Note: Descriptions are shown in the official language in which they were submitted.
WO 94/24306 = ~ .. pCT/US94/04098
1
REPORTER ENZYME RELEASE TECHNOLOGY: METHODS OF ASSAYING FOR
THE PRESENCE OF ASPARTIC PROTEASES AND OTHER HYDROLYTIC
ENZYME ACTIVITIES
FIELD OF THE INVENTION
This invention relates generally to methods of assaying for the presence of
hydrolase activity (i.e., hydrolytic enzymes) in a sample or specimen. In
particular, this
invention relates to a method for detecting candidiasis by assaying for the
presence of
enzymatically active aspartic protease in a sample.
BACKGROUND OF THE INVENTION
Candida albicans and other Candida species cause a number of common,
medically important infections. Oral candidiasis, for example, is very common
in
patients with immunodeficiency. Moreover, wlvovaginal candidiasis is one of
the most
frequent disorders in obstetrics and gynecology. It has been estimated that
approximately
three-quarters of all adult women suffer from at least one attack of this
disease. (De
Bernardis, et al., J. Clin. Microbiol. 27(11):2598-2603 (1989)). As a result
of its wide-
spread occurrence, extensive amounts of research have gone into understanding
the
etiology of candidiasis.
Research has demonstrated that Candida albicans and other Candida species
have an etiological involvement in human candidiasis, and it is now generally
believed
that candidiasis is caused primarily by the presence of Candida albicans.
Further, there
is now considerable evidence for a role of an aspartic protease or
(interchangeably) acid
proteinase as a virulence factor of Candida albicans. It is known that pure
cultures of
Candida albicans secrete an aspartic protease when grown under precisely
defined
conditions. Similarly, it is known that pure strains of Candida albicans
isolated from
women with symptomatic wlvovaginitis release this enzyme when they are
subsequently
grown in specifically defined culture medium.
Candida albicarrs acid proteinase antigen, i.e., aspartic protease antigen,
has
been detected immunologically in the vaginal fluid of all women from which
wlvovaginal
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Candida albicans was isolated.
The concentration of Candida albicans acid proteinase antigen, however, was
significantly higher in patients with symptomatic vulvovaginal candidiasis
than in
asymptomatic carriers. The vaginal fluid concentration of this antigen in
women with
candidiasis is approximately 176 t 15.2 ng/mL,whereas the vaginal fluid
concentration
of this antigen in women without isolation of Candida albicans, i. e. ,
without clinical
candidiasis, was less than 2 ng/ml. Asymptomatic Candida albicans carriers had
intermediate antigen levels (94 t 18.5 nglml). These findings are a strong
indication
that acid proteinase (i.e., aspartic protease) is involved in the pathogenesis
cf
wlvovaginal candidiasis. Candida albicans aspartic protease, however, is known
to be
unstable at body temperatures. Moreover, detection of the aspartic proteinase
antigen
immunologically did not indicate whether the enzyme was present in an
enzymatically
active form.
Candida albicans acid proteinase is an extracellular aspartic protease.
Aspartic proteases are one of the major classes of proteases. They contain one
or mom
key aspartic acid residues which are required for activity. Candida albicans
aspartic
protease has a broad protein substrate specificity which includes, for
example, albur~Ain,
hemoglobin, casein, immunoglobin A, and many other proteins. This enzyme
performs
optimally under acidic conditions (i.e., pH 2.5-5.5), and it is rapidly
inactivated at a high
pH (i.e, at pH 7.5). Candida albicarrs aspartic protease is strongly inhibited
by
pepstatin, but it is not inhibited by thiol reagents, chelators or serine
protease inhibitors.
Marry pharmaceutical companies and leading academicians are studying
aspartic protease inhibitors for potential therapeutic use. Their efforts,
however, are
made di~cult due to a lack of a suitable enzyme assay for aspartic proteases.
While
simple colorimetric assays are available for some serine, thiol, metallo,
acid, and alkaline
proteases and peptidases, they are not available for aspartic proteases. The
substrate
specificity of this particular class of enzymes requires the presence of
several
hydrophobic amino acids. This property has greatly hindered the search for
simple
synthetic chromogenic substrates because the hydrophobic amino acids which
serve as the
substrate for aspartic proteases are notoriously difficult to dissolve in
water. As a result,
chromogenic substrates for aspartic protease are not commercially available,
are difficult
to synthesize and characterize, and are poorly water soluble. The net effect
of these
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limitations is that the enzyme activity as defined by these chromogenic
substrates is
extremely low and thus, colorimetric assays for aspartic protease are quite
insensitive.
Similarly, fluorogenic substrates have been described for aspartic proteases,
but they,
too, are of limited utility. First, as previously mentioned, the substrate
specificity of this
particular class of enzymes requires the presence of several hydrophobic amino
acids,
rendering the substrates relatively insoluble. Second, at the low achievable
concentrations of these substrates, the fluorogenic substrates are hydrolyzed
very slowly
and thus, fluorogenic assays are time-consuming. In addition, many biological
specimens
contain fluorescent materials which can interfere with fluorogenic assays for
aspartic
proteases.
Due to the lack of suitable colorimetric or fluorogenic assays for the
detection
of aspartic proteases, ultraviolet (UV) spectrophotometric assays are
generally used to
assay for the presence of this particular class of enzymes. In typical UV
spectrophotometric assays, aspartic protease is added to a solution of protein
(such as, for
example, hemoglobin or albumin) and the mixture is incubated at 30-37
°C for 0.5 to 4
hours. After incubation, cold, concentrated trichloroacetic acid (TCA) is
added to the
chilled incubation mixture to precipitate the undigested protein, leaving
ultraviolet light
absorbing peptides in solution. Finally, the precipitated, undigested protein
is pelleted by
centrifugation for approximately 1 hour at refrigerated temperatures, the
supernatant
aspirated, and its Optical Density at 280 nm is determined to reflect the
amount of
protein hydrolysis. Although this assay can be used for the detection of
aspartic
proteases, it is both time-consuming and laborious.
To date, therefore, no convenient, simple, on-site assay has been developed
for detecting the presence of enzymatically active aspartic proteases.
Accordingly, the
present invention is directed to a method of assaying for the presence of
enzymatically
active aspartic proteases which overcomes the problems and disadvantages of
the prior
art. Further, the methods of the present invention are also useful for
assaying for the
presence of other known hydrolytic enzymes, i. e. , hydrolases.
~ SUMMARY OF THE INVENTION
It has now been discovered that enzymatically active Candida albicans aspartic
protease is present in the vaginal fluid of women with wlvovaginal
candidiasis. It has
further been discovered that the presence of enzymatically active aspartic
protease in a
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sample or specimen can serve as a marker for the detection and diagnosis of
candidiasis.
Accordingly, a method has now been developed for detecting candidiasis by
assaying for
the presence of enzymatically active aspartic protease in a sample.
In this method, a sample, e.g., vaginal fluid, is contacted with a solid
support.
The solid support with which the sample is contacted has a reporter enzyme
(i.e., a signal
generating enzyme) immobilized thereon. The reporter enzyme is immobilized on
the
solid support in a manner such that it is released from the solid support upon
action of
the enzymatically active aspartic protease if the enzymatically active
aspartic protease is,
in fact, present in the sample. The sample after having been contacted with
the solid
support is combined with an indicator. The indicator is any chemical species
which is
susceptible to a visible or detectable change (such as, for example, a change
in color)
upon action of the reporter enzyme. If after contact with the sample the
indicator
undergoes a detectable change, enzymatically active aspartic protease is
present in the
sample and, hence, it can be said that candidiasis is present.
Prior to the present invention, there was no rapid and straightforward means
of assaying for the presence of enzymatically active aspartic proteases or,
more
importantly, candidiasis. Spectrophotometric, fluorogenic and immunological
assays have
been used to assay for the presence of aspartic protease activity, but these
assays are
time-consuming, laborious and not suitable for use by untrained, on-site
clinical
personnel. Similarly, Candida albicans can be detected by culturing a specimen
in a
defined media or by wet mount microscopy. The culturing procedure is very time-
consuming (i.e., it takes approximately 48 hours), and both procedures require
expensive
equipment and extensive training. In contrast to previously used assays, the
presently
claimed method of assaying for the presence of enzymatically active aspartic
protease
and, in turn, candidiasis is rapid, accurate, cost-effective, and simple to
use.
The reporter enzyme release technology upon which the aspartic protease
assay is based can also be used to assay for the presence of any active
hydrolytic enzyme
including, but not limited to, the following: proteases or (interchangeably)
proteinases,
peptidases, lipases, nucleases, homo-oligosaccharidases, hetero-
oligosaccharidases, homo-
polysaccharidases, hetero-polysaccharidases, phosphatases, sulfatases,
neuraminidases and
esterases. Accordingly, methods of assaying for the presence of an
enzymatically active
hydrolase in a sample have now been developed.
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WO 94/24306 . PCT/US94/04098
In these methods, a sample or specimen is contacted with a solid support. The
solid support with which the sample is contacted has a reporter enzyme (i.e.,
a signal
generating enzyme) immobilized thereon. The reporter enzyme is immobilized on
the
solid support in a manner such that it is released from the solid support upon
action of
5 the hydrolase if the enzymatically active hydrolase is, in fact, present in
the sample. The
sample after it has been contacted with the solid support is combined with an
indicator.
The indicator is any chemical species which is susceptible to a detectable
change, usually
a change in color, upon action of the reporter enzyme. A detectable change in
the
indicator is an indication that the enzymatically active hydrolase is present
in the sample.
Conversely, the lack of a detectable change in the indicator is an indication
that the
enzymatically active hydrolase is absent from the sample. As with the assay
for
enzymatically active aspartic protease and, in turn, candidiasis, the
presently claimed
methods of assaying for the presence of an enzymatically active hydrolase in a
sample are
rapid, accurate, cost-effective, and simple to use.
Moreover, the reporter enzyme release technology upon which the above
methods are based can also be used to assay for the presence of an inhibitor
of any
known hydrolytic enzyme, including, but not limited to, the following:
inhibitors of the
proteases or (interchangeably) proteinases, peptidases, lipases, nucleases,
homo-
oligosaccharidases, hetero-oligosaccharidases, homo-polysaccharidases, hetero-
polysaccharidases, phosphatases, sulfatases, neuraminidases and esterases.
Accordingly,
methods of assaying for the presence of a hydrolase inhibitor in a sample have
now been
developed.
In these methods, a sample or specimen is contacted with a target hydrolase
and a solid support. The solid support with which the sample is contacted has
a reporter
enzyme (i. e. , a signal generating enzyme) immobilized thereon. The reporter
enzyme is
immobilized on the solid support in a manner such that it is released from the
solid
support upon action of the target hydrolase provided the target hydrolase is
not
inactivated due to the presence of the hydrolase inhibitor. The sample after
it has been
contacted with the target hydrolase and the reporter enzyme is combined with
an
. 30 indicator. The indicator is any chemical species which is susceptible to
a detectable
change, usually a change in color, upon action of the reporter enzyme if the
reporter
enzyme has been released from the solid support by the target hydrolase. In
the event
that the target hydrolase inhibitor is not present in the sample, the target
hydrolase will
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release the reporter enzyme from the support, thereby producing a detectable
change in
the indicator. Conversely, if the target hydrolase inhibitor is present in the
sample, the
target hydrolase will be inhibited, the reporter enzyme will not be released
from the solid
support, and a detectable change or response will not be produced in the
indicator. As
with the previously described methods, the presently claimed methods of
assaying for the
presence of a hydrolase inhibitor in a sample are rapid, accurate, cost-
effective, and
simple to use.
In addition to the methods of assaying for the presence of enzymatically
active
aspartic protease and the activities of other hydrolytic enzymes, a dry, self-
contained test
device has now been developed for testing a sample for the presence of
candidiasis by
assaying for the presence of enzymatically active aspartic protease.
Furthermore, a dry,
self-contained test device for assaying for the presence of an enzymatically
active
hydrolase in a sample has also been developed. These test devices combine a
reporter
enzyme immobilized on a solid support, an indicator, and one or more other
reagents in
dry form in a laminated panel with an internal chamber, the chamber being a
void space
until the sample is placed inside. For convenience, the parts of the panel and
the
locations of the functional chemicals in the panel will be described from a
frame of
reference in which the panel is in a horizontal position, since this is the
most likely
position which the panel will occupy during use. With the panel in this
position,
particularly for the preferred panels of this invention which are thin, flat
structures, the
sample will be placed in the chamber through an opening at the top of the
panel. Of the
laminae forming the panel, the uppermost lamina in this position, this lamina
being the
one through which the sample is introduced, will be referred to as the top
lamina of the
panel, the lower surface of this lamina forming the upper surface of the
chamber.
Likewise, the lowermost lamina of the panel will be referred to as the bottom
lamina of
the panel, the upper surface of this bottom lamina forming the lower surface
of the
chamber. The thin edges along the perimeters of these top and bottom laminae
will be
referred to as the side edges of the panel, and the thin lateral extremities
of the chamber
along the edges of its upper and lower surfaces will be referred to as the
side walls of the
chamber. Regions of any given surface which are adjacent to each other in the
same
horizontal plane will be referred to as horizontally adjacent, whereas lamina
applied
directly over other laminae to form parallel horizontal planes will be
referred to as
vertically adjacent.
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The top, bottom, or both laminae of the panel are fabricated of a light-
transmitting, preferably transparent, material. The reporter enzyme
immobilized on a
solid support, indicator and other,components and reagents needed for the test
are
arranged in one or more laminae within the chamber, either as coatings on the
upper
surface of the chamber, as coatings on the lower surface of the chamber, or on
both.
The indicator is any chemical species which is susceptible to a detectable
change, usually
a color change, upon action of the reporter enzyme when it is released from
the solid
support by the enzymatically active hydrolase whose presence is being
detected. The
lamina containing the indicator may be on the upper or lower surface of the
chamber.
One or more of the reagents needed for the test may be included in the same
lamina as
the indicator, or in separate laminae on the same surface or on the opposite
surface. In
certain preferred embodiments of the invention, the indicator is contained in
the lamina
applied directly underneath a light-transmitting wall and the reporter enzyme
immobilized
on the solid support is contained in the lamina applied to the opposing wall.
The reagents occupying the laminae may be selected such that all that is
needed to complete the test is the addition of the sample plus a minimal
number of
additional reagents such as, for example, a developer. In particularly
preferred
embodiments, however, the laminae contain all reagents needed other than the
sample, so
that performance of the test requires nothing more than addition of the
sample.
All laminae are solid layers prior to contact with the sample, and the lamina
containing the indicator is preferably of a composition which is insoluble in
the liquid
sample for which the test is designed, so that the indicator remains in the
lamina
throughout the duration of the test. For samples in either aqueous or water-
soluble
media, therefore, the preferred indicator lamina is either an indicator which
is insoluble
in water or an indicator held in a matrix which is insoluble in water. With
the indicator
thus retained in a thin concentrated lamina directly underneath a light-
transmitting wall, a
change in the indicator which is detectable through the light-transmitting
wall occurs in a
short period of time, resulting in both high sensitivity and a fast result.
This invention may be adapted and used for tests for a wide variety of
hydrolases in test samples from a wide variety of sources. Moreover, this
invention may
be adapted and used for tests for a wide variety of hydrolase inhibitors. A
test may
involve either a single reaction or a sequence of reactions culminating in a
detectable
change in the indicator, and the number and types of reagents and reactions
will
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WO 94/2-1306 216 0 2 6 2 PCT/US94/04098
accordingly vary from one test to the next depending on which hydrolase is
being
detected. In some cases, best results are obtained when the pre-applied
reacting species
are distributed between the upper and lower surfaces of the chamber such that
they are
separated by a gap until the gap is filled with the test sample. In other
cases, the reacting
species may be placed in a common lamina or in two or more distinct but
vertically
adjacent laminae on the upper or lower surface of the chamber with no loss in
the
reliability of the test. In all cases, however, the laminae are constituted
and arranged
such that the reactions which culminate in the detectable indicator change
occur only
when the chamber is filled with the test sample, and such that when the
indicator change
does occur, it is at least concentrated in, and preferably restricted to, the
lamina
immediately adjacent to a light-transmitting wall.
