety for an esterase.
[0246] 23. The hydrolytic enzyme substrate of embodiment 22,
wherein the esterase is selected from the group consisting of a
carboxylesterase, an acetyl esterase and an alpha-amino acid
esterase.
[0247] 24. The hydrolytic enzyme substrate of embodiment 1, wherein
X comprises a substrate moiety for a phosphatase.
[0248] 25. The hydrolytic enzyme substrate of embodiment 24,
wherein the phosphatase is an alkaline phosphatase.
[0249] 26. A combination, which comprises:
[0250] a) a hydrolytic enzyme substrate of any of the embodiments
1-25; and
[0251] b) a hydrolytic enzyme that is capable of cleaving said
hydrolytic enzyme substrate to produce an aryl alcohol molecule or
unsaturated aliphatic alcohol molecule as a product of said
cleavage reaction catalyzed by said hydrolytic enzyme, wherein said
aryl alcohol molecule or unsaturated aliphatic alcohol molecule has
a structure of said formula (II):
##STR00029##
[0252] wherein A, R and n are as defined in claim 1.
[0253] 27. The combination of embodiment 26, wherein the hydrolytic
enzyme is an esterase, a phosphatase, or a glycosidase.
[0254] 28. The combination of embodiment 27, wherein the hydrolytic
enzyme is selected from the group consisting of an acetylesterase,
an amino acid esterase, a carboxylesterase, a nuclease, a
phosphodiesterase, a lipase and a phosphatase.
[0255] 29. The combination of embodiment 27, wherein the hydrolytic
enzyme is an alkaline phosphatase.
[0256] 30. The combination of embodiment 28, wherein the hydrolytic
enzyme is an .alpha.-amino acid esterase.
[0257] 31. The combination of embodiment 27, wherein the hydrolytic
enzyme is a beta-galactosidase.
[0258] 32. The combination of embodiment 27, wherein the hydrolytic
enzyme is a .beta.-glycosidase.
[0259] 33. The combination of embodiment 26, which further
comprises an oxidizing reagent that is capable of oxidizing the
aryl alcohol molecule or unsaturated aliphatic alcohol molecule
produced by the cleavage reaction catalyzed by the hydrolytic
enzyme.
[0260] 34. The combination of embodiment 33, wherein the oxidizing
reagent is an aryl alcohol oxidase or an aliphatic alcohol oxidase
that is capable of oxidizing the aryl alcohol molecule or
unsaturated aliphatic alcohol molecule in the presence of oxygen to
produce an aryl aldehyde molecule or unsaturated aliphatic aldehyde
molecule and H.sub.2O.sub.2.
[0261] 35. The combination of embodiment 34, which further
comprises a reagent for measuring the H.sub.2O.sub.2.
[0262] 36. The combination of embodiment 35, wherein the reagent
for measuring the H.sub.2O.sub.2 comprises a peroxidase, 4-AA
and/or an aniline analog.
[0263] 37. The combination of embodiment 33, wherein the oxidizing
reagent is an aryl alcohol dehydrogenase or an alcohol
dehydrogenase that is capable of oxidizing the aryl alcohol
molecule or unsaturated aliphatic alcohol molecule in the presence
of NAD.sup.+ or NADP.sup.+ to produce NADH or NADPH.
[0264] 38. The combination of embodiment 37, which further
comprises NAD.sup.+ or NADP.sup.+.
[0265] 39. The combination of embodiment 38, which further
comprises a reagent for measuring the NADH or NADPH.
[0266] 40. The combination of embodiment 34, which further
comprises NADH or NADPH and an aryl alcohol dehydrogenase or an
alcohol dehydrogenase that is capable of reducing the aryl aldehyde
molecule or unsaturated aliphatic aldehyde molecule in the presence
of NADH or NADPH.
[0267] 41. The combination of embodiment 40, which further
comprises a reagent for measuring the H.sub.2O.sub.2.