In preferred embodiments of the invention, the test device includes a built-in
positive control, a built-in negative control, or both, all of which are
activated by the
addition of a single specimen. The activation of these controls occurs
simultaneously
with the performance of the test, and detectable indications (such as, for
example, color
changes or the lack thereof) representing both the controls and the test, are
achieved with
a single application of the specimen to the device and are detectable through
a light-
transmitting wall. The controls occupy positions on the device which are
horizontally
adjacent to the test area, with appropriate indicia on the upper or lower
surface of the
device, preferably the upper, to identify the controls and differentiate them
from the test.
The controls themselves generally consist of further laminae containing
reagents or other
appropriate species which will either induce the detectable change in the
indicator by
themselves or prevent the change from occurring, and will do so only when the
test
sample is present and yet independently of the presence or absence of the
suspect
hydrolase in the test sample. Again, the choice of these controls and the
chemical
mechanisms by which they function, as well as the choice between placing these
laminae
on the same surface of the chamber as the indicator or on the opposing
surface, will vary
from one test to the next.
Further preferred embodiments of the invention contain additional features to
enhance the performance of the test. For water-based samples, the
incorporation of a
surface-active agent in the laminae immediately adjacent to the gap to be
filled with the
sample will promote the wetting of the laminae with the sample and the rapid
and
uniform filling of the chamber. The surface-active agent may be the sole
functional
SUBSTITUTE SHEET (RULE 26j
CA 02160262 2001-04-17
9
ingredient in the lamina or combined in the lamina with test reagents.
Preferably, both
sides of the gap are lined with laminae bearing the surface-active agent. A
sample
introduction port is included in the device to permit direct insertion of the
sample into the
chamber, and preferred embodiments include one or more vent holes in the
chamber,
spaced apart from the sample introduction port, to further facilitate the
filling of the
chamber.
The invention provides a method for assaying for the presence of an
enzymatically
active hydrolase in a sample, said method comprising:
(a) placing said sample in a test device which contains first and second
solid supports, said first solid support having a reporter enzyme covalently
attached
thereto in such a manner whereby said reporter enzyme is released upon action
of
said hydrolase, said second solid support, which is not in contact with said
first
solid support, having immobilized thereon an indicator, said indicator being
one
which is susceptible to a detectable change upon action of said reporter
enzyme
released from said solid support, said sample being placed in~said device in
such a
manner that said sample contacts said first and second solid supports such
that any
reporter enzyme released by any hydrolase activity present in said sample is
permitted to diffuse through said sample to said second solid support; and
observing whether said indicator undergoes a detectable change, said
detectable
change being an indication of the presence of said enzymatically active
hydrolase in
said sample.
The invention also provides a method for detecting candidiasis by assaying for
the
presence of enzyrnatically active aspartic protease in a sample, said method
comprising:
(a) contacting said sample with a solid support, said solid,support having
a reporter enzyme immobilized thereon in such a manner whereby said reporter
enzyme is released from said solid support upon action of said aspartic
protease;
(b) combining said sample after, having been contacted with said solid
support with an indicator, said indicator being one which is susceptible to a
detectable change upon action of said reporter enzyme released from said solid
support; and
CA 02160262 2001-04-17
9a
(c) observing whether said indicator undergoes a detectable change, said
detectable change being an indication of the presence of said enzymatically
active
aspartic protease in said sample and thus, candidiasis.
The invention also provides a method for detecting Trichomonas vaginalis by
assaying for the presence of enzymatically active thiol protease in a sample,
said
method comprising:
(a) contacting said sample with a solid support, said solid support having a
reporter enzyme immobilized thereon in such a manner whereby said reporter
enzyme is released from said solid support upon action of said thiol protease;
(b) combining said sample after having been contacted with said solid support
with an indicator, said indicator being one which is susceptible to a
detectable
change upon action of said reporter enzyme released from said solid support;
and
(d) observing whether said indicator undergoes a detectable change, said
detectable change being an indication of the presence of said enzymatically
active
thiol protease in said sample and thus, Trichomonas vaginalis. ,
The invention also provides a test device for testing a sample for the
presence of
candidiasis by assaying for the presence of enzymatically active aspartic
protease,
said test device comprising:
(a) a receptacle defined at least in part by first and second opposing walls
having interior-facing surfaces with a gap therebetween, said first wall, said
second
wall, or both being of light-transmitting material;
(b) a reporter enzyme immobilized on a solid support on said interior-
facing surface of one of said first and second walls in such a manner whereby
said
reporter enzyme is released upon action of said aspartic protease;
(c) an indicator immobilized on said interior-facing surface of one of
said first and second walls, said indicator being one which undergoes a
detectable
change upon action of said reporter enzyme released from said solid support;
and
(d) an opening in said receptacle for introduction of said sample.
The invention also provides a test device for testing a sample for the
presence of
Trichomonas vaginalis by assaying for the presence of enzymatically active
thiol
protease, said test device comprising:
CA 02160262 2001-04-17
9b
(a) a receptacle defined at least in part by first and second opposing walls
having
interior-facing surfaces with a gap therebetween, said first wall, said second
wall, or
both being of light-transmitting material;
(b) a reporter enzyme immobilized on a solid support on said interior-facing
surface of one of said first and second walls in such a manner whereby said
reporter
enzyme is released upon action of said thiol protease;
(c) an indicator immobilized on said interior-facing surface of one of said
first
and second walls, said indicator being one which undergoes a detectable change
upon action of said reporter enzyme released from said solid support; and
(d) an opening in said receptacle for introduction of said sample.
Other features, objects and advantages of the invention and its preferred
embodiments will become apparent from the description which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a view in perspective of an illustrative test device in accordance
with
the invention.
FIG. 2 is a side view in cutaway of a portion of the test device shown in
FIG. 1.
DETAILED DESCRIPTION OF THE INVENTION
AND PREFERRED EMBODIMENT
In one aspect of the present invention, a method of assaying for the presence
of an enzymatically active hydrolase in a sample is provided, the method
comprising: (a)
contacting the sample with a solid support, the solid support having a
reporter enzyme
immobilized thereon in such a manner whereby the reporter enzyme is released
upon
action of the hydrolase; (b) combining the sample after it has been contacted
with the
solid support with an indicator, the indicator being one which is susceptible
to a
detectable change upon action of the reporter enzyme; and (c) observing
whether the
indicator undergoes a detectable change, the detectable change being an
indication of the
presence of the enzymatically active hydrolase in the sample.
CA 02160262 2001-04-17
9c
The term "hydrolase" is used herein to refer to an enzyme which catalyzes
hydrolytic reactions. The method of the present invention can be used to assay
for the
presence of any known hydrolytic enzyme. Such hydrolytic enzymes, i. e. ,
hydrolases,
include, but are not limited to, the following: proteases or (interchangeably)
proteinases,
peptidases, lipases, nucleases, homo- or hetero-oligosaccharidases, homo- or
hetero-
polysaccharidases, phosphatases, sulfatases, neuraminidases and esterases. In
a preferred
embodiment, the method of the present invention can be used to assay for the
presence of
WO 94/24306 PCT/US94/04098
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proteases, including, but not limited to, the following: aspartic proteases,
serine
proteases, thiol proteases, metallo proteases, acid proteases, and alkaline
proteases. In
another preferred embodiment, the method of the present invention can be used
to assay
for the presence of homo- or hetero-oligosaccharidases or homo- or hetero-
polysaccharidases, including, but not limited to, chitinase, amylases,
cellulase and
lysozyme.
The term "reporter enzyme" or (interchangeably) "marker enzyme" is used
herein to refer to a signal generating enzyme, i.e., an enzyme whose activity
brings about
a detectable change. Such reporter enzymes include, but are not limited to,
the
following: perozidases, phosphatases, ozidoreductases, dehydrogenases,
transferases,
isomerases, kinases, reductases, deaminases, catalases, unease and
glucuronidase. In
selecting a reporter enzyme to be used in the presently claimed method, it is
imperative
that the reporter enzyme is not subject to inactivation by any agent in the
sample,
including inactivating hydrolysis by any hydrolase activity present in the
sample. The
selection of an appropriate reporter or marker enzyme will be readily apparent
to those
skilled in the art. Presently preferred reporter enzymes are the perozidases,
such as, for
example, horseradish perozidase.
The reporter enzyme is immobilized on a solid support, i. e. , an insoluble
polymeric material, inorganic or organic matrix, gel, aggregate, precipitate
or resin, in
such a manner whereby the reporter enzyme is released upon action of the
hydrolase
whose presence is being assayed. Preferred solid supports in accordance with
the present
invention include, but are not limited to, the following: cellulose, agarose,
deztran,
polyacrylate, polyacrylamide, or their derivatives, chitin, sepharose, ozirane
acrylic
beads and polymeric dialdehyde, starch, collagen, keratin, elastin, bovine
hide powder,
bacterial cell wall peptidoglycan or fragments thereof, nylon, polyethylene
terephthalates,
polycarbonates, and controlled pore glass. Immobilization of the reporter
enzyme on the
solid support is carried out using conventional methods and procedures known
to and
understood by those skilled in the art.
The reporter enzyme can be attached .directly to the solid support. In this
case, the insoluble support serves directly as a substrate for the hydrolase.
For example,
when chitinase is the hydrolase being assayed, the reporter or marker enzyme
(such as,
for example, horseradish perozidase) can be attached directly to the insoluble
chitin. In
the presence of chitinase, horseradish perozidase will be released from the
solid support.
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11
Similarly, if cellulase is the hydrolase being detected, the reporter enzyme,
e.g.,
horseradish peroxidase, can be directly attached to cellulose, and in the
presence of the
hydrolase cellulase, horseradish peroxidase will be released from the solid
support.
Finally, if lysozyme is the hydrolase being detected, the reporter enzyme,
e.g.,
horseradish peroxidase, can be directly attached to bacterial cell wall
peptidoglycan, and
in the presence of the hydrolase lysozyme, horseradish peroxidase will be
released from
the solid support:
Alternatively, the reporter enzyme can be immobilized on the solid support
through the use of a linker molecule which is a hydrolyzable substrate for the
hydrolase
being detected. Such linker molecules include, but are not limited to, the
following:
proteins, carbohydrates, lipids, peptides, esters and nucleic acids. The
particular linker
molecule used to attach the reporter enzyme to the solid support will depend
on which
hydrolase is being detected, and the selection in any given case will be
readily apparent
to those skilled in the art.
The term "indicator" is used herein to refer to any chemical species which
undergoes a detectable change as a result of the reaction or as a result of
the culmination
of reactions occurring when the enzymatically active hydrolase is present in
the sample or
specimen. The resulting detectable change is an indication that the
enzymatically active
hydrolase is present in the sample or specimen.
Preferred indicators are visual indicators and, in particular, chromogenic
indicators, i.e., those in which the visible change is a change in color,
including the
formation of color in an otherwise colorless material, upon action of the
reporter or
marker enzyme when it is released from the solid support by the enzymatically
active
hydrolase whose presence is being detected. Alternatively, the reporter enzyme
may be
capable of catalyzing the formation of a fluorescent signal, a phosphorescent
signal, a
bioluminescent signal, a chemiluminescent signal, or an electrochemical signal
upon its
release from the solid support by the action of the hydrolase. Additionally,
the reporter
enzyme may be capable of producing other visible or detectable signals, such
as, for
example, a clot, an agglutination, a precipitation, or a clearing zone. In
these cases, the
indicator would be the chemical species or substrate required by the reporter
or marker
enzyme in order to bring about the desired detectable change.
A wide variety of chromogenic indicators (i. e. , chromogens) and other
species
having a similar effect may be used as visual indicators with horseradish
peroxidase as
SUBSTITUTE SHEET (RULE 26j
WO 94/24306 216 0 2 6 2 . PCT/US94/04098
12
the reporter enzyme. Preferred chromogenic indicators in accordance with the
present
invention comprise a hydroperozide and a chromogen including, but not limited
to, one
of the following: guaiac, 2-2'-azino-bis(3-ethyl-benthiazoline-6-sulfonic
acid),
tetramethylbenzidine, phenol, 4-aminoantipyrine, and 4,5-dihydrozynaphthalene-
2,7-
disulfonic acid. A particularly preferred chromogenic indicator is comprised
of a
hydroperozide and guaiac, a chromogen which is colorless in its reduced state
and deep
blue in its oxidized state. Optionally, guaiac may be purified prior to use,
e.g., by
solvent extraction. The most appropriate chromogenic indicator for any given
reporter
enzyme will depend upon the reaction or reactions which the reporter enzyme is
capable
of catalyzing or initiating, and the selection in any given case will be
readily apparent to
those skilled in the art.
If the visual indicator is a chromogenic indicator, it is possible to employ
either a liquid chromogen system or a solid chromogen system. If a liquid
chromogen
system for detecting perozidases is used, such a system would comprise a
solvent, a
hydroperozide and a chromogen capable of being oxidized by hydroperozides in
the
presence of a perozidase, such as, for example, horseradish perozidase.
Alternatively, if
a solid chromogen system is used, such a system would comprise a
hydroperozide, a
chromogen capable of being oxidized by hydroperozides in the presence of a
perozidase,
and a solid support onto which the chromogen has been impregnated or
immobilized and
dried. In this system, the chromogen can be impregnated onto a bibulous paper
or
support as is done with Hemoccult~ slides or, alternatively, the chromogen can
be
deposited onto a plastic or other sheet in the form of a thin layer. In this
latter format,
the chromogen could be layered as a solution containing a polymeric material
(such as,
for example, hydrozypropyl cellulose, ethyl cellulose, etc.). If the chromogen
itself is a
water soluble chromogen, it can be trapped in a matrix of material which is
insoluble in
water. Alternatively, if the chromogen itself is insoluble in water, it can be
layered as a
solution in an organic solvent either alone or in combination with a water
soluble or
water insoluble polymer.
In the solid chromogen system for detecting perozidases, the hydroperozide
can be provided either in a solid form (such as, for example, titanium
hydroperozide) or
it can be generated in situ. Hydrogen peroxide, for example, can be generated
in situ
with glucose, ambient oxygen and glucose ozidase. Alternatively, hydrogen
peroxide can
be generated in situ through the use of a dried layer formed after depositing
a suspension
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 -. PCT/US94/04098
13
of sodium perborate in alcohol. At low pH, sodium perborate releases hydrogen
peroxide spontaneously. In the presence of the hydrogen peroxide and the
peroxidase
upon its release from the solid support by the hydrolase, the chromogen will
be oxidized
and a visually detectable change in color will result. This resulting change
in color is an
indication that the enzymatically active hydrolase is present
in the sample or specimen.
This method as well as the other methods of the presently claimed invention
can be used to simultaneously assay for the presence of two or more active
hydrolases in
a sample or specimen. Such a method would employ a mixture of two or more
reporter
enzymes immobilized on a solid supports) through substrate linkages which are
susceptible to specific hydrolases, and two or more indicator and reagent
systems to
generate a detectable response to each of the reporter enzymes. For example,
it would
be possible using the methods of the present invention to detect
simultaneously a mixture
of proteases, lipases and polysaccharidases, thereby obtaining a hydrolytic
profile of a
given pathogen or disease process.
Additionally, it will be readily apparent to those skilled in the art that the
reactions conditions (such as, for example, the choice of the linker molecule
or bridge,
solid supports, pH, buffer capacity, buffer identity, salts, etc. ) of the
presently claimed
methods can be modified and regulated to increase hydrolase specificity and to
differentiate between the different hydrolases present in a sample. For
example, it is
known that certain hydrolase function at a low pH and are inhibited at a high
pH. In
contrast, other hydrolases function at a high pH and are inhibited at a low
pH. By
regulating the pH of the assay, one will be able to selectively detect the
presence of a
particular hydrolase. Additionally, it will be readily apparent to the skilled
artisan that
specific enzyme inhibitors can also be used in the presently claimed methods
to increase
hydrolase activity and specificity and to differentiate between the different
hydrolases.