[0268] 42. The combination of embodiment 41, wherein the reagent
for measuring the H.sub.2O.sub.2 comprises at least one of a
peroxidase, an antipyrine, a phenol, and/or an aniline analog.
[0269] 43. The combination of embodiment 26, wherein the hydrolytic
enzyme substrate comprises at least a part of a .beta.-glycosidase
substrate molecule, and the hydrolytic enzyme is a
.beta.-glycosidase or beta-galactosidase.
[0270] 44. The combination of embodiment 43, which further
comprises:
[0271] a) an aryl alcohol oxidase or an aliphatic alcohol oxidase
that is capable of oxidizing the aryl alcohol molecule or
unsaturated aliphatic alcohol molecule in the presence of oxygen to
produce an aryl aldehyde molecule or unsaturated aliphatic aldehyde
molecule and H.sub.2O.sub.2;
[0272] b) NADH or NADPH;
[0273] c) an aryl alcohol dehydrogenase or an alcohol dehydrogenase
that is capable of reducing the aryl aldehyde molecule or
unsaturated aliphatic aldehyde molecule in the presence of NADH or
NADPH.
[0274] 45. The combination of embodiment 44, which further
comprises a reagent for measuring H.sub.2O.sub.2.
[0275] 46. The combination of embodiment 26, wherein the hydrolytic
enzyme substrate comprises at least a part of an alkaline
phosphatase substrate molecule, and the hydrolytic enzyme is an
alkaline phosphatase.
[0276] 47. The combination of embodiment 46, which further
comprises:
[0277] a) an aryl alcohol oxidase or an aliphatic alcohol oxidase
that is capable of oxidizing the aryl alcohol molecule or
unsaturated aliphatic alcohol molecule in the presence of oxygen to
produce oxidized aryl alcohol molecule or unsaturated aliphatic
alcohol molecule and H.sub.2O.sub.2;
[0278] b) NADH or NADPH;
[0279] c) an aryl alcohol dehydrogenase or an alcohol dehydrogenase
that is capable of reducing the oxidized aryl alcohol molecule or
unsaturated aliphatic alcohol molecule in the presence of NADH or
NADPH.
[0280] 48. The combination of embodiment 47, which further
comprises at least one reagent for measuring H.sub.2O.sub.2.
[0281] 49. The combination of any of the embodiments 26-48, wherein
the components of the combination are comprised in a kit.
[0282] 50. The combination of any of the embodiments 26-49, which
combination is comprised in an assay, isolation and/or production
system for a target.
[0283] 51. The combination of embodiment 50, wherein the target is
an inorganic molecule, an organic molecule and/or a complex
thereof.
[0284] 52. The combination of embodiment 51, wherein the target is
an organic molecule selected from the group consisting of an amino
acid, a peptide, a protein, a nucleoside, a nucleotide, an
oligonucleotide, a nucleic acid, a vitamin, a monosaccharide, an
oligosaccharide, a carbohydrate, a lipid and a complex thereof.
[0285] 53. The combination of embodiment 52, wherein the system is
a system for immunoassay, protein sequencing, nucleic acid
amplification, hybridization and/or sequencing.
[0286] 54. A method for assessing activity and/or amount of a
hydrolytic enzyme in a sample, which method comprises:
[0287] a) contacting a hydrolytic enzyme substrate of any of the
embodiments 1-25 with a sample containing or suspected of
containing a hydrolytic enzyme with a hydrolytic enzyme substrate
having a structure of formula (I):
##STR00030##
[0288] under conditions where said hydrolytic enzyme, if present in
said sample, cleaves said substrate to produce an aryl alcohol
molecule or unsaturated aliphatic alcohol molecule having a
structure of formula (II) and a compound having a structure of
formula (III):
##STR00031##
[0289] wherein A, R, n and X are as defined in claim 1; and
[0290] b) assessing the presence and/or amount of said aryl alcohol
molecule or unsaturated aliphatic alcohol molecule to assess
activity and/or amount of said hydrolytic enzyme in said
sample.
[0291] 55. The method of embodiment 54, wherein the hydrolytic
enzyme is an esterase, a beta-galactosidase, or a glycosidase.
[0292] 56. The method of embodiment 55, wherein the hydrolytic
enzyme is an esterase selected from the group consisting of an
acetylesterase, an amino acid esterase, a carboxylesterase, a
nuclease, a phosphodiesterase, a lipase and a phosphatase.
[0293] 57. The method of embodiment 56, wherein the hydrolytic
enzyme is an alkaline phosphatase.
[0294] 58. The method of embodiment 56, wherein the hydrolytic
enzyme is an .alpha.-amino acid esterase.
[0295] 59. The method of embodiment 55, wherein the hydrolytic
enzyme is a beta-galactosidase.
[0296] 60. The method of embodiment 55, wherein the hydrolytic
enzyme is a .beta.-glycosidase.
[0297] 61. The method of embodiment 54, wherein the step of
assessing the presence and/or amount of the aryl alcohol molecule
or unsaturated aliphatic alcohol molecule comprises oxidizing the
aryl alcohol molecule or unsaturated aliphatic alcohol molecule
with an oxidizing reagent.
[0298] 62. The method of embodiment 61, wherein the presence and/or
amount of the aryl alcohol molecule or unsaturated aliphatic
alcohol molecule is assessed by oxidizing the aryl alcohol molecule
or unsaturated aliphatic alcohol molecule with an aryl alcohol
oxidase or an aliphatic alcohol oxidase in the presence of oxygen
to produce H.sub.2O.sub.2 and assessing the presence and/or amount
of the H.sub.2O.sub.2.
[0299] 63. The method of embodiment 62, wherein the presence and/or
amount of the H.sub.2O.sub.2 is assessed by contacting the
H.sub.2O.sub.2 with a peroxidase, a phenol, an antipyrine, and/or
an aniline analog.
[0300] 64. The method of embodiment 54, wherein the presence and/or
amount of the aryl alcohol molecule or unsaturated aliphatic
alcohol molecule is assessed by oxidizing the aryl alcohol molecule
or unsaturated aliphatic alcohol molecule with an aryl alcohol
dehydrogenase or an alcohol dehydrogenase in the presence of
NAD.sup.+ or NADP.sup.+ to produce NADH or NADPH, and assessing the
presence and/or amount of the NAD+, NADP+, NADH or NADPH.
[0301] 65. The method of embodiment 54, wherein the presence and/or
amount of the aryl alcohol molecule or unsaturated aliphatic
alcohol molecule is assessed by:
[0302] a) oxidizing the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule with an aryl alcohol oxidase or an
aliphatic alcohol oxidase in the presence of oxygen to produce aryl
aldehyde molecule or unsaturated aliphatic aldehyde molecule and
H.sub.2O.sub.2;
[0303] b) reducing the aryl aldehyde molecule or unsaturated
aliphatic aldehyde molecule with an aryl alcohol dehydrogenase or
an alcohol dehydrogenase in the presence of NADH or NADPH to form a
reaction cycle in which the reduced aryl alcohol molecule or
unsaturated aliphatic alcohol molecule is oxidized by the aryl
alcohol oxidase or an aliphatic alcohol oxidase in the presence of
oxygen to produce additional aryl aldehyde molecule or unsaturated
aliphatic aldehyde molecule and H.sub.2O.sub.2; and
[0304] c) assessing the presence and/or amount of the
H.sub.2O.sub.2, or the amount of NADH, NADPH, NAD+, or NADP+.
[0305] 66. The method of embodiment 65, wherein the presence and/or
amount of the H.sub.2O.sub.2 is assessed by contacting the
H.sub.2O.sub.2 with a peroxidase, an antipyrine, a phenol, and/or
an aniline analog.