In another aspect of the present invention, a method for assaying for the
presence of an enzymatically active hydrolase in a sample is provided, the
method
comprising: placing the sample in a device which contains first and second
solid
supports, -the first solid support having a reporter enzyme immobilized
thereon in such a
manner whereby the reporter enzyme is released upon action of the hydrolase,
the second
solid support, which is not in contact with the first solid support, having
immobilized
thereon an indicator, the indicator being one which is susceptible to a
detectable change
SUBSTITUTE SHEET (RULE 26)
WO 94/24306
. PCT/US94/04098
14
upon action of the reporter enzyme, the sample being placed in the device in
such a
manner that the sample contacts the first and second solid supports such that
any reporter
enzyme released by any hydrolase activity present in the sample is permitted
to diffuse
through the sample to the second solid support; and observing whether the
indicator
undergoes a detectable change, the detectable change being an indication of
the presence
of the enzymatically active hydrolase in the sample.
This method of the present invention can also be used to assay for the
presence of any known hydrolytic enzyme, including, but not limited to, the
following
hydrolases: proteases or (interchangeably) proteinases, peptidases, lipases,
nucleases, homo-
or hetero-oligosaccharidases, homo- or hetero-polysaccharidases, phosphatases,
sulfatases, neuraminidases and ester3ses. In a preferred embodiment, this
method is used
to assay for the presence of proteases, including, but not limited to, the
following:
aspartic proteases, serine proteases, thiol proteases, metallo proteases, acid
proteases and
alkaline proteases.
The reporter enzyme or marker enzyme used in this method can be any signal
generating enzyme not subject to inactivation by any agent in the sample,
including
inactivating hydrolysis by any hydrolase activity present in the sample. Such
reporter
enzymes include, but are not limited to, the following: peroxidases,
phosphatases,
oxidoreductases, dehydrogenases, transferases, isomerases, kinases,
reductases,
deaminases, catalases, urease and glucuronidase. Presently preferred reporter
or marker
enzymes are the peroxidases, such as, for example, horseradish peroxidase.
The reporter enzyme is immobilized on a first solid support, i.e., an
insoluble
matrix, gel or resin, in such a manner whereby the reporter enzyme is released
from the
solid support upon action of the enzymatically active hydrolase whose presence
is being
assayed. The reporter enzyme can be attached directly to the first solid
support if the
solid support is a substrate for the hydrolase or, alternatively, the reporter
enzyme can be
immobilized on the first solid support through the use of a linker molecule
which is a
substrate for the enzymatically active hydrolase which is being detected. Such
linker
molecules include, but are not limited to, the following: proteins,
carbohydrates, lipids,
peptides, esters and nucleic acids. The particular linker molecule used to
attach the
reporter enzyme to the first solid support will depend on which hydrolase is
being
detected, and the selection in any given case will be readily apparent to
those skilled in
the art. Immobilization of the reporter enzyme on the first solid support is
carried out
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 _ PCT/US94/04098
using conventional methods and procedures known to and understood by those
skilled in
the art.
In this method of the present invention, the indicator is attached to the
second
solid support which is not in contact with the first solid support. Preferred
first and
5 second solid supports in accordance with the present invention include, but
are not
limited to, the following: cellulose, agarose, dextran, polyacrylate,
polyacrylamide, or
their derivatives chitin, sepharose, oxirane acrylic beads, polymeric
dialdehyde, starch,
collagen, keratin, elastin, bovine hide powder, bacterial cell wall
peptidoglycan or
fragments thereof, nylon, polyethylene terephthalates, polycarbonates, and
controlled pore
10 glass. Immobilization of the visual indicator on the second solid support
is carried out
using conventional methods and procedures known by those skilled in the art.
The indicator can be any chemical species which undergoes a detectable
change as a result of the reaction or as a result of the culmination of
reactions occurring
when the enzymatically active hydrolase is present in the sample or specimen.
The
15 resulting detectable change in the indicator is an indication that the
enzymatically active
hydrolase is present in the sample. Preferred indicators are visual indicators
and, in
particular, chromogenic indicators, i. e. , those in which the visible change
is a change in
color, including the formation of color in an otherwise colorless material,
upon action of
the reporter or marker enzyme when it is released from the solid support by
the
enzymatically active hydrolase whose presence is being detected.
A wide variety of chromogenic indicators (i. e. , chromogens) and other
species
having a similar effect may be used as visual indicators. Preferred
chromogenic
indicators for peroaidase-like reporter enzymes in accordance with the present
invention
comprise a hydroperoxide and a chromogen including, but not limited to, one of
the
following: guaiac, 2-2'-azino-bis(3-ethyl-benthiazoline-~sulfonic acid),
tetramethylbenzidine, phenol, 4-aminoantipyrine, and 4,5-dihydroxynaphthalene-
2,7-
disulfonic acid. A particularly preferred chromogenic indicator is comprised
of a
hydroperoxide and guaiac, a chromogen which is colorless in its reduced state
and deep
blue in its oxidized state. The most appropriate chromogenic indicator for any
given
reporter enzyme will depend on the reaction or reactions which the reporter
enzyme is
capable of catalyzing or initiating, and the selection in any given case will
be readily
apparent to those skilled in the art.
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 PCT/US94/04098
2isozs~
16
If the visual indicator is a chromogenic indicator for perozidative reactions,
a
solid chromogen system is employed, and such a system comprises a
hydroperozide, a
chromogen capable of being oxidized by hydroperozides in the presence of a
peroxidase,
and a solid support onto which the chromogen has been impregnated or
immobilized and
dried. In this system, the chromogen can be impregnated onto a bibulous paper
or
support as is done with Hemoccultm slides or, alternatively, the chromogen can
be
deposited onto a plastic or other sheet in the form of a thin layer. In this
latter format,
the chromogen could be layered as a solution containing a polymeric material
(such as,
for example, hydrozypropyl cellulose, ethyl cellulose, etc.). If the chromogen
itself is a
water soluble chromogen, it can be trapped in a matrix of material which is
insoluble in
water. Alternatively, if the chromogen itself is insoluble in water, it can be
layered as a
solution in an organic solvent either alone or in combination with a water
soluble or
water insoluble polymer.
In the solid chromogen system, the hydroperozide can be provided either in a
solid form (such as, for example, titanium hydroperozide) or it can be
generated in situ
in the device. In the presence of the hydroperozide and the perozidase upon
its release
from the first solid support by the hydrolase, the chromogen will be oxidized
and a
visually detectable change in color will result. This resulting change in
color is an
indication of the presence of the enzymatically active hydrolase in the sample
or
specimen.
In a presently preferred embodiment of this method of the present invention,
enzymatically active aspartic protease is the hydrolase being detected, the
method
comprising: placing the sample in a device which contains first and second
solid
supports, the first solid support being polyacrylate and having horseradish
perozidase
immobilized thereon through a myoglobin molecule which is a substrate for
aspartic
protease, the second solid support, which is not in contact with the first
solid support,
being a cellulose derivative and having immobilized thereon a hydroperozide
and guaiac,
a chromogenic substrate which undergoes a color change upon action of
horseradish
perozidase in the presence of a hydroperozide, the sample being placed iwthe
device in
such a manner that the sample contacts the first and second solid supports
such that any
horseradish perozidase released by any enzymatically active aspartic protease
present in
the sample is permitted to diffuse through the sample to the second solid
support; and
SUBSTITUTE SHEET (RULE 26)
2160262
WO 94/24306 PCT/US94/04098
17
observing whether guaiac undergoes a color change, the color change being an
indication
of the presence of enzymatically active aspartic protease in the sample.
As previously mentioned, it will be readily apparent to the skilled artisan
that
particular reaction conditions (such as, for example, the choice of linker
molecule, solid
support, pH, buffer capacity, buffer identity, salts, etc. ) and specific
inhibitors can used
to increase aspartic protease specificity. For example, by assaying for
aspartic protease
at a pH of about 2.5 to about 5.0, one can selectively detect aspartic
proteases over many
thiol proteases, serine proteases, metallo proteases, and alkaline proteases.
Further, by
adding inhibitors of the metallo-proteases, the thiol proteases, the serine
proteases, and
the acid or alkaline proteases, one can selectively detect aspartic proteases.
In still another aspect of the present invention, a method for detecting
candidiasis by assaying for the presence of enzymatically active aspartic
protease in a
sample is provided, the method comprising: (a) contacting the sample with a
solid
support, the solid support having a reporter enzyme immobilized thereon in
such a
manner whereby the reporter enzyme is released upon action of the aspartic
protease; (b)
combining the sample after having been contacted with the solid support with
an
indicator, the indicator being one which is susceptible to a detectable change
upon action
of the reporter enzyme; and (c) observing whether the indicator undergoes a
detectable
change, the detectable change being an indication of the presence of
enzymatically active
aspartic protease in the sample and thus, candidiasis.
The reporter enzyme or marker enzyme used in this method of the present
invention can be any signal generating enzyme, i.e., an enzyme whose activity
brings
about a visible or detectable change, not subject to inactivation by any agent
in the
sample, including inactivating hydrolysis by any aspartic protease or any
other hydrolase
activity present in the sample. Such reporter enzymes include, but are not
limited to, the
following: peroxidases, phosphatases, oxidoreductases, dehydrogenases,
transferases,
isomerases, kinases, reductases, deaminases, catalases, unease and
glucuronidase. The
selection of an appropriate reporter or marker enzyme will be readily apparent
to those
skilled in the art. In this method of the present invention, the preferred
reporter enzymes
are the peroxidases. In particular, horseradish peroxidase is the presently
preferred
reporter enzyme because horseradish peroxidase is not readily hydrolyzed by
aspartic
protease or many other well-known proteases which may be present in the
sample.
SUBSTITUTE SHEET (RULE 26)
CA 02160262 2001-04-17
18
The reporter enzyme is immobilized on a solid support, i.~., an insoluble
matrix, gel or resin, in such a manner whereby the reporter enzyme is released
upon
action of asparric protease. Solid supports in accordance with the present
invention
include, but are not limited to, the following: cellulox, agarox, deatran,
polyacrylate,
TM
polyacrylamide, or their derivatives, chitin, sepharosc, oxirane acrylic
beads, polymeric
dialdehyde, starch, collagen, keratin, elastin, bovine hide powder, bacterial
cell wall
peptidoglycan or fragments thereof, nylon, polyethylene terephthalates,
polycarbonates,
and controlled pore glass. Presently preferred solid supports in this method
of the
claimed invention include chitin, polyacrylate, cellulox, and their
derivatives, and
xpharox. Immobilization of the reporter enzyme on the solid support is carried
out
using conventional methods and procedures known to and understood by thox
skilled in
the art.
The reporter enzyme can be immobilized on the solid support through the use
of a linker molecule which is a hydrolyzable substrate for the enzymatically
active
aspartic protease. In this method of the present invention, such linker
molecules include
proteins and p~tides, with proteins being the preferred linker molecules. If
the linker
molecule is a protein, preferred proteins include, but are not limited to, the
following:
azocasein, casein, K-caxin, immunoglobulins, hemoglobin, myoglobin, albumin,
elastin,
keratin and collagen. In preferred embodiments of this method of the claimed
invention,
the linker molecule is K-caxin, casein, hemoglobin, or myoglobin.
The indicator can be any chemical species which undergoes a detectable
change as a result of the reaction or as a result of the culmination of
reactions occurring
when enzymatically active aspar<ic protease is present in the sample or
specimen. The
resulting detectable change is an indication of the presence of enzymatically
active
aspartic protease in the sample and, in turn, the presence of enzymatically
active aspartic
proteax in the sample indicates the presence of candidiasis.
Preferred indicators are visual indicators and, in particular, chmmogenic
inaicators, i.t., thox in which the detectable change is a change in color,
including the
formation of color in an otherwix colorless material, upon action of the
reporter or
marker enzyme when it is releaxd from the solid support by enzymaacally active
aspartic protease. Alternatively, the reporter enzyme may be capable of
catalyzing the
formation of a fluorescent signal, a phosphorescent signal, a bioluminescent
signal, a
chemiluminescent signal, or an electrochemical signal upon its releax from the
solid
~ ~l fifl2~2
WO 94/24306 PCT/US94/04098
19
support by the action of aspartic protease. Additionally, the reporter enzyme
may be
capable of producing other visible or detectable signals, such as, for
example, a clot, an
agglutination, a precipitation, or a clearing zone. In these cases, the
indicator would be
the chemical species or substrate required by the reporter enzyme in order to
bring about
the desired detectable change.
A wide variety of chromogenic indicators (i. e. , chromogens) and other
species
having a similar effect may be used as visual indicators when perozidases are
used as the
reporter or marker enzymes. Preferred chromogenic indicators in accordance
with the
present invention comprise a hydroperozide and a chromogen including, but not
limited
to, one of the following: guaiac, 2-2'-azino-bis(3-ethyl-benthiazoline-b-
sulfonic acid),
tetramethylbenzidine, phenol, 4-aminoantipyrine, and 4,5-dihydrozynaphthalene-
2,7-
disulfonic acid. A particularly preferred chromogenic indicator is comprised
of a
hydroperozide and guaiac, a chromogen which is colorless in its reduced state
and deep
blue in its oxidized state. Optionally, the guaiac may be purified prior to
use, e.g., by
solvent extraction. The most appropriate chromogenic indicator for any given
reporter
enzyme will depend on the reaction or reactions which the reporter enzyme is
capable of
catalyzing or initiating, and the selection in any given case will be readily
apparent to
those skilled in the art. As previously described, if the visual indicator is
a chromogenic
indicator, it is possible to employ either a liquid chromogen system or a
solid chromogen
system.
In yet another aspect of the present invention, a method is provided for
detecting Trichomonas vaginalis by assaying for the presence of enzymatically
active thiol
protease in a sample, this method comprising: (a) contacting the sample with a
solid
support, the solid support having a reporter enzyme immobilized thereon in
such a
manner whereby the reporter enzyme is released upon action of the
enzymatically active
thiol protease; (b) combining the sample after having been contacted with the
solid
support with an indicator, the indicator being one which is susceptible to a
detectable
change upon action of the reporter enzyme; and (c) observing whether the
indicator
undergoes a detectable change, the detectable change being an indication of
the presence
~ of enzymatically active thiol protease in the sample and thus, Trichomonas
vaginalis.
As with the previously described methods, the reporter enzyme or marker
enzyme used in this method of the present invention can be any signal
generating
enzyme, i. e. , an enzyme whose activity brings about a visible or detectable
change, not
SUBSTITUTE SHEET (RULE 26)
WO 94/2.1306 - PCTlUS94/04098
subject to inactivation by any agent in the sample, including inactivating
hydrolysis by
any hydrolase activity present in the sample. Such reporter enzymes include,
but are not
limited to, the following: perozidases, phosphatases, ozidoreductases,
dehydrogenases,
transferases, isomerases, kinases, reductases, deaminases, catalases, urease
and
5 glucuronidase. The selection of an appropriate reporter or marker enzyme
will be readily
apparent to those skilled in the art. In this method of the present invention,
the preferred
reporter enzymes are the perozidases and, in particular, horseradish
perozidase.
The reporter enzyme is immobilized on a solid support, i. e. , an insoluble
matrix, gel or resin, in such a manner whereby the reporter enzyme is released
from the
10 solid support upon action of the enzymatically active thiol protease. Solid
supports in
accordance with the present invention include, but are not limited to, the
following:
cellulose, agarose, deztran, polyacrylate, polyacrylamide, or their
derivatives, chitin,
sepharose, ozirane acrylic beads, polymeric dialdehyde, starch, collagen,
keratin, elastin,
bovine hide powder, bacterial cell wall peptidoglycan or fragments thereof;
nylon,
15 polyethylene terephthalates, polycarbonates, and controlled pore glass.
Presently
preferred solid supports in this method of the claimed invention include
chitin,
polyacrylate, cellulose, and their derivatives, and sepharose. Immobilization
of the
reporter enzyme on the solid support is carried out using conventional methods
and
procedures known to and understood by those skilled in the art.
20 The reporter enzyme can be immobilized on the solid support through the use
of a linker molecule which is a hydrolyzable substrate for the enzymatically
active thiol
protease. In this method of the present invention, such linker molecules
include proteins
and peptides, with proteins being the preferred linker molecules. If the
linker molecule is
a protein, preferred proteins include, but are not limited to, the following:
azocasein,
casein, K-casein, immunoglobulins, hemoglobin, myoglobin, albumin, elastin,
keratin and
collagen. In preferred embodiments of this method of the claimed invention,
the linker
molecule is K-casein, casein, hemoglobin, or myoglobin.