[0306] 67. The method of embodiment 54, wherein the hydrolytic
enzyme substrate comprises at least a part of a .beta.-glycosidase
substrate molecule, and the hydrolytic enzyme is a
.beta.-glycosidase or beta-galactosidase.
[0307] 68. The method of embodiment 67, wherein the step of
assessing the presence and/or amount of the aryl alcohol molecule
or unsaturated aliphatic alcohol molecule comprises oxidizing the
aryl alcohol molecule or unsaturated aliphatic alcohol molecule
with an oxidizing reagent.
[0308] 69. The method of embodiment 67, wherein the presence and/or
amount of the aryl alcohol molecule or unsaturated aliphatic
alcohol molecule is assessed by oxidizing the aryl alcohol molecule
or unsaturated aliphatic alcohol molecule with an aryl alcohol
oxidase or an aliphatic alcohol oxidase in the presence of oxygen
to produce oxidized aryl alcohol molecule or unsaturated aliphatic
alcohol molecule and H.sub.2O.sub.2 and assessing the presence
and/or amount of the H.sub.2O.sub.2.
[0309] 70. The method of embodiment 69, wherein the presence and/or
amount of the H.sub.2O.sub.2 is assessed by contacting the
H.sub.2O.sub.2 with a peroxidase, 4-AA and/or an aniline
analog.
[0310] 71. The method of embodiment 67, wherein the presence and/or
amount of the aryl alcohol molecule or unsaturated aliphatic
alcohol molecule is assessed by oxidizing the aryl alcohol molecule
or unsaturated aliphatic alcohol molecule with an aryl alcohol
dehydrogenase or an alcohol dehydrogenase in the presence of
NAD.sup.+ or NADP.sup.+ to produce NADH or NADPH, and assessing the
presence and/or amount of the NAD+, NADP+, NADH or NADPH.
[0311] 72. The method of embodiment 67, wherein the presence and/or
amount of the aryl alcohol molecule or unsaturated aliphatic
alcohol molecule is assessed by:
[0312] a) oxidizing the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule with an aryl alcohol oxidase or an
aliphatic alcohol oxidase in the presence of oxygen to produce
oxidized aryl alcohol molecule or unsaturated aliphatic alcohol
molecule and H.sub.2O.sub.2;
[0313] b) reducing the oxidized aryl alcohol molecule or
unsaturated aliphatic alcohol molecule with an aryl alcohol
dehydrogenase or an alcohol dehydrogenase in the presence of NADH
or NADPH to form a reaction cycle in which the reduced aryl alcohol
molecule or unsaturated aliphatic alcohol molecule is oxidized by
the aryl alcohol oxidase or an aliphatic alcohol oxidase in the
presence of oxygen to produce additional oxidized aryl alcohol
molecule or unsaturated aliphatic alcohol molecule and
H.sub.2O.sub.2; and
[0314] c) assessing the presence and/or amount of the
H.sub.2O.sub.2.
[0315] 73. The method of embodiment 72, wherein the presence and/or
amount of the H.sub.2O.sub.2 is assessed by contacting the
H.sub.2O.sub.2 with a peroxidase, a phenol, an antipyrine, and/or
an aniline analog.
[0316] 74. The method of embodiment 54, wherein the hydrolytic
enzyme substrate comprises at least a part of an alkaline
phosphatase substrate molecule, and the hydrolytic enzyme is an
alkaline phosphatase.
[0317] 75. The method of embodiment 74, wherein the step of
assessing the presence and/or amount of the aryl alcohol molecule
or unsaturated aliphatic alcohol molecule comprises oxidizing the
aryl alcohol molecule or unsaturated aliphatic alcohol molecule
with an oxidizing reagent.
[0318] 76. The method of embodiment 74, wherein the presence and/or
amount of the aryl alcohol molecule or unsaturated aliphatic
alcohol molecule is assessed by oxidizing the aryl alcohol molecule
or unsaturated aliphatic alcohol molecule with an aryl alcohol
oxidase or an aliphatic alcohol oxidase in the presence of oxygen
to produce oxidized aryl alcohol molecule or unsaturated aliphatic
alcohol molecule and H.sub.2O.sub.2, and assessing the presence
and/or amount of the H.sub.2O.sub.2.
[0319] 77. The method of embodiment 76, wherein the presence and/or
amount of the H.sub.2O.sub.2 is assessed by contacting the
H.sub.2O.sub.2 with a peroxidase, a phenol, an antipyrine, and/or
an aniline analog.
[0320] 78. The method of embodiment 74, wherein the presence and/or
amount of the aryl alcohol molecule or unsaturated aliphatic
alcohol molecule is assessed by oxidizing the aryl alcohol molecule
or unsaturated aliphatic alcohol molecule with an aryl alcohol
dehydrogenase or an alcohol dehydrogenase in the presence of
NAD.sup.+ or NADP.sup.+ to produce NADH or NADPH, and assessing the
presence and/or amount of the NAD+, NADP+, NADH or NADPH.
[0321] 79. The method of embodiment 74, wherein the presence and/or
amount of the aryl alcohol molecule or unsaturated aliphatic
alcohol molecule is assessed by
[0322] a) oxidizing the aryl alcohol molecule or unsaturated
aliphatic alcohol molecule with an aryl alcohol oxidase or an
aliphatic alcohol oxidase in the presence of oxygen to produce
oxidized aryl alcohol molecule or unsaturated aliphatic alcohol
molecule and H.sub.2O.sub.2;
[0323] b) reducing the oxidized aryl alcohol molecule or
unsaturated aliphatic alcohol molecule with an aryl alcohol
dehydrogenase or an alcohol dehydrogenase in the presence of NADH
or NADPH to form a reaction cycle in which the reduced aryl alcohol
molecule or unsaturated aliphatic alcohol molecule is oxidized by
the aryl alcohol oxidase or an aliphatic alcohol oxidase in the
presence of oxygen to produce additional oxidized aryl alcohol
molecule or unsaturated aliphatic alcohol molecule and
H.sub.2O.sub.2; and
[0324] c) assessing the presence and/or amount of the
H.sub.2O.sub.2.
[0325] 80. The method of embodiment 79, wherein the presence and/or
amount of the H.sub.2O.sub.2 is assessed by contacting the
H.sub.2O.sub.2 with a peroxidase, a phenol, an antipyrine, and/or
an aniline analog.
[0326] 81. The method of any of the embodiments 54-80, which is
conducted as part of an assay, isolation and/or production of a
target.
[0327] 82. The method of embodiment 81, wherein the target is an
inorganic molecule, an organic molecule and/or a complex
thereof.
[0328] 83. The method of embodiment 82, wherein the organic
molecule is selected from the group consisting of an amino acid, a
peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide,
a nucleic acid, a vitamin, a monosaccharide, an oligosaccharide, a
carbohydrate, a lipid and a complex thereof.
[0329] 84. The method of embodiment 82, which is conducted as part
of an immunoassay, protein sequencing, nucleic acid amplification,
hybridization or sequencing.
[0330] 85. The method of embodiment 84, wherein the method is used
to monitor RNA or DNA sequencing.
[0331] 86. The method of embodiment 85, wherein the hydrolytic
enzyme is alkaline phosphatase.
[0332] The present invention is further illustrated by the
following exemplary embodiments.
EXAMPLES
Example 1
Synthesis of Substrates
[0333] Suitable hydrolytic enzyme substrates for use in the
compositions and methods of the invention can be made by
conventional methods using known starting materials. Examples of
such hydrolytic enzyme substrates include p-MOBG (p-methoxybenzyl
galactose) and HDEGP, which can be made from the known galactosyl
bromide in protected form (tetra-acetate) using silver (I) oxide in
dichloromethane, followed by hydrolysis with methoxide in methanol
to remove the acetate protecting groups.