The indicator can be any chemical species which undergoes a detectable
change as a result of the reaction or as a result of the culmination of
reactions occurring
30, when enzymatically active thiol protease is present in the sample or
specimen. The
resulting detectable change is an indication of the presence of enzymatically
active thiol
protease in the sample and, in turn, the presence of enzymatically active
thiol protease in
the sample indicates the presence of Trichomonas vaginalis.
SUBSTITUTE SHEET (RULE 26j
2160262
WO 94/24306 _ PCT/US94/04098
21
Preferred indicators are visual indicators and, in particular, chromogenic
indicators, i.e., those in which the detectable change is a change in color,
including the
formation of color in an otherwise colorless material, upon action of the
reporter or
marker enzyme when it is released from the solid support by the enzymatically
active
aspartic protease. Alternatively, the reporter enzyme may be capable of
catalyzing the
formation of a fluorescent signal, a phosphorescent signal, a bioluminescent
signal, a
chemiluminescent signal, or an electrochemical signal upon its release from
the solid
support by the action of aspartic protease. Additionally, the reporter enzyme
may be
capable of producing other visible or detectable signals, such as, for
example, a clot, an
agglutination, a precipitation, or a clearing zone. In these cases, the
indicator would be
the chemical species or substrate required by the reporter enzyme in order to
bring about
the desired detectable change.
A wide variety of chromogenic indicators (i. e. , chromogens) and other
species
having a similar effect may be used as visual indicators when perozidases are
used as the
reporter or marker enzymes. Preferred chromogenic indicators in accordance
with the
present invention comprise a hydroperozide and a chromogen including, but not
limited
to, one of the following: guaiac, 2-2'-azino-bis(3-ethyl-benthiazoline-6-
sulfonic acid),
tetramethylbenzidine, phenol, 4-aminoantipyrine, and 4,5-dihydrozynaphthalene-
2,7-
disulfonic acid. A particularly preferred chromogenic indicator is comprised
of a
hydroperozide and guaiac, a chromogen which is colorless in.its reduced state
and deep
blue in its oxidized state. The most appropriate chromogenic indicator for any
given
reporter enzyme will depend on the reaction or reactions which the reporter
enzyme is
capable of catalyzing or initiating, and the selection in any given case will
be readily
apparent to those skilled in the art. As previously described, if the visual
indicator is a
chromogenic indicator, it is possible to employ either a liquid chromogen
system or a
solid chromogen system.
As with the aspartic protease assays, certain reaction conditions and specific
inhibitors can used to increase thiol protease activity and specificity. For
example, one
could add inhibitors (such as, for example, pepstatin to inhibit aspartic
proteases, soybean
trypsin inhibitor to inhibit trypsin, EDTA or other chelators to inhibit
metallo-proteases,
or any of the many known naturally occurring inhibitors of non-thiol protein
proteases) to
the test system so that only thiol proteases would function and thus, be
detected.
SUBS~fITUTE SHEET (RULE 26)
WO 94/24306 _ ~ . PCT/US94/04098
22
Additionally, by assaying at a pH of about 7.4, many thiol proteases will be
active, but
aspartic proteases will be inactivated.
It is important to note that the reporter enzyme release technology of the
present invention can also be employed to detect the presence or absence of a
hydrolase
inhibitor in a sample. Many biological processes, including regulation of
blood pressure,
blood clotting, bacterial replication, etc. , involve the use of very
specific, carefully
modulated hydrolases. Moreover, numerous drugs, pesticides and herbicides,
etc. , are
known to function by virtue of inhibiting specific hydrolases. Under certain
circumstances, it is highly desirable to determine the blood concentration of
a hydrolase-
inhibiting therapeutic drug or to determine the presence of a potential
pesticide hydrolase
inhibitor contamination in produce, etc. In these cases, it is, therefore,
necessary to
detect the inhibitor of a hydrolase, rather than the hydrolase itself.
Accordingly, in another aspect of the present invention, a method for assaying
for the presence of an inhibitor of a target hydrolase in a sample is
provided, the method
comprising: (a) contacting the sample with the target hydrolase and a solid
support, the
solid support having a reporter enzyme immobilized thereon in such a manner
whereby
the reporter enzyme is released upon action of the target hydrolase if the
target hydrolase
is not inactivated by the presence of the inhibitor; (b) combining the sample
after having
been contacted with the target hydrolase and the solid support with an
indicator, the
indicator being one which is susceptible to a detectable change upon action of
the
reporter enzyme; and (c) observing whether the indicator undergoes a
detectable change,
the detectable change being an indication of the absence of the inhibitor of
the target
hydrolase in the sample.
To detect the presence of a hydrolase inhibitor in a sample, a defined
quantity
of the target hydrolase is incorporated into the test system, and test
performance involves
detecting the ability of the sample to inhibit the target hydrolase. In the
event that the
target hydrolase inhibitor is not present in the sample, the target hydrolase
will release
the reporter enzyme from the solid support, thereby producing a detectable
change in the
indicator. Conversely, if the target hydrolase inhibitor is present in the
sample, the target
hydrolase will be inhibited, the reporter enzyme will not be released from the
solid
support, and a detectable change or response will not be produced in the
indicator. It is
not essential that the inhibitor in the sample completely inhibit the target
hydrolase added
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 ~~~ 2 i ~ p 2 6 2 ~ PCT/LTS94/04098
23
to the test system. It is only required that a sufficient amount of target
hydrolase
inhibition occurs to produce a noticeable difference in the anticipated
detectable response.
This method of the present invention can be used to assay for the presence or
absence of any known inhibitor of a hydrolytic enzyme, including, but not
limited to, the
following: inhibitors of the proteases or (interchangeably) proteinases,
peptidases,
lipases, nucleases, homo- or hetero-oligosaccharidases, homo- or hetero-
polysaccharidases, phosphatases, sulfatases, neuraminidases and esterases. In
a preferred
embodiment, this method is used to assay for the presence protease inhibitors,
including,
but not limited to, the following: aspartic protease inhibitors, serine
protease inhibitors,
thiol protease inhibitors, metallo protease inhibitors, acid protease
inhibitors and alkaline
protease inhibitors. In a further preferred embodiment, this method is used to
assay for
the presence of aspartic protease inhibitors such as, for example, pepstatin,
ovomacroglobulin, haloperidol, transition state mimetics, U-81749, H-261, MV7-
101, A-
75925, A-76928 and A-7003. U-81749, H-261, MV7-101, A-75925, A-76928 and A-
7003 are experimental drugs which have previously been described in the
literature. In
an even further preferred embodiment of this method of the present invention,
pepstatin is
the aspartic protease inhibitor whose presence is being detected.
In accordance with this method of the present invention, target hydrolases
include, but are not limited to, the following: proteases, proteinases,
peptidases, lipases,
nucleases, homo- or hetero-oligosaccharidases, homo- or hetero-
polysaccharidases,
phosphatases, sulfatases, neuraminidases and esterases. The particular target
hydrolase
used in the above method will depend upon which inhibitor is being detected,
and the
selection in any given case will be readily apparent to those skilled in the
art. If, for
example, pepstatin is the inhibitor being detected, then aspartic protease
would be the
target hydrolase used in the test system described above.
The reporter enzyme or marker enzyme used in this method of the present
invention can be any signal generating enzyme, i.e., an enzyme whose activity
brings
about a visible or detectable change, not subject to inactivation by any agent
in the
sample, including inactivating hydrolysis by the target hydrolase which is
incorporated
into the test system. Such reporter enzymes include, but are not limited to,
the
following: peroxidases, phosphatases, oxidoreductases, dehydrogenases,
transferases,
isomerases, kinases, reductases, deaminases, catalases, un"ase and
glucuronidase. The
selection of an appropriate reporter or marker enzyme will be readily apparent
to those
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 PCT/US94/04098
24
skilled in the art. In this method of the present invention, the preferred
reporter enzymes
are the peroxidases, and, in particular, horseradish peroxidase.
The reporter enzyme is immobilized on a solid support, i.e., an insoluble
matrix, gel or resin, in such a manner whereby the reporter enzyme is released
from the
solid support upon action of the target hydrolase if the target hydrolase is
not inactivated
by the presence of the inhibitor. Solid supports in accordance with the
present invention
include, but are not limited to, the following: cellulose, agarose, dextran,
polyacrylate,
polyacrylamide, or their derivatives, chitin, sepharose, oxirane acrylic
beads, polymeric
dialdehyde, starch, collagen, keratin, elastin, bovine hide powder, bacterial
cell wall
peptidoglycan or fragments thereof, nylon, polyethylene terephthalates,
polycarbonates,
and controlled pore glass.
The reporter enzyme can be immobilized on the solid support through the use
of a linker molecule which is a hydrolyzable substrate for the enzymatically
active target
hydrolase. Such linker molecules include, but are not limited to, the
following:
proteins, carbohydrates, lipids, peptides, esters and nucleic acids. The
particular linker
molecule used to attach the reporter enzyme to the solid support will depend
on which
target hydrolase is added to the test system, and the selection in any given
case will be
readily apparent to those skilled in the art.
The indicator can be any chemical species which undergoes a detectable
change as a result of the reaction or as a result of the culmination of
reactions occurring
if the enzymatically active target hydrolase is not inactivated by the
presence of the
inhibitor in the sample or specimen. Preferred indicators are visual
indicators and, in
particular, chromogenic indicators, i.e., those in which the visible change is
a change in
color, including the formation of color in an otherwise colorless material,
upon action of
the reporter or marker enzyme when it is released from the solid support by
the
enzymatically active target hydrolase provided it is not inactivated by the
presence of the
inhibitor in the sample or specimen.
A wide variety of chromogenic indicators (i. e. , chromogens) and other
species
having a similar effect may be used as visual indicators. Preferred
chromogenic
indicators for peroxidase-like reporter enzymes in accordance with the present
invention
comprise a hydroperoxide and a chromogen including, but not limited to, one of
the
following: guaiac, 2-2'-azino-bis(3-ethyl-benthiazoline-6-sulfonic acid),
tetramethylbenzidine, phenol, 4-atninoantipyrine, and 4,5-dihydroxynaphthalene-
2,7-
SUB~fITUTE SHEET (RULE 26)
WO 94/24306 ~ a PCTIUS94/04098
disulfonic acid. A particularly preferred chromogenic indicator is comprised
of a
hydroperozide and guaiac, a chromogen which is colorless in its reduced state
and deep
blue in its oxidized state. As previously described, if the visual indicator
is a
chromogenic indicator, it is possible to employ either a liquid chromogen
system or a
5 solid chromogen system.
In yet another aspect of the present invention, a test device for assaying for
the
presence of an enzymatically active hydrolase in a sample is provided, the
test device
comprising: a receptacle defined at least in part by first and second opposing
walls
having interior-facing surfaces with a gap therebetween, the first, second, or
both walls
10 being of light-transmitting material; a reporter enzyme immobilized on a
solid support on
the interior-facing surface of one of the first and second walls in such a
manner whereby
the reporter enzyme is released upon action of the hydrolase; an indicator
contained on
the interior-facing surface of one of the first and second walls, the
indicator being one
which is susceptible to a detectable change upon action of the reporter
enzyme; and an
15 opening in the receptacle for introduction of the sample.
In accordance with this aspect of the present invention, the receptacle is
preferably flat and thin and of a size which can be easily held by hand.
Accordingly, the
chamber is preferably flat and shallow as well, with a width and length much
greater than
its depth, the depth being substantially constant. The chamber is preferably
shallow
20 enough to promote spontaneous wetting of the chamber walls with the
specimen to
achieve the maximum contact between the specimen and the dry reagent coatings
on the
upper and lower surfaces. This is of particular interest when reagent coatings
are present
on both the upper and lower surfaces of the chamber. In such cases, a small
constant
distance between these surfaces will also minimize the distance over which the
reagents
25 on the surface opposite that to which the visual indicator has been applied
will need to
diffuse in order to reach the indicator.
Within these considerations, the chamber depth is not critical to the
invention
and may vary. In most cases, a chamber ranging from about 3 mil to about 50
mil
(0.003-0.050 inch; 0.0076-0.127 cm) in depth, preferably from about 5 mil to
about 15
mil (0.005-0Ø015 inch; 0.0127-0.0381 cm), will give the best results. For
any given
depth, the lateral dimensions of the chamber (i.e., the spacing between its
side walls) will
define the size of the sample which the device will accommodate, and are
otherwise
unimportant except to define the size and shape of the visible test area on
the outer
SUBSTITUTE SHEET (RULE 26)
WO 94/2.1306 216 0 2 G 2 PCT/US94104098
26
surface of the device. The lateral dimensions should thus provide a test area
which is
large enough to be seen, and yet small enough that the chamber which will be
completely
filled by a specimen of reasonable size. The specimen size will vary with the
type of
specimen and its source and method of sampling. In typical structures, it is
contemplated
that the lateral area of the chamber will range from about 0.1 cm2 to about 10
cmz, or
preferably from about 0.3 cmZ to about 3 cm2. The internal volume of the
chamber in
typical structures will likewise vary, and for most types of samples, volumes
ranging
from about 3 ~,L to about 300 ~cL will be the most appropriate and convenient.
The test device of the present invention can be used to assay for the presence
of any known hydrolytic enzyme including, but not limited to, the following:
proteases
or (interchangeably) proteinases, peptidases, lipases, nucleases, homo- or
hetero-
oligosaccharidases, homo- or hetero-polysaccharidases, phosphatases,
sulfatases,
neuraminidases and esterases. In a preferred embodiment, the test device of
the present
invention can be used to assay for the presence of proteases, including, but
not limited to,
the following: aspartic proteases, serine proteases, thiol proteases, metallo
proteases, and
acid or alkaline proteases. In another preferred embodiment, the test device
of the
present invention can be used to assay for the presence of homo- or hetero-
oligosaccharidases or homo- or hetero-polysaccharidases, including, but not
limited to,
chitinase, cellulase, amylase and lysozyme.
The reporter enzyme or marker enzyme used in the test device can be any
signal generating enzyme, i. e. , an enzyme whose activity brings about a
detectable
change, not subject to inactivation by any agent in the sample, including
inactivating
hydrolysis by any hydrolase activity present in the sample. Such reporter
enzymes
include, but are not limited to, the following: perozidases, phosphatases,
ozidoreductases, dehydrogenases, transferases, isomerases, kinases,
reductases,
deaminases, catalases, urease and glucuronidase. The selection of an
appropriate reporter
or marker enzyme will be readily apparent to those skilled in the art.
Presently preferred
reporter enzymes are the perozidases, such as, for example, horseradish
perozidase.
The reporter enzyme is immobilized on a first solid support, i.e., an
insoluble
matrix, gel or resin, in such a manner whereby the reporter enzyme is released
from the
solid support upon action of the hydrolase whose presence is being detected.
Preferred
solid supports in accordance with the present invention include, but are not
limited to, the
following: cellulose, agarose, deztran, polyacrylate, polyacrylamide, or their
derivatives,
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 PCT/US94104098
2a.sozsz
27
chitin, sepharose, oxirane acrylic beads, polymeric dialdehyde, starch,
collagen, keratin,
elastin, bovine hide powder, bacterial cell wall peptidoglycan or fragments
thereof,
nylon, polyethylene terephthalates, polycarbonates, and controlled pore glass.
Immobilization of the reporter enzyme on the first solid support is carried
out using
conventional methods and procedures known to and understood by those skilled
in the
art.
The reporter erizyme can be attached directly to the first solid support or,
alternatively, the reporter enzyme can be immobilized on the first solid
support through
the use of a linker molecule having a hydrolyzable linkage which is a
substrate for the
enzymatically active hydrolase being detected. Such linker molecules include,
but are not
limited to, the following: proteins, carbohydrates, lipids, peptides, esters
and nucleic
acids. The particular linker molecule used to attach the reporter enzyme to
the first solid
support will depend on which hydrolase is being detected, and the selection in
any given
case will be readily apparent to those skilled 'in the art.
In the test device of the present invention, the indicator is immobilized on a
second solid support which is not in contact with the first solid support.
Preferred solid
supports in accordance with the present invention include, but are not limited
to, the
following: cellulose, agarose, dextran, polyacrylate, or their derivatives,
chitin,
sepharose, oxirane acrylic beads, polymeric dialdehyde, starch, collagen,
keratin, elastin,
bovine hide powder, bacterial cell wall peptidoglycan, or fragments thereof,
polyacrylamide, nylon, polyethylene terephthalates, polycarbonates, and
controlled pore
glass. Immobilization of the indicator on the second solid support is carried
out using
conventional methods and procedures known to those skilled in the art.
The indicator can be any chemical species which undergoes a detectable
change as a result of the reaction or as a result of the culmination of
reactions occurring
when the enzymatically active hydrolase is present in the sample or specimen.
This
detectable change is, therefore, an indication that the enzymatically active
hydrolase
being assayed is present in the sample or specimen. Preferred indicators are
visual
indicators and, in particular, chromogenic indicators, i.e., those in which
the visible
change is a change in color, including the formation of color in an otherwise
colorless
material, upon action of the reporter or marker enzyme when it is released
from the solid
support by the hydrolase whose presence is being detected. Alternatively, the
reporter
enzyme may be capable of catalyzing the formation of a fluorescent signal, a
SUBSTITUTE SHEET (RULE 26)
WO 94/2.1306 PCT/L1S94/04098
216262
- 2g
phosphorescent signal, a bioluminescent signal, a chemiluminescent signal, or
an
electrochemical signal upon its release from the solid support by the action
of the
hydrolase. Additionally, the reporter enzyme may be capable of producing other
visible
or detectable signals, such as, for example, a clot, an agglutination, a
precipitation, or a
S clearing zone. In these cases, the indicator would be the chemical species
or substrate
required by the reporter or marker enzyme in order to bring about the desired
detectable
change.
A wide variety of chromogenic indicators (i. e. , chromogens) and other
species
having a similar effect may be used as visual indicators. When perozidases are
used as
reporter or marker enzymes, preferred chromogenic indicators comprise a
hydroperozide
and a chromogen including, but not limited to, one of the following: guaiac, 2-
2'-azino-
bis(3-ethyl-benthiazoline-6-sulfonic acid), tetramethylbenzidine, phenol, 4-
aminoantipyrine, and 4,5-dihydrozynaphthalene-2,7-disulfonic acid. A
particularly
preferred chromogenic indicator is comprised of a hydroperozide and guaiac, a
chromogen which is colorless in its reduced state and deep blue in its
oxidized state.
Optionally, the guaiac may be purified prior to use, e.g., by solvent
extraction. The
most appropriate chromogenic indicator for any given reporter enzyme will
depend on the
reaction or reactions which the reporter enzyme is capable of catalyzing or
initiating, and
the selection in any given case will be readily apparent to those skilled in
the art.
If the visual indicator is a chromogenic indicator, a solid chromogen system
is
employed. This solid chromogen system comprises a hydroperozide, a chromogen
capable of being oxidized by hydroperozides in the presence of a perozidase,
and a solid
support onto which the chromogen has been impregnated or immobilized and
dried. In
this system, the chromogen can be impregnated onto a bibulous paper or support
as is
done with Hemoccultm slides or, alternatively, the chromogen can be deposited
onto a
plastic or other sheet in the form of a thin layer. In this latter format, the
chromogen
could be layered as a solution containing a polymeric material (such as, for
example,
hydrozypropyl cellulose, ethyl cellulose, etc.). If the chromogen itself is a
water soluble
chromogen, it can be trapped in a matrix of material which is insoluble in
water.
Alternatively, if the chromogen itself is insoluble in water, it can be
layered as a solution
in an organic solvent either alone or in combination with a water soluble or
water
insoluble polymer.
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 216 0 2 6 2 PCT/US94/04098
29
In this particular chromogen system, the hydroperozide can be provided either
in a solid form (such as, for example, titanium hydroperozide) or it can be
generated in
situ in the device. Hydrogen peroxide, for example, can be generated in situ
by layering
dried glucose and glucose ozidase on the interior-facing surfaces of the test
device.
Alternatively, hydrogen peroxide can be generated in situ by layering a
suspension of
sodium perborate in alcohol on the interior-facing surfaces of the test
device. At low pH,
sodium perborate releases hydrogen peroxide spontaneously. In the presence of
the
hydrogen peroxide and the perozidase (e.g., horseradish perozidase) upon its
release
from the first solid support by the hydrolase, the chromogen will be oxidized
and a
visually detectable change in color will result. As previously mentioned, this
resulting
change in color is an indication that the enzymatically active hydrolase is
present in the
sample or specimen.
It will be readily apparent to those skilled in the art that the reactions
conditions (such as, for example, the selection of the solid support and
linker molecule,
pH, buffer capacity, buffer identity, salts, etc. ) of the presently claimed
test device can
be modified and regulated to increase hydrolase activity and specificity, and
to
differentiate between the different hydrolases which may be present in a
sample.
Additionally, it will be readily apparent to the skilled artisan that specific
enzyme
inhibitors can be added to the presently claimed test devices to increase
hydrolase activity
and specificity, and to differentiate between the different hydrolases in a
sample.
The test device is provided with a sample introduction port by which the
specimen is placed in the chamber. The port is preferably in the same wall
through
which changes in the visual indicator are observed, i.e., the light-
transmitting wall. The
port will be shaped to accommodate the transfer device which is used to convey
the
sample from its source, and the port may thus be varied to suit any of the
various types
of transfer devices which might be used. Examples of transfer devices are
syringes,
pipettes, swabs and specula. Others will readily occur to those skilled in the
art. A
circular port is generally adequate, although for transfer devices such as
swabs, the port
may contain a straight edge along which the transfer device can be scraped to
more easily
release the specimen.
Preferred embodiments of the test device contain additional features which
further promote the fluid migration needed to fill the chamber and thereby
place all
reagents in contact with the specimen. One such feature is the inclusion of
one or more
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 ~ PCT/US94/04098
vent holes in the chamber to permit the escape of air. The vent holes will be
adequately
distanced from the sample introduction port to maximize the surface area
wetted by the
specimen. In devices where specimen-activated positive and negative controls
are
included inside the chamber in positions horizontally adjacent to the test
area, the vent
5 holes will be arranged to assure that the specimen reaches both controls and
fills them to
avoid any false or ambiguous readings. As discussed below, one preferred
arrangement
of the device is the placement of the test area between the control areas such
that the
positive and negative control areas do not share a common boundary although
each does
share a common boundary with the test area. In this arrangement, the sample
10 introduction port is most conveniently placed at a location in the wall
directly above the
test area, and one vent hole is placed above each of the two control areas at
or near the
outer extremities of these areas, thereby causing the specimen to fill first
the test area and
then both control areas.
Another feature promoting fluid migration in preferred embodiments of the
15 invention is the placement of a surface-active agent along the interior
surface of the
chamber. The agent may be along one or the other of the upper and lower
surfaces of
the chamber, preferably both, and may be included as a dry solute in a support
matrix
comprising the innermost lamina or coating on the surface. In some cases, the
lamina
will also contain one or more reagents taking part in the test reactions. In
other cases,
20 the surface-active agent will be the sole functional component of the
lamina.
Surface-active agents will be useful for specimens which are water-based, as
most biological specimens are. Suitable surface-active agents will be those
which can be
rendered in solid form, and a wide variety of substances which have a surface-
active
effect may be used. The substances will generally be detergents, wetting
agents or
25 emulsifiers, and will vary widely in chemical structure and electronic
character, including
anionic, cationic, zwitterionic and nonionic substances. Examples are alkyl-
alkozy
sulfates, alkyl aryl sulfonates, glycerol fatty acid esters, lanolin-based
derivatives,
polyozyethylene alkyl phenols, polyozyethylene amines, polyozyethylene fatty
acids and
esters, polyozyethylene fatty alcohols and ethers, polyethylene glycol) fatty
acids and
30 , esters, polyozyethylene fatty esters and oils,
polyozypropylene/polyozyethylene
condensates and block polymers, sorbitan fatty acid esters, sulfo derivatives
of succinates,
alkyl glucosides, and cholic acid derivatives. Trade names of products falling
within
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 ~ PCT/LTS94/04098
31
some of these classes are Lubrol, Brij, Tween, Tergitol, Igepal, Triton,
Teepol and many
others.
Formation of the solid laminae, both indicator and reagent laminae, may be
done by applying the lamina material in liquid form followed by drying or
other
solidification. The liquid form of the substance may be, for example, a
solution, a
suspension, or an uncured liquid state of the substance, and the
solidification step may
thus be an evaporation of the solvent or a curing of the substance. The
substance of
interest may be combined with additional materials for any of a variety of
purposes, such
as for example:
(1) to facilitate the application of the liquid to the surface by modifying
the
viscosity of the liquid,
(2) to help form a continuous smooth solid layer which remains uniform and
does not disintegrate or granulate over time or upon the application of
additional layers over it,
(3) to modify the solubility of the layer with solvents used in layers to be
applied over it or to make the layer soluble in solvents which do not
dissolve layers applied underneath,
or all of these at the same time. Soluble polymeric materials are preferred
additives to
serve one or all of these purposes. Examples are cellulose and various
cellulose
derivatives, with the substitutions appropriately selected to achieve the
desired solubility
characteristics. For those test devices designed for aqueous or other water-
based
samples, the indicator lamina preferably contains the indicator retained in a
matrix of
solid material which is insoluble in water. This prevents the indicator from
migrating out
of the lamina and away from the light-transmitting surface. Alternatively,
however, the
indicator itself is insoluble in water and will by itself form a coherent
lamina which will
remain intact.
For those embodiments of the invention in which a positive control indicator,
a
negative control indicator or both are included in the device, one or more
additional
reagents will be included for each control. These additional reagents will
either be
. incorporated within one of the existing laminae in a horizontally defined
portion of that
lamina or applied as a separate, vertically adjacent lamina over a
horizontally defined
portion of the existing lamina. By virtue of their position in the chamber,
therefore,
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 216 0 2 6 2
PCT/US94104098
32
these additional reagents define control areas which are horizontally
separated from each
other and from the test area.
The selection of an appropriate reagent for a positive or negative control
will
depend on the hydrolase toward which the overall test is directed, the type of
visual
indicator used to detect the presence of the hydrolase, and whether the
reagent is intended
to serve as a positive control or a negative control. By utilizing known
chemistries, the
selection of an appropriate reagent will in most cases be apparent to those
skilled in the
art. The reagent for a positive control, for example, may be a sample of the
hydrolase
itself, an analogue of the hydrolase, or any other species with a parallel
mode of action
which initiates or induces the reaction or reaction sequence which culminates
in a
detectable change in the indicator. The lamina containing this reagent will be
on either
the upper or lower surface of the chamber provided that the reagent will not
initiate or
induce the detectable change until the specimen is present, but will do so
independently
of the presence or absence of the enzymatically active hydrolase in the
specimen. The
reagent for a negative control may likewise be an inhibiting species such as a
denaturing,
inhibiting or otherwise inactivating agent which prevents or blocks the
reaction or
reaction sequence, and thereby prevents the detectable change from occurring
regardless
of whether or not the enzymatically active hydrolase is present in the sample
or
specimen.
Both controls are activated when the specimen is applied to the test device.
In
some cases, this is achieved most effectively by placing the control reagents
in laminae
on the same surface as the laminae) containing the other reagent(s). In
others, best
results are achieved when the control reagents are placed in laminae on the
chamber
surface opposite that which bears the other reagent(s), such that the control
reagent and
the remaining reagents) are separated by the air gap. In preferred
embodiments, the
control areas of the device will contain all components and reagents used in
the test area
with the addition of the control reagents, either incorporated in horizontally
delineated
sections of one or more of the same laminae used in the test area or applied
as separate
laminae over such horizontally delineated sections. To achieve sharp
boundaries for the
control areas and to prevent the control reagents from activating or
deactivating the test
area, it is often beneficial to place discontinuities in the laminae at the
boundaries
separating the control areas from the test area to minimize or eliminate the
possibility of
SUBSTITUTE SHEET (RULE 26)
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WO 94/24306 ' PCT/US94/04098
33
lateral diffusion of the control reagents out of their respective control
areas. These
discontinuities may be in laminae along the upper surface, the lower surface,
or both.
As indicated above, the controls are preferably activated by the same specimen
sample used for the test. This is conveniently done by arranging the control
areas as
extensions of the test area, all contained in the same chamber in the test
device, with
unobstructed fluid communication between the various areas. In preferred
embodiments
where both positive and negative control areas are included, the control areas
are isolated
from each other by the test area which is positioned in between the two.
Filling of all
areas with a single application can be accomplished with the arrangement of
the sample
introduction port and vent holes described above. Since the detectable
changes, or
absence thereof, are detectable through the light-transmitting wall of the
device, the
identification of areas as positive and negative controls is conveniently
achieved by
placing appropriate indicia on the outer surface of the device.
The light-transmitting wall may be any material which is inert and
sufficiently
rigid to support the indicator lamina, and yet sufficiently transmissive of
light to show the
change in the indicator as soon as it occurs. Translucent or transparent
materials,
preferably nonabsorptive materials, may be used; transparent materials are
preferred.
Examples of transparent polymeric materials suitable for this use are
polyethylene
terephthalates (such as, for example, Mylarm ) and polycarbonates (such as,
for example,
Lexan~. The opposing (i. e. , bottom) wall of the device may likewise be made
of
transparent or translucent material, although it may also be of opaque
material since
visualization of the test results as well as the positive and negative
controls is required
only from one side of the device. When the bottom wall is transparent,
detection of the
change in the test area, control areas or both through the top wall can be
enhanced by
applying a printing or coating to either surface of the bottom wall with a
colored or
reflective material to heighten the color contrast.
The test device may be formed in a variety of ways. Sheets of polymeric
material may be laminated together, with appropriate cutouts to define the
shape of the
chamber and holes for the sample introduction port and the vent holes. The
depth of the
chamber as well as its shape and lateral dimensions will then be defined by
the thickness
of the central sheet, while the placement of the holes will be controlled by
the top sheet.
The indicator and reagent coatings may be applied to the top sheet, bottom
sheet or both,
as required, before the sheets are assembled into the laminate. The sheets may
then be
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 ~ 16 0 ~ ~ ~ PCT/US94/04098
34
secured together by any conventional means, such as, for example, by heat
sealing or
through the use of adhesives.
A particularly preferred method of forming the device is by the use of a
single
sheet of transparent or otherwise light-transmitting polymeric material, with
a section of
the sheet embossed or otherwise processed, mechanically or chemically, to
contain a
depression or indentation of constant depth in the inner surface of the
chamber. The
depression is located on one half of the sheet, with the holes for sample
introduction and
venting on the other half. The indicator and reagent coatings are applied at
appropriate
locations on the sheet, and the half containing the holes is then folded over
the other half
to form the enclosed chamber and to achieve correct alignment of the areas
representing
the upper and lower surfaces of the chamber. The facing surfaces of the sheet
are
bonded together as in the laminate of the preceding paragraph.
A preferred method for bonding the two halves together is through the use of
a heat-sensitive, pressure-sensitive, water-based or solvent-based adhesive.
The adhesive
may be restricted to the areas peripheral to the chamber to avoid contact with
the test
reagents, or it may cover the entire surface of the sheet, having been applied
prior to
application of the indicator and reagent coatings. In the latter case,
appropriate adhesives
will be those which are transparent, inert, wettable by, and otherwise
compatible with the
layers to be applied over it. Many types of adhesives suitable for this
application exist,
and the most appropriate choice will vary from one system to the next
depending on the
layers to be applied above it.
In yet another aspect of the present invention, a test device for testing a
sample for the presence of candidiasis by assaying for the presence of
enzymatically
active aspartic protease is provided, the test device comprising: a receptacle
defined at
least in part by first and second opposing walls having interior-facing
surfaces with a gap
therebetween, the first, second, or both walls being of light-transmitting
material; a
reporter enzyme immobilized on a solid support on the interior-facing surface
of one of
the first and second walls in such a manner whereby the reporter enzyme is
released
upon action of the aspartic protease; an indicator immobilized on the interior-
facing
surface of one of the first and second walls, the indicator being one which
undergoes a
detectable change upon action of the reporter enzyme; and an opening in the
receptacle
for introduction of the sample.