Synthesis of p-Methoxybenzyl-.beta.-D-Galactopyranoside (P-MOBG)
(MW: 300.3)
1). p-Methoxybenzyl
2,3,4,6-Tetra-O-Acetyl-.beta.-D-Galactopyranoside (p-MBAGP) (MW:
468.38)
[0334] To a solution of 2.64 g (6.42 mmol) of
acetobromo-.alpha.-D-galactose and 1.38 mL (11.06 mmol) of
p-methoxybenzyl alcohol in 20 mL of dichloromethane was added 1.5 g
of molecular sieves (4A.degree.). After stirring for 20 min at room
temperature, 1.5 g (6.48 mmol) of silver(I) oxide was added. The
reaction mixture was stirred overnight at room temperature and then
filtered and concentrated. Chromatography of the residue on silica
gel yielded 1.0 g (80.64%) of the product as a colorless syrup. 1H
NMR (500 MHz, CDCl3): .delta.=7.22, 6.88 (2 d, 4H, phenyl),
C.sub.22H.sub.28O.sub.11:calculated 468.5. found 491.6
(M+.sup.23Na).
2). p-Methoxybenzyl-.beta.-D-Galactopyranoside (P-MOBG) (MW:
300.3)
[0335] After 1.0 g (2.14 mmol) of P-methoxybenzyl
2,3,4,6-tetra-O-acetyl-.beta.-D-galactopyranoside (p-MBAGP) was
co-evaporated with anhydrous methanol (3.times.10 mL), 10 mL of
anhydrous methanol and 1 mL of 0.5 M sodium methoxide were added.
The reaction mixture was stirred for 1 hr at room temperature and
then was neutralized to pH 7.0 by addition of 2 N HCl. After the
solvents were removed by evaporation at 20.degree. C. to 30.degree.
C., the residue was purified by silica gel chromatography eluted by
ethyl acetate and then ethyl acetate/ethanol (5/1). Product: 0.4 g
of white solid. Yield: 62.2%. .sup.1H NMR (500 MHz,
CDCl.sub.3/CD.sub.3OD=90/10): .delta.=7.2, 6.76 (2d, 4H, phenyl),
4.74, 4.46 (2d, 2H, CH.sub.2-phenyl), 4.18 (D, 1H, J1,2=7.6 Hz,
1-H), 3.70 (s, 3H, CH.sub.3O--), 3.30-3.78 (m, 6H).
C.sub.14H.sub.20O.sub.7: Calculated 300.3, found 299.2 (M-1) and
323.2 (M+.sup.23Na).
##STR00032##
[0336] The substrate HDEG was synthesized by a similar
procedures.
Example 2
Hydrolysis by Beta-Galactosidase
[0337] Hydrolytic enzyme substrates of the invention can be
hydrolyzed by suitable enzymes matched with the substrate; thus
PMOBG or HDEGP as shown above can be hydrolyzed by
beta-galactosidase as illustrated below.
##STR00033##
[0338] Incubation of 10 mM of p-MOBG with 10 unit/ml of
beta-galactosidase and 20 unit/ml of aryl alcohol oxidase at
37.degree. C. for 30 mM resulted in generation of H.sub.2O.sub.2,
which was detected by peroxidase in the presence of 4-AA and TOOS
at 560 nm. When p-MOBG was incubated with aryl alcohol oxidase
alone, there was no H.sub.2O.sub.2 detected, indicating p-MOBG is
not a substrate for aryl alcohol oxidase but becomes the substrate
for aryl alcohol oxidase only after hydrolysis of p-MOBG by
beta-galactosidase. The aryl alcohol dehydrogenase used in this
example was recombinantly produced based on the description in
Guillen and Evans, Appl. Environmental Microbiol., 60(8):2811-17
(1994) and Reiser et al., J. Biol. Chem., 269(45):28152-28159
(1994).
[0339] Some specific examples of the hydrolysis reaction coupled
with the enzymatic cycling reaction.