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 PCT/LTS94/04098
The reporter enzyme or marker enzyme used in this test device can be any
signal generating enzyme not subject to inactivation by any agent in the
sample, including
hydrolysis by any aspartic protease activity or any other hydrolase activity
present in the
sample. Such reporter enzymes include, but are not limited to, the following:
5 perozidases, phosphatases, ozidoreductases, dehydrogenases, transferases,
isomerases,
ltinases, reductases, deaminases, catalases, urease and glucuronidase.
Presently preferred
reporter enzymes are the perozidases, such as, for ezample, horseradish
perozidase.
The reporter enzyme is immobilized on a first solid support, i. e. , an
insoluble
matriz, gel or resin, in such a manner whereby the reporter enzyme is released
upon
10 action of aspartic protease. The reporter enzyme can be immobilized on the
solid support
through the use of a linker molecule having a hydrolyzable linkage which is a
substrate
for aspartic protease. In this test device, such linker molecules include
proteins and
peptides, with proteins being the preferred linker molecules. If the linker
molecule is a
protein, preferred proteins include, but are not limited to, the following:
azocasein,
15 casein, K-casein, immunoglobulins, hemoglobin, myoglobin, albumin, elastin,
keratin and
collagen. In preferred embodiments of this test device, the linker molecule is
K-casein,
casein, hemoglobin, or myoglobin.
In this test device of the present invention, the indicator is immobilized on
a
second solid support which is not in contact with the first solid support.
Preferred solid
20 supports in accordance with the present invention include, but are not
limited to, the
following: cellulose, agarose, deztran, polyacrylate, or their derivatives
chitin,
sepharose, ozirane acrylic beads, polymeric dialdehyde, starch, collagen,
keratin, elastin,
bovine hide powder, bacterial cell wall peptidoglycan or fragments thereof,
polyacrylamide, nylon, polyethylene terephthalates, polycarbonates, and
controlled pore
25 glass. Immobilization of the indicator on the second solid support is
carried out using
conventional methods and procedures known by those skilled in the art.
The indicator can be any chemical species which undergoes a detectable
change as the result of the reaction or as a result of the culmination of
reactions
occurring when the enzymatically active hydrolase is present in the sample or
specimen.
30 The resulting detectable change is an indication of the presence of
enzymatically active
aspartic protease in the sample and, in turn, the presence of enzymatically
active aspartic
protease in the sample indicates the presence of candidiasis. Preferred
indicators are
visual indicators and, in particular, chromogenic indicators, i. e. , those in
which the
SUB~fITUTE SHEET (RULE 26)
WO 94/24306 216 Q 2 6 2 PCT/US94/04098
36
visible change is a change in color, including the formation of color in an
otherwise
colorless material, upon action of the reporter or marker enzyme when it is
released from
the solid support by the enzymatically active aspartic protease whose presence
is being
detected.
A wide variety of chromogenic indicators (i. e. , chromogens) and other
species
having a similar effect may be used as visual indicators. Preferred
chromogenic
indicators in accordance with the present invention when peroxidase is the
releasable
reporter enzyme being used, comprise a hydroperoxide and a chromogen
including, but
not limited to, one of the following: guaiac, 2-2'-azino-bis(3-ethyl-
benthiazoline-6-
sulfonic acid), tetramethylbenzidine, phenol, 4-aminoantipyrine, and 4,5-
dihydroxynaphthalene-2,7-disulfonic acid. A particularly preferred chromogenic
indicator
is comprised of a hydroperoxide and guaiac, which is colorless in its reduced
state and
deep blue in its oxidized state.
If the visual indicator is a chromogenic indicator for peroxidases or
pseudoperoxidases, a solid chromogen system is employed, and such a system
comprises
a hydroperoxide, a chromogen capable of being oxidized by hydroperoxides in
the
presence of a peroxidase, and a solid support onto which the chromogen has
been
impregnated or immobilized and dried. In this system, the chromogen can be
impregnated onto a bibulous paper or support as is done with Hemoccult~ slides
or,
alternatively, the chromogen can be deposited onto a plastic or other sheet in
the form of
a thin layer. In this latter format, the chromogen would be layered as a
solution
containing a polymeric material (such as, for example, hydroxypropyl
cellulose, ethyl
cellulose, etc.). If the chromogen itself is a water soluble chromogen, it can
be trapped
in a matrix of material which is insoluble in water. Alternatively, if the
chromogen itself
is insoluble in water, it can be layered as a solution in an organic solvent
either alone or
in combination with a water soluble or water insoluble polymer.
In this solid chmmogen system, the hydroperoxide can be provided either in a
solid form (such as, for example, titanium hydroperoxide) or it can be
generated in situ
in the test device. In the presence of the hydroperoxide and the peroxidase
upon its
release from the first solid support by the enzymatically active aspartic
protease, the
chromogen will be oxidized and a visually detectable change in color will
result. As
previously mentioned, this resulting change in color is an indication of the
presence of
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 PCT/US94/04098
37
enzymatically active aspartic protease in the sample or specimen and, in turn,
the
presence of enzymatically active aspartic protease indicates the presence of
candidiasis.
In a final aspect of the present invention, a test device for testing a sample
for
the presence of an inhibitor of a target hydrolase is provided, the test
device comprising:
a receptacle defined at least in part by first and second opposing walls
having interior
facing surfaces with a gap therebetween, the first wall, the second wall, or
both being of
light-transmitting material; a target hydrolase contained on the interior-
facing surface of
one of the first and second walls, the target hydrolase being susceptible to
inactivation by
the presence of the inhibitor; a reporter enzyme immobilized on a solid
support on the
interior-facing surface of one of the first and second walls in such a manner
whereby the
reporter enzyme is released upon action of the target hydrolase if the target
hydrolase is
not inactivated by the presence of the inhibitor; an indicator contained on
the interior-
facing surface of one of the first and second walls, the indicator being one
which is
susceptible to a detectable change upon action of the reporter enzyme; and an
opening in
the receptacle for introduction of the sample.
To detect the presence of a hydrolase inhibitor in a sample, a defined
quantity
of the target hydrolase is incorporated into the test device either
immediately prior to
performing the test, or, preferably, during the manufacture of the test
device. Test
performance involves detecting the ability of the sample to inhibit the target
hydrolase.
In the event that the target hydrolase inhibitor is not present in the sample,
the target
hydrolase will release the reporter enzyme from the solid support, thereby
producing a
detectable response in the indicator. Conversely, if the target hydrolase
inhibitor is
present in the sample, the target hydrolase will be inhibited, the reporter
enzyme will not
be released from the solid support, and a detectable change or response will
not be
produced in the indicator. It is not essential that the inhibitor in the
sample completely
inhibit the target hydrolase added to the test device. It is only required
that a sufficient
amount of target hydrolase inhibition occurs to produce a noticeable
difference in the
anticipated detectable response. The positive and negative control elements of
the test
device provide comparison responses to illustrate the appearances of
completely inhibited
target hydmlase and target hydrolase free of inhibition.
The sensitivity of this enzyme release technology can be adjusted as needed by
incorporating defined quantities of the target hydrolase into the test device.
Similarly, if
desired, exposure time of the target hydrolase to any inhibitor in the sample
can be
SUBSTITUTE SHEET (RULE 26)
WO 94/2x306 ~ PCT/LTS94104098
38
regulated by physically or chemically separating the target hydrolase from the
immobilized reporter enzyme. For example, by coating the immobilized reporter
enzyme
in a timed-release matrix, a pH degradable coating, or other controlled-
release material
which dissolves comparatively slowly, temporal access of the target hydrolase
to the
immobilized reporter enzyme can be controlled. Alternatively, the target
hydrolase can
be present in a location or chemical form which is immediately accessible to
any inhibitor
which may be present in the sample. Hence, prior to its gaining access to the
immobilized reporter enzyme, the target hydrolase can first be exposed to the
inhibitor
for sufficient time for inhibition to take place. Since access to the
immobilized reporter
enzyme can be temporally regulated, the test results can detect the presence
of even
slowly acting inhibitors.
This test device of the present invention can be used to test a sample for the
presence or absence of any known inhibitor of a hydrolytic enzyme, including,
but not
limited to, the following: inhibitors of the proteases or (interchangeably)
proteinases,
peptidases, lipases, nucleases, homo- or hetero-oligosaccharidases, homo- or
hetero-
polysaccharidases, phosphatases, sulfatases, neur3minidases and esterases. In
a preferred
embodiment, this test device is used to assay for the presence protease
inhibitors,
including, but not limited to, the following: aspartic protease inhibitors,
serine protease
inhibitors, thiol protease inhibitors, metallo protease inhibitors, acid
protease inhibitors
and alkaline protease inhibitors. In a further preferred embodiment, this test
device is
used to assay for the presence of aspartic protease inhibitors such as, for
example,
pepstatin, ovomacroglobulin, haloperidol, transition state mimetics, U-81749,
H-261,
MV7-101, A-75925, A-76928 and A-7003. U-81749, H-261, MV7-101, A-75925, A-
76928 and A-7003 are experimental drugs which have previously been described
in the
literature. In an even further preferred embodiment of this test device of the
present
invention, pepstatin is the aspartic protease inhibitor whose presence is
being detected.
In accordance with this test device of the present invention, target
hydrolases
include, but are not limited to, the following: proteases, proteinases,
peptidases, lipases,
nucleases, homo- or hetero-oligosaccharidases, homo- or hetero-
polysaccharidases,
phosphatases, sulfatases, neuraminidases and esterases. The particular target
hydrolase
used in this test device will depend upon which inhibitor is being detected,
and the
selection in any given case will be readily apparent to those skilled in the
art. If, for
example, pepstatin is the inhibitor being detected, then aspartic protease
would be the
SUBSTITUTE SHEET (RULE 26)
21~~12G2
WO 94/24306 - PCT/US94/04098
39
target hydrolase used in the test device described above. Moreover, the
appropriate
concentrations of the target hydrolase, the geometric location of the target
hydrolase in
the test device, and the appropriate timed- and controlled-release
technologies and
matrices are known to those skilled in the art.
As with the other test devices presently claimed, the reporter enzyme or
marker enzyme used in this particular test device can be any signal generating
enzyme,
i.e., an enzyme whose activity brings about a visible or detectable change,
not subject to
inactivation by any agent in the sample, including inactivating hydrolysis by
the target
hydrolase present in the test device. Such reporter enzymes include, but are
not limited
to, the following: peroxidases, phosphatases, oxidoreductases, dehydrogenases,
transferases, isomerases, kinases, reductases, deaminases, catalases, urease
and
glucuronidase. The selection of an appropriate reporter or marker enzyme will
be readily
apparent to those skilled in the art. In this test device of the present
invention, the
preferred reporter enzymes are the peroxidases, and, in particular,
horseradish
peroxidase.
The reporter enzyme is immobilized on a solid support, i.e., an insoluble
matrix, gel or resin, in such a manner whereby the reporter enzyme is released
upon
action of the target hydrolase if the target hydrolase is not inactivated by
the presence of
the inhibitor in the sample. Solid supports in accordance with the present
invention
include, but are not limited to, the following: cellulose, agarose, dextran,
polyacrylate,
polyacrylamide, or their derivatives, chitin, sepharose, oxirane acrylic
beads, polymeric
dialdehyde, starch, collagen, keratin, elastin, bovine hide powder, bacterial
cell wall
peptidoglycan or fragments thereof, nylon, polyethylene terephthalates,
polycarbonates,
and controlled pore glass.
As previously explained, the reporter enzyme can be immobilized on the solid
support through the use of a linker molecule which is a hydrolyzable substrate
for the
enzymatically active target hydrolase. Such linker molecules include, but are
not limited
to, the following: proteins, carbohydrates, lipids, peptides, esters and
nucleic acids. The
particular linker molecule used to attach the reporter enzyme to the solid
support will
depend on which target hydrolase is incorporated in the test device, and the
selection in
any given case will be readily apparent to those skilled in the art.
The indicator can be any chemical species which undergoes a detectable
change as a result of the reaction or as a result of the culmination of
reactions occurring
SUB~fITUTE SHEET (RULE 26)
WO 94/24306 216 0 2 6 2 . PCT/US94/04098
if the enzymatically active target hydrolase is not inactivated by the
presence of the
inhibitor in the sample or specimen. Preferred indicators are visual
indicators and, in
particular, chromogenic indicators, i. e. , those in which the visible change
is a change in
color, including the formation of color in an otherwise colorless material,
upon action of
5 the reporter or marker enzyme when it is released from the solid support by
the
enzymatically active target hydrolase provided it is not inactivated by the
presence of the
inhibitor in the sample or specimen.
A wide variety of chromogenic indicators (i.e., chromogens) and other species
having a similar effect may be used as visual indicators. Preferred
chromogenic
10 indicators for peroxidase-like reporter enzymes in accordance with the
present invention
comprise a hydroperoxide and a chromogen including, but not limited to, one of
the
following: guaiac, 2-2'-azino-bis(3-ethyl-benthiazoline-6-sulfonic acid),
tetramethylbenzidine, phenol, 4-aminoantipyrine, and 4,5-dihydroxynaphthalene-
2,7-
disulfonic acid. A particularly preferred chromogenic indicator is comprised
of a
15 hydroperozide and guaiac, a chromogen which is colorless in its reduced
state and deep
blue in its oxidized state. As previously described, if the visual indicator
is a
chromogenic indicator, it is possible to employ either a liquid chromogen
system or a
solid chromogen system.
It should be understood that the prior discussion pertaining to the structure
of
20 the test device for assaying for the presence of a hydrolase, its
construction and its
preferred embodiments is fully applicable to the test device for testing a
sample for the
presence of candidiasis by assaying for the presence of enzymatically active
aspartic
protease, and to the test device for testing a sample for the presence of an
inhibitor of a
target hydrolase.
25 While the invention is not intended to be limited to any particular
construction
of a test device, the attached Figures, which are not drawn to scale,
illustrate how one
such device may be constructed.
FIG. 1 depicts the support structure of the device in a perspective view,
prior
to the indicator and reagents being applied and the chamber being enclosed.
The support
30 structure consists of a single sheet 11 of relatively stiff, transparent,
chemically inert
plastic material, with a score line 12 defining a fold separating the sheet
into two halves
13, 14, each having the same length and width. The lower half 13 contains an
indentation of a composite shape consisting of a circle 15 at the center with
two
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 PCT/U894/04098
41
rectangular extensions 16, 17 extending to opposite sides. The upper half 14
contains
three holes including a central hole 18 which serves as the sample
introduction port, and
two side holes 19, 20 which serve as vent holes. The two vent holes 19, 20 are
circular,
while the sample introduction port 18 is circular with one straight edge to
facilitate
scraping of the specimen from the swab which is used as a transfer device. The
holes
are positioned such that when the plastic is folded at the score line 12 and
the top half 14
is placed in contact with the bottom half 13, the sample introduction port 18
is above the
center of the circular part 15 of the indentation, and the vent holes 19, 20
are above the
two rectangular extensions 16, 17 at the outermost edge of each. The two
rectangular
extensions 16, 17 represent the positive and negative control areas of the
device.
Many variations of the device of FIG. 1 may be made. The two halves 13, 14
may be of differing lengths, widths or both for various reasons. The only
critical feature
is that the indentations in the lower half and the holes in the upper half be
positioned
relative to the score line such that the holes and indentations are in proper
registration
when the two halves are folded at the score line. As another example, the
rectangular
extensions 16, 17 in the lower half of the structure may terminate in circular
(or half
circular) areas to match the vent holes 19, 20 in the top half. The vent holes
themselves
may be of any shape. In fact, vent holes which are shaped differently from the
sample
introduction hole 18 have the advantage of preventing user confusion as to
where to
introduce the sample.
FIG. 2 is a side cutaway view of the device of FIG. 1, showing the chamber
31 in cutaway after the coatings have been applied and the two halves folded
over and
sealed to one another. The inner surfaces of each of the two halves 13, 14 of
the
transparent poiymer are coated with an adhesive 32, 33, respectively. Directly
underneath the upper adhesive layer 33 is the layer containing the visual
indicator 34, and
beneath the latter is a layer of reagent 35. It will be noted that both the
visual indicator
layer 34 and the reagent layer 35 can extend the full length and width of the
chamber,
surrounding the sample introduction port 18 and extending into all areas of
the chamber.
The test and control areas of the chamber are defined by the horizontal
. locations of the coatings on the lower wall 13 of the chamber. A reagent for
the negative
control is contained in one coating 36 which occupies the lower surface of one
of the two
rectangular extensions 16 of the chamber (see FIG. 1), and a reagent for the
positive
control is contained in a second coating 37 similarly situated in the other
rectangular
SUBSTITUTE SHEET (RULE 26j
CA 02160262 2001-04-17
42
extension 17. Alternatively, reagents for the controls may be placed on the
upper surface
of the chamber rather than the lower. This is in fact preferred for certain
assays. The
portion of the lower surface under the cxntral circular portion 15 of the
chamber (see
FIG. 1) is coated with a layer 38 which may either contain an additional
reagent used in
the test reaction or no reagent at all. Thus, as viewed from the top of the
closed device,
the circular test area 41 is flanked by a rectangular negative control area 42
and a
rectangular positive control area 43. The three xgments 36, 37, 38 can be
separated by
gaps or discontinuities 44, 45 to retard or minimize diffusion between, or
contact of, the
contents of thex xgments. Similar discontinuities may also be placed in either
or both
of the visual indicator and reagent layers 34, 35, directly above the
discontinuities 44, 45
in the lower layer. The discontinuities in the visual indicator and reagent
layers will
further prevent diffusion of control components or other reagents from the
control areas
into the test area. The most inward-facing of the layers 35, 36, 37, 38 all
may contain,
in addition to any reagents prexnt, a wetting agent or detergent to promote
the rapid and
complete spreading of the specimen along the upper and lower surfaces to fill
the
chamber. In some cases, the same effect is achieved by a layer of protein.
In certain embodiments of the invention, the reagents tend to deteriorate upon
prolonged exposure to air or to air-borne moisture. In the device shown in
FIG. 2, this
is prevented by a thin sheet of material 46 which is both moisture-impermeable
and air-
impermeable. The sheet covers the sample injection port and both vent holes,
sealing the
chamber interior from the environment until the device is ready for ux,
whereupon the
sheet is readily peeled off. For materials which are particularly water-
xnsitive or air-
xnsitive, it may also be desirable to place a moisture- and air-impermeable
sheet on the
bottom of the device, the sheet being either permanently attached or capable
of being
peeled off. Further protection against moisture and air can be achieved by
placing the
device in a pouch which completely surrounds the device.
As indicated above, each of the dimensions of the device shown in Figures 1
and 2 may vary, as may their arrangements and shapes. A typical example,
however, is
one in which the support sheet is Mylar 5 mil in thickness (0.005 inch, 0.0127
cm), and
the adhesive layer is low density polyethylene 2 mil in thickness (0.002 inch,
0.0051
cm), the gap width 47 is 7.5 mil (0.0075 inch, 0.019 cm), the test area is a
circle 5/16
inch in diameter (area: 0.0766 square inch, 0.494 cm~), and the Negative and
positive
control areas each measure 1/8 inch x 1/16 inch (area: 0.0078 square inch,
0.0504
WO 94/24306 _ PCT/US94/04098
43
cmz). The air vents in this example are each circular, and they and the sample
introduction port are each 1/8 inch (0.32 cm) in diameter. The chamber volume
is
approximately 12 ~,L.
The test device of the present invention is useful for testing samples for the
presence of a hydrolase from a wide range of sources, including biological
sources and
otherwise. Bodily fluids such as blood, serum, plasma, urine, urethral
discharge, tears,
vaginal fluid, cervical exudate, spinal fluid and saliva, as well as non-
bodily fluids such
as foods, pond or swimming pool water and liquid wastes are examples.
EXAMPLES
I. PREPARATIONS
A. PREPARATION OF CYANOGEN BROMIDE ACTIVATED SOLID SUPPORTS
1. MATERIALS
a. Solid, insoluble supports (Sepharose 4B, chitin, and Sigmacell~ 20
[purchased
from Sigma Chemical Co.])
b. Distilled water and ice made with distilled water
c. 4.0 M NaOH
d. Solid CNBr
e. Coupling buffer (0.1 M NaHC03 containing 0.5 M NaCI)
f. Magnetic stirring motor and stirring bar; pH meter; chemical fume hood
2. PROCEDURE
Ten milliliters (10 mL) of cold distilled water were added to 5 grams moist,
washed solid support, and the mixture was chilled to 10°C -
15°C. The pH of the
suspension was adjusted to 10.8 with 4.0 M NaOH, and 100 mg solid, crushed
CNBr
were added per gram moist solid support. The pH was maintained at 10.8 by
adding 4.0
M NaOH as necessary, and the temperature of the suspension was allowed to
increase to
18°C - 20°C during the activation process. Activation was
considered complete when no
further additions of 4.0 M NaOH were needed to maintain pH at 10.8. At this
time,
crushed ice was added to cool the reaction mixture, and the suspension
filtered on a pre-
chilled sintered funnel. The filtrate was collected in a suction flask
containing solid
ferrous sulfate which inactivated residual CNBr and cyanide remaining in the
reaction
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 ~ PCT/US94/04098
44
mixture. The solid support was washed with 1 liter cold distilled water and 1
liter
coupling buffer under suction and stored as a moist paste at 4°C.
B. PREPARATION OF [KAPPA-CASEIN-HItPO] CONJUGATES (HYDRAZIDE
S METHOD)
1. MATERIALS
a. Kappa-Casein and Horse Radish Peroxidase (HRPO) Hydrazide [purchased
from Sigma Chemical Co.]
b. Distilled water
c. Buffers:
i. 50 mM 2-[Morpholino] ethane sulfonic acid] (MES) buffer, pH 6.0
ii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCI
iii. 100 mM Sodium borate, pH 9.0 containing 0.5 M NaCI
d. Solid sodium periodate
e. 100 mM Formaldehyde
2. PROCEDURE
a. Periodate Activation of Kappa-Casein
Twenty mg of Kappa-Casein were dissolved in 2 mL MES buffer and 8.6 mg
solid sodium periodate were added to the solution. The mixture was incubated
on a rotor
for 30 minutes in the dark at room temperature. The reaction mixture was then
dialyzed
against approximately 300 mL 50 mM MES, pH 6.0 for approximately 45 minutes at
room temperature, after which the dialysis fluid was replaced and dialysis
continued for
an additional 45 minutes.
b. Covalent Linking of HRPO to Kappa-Casein
Five mg HRPO hydrazide (approximately 175 Units/mg protein) were added
to the activated, dialyzed Kappa-Casein, and the mixture incubated with
agitation for 4
hours at room temperature. Two hundred microliters of 100 mM formaldehyde were
added to the mixture, and incubation continued at room temperature for and
additional 30
minutes. Two mL 1 M cold acetate buffer, pH 4.0 were added, and the conjugate
allowed to precipitate for 30 minutes at 4°C. The pellet was removed by
centrifugation,
redissolved in 2 mL 100 mM borate buffer, pH 9.0, and stored at 4°C
until used.
SUBSTITUTE SHEET (RULE 26)
WO 9-l/24306 216 ~ 2 G ? PCT/US94/04098
C. ATTACHIVVIENT OF [KAPPA-CASEIN-HItPO] CONJUGATES (HYDRAZmE
METHOD) TO CYANOGEN BR011~E ACTIVATED SUPPORTS
1. MATERIALS
a. [Kappa-Casein-HRPO] conjugates (PREPARATION B)
5 b. Distilled water
c. Buffers:
i. Coupling Buffer (0.1 M NaHC03 containing 0.5 M NaCI)
ii. 1.0 M Tris buffer, pH 8.0
iii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCI
10 iv. 100 mM Sodium borate buffer, pH 9.0, containing 0.5 M NaCI
d. Cyanogen bromide activated Sepharose 4B and Sigmacell~ 20
(PREPARATION A)
e. Cyanogen bromide activated Sepharose 6 MB (from Sigma Chemical Co.)
15 2. PROCEDURE
a. Cyanogen Bromide Activated Sepharose 4B and Sigmacell~ 20
Two milliliters [Kappa-Casein-HRPO] conjugate prepared from the hydrazide
of HRPO as described in PREPARATION B were diluted with 3 mL coupling buffer
and
added to one gram moist cyanogen bromide-activated Sepharose 4B or Sigmacell~.
The
20 suspension was mixed end-over-end at room temperature for two hours. The
solid
support was washed with coupling buffer and water and 3 mL 1.0 M Tris buffer,
pH 8
were added. The suspension was incubated for 2 hours at room temperature to
inactivate
remaining active sites on the solid support. Three washing cycles, each
consisting of pH
4.0 acetate buffer followed by coupling buffer, were used to remove unbound
materials
25 from the support. A final wash with distilled water was used, and the moist
suspension
stored at 4°C until used.
b. Commercially Activated Sepharose 6 MB (Sigma Chemical Co.)
The moist gel (1 gram moist weight) was washed with 200 mL 1 mM HCl
prior to use as described above.
30 ~ c. Activated Chitin
The procedure described in A above was employed, except that 500 mg of
moist, activated chitin were used, instead of 1 gram.
SUBSTITUTE SHEET (RULE 26)
WO 9~/2a306 216 4 2 ~ ~ PCT/LTS9=1/04098
46
D. PREPARATION OF HORSE RADISH PEROXIDASE ()EHtPO) ALDEHYDE
1. MATERIALS
a. HRPO, Type II, 200 Units/mg (from Sigma Chemical Co.)
b. 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH S.0
c. Solid sodium periodate
d. BiogelR P-30 spherical polyacrylamide gel purchased from Bio-Rad
2. PROCEDURE
25.7 mg solid sodium periodate (40 millimoles) were added to 30 mg HRPO
in 3 mL MES buffer. The solution was incubated in the dark for 30 minutes at
room
temperature with continuous rotation. The solution was passed through a P-30
column in
MES buffer to remove excess sodium periodate, and the colored HRPO collected
in a
total volume of 3 ml. The HRPO aldehyde was immediately coupled to the desired
protein (hemoglobin, myoglobin, or casein).
E. ATTACHIVVIENT OF HEMOGLOBIN AND MYOGLOBIN TO CYANOGEN
BROMIDE ACTIVATED SOLID SUPPORTS
1. MATERIALS
a. Solid, insoluble supports
i. Cyanogen bromide activated sepharose 6MB (from Sigma Chemical Co.)
ii. Sigmacell~ 20 (as described in PREPARATION A)
b. Distilled water
c. Buffers:
i. Coupling Buffer (0.1 M NaHC03 containing 0.5 M NaCI)
ii. 1.0 M Tris buffer, pH 8.0
iii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCI
iv. 100 mM Sodium borate buffer, pH 8.0 containing 0.5 M NaCI
v. 0.1 Percent (v/v) glutaraldehyde solution in water
2. PROCEDURES
One hundred milligrams of either hemoglobin or myoglobin dissolved in 5 mL
of coupling buffer were added to 1 gram moist solid support. The suspension
was mixed
end-over-end at room temperature for two hours. The solid support was washed
with
coupling buffer and water and 3 mL 1.0 M Tris buffer, pH 8 were added and the
suspension incubated for 2 hours at room temperature to inactivate remaining
active sites
SUB~fiTUTE SHEET (RULE 26)
WO 94/24306 216 0 2 fi 2 PCT/US94104098
47
on the solid support. Three washing cycles, each consisting of pH 4.0 acetate
buffer
followed by borate buffer, both containing sodium chloride, were used to
remove
unbound materials from the support. For myoglobin derivatized supports, a
final wash
with distilled water was used, and the moist suspension stored at 4°C
until used. For
hemoglobin derivatized supports, 5 mL 0.1 °~ glutaraldehyde solution
were added to the
moist suspension resulting from the above procedure, and the suspension was
incubated
overnight at 4°C. Three washing cycles, each consisting of pH 4.0
acetate buffer
followed by borate buffer at pH 8.0, were used to remove unbound materials
from the
support. A final wash was done with distilled water, and the moist suspension
stored at
4°C until used.
F. ATTACHMENT OF HORSE RADISH PERORIDASE (HItPO) ALDEHYDE TO
HEMOGLOBIN OR MYOGLOBIN DERIVATIZED SUPPORTS
1. MATERIALS
a. HRPO aldehyde prepared as described in PREPARATION D
b. Hemoglobin or myoglobin derivatized solid supports prepared as described in
PREPARATION E
c. 100 mM Sodium cyanoborohydride
d. 1.0 M NaCI
e. distilled water
2. PROCEDURE
The three mL HRPO aldehyde (30 mg) recovered from the P-30 column in
PREPARATION D were added to 1 gram of the hemoglobin or myoglobin derivatized
solid supports described in PREPARATION E. The suspension was incubated for 16
hours at 4°C followed by 4 hours at room temperature. Three washing
cycles, each
consisting of distilled water and 1.0 M NaCI, were used to remove unbound
materials
from the support. The solid support was then incubated with 5 mL 100 mM
cyanoborohydride overnight at 4°C. A final wash was done with distilled
water, and the
moist suspension stored at 4°C until used.
G. PREPARATION OF ALDEHYD~ACTIVATED Sigmacell~ 20
1. MATERIALS
a. Sigmacell~ 20 (from Sigma Chemical Co.)
SUBSTITUTE SHEET (RULE 26j
WO 94/24306 2 I 6 0 2 6 ~ PCT/US94/04098
48
b. Distilled Water
c. Solid Sodium periodate
d. 20 mM Sodium bicarbonate, pH 9.5
2. PROCEDURE
Solid sodium periodate (428 mg) was added to one gram Sigmacell~ suspended
in 10 mL distilled water. The mixture was rotated continuously at room
temperature for
2 hours. The pellet was removed by centrifugation, washed five times with 10
mL
distilled water and once with 10 mL 20 mM sodium bicarbonate, pH 9.5. The
resin was
used immediately after preparation.
H. COVALENT ATTACHMENT OF HEMOGLOBIN AND MYOGLOBIN TO
ALDEHYDE-ACTIVATED SIGMACELL~ 20
1. MATERIALS
a. Aldehyde-activated Sigmacell~ 20 (PREPARATION G)
b. Distilled Water
c. 100 mM Sodium cyanoborohydride
d. Human hemoglobin and horse heart Myoglobin (both from Sigma Chemical
Co. )
e. Buffers
i. 20 mM Sodium bicarbonate, pH 9.5
ii. 50 mM Ethanolamine, pH 9.5
iii. 100 mM M Tris buffer, pH 8.0 containing 0.5 M NaCI
iv. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCI
v. 20 mM MES buffer, pH 5.0
2. PROCEDURE
Fifty mg of either myoglobin or hemoglobin dissolved in 9 mL sodium
bicarbonate, pH 9.5 were added to 1 mL of aldehyde-activated Sigmacell~ 20
(PREPARATION G). The suspension was rotated overnight at room temperature, and
the pellet removed by centrifugation. The pellet was washed with 10 mL water.
Nine
. mL 50 mM, ethanolamine, pH 9.5 were added, and the mixture rotated
continuously at
room temperature for one hour. The pellet was isolated by centrifugation and
washed
sequentially with 10 mL each of acetate buffer, Tris buffer, and MES buffer.
Twenty
mL of an aqueous solution of 100 mM sodium cyanoborohydride were added, and
the
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 PCT/US94/04098
49
mixture incubated for one hour with continuous rotation at room temperature,
followed
by overnight incubation at 4°C. After the overnight incubation, the
derivatized support
was washed five times with 10 mL aliquots of distilled water and twice with 10
mL
aliquots MES buffer.
I. COVALENT ATTACHIVViENT OF HItPO ALDEHYDE TO HEMOGLOBIN
OR MYOGLOBIN IIVVIMOBILIZED ON ALDEHYD~ACTIVATED
SIGMACELL~ 20
1. MATERIALS
a. Hemoglobin or myoglobin derivatized Sigmacell~ 20 (PREPARATION H)
b. Distilled Water
c. 100 mM sodium cyanoborohydride
d. Buffers
i. 100 mM M Tris buffer, pH 8.0 containing 0.5 M NaCI
ii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCI
iii. 20 mM MES buffer, pH 5.0
e. Aldehyde-Activated HRPO (PREPARATION D)
2. PROCEDURE
Twenty milligrams desalted HRPO aldehyde (PREPARATION D) dissolved in
2 mL MES buffer, pH 5.0 were added to 1 gram hemoglobin or myoglobin
derivatized
Sigmacellm 20 (PREPARATION H), and the suspension rotated for 1 hour at room
temperature followed by overnight incubation at 4°C. The support was
isolated by
centrifugation, and washed twice with 10 mL aliquots of distilled water.