Example 3
TABLE-US-00001 [0340] Reagent 1: Reagent 2: Beta-galactosidase: 50
unit/ml Phosphate buffer, 100 mM pH 6.3: Substrate p-MOBG: 3 mM
4-AA: 5 mM Aryl alcohol oxidase: 20 unit/ml Toos: 5 mM Aryl alcohol
30 unit/ml Horse radish 10 unit/ml dehydrogenase: peroxidase: NADH:
2 mM Tris-HCl buffer, pH 9.0 50 mM ##STR00034##
[0341] In this study, 180 .mu.l of reagent 1 is mixed with 20 .mu.l
of a sample to be tested and the mixture is incubated at 37.degree.
C. for 5 minutes. Sixty (60) .mu.l of reagent 2 is then added to
the mixture and is incubated at 37.degree. C. for another 5
minutes. The change of absorbance at 560 nm is measured for 2-5
minutes after the reagent 2 is added.
Example 4
TABLE-US-00002 [0342] Reagent 1: Reagent 2: Beta-galactosidase: 30
unit/ml Citric buffer, pH 6.0: 100 mM Substrate HDEG: 3 mM 4-AA: 3
mM Aryl alcohol oxidase: 15 unit/ml Aniline analog: 5 mM Alcohol
dehydrogenase: 40 unit/ml Horse radish 15 unit/ml peroxidase:
NADPH: 1 mM Borate buffer, pH 9.1 30 mM ##STR00035##
[0343] In this study, 180 .mu.l of reagent 1 is mixed with 20 .mu.l
of a sample to be tested and the mixture is incubated at 37.degree.
C. for 5 minutes. Sixty (60) .mu.l of reagent 2 is then added to
the mixture and is incubated at 37.degree. C. for another 4
minutes. The change of absorbance at 560 nm is measured for 1-4
minutes after the reagent 2 is added.
Example 5
TABLE-US-00003 [0344] Reagent 1: Reagent 2: Alkaline phosphatase:
50 unit/ml Phosphate buffer, 100 mM pH 6.3: Substrate AP: 3 mM
4-AA: 5 mM Aryl alcohol oxidase: 20 unit/ml Toos: 5 mM Aryl alcohol
30 unit/ml Horse radish 10 unit/ml dehydrogenase: peroxidase: NADH:
2 mM Tris-HCl buffer, pH 9.0 50 mM ##STR00036##
[0345] The structure of AP is shown in FIG. 4. In some examples,
R1, R2, R4 and R5 are hydrogen, and R3 is --OCH3. In this study,
180 .mu.l of reagent 1 is mixed with 20 .mu.l of a sample to be
tested and the mixture is incubated at 37.degree. C. for 5 minutes.
Sixty (60) .mu.l of reagent 2 is then added to the mixture and is
incubated at 37.degree. C. for another 5 minutes. The change of
absorbance at 560 nm is measured for 2-5 minutes after the reagent
2 is added.
Example 6
TABLE-US-00004 [0346] Reagent 1: Reagent 2: Acetylesterase: 30
unit/ml Citric buffer, pH 6.0: 100 mM Substrate AE: 3 mM 4-AA: 3 mM
Aryl alcohol oxidase: 15 unit/ml Aniline analog: 5 mM Alcohol
dehydrogenase: 40 unit/ml Horse radish 15 unit/ml peroxidase:
NADPH: 1 mM Borate buffer, pH 9.1 30 mM ##STR00037##
[0347] The structure of AE is shown in FIG. 5. In some examples,
R1, R2, R4 and R5 are hydrogen, and R3 is --OCH3. In this study,
180 .mu.l of reagent 1 is mixed with 20 .mu.l of a sample to be
tested and the mixture is incubated at 37.degree. C. for 5 minutes.
Sixty (60) .mu.l of reagent 2 is then added to the mixture and is
incubated at 37.degree. C. for another 4 minutes. The change of
absorbance at 560 nm is measured for 1-4 minutes after the reagent
2 is added.
Example 7
[0348] An exemplary AAO/AAD cycling system using an aryl alcohol
oxidase (AAO) and an aryl alcohol dehydrogenase (AAD) was used to
detect an aryl alcohol (see below).
##STR00038##
[0349] AAO was purified from Pleurotus eryngii using the procedures
described in Guillen, F. et al., "Substrate specificity and
properties of aryl-alcohol oxidase from the ligninolytic fungus
Pleurotus eryngii," Eur. J. Biochem., 209:603-611 (1992). The
purification steps include concentration, anion exchange
chromatography and hydrophobic interaction chromatography. DNA
construct for recombinant production of Pseudomonas putida AAD was
made based on the DNA sequence disclosed in Shaw, J. P. et al.,
Kinetic studies on benzyl alcohol dehydrogenase encoded by TOL
plasmid pWW0," J. Biol. Chem., 268:10842-10850 (1993), and the
recombinant Pseudomonas putida AAD was made by overexpression in E.
coli. The cycling system was combined with chemiluminescent
(luminol-hydrogen peroxide-horseradish peroxidase) reaction to form
a sensitive assay.
[0350] For AAO, K.sub.p-anisyl-OH=37.+-.8 .mu.M; and
k.sub.cat=117/s. For AAD, K.sub.NADH=9.1.+-.2 .mu.M; K.sub.p-Anisyl
aldehyde=6.6.+-.0.8 .mu.M; and k.sub.cat=30/s.
[0351] Reaction condition: Tris-HCL, pH 7, 100 mM; NADH 100 .mu.M;
EDTA 100 mM; HRP, 1.5 U; G6PH, 1 mM; G6PDH, 2 U; Luminol/enhancer,
5 .mu.l; AAO, 20 .mu.l (6 mg/ml); AAD, 20 .mu.l (25 mg/ml) in a
total volume 200 .mu.l. AAD was added last to start the cycling
reaction. The reaction was monitored for 10 minutes using impulse
2, CLIA reader from Monobind Inc.
[0352] As shown in FIG. 10, the aryl alcohol can be detected as low
as 0.2 nM with the AAO/AAD cycling system. Compared with the
reaction only catalyzed by AAO, the detection limit for aryl
alcohol with the AAO/AAD cycling system can be increased at least
1,000 fold higher.
Example 8
[0353] An exemplary AAO/AAD cycling system using an aryl alcohol
oxidase (AAO) and an aryl alcohol dehydrogenase (AAD) as described
in Example 7 was used to detect alkaline phosphatase (ALP).
[0354] Reaction condition: 50 .mu.l ALP reaction system; Tris-HCl,
pH 10.0 (100 mM); Anisyl phosphate (40 .mu.M), 5 .mu.l ALP reaction
buffer. ALP reaction was kept for 5 mins.
[0355] 200 .mu.l cycling system: 50 .mu.l ALP reaction system; AAO
10 .mu.l (2.5 mg/ml); AAD 10 .mu.l (20 mg/ml); NADH, 20 .mu.M;
G6PH, 2 mM; G6PDH, 2 U; HRP, 1.5 U; Hyperblue, 10 .mu.l; and
Tris-HCl, pH 7.0 (200 mM). AAD was added last to start the cycling
reaction. The reaction was monitored for 30 minutes using impulse
2, CLIA reader from Monobind Inc.
[0356] As shown in FIG. 11, when the assay was used to detect
alkaline phosphatase in solution using 40 .mu.M of anisyl phosphate
as the substrate, 2.6 attomole of ALP can be detected. The
detection sensitivity reached to sub attomole, and can be further
improved with the optimization of ALP substrate concentration and
increase of enzyme concentration and reaction time.
[0357] The above examples are included for illustrative purposes
only and are not intended to limit the scope of the invention. Many
variations to those described above are possible. Since
modifications and variations to the examples described above will
be apparent to those of skill in this art, it is intended that this
invention be limited only by the scope of the appended claims.
* * * * *