Twenty mL 100
mM sodium cyanoborohydride were added, and the suspension incubated for 60
hours at
4°C. The pellet was washed sequentially twice with each of the
following: distilled
water; acetate buffer; Tris buffer; and MES buffer.
J. COVALENT ATTACHrZENT OF HEMOGLOBIN TO CONnViERCIAL
EUPERGITm C ACRYLIC BEADS
1. MATERIALS
a. Ozirane acrylic beads (Eupergit~ C, from Sigma Chemical Co.)
b. Human hemoglobin (type IV from Sigma Chemical Co.)
c. Buffers and solutions
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 216 0 2 6 2 . PCT/US94/04098
i. 1.0 M Potassium Phosphate buffer, pH 7.5 containing 0.1 °& sodium
azide
(w/v)
ii. 1.0 M NaCI
iii. 5 °.b (v/v) Mercaptoethanol, adjusted to pH 8.0 with 0.5 M NaOH
5 iv. 100 mM Potassium phosphate buffer, pH 7.4
v. 500 mM Potassium phosphate buffer, pH 7.4
vi. 3.5 M Sodium thiocyanate
vii. Phosphate buffered saline (PBS) (0.01 M sodium phosphate, pH 7.2
containing 0.15 M NaCI)
10 d. Distilled water
2. PROCEDURE
Hemoglobin (125 mg) was dissolved in 5 mL 1.0 M phosphate buffer, pH 7.5
containing sodium azide and added to 1 gram Eupergit~ C. The mixture was
allowed to
incubate without agitation for 72 hours at room temperature. The support was
washed
15 three times with 1.0 M NaCI and five times with distilled water. The
support was mixed
with 2.5 mL mercaptoethanol previously adjusted to pH 8.0, and the suspension
allowed
to stand overnight at room temperature. The beads were washed 10 times with
distilled
water, placed on a small sintered glass funnel, and washed sequentially with
50 mL of
each of the following: 0.5 M potassium phosphate buffer, pH 7.5; 0.1 M
potassium
20 phosphate buffer pH 7.5; 3.5 M sodium thiocyanate; and finally with large
volumes of
phosphate buffered saline (PBS) (0.01 M sodium phosphate, pH 7.2 containing
0.15 M
NaCI). The derivatized beads were treated with 0.1 ~ (v/v) glutaraldehyde with
rotation
for 2 hours at room temperature, overnight at 4°C, and then washed with
distilled water.
25 K. COVALENT ATTACHMENT OF HRPO ALDEHYDE (PREPARATION D) TO
HEMOGLOBIN DERIVATIZED Eupergit~ C (PREPARATION ~
1. MATERIALS
a. HRPO aldehyde (PREPARATION D)
b. Hemoglobin derivatized Eupergit~ C (PREPARATION .>)
30 c. 100 mM Sodium cyanoborohydride
d. 1.0 M NaCI
e. Distilled water
SUB~fITUTE SHEET (RULE 26)
WO 94/24306 . PCT/LTS94/04098
51
2. PROCEDURES
Thirty milligrams of HRPO aldehyde (PREPARATION D) in three mL MES
buffer were added to 1 gram hemoglobin derivatized Eupergit~ C (PREPARATION
J).
The suspension was incubated for 16 hours at 4°C followed by 4 hours
at room
temperature. Three washing cycles, each consisting of distilled water and 1.0
M NaCI
were used to remove unbound materials from the support. The solid support was
then
incubated with 5 mL 100 mM cyanoborohydride for 60 hours at 4°C. A
final wash was
done with distilled water, and the moist suspension stored at 4°C until
used.
L. COVALENT ATTAC~VVIENT OF CASEIN-HItPO TO POLYMERIC
DIA~LDEHYDE
1. MATERIALS
a. 25 mg Kappa-Casein-HRPO conjugate prepared by the hydrazide method
(PREPARATION B) dissolved in 2 mL 100 mM borate buffer, pH 9.0
b. Polymeric dialdehyde (from Sigma Chemical Co.)
c. Buffers:
i. 100 mM Ethanolamine, pH 9.5
ii. 100 mM Sodium borate buffer, pH 9.0 containing 0.5 M NaCI
iii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCI
iv. 50 mM MES buffer, pH 6.0
d. 100 mM Formaldehyde
e. 100 mM Sodium cyanoborohydride
2. PROCEDURE
Two mL of Kappa-Casein-HRPO conjugate were prepared as described in
PREPARATION B with one modification: the formaldehyde treatment was omitted.
This solution was mined with three mL MES buffer, and the solution added to 1
gram
polymeric dialdehyde. The suspension was rotated at room temperature for 6
hours, and
incubated overnight at 4°C. Two hundred microliters of 100 mM
formaldehyde were
added to the suspension and the mizture incubated for 1 hour at room
temperature. The
resin was removed by centrifugation, washed with water, and resuspended in 3
mL 100
mM ethanolamine, pH 8.5. After a two hour incubation with rotation at room
temperature, the resin was sequentially washed acetate buffer, borate buffer,
and water.
The solid support was then incubated with 5 mL 100 mM cyanoborohydride
overnight at
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 _ ~ PCT/US94I04098
52
4°C. A final wash was done with distilled water, and the moist
suspension stored at 4°C
until used.
M. ATTACH11ZENT OF HItPO-LABELED (ALDEHYDE METHOD) WHOLE
CASEIN OR KAPPA-CASEIN TO EUPERGITm C ACRYLIC BEADS
1. MATERIALS
a. Oxirane acrylic beads (Eupergit~ C, from Sigma Chemical Co.)
b. Bovine whole Casein or Kappa-Casein (from Sigma Chemical Co.)
c. Buffers and solutions
i. 100 mM Sodium bicarbonate, pH 8.5 containing 0.5 M NaCI
ii. 100 mM Sodium acetate buffer pH 4.0 containing 0.5 M NaCI
iii. 100 mM Tris buffer pH 8.0, containing 0.5 M NaCI
iv. 20 mM MES buffer, pH 5.0
v. 5 ~ (v/v) mercaptoethanol in water
vi. 200 mM Sodium borohydride in water
d. Distilled water
2. PROCEDURES
a. Covalent Attachment of Casein or Kappa-Casein to Eupergit~ C Beads
2 mL of a solution containing either whole casein or Kappa-Casein (10
mg/mL) in 100 mM sodium bicarbonate buffer, pH 8.5 and containing 0.5 M NaCI
were
added to 1 mL of Eupergit~ C beads suspended in water. The mixture was
incubated
with rotation for 48 hours at room temperature. The pellet was isolated by
centrifugation, and washed sequentially with 9 mL aliquots of Acetate and Tris
buffers,
followed by two washings with 9 mL aliquots of MES buffer. Ten mL 5
mercaptoethanol were added and the mixture incubated with rotary mixing
overnight at
room temperature. The casein-derivatized support was isolated by
centrifugation, and
washed four times with 9 mL MES buffer.
b. Labeling of Casein immobilized on Eupergit~ C with HRPO
Three mL aldehyde activated HRPO (PREPARATION D) were added to the
casein-derivatized Eupergitm C from section A above, and the mixture incubated
with
rotation for 1 hour at room temperature, and 60 hours at 4°C. The
pellet was isolated by
centrifugation, and washed with two 10 mL aliquots of water. An aqueous
solution of
sodium borohydride (5 mL) was added to the pellet, and the suspension
incubated for 6
SUBSTITUTE SHEET (RULE 26)
21fi~262
WO 9/24306 - PCTIUS94/04098
53
hours at room temperature. The pellet was isolated by centrifugation, and
washed
sequentially with 9 mL aliquots of Acetate, Tris, Acetate, Tris, and MES
buffer.
N. COVALENT ATTACHMENT OF MYOGLOBIN-HItFO TO POLYMERIC
DIALDEHYDE
1. MATERIALS
a. 30 mg horse heart myoglobin dissolved in 2 mL 20 mM sodium bicarbonate
buffer, pH 9.5
b. Polymeric dialdehyde (cellulose dialdehyde, purchased from Sigma Chemical
Co. )
c. Buffers:
i. 50 mM Ethanolamine, pH 9.5
ii. 100 mM Sodium borate, pH 9.0 containing 0.5 M NaCI
iii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCI
iv. 50 mM MES buffer, pH 6.0
v. 100 mM Tris buffer, pH 8.0 containing 0.5 M Nacl
d. 100 mM Formaldehyde
e. 100 mM Sodium cyanoborohydride in water
2. PROCEDURE
a. Covalent Binding of Myoglobin to Polymeric Dialdehyde
Two mL of the myoglobin solution were mixed with 1 mL of a suspension of
polymeric dialdehyde and the suspension was rotated at room temperature
overnight.
The resin was separated by centrifugation and washed twice with 10 mL aliquots
of
water. Nine mL ethanolamine solution were added and the suspension incubated
with
rotation for 1 hour at room temperature. The resin was isolated by
centrifugation, and
washed sequentially with 10 mL aliquots of acetate, Tris, and MES buffer.
Twenty mL
of sodium cyanoborohydride were added. The suspension incubated overnight at
4°C.
The Myoglobin-derivatized polymeric dialdehyde was washed five times with 10
mL
aliquots of water, and twice with MES buffer.
b. Labelling of Myoglobin-Derivatized Beads with HRPO Aldehyde
(PREPARATION D).
Two mL desalted HRPO aldehyde (PREPARATION D) were added to the
resin, and the suspension incubated with rotation at room temperature for 1
hour, and
SUBSTITUTE SHEET (RULE 26)
WO 94/2.1306 _ 2 I 6 0 2 6 ~ PCT/US94/04098
54
overnight a 4°C. The resin was then isolated by centrifugation, washed
twice with 10
mL aliquots of water, suspended in 20 mL sodium cyanoborohydride, and
incubated 60
hours at 4°C. The resin was isolated by centrifugation, washed twice
with 10 mL
aliquots of water, then twice sequentially with four 10 mL aliquots of
acetate/NaCI and
Tris/NaCI buffers. Finally, the resin was washed with 10 mL MES buffer and
stored at
4°C, until used.
O. PREPARATION OF HRPO LABELED CHITIN AND ITS USE TO ASSAY
CHITINASE
1. MATERIALS
a. Cyanogen bromide activated chitin (PREPARATION A)
b. HRPO (Type II from Sigma Chemical Co:, 78 units/mg)
c. Chitinase (EC 3.2.1.14, isolated from Streptomyces grisius and purchased
from Sigma Chemical Co.)
d. Buffers:
i. 500 mM Tris/MES buffer, pH 5.4
ii. Coupling buffer, 100 mM sodium bicarbonate containing 0.5 M NaCI
iii. 100 mM Sodium acetate buffer, pH 4.0, containing 0.5 M NaCI
iv. 100 mM Sodium borate buffer, pH 8.0, containing 0.5 M NaCI
v. 1.0 M Tris buffer, pH 8.0
e. ABTS assay mix: 40 microliters 25 mg/mL ABTS (2,2'-Azino-di[3-
ethylbenzthiazoline sulfonic acid, diammonium salt]; 10 microliters 1 % (v/v)
hydrogen peroxide; 950 microliters water
2. Procedure
a. Preparation of HRPO-Derivatized Chitin
Five hundred mg cyanogen bromide activated chitin (PREPARATION A) were
suspended in 5 mL coupling buffer, 5 mg HRPO were added, and the suspension
mixed
end-over-end for 2 hours at room temperature. The solid material was washed
with
distilled water and coupling buffer. Three mL 1.0 M Tris, pH 8.0 buffer were
added to
the support, and the suspension stirred at room temperature for 2 hours before
being
washed 3 times sequentially with acetate and borate buffers, and finally with
water.
b. Chitinase Assay
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 PCT/US94/04098
$S
Fifty mg HRPO labeled chitin were suspended in 200 microliters MES/Tris
buffer. Fifty microliters of a solution of Chitinase (50 units/mL) were added,
and the
mixture incubated for 4 hours at 37°C. The reaction mixture was
centrifuged to
sediment the chitin, and a 10 microliter aliquot of the supernatant was mined
with 100
microliters of the ABTS assay mix. The chitinase-catalyzed release of chitin-
bound
HRPO was detected by the generation of a green color in the solution.
P. PREPARATION OF CYANOGEN BROMIDE ACTIVATED SEPHAROSE-
CASEIN OR KAPPA-CASEIN-HItPO ALDEHYDE
1. MATERIALS
a. Cyanogen bromide activated Sepharose (PREPARATION A)
b. Casein or Kappa-Casein at 10 mg/mL in coupling buffer (0.1 M sodium
bicarbonate containing 0.5 M NaCI)
c. Buffers and Solutions:
i. 50 mM Ethanolamine, pH 9.5
ii. 100 mM Sodium borate, pH 9.0 containing 0.5 M NaCI
iii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCI
iv. 20 mM MES buffer, pH 5.0
v. 100 mM Tris buffer, pH 8.5 containing 0.5 M NaCI
vi. 100 mM sodium borohydride in water
d. HRPO aldehyde (PREPARATION D)
2. PROCEDURE
Two mL Casein or Kappa-Casein solution were added to one mL cyanogen
bromide activated Sepharose and the suspension incubated for 2 hours with
rotation at
room temperature. Seven mL ethanolamine solution were added, and the mixture
incubated for 1 hour at room temperature. The resin was isolated by
centrifugation, and
washed sequentially with 9 mL aliquots of acetate and Tris buffers containing
sodium
chloride, followed by two washes with 9 mL of MES buffer.
3 mL desalted HRPO aldehyde (PREPARATION D) were added, and the
mixture rotated for 1 hour at room temperature, followed by overnight
incubation at 4°C.
The resin was isolated by centrifugation, and washed twice with 10 mL water.
The resin
was suspended in 10 mL sodium borohydride, and the mixture incubated for 2
hours at
room temperature. Finally, the resin was isolated by centrifugation, and
washed
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 _ ~ PCT/US94/04098
56
sequentially with 9 mL of each of the following buffers containing 0.5 M NaCI:
acetate;
Tris; acetate; and Tris. The resin was finally washed with 9 mL MES buffer,
and stored
in MES buffer at 4°C until used.
Q. COVALENT ATTACHrZENT OF MYOGLOBIN AND BOVINE SERUM
ALBUMIhT TO FINELY PULVERIZED EUPERGIT~ C ACRYLIC BEADS
(Eupergitm C) AND SUBSEQUENT COVALENT LABELING WITH HORSE
RADISH PEROXIDASE (ALDEHYDE METHOD)
Step 1: Covalent Attachment of Myoglobin and Bovine Serum Albumin to Eupergit~
C
Acrylic Beads (Eupergit~ C)
1. MATERIALS
a. Ozirane acrylic beads (Eupergit~ C, 150 micron beads from Sigma Chemical
Co. )
b. Horse heart myoglobin (type IV) or bovine serum albumin (from Sigma
Chemical Co.)
c. Buffers and solutions
i. 1.0 M Potassium Phosphate buffer, pH 7.5 containing 0.1 ~ sodium azide
(w/v)
ii. 1.0 M NaCI
iii. 5 ~ (v/v) Mercaptoethanol, adjusted to pH 8.0 with 0.5 N NaOH
iv. 100 mM Potassium phosphate buffer, pH 7.5
v. 500 mM Potassium phosphate buffer, pH 7.5
vi. 3.5 M Sodium thiocyanate
vii. Phosphate buffered saline (PBS) (0.01 M sodium phosphate, pH 7.2
containing 0.15 M NaCI
d. Distilled water
2. PROCEDURE
One gram of oxirane acrylic beads were ground to a fine powder manually
with a mortar and pestle. Myoglobin or bovine serum albumin (125 mg) were
dissolved
in 5 mL 1.0 M phosphate buffer, pH 7.5 containing sodium azide and added to 1
gram
finely ground Eupergit~ C. The mixture was allowed to incubate without
agitation for 72
hours at room temperature. Using centrifugation, the support was washed three
times
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 _
216 0 2 ~ 2, pCT/US94/04098
S~
with 1.0 M NaCI and five times with 20 mL distilled water. The support was
mined
with 2.5 mL mercaptoethanol previous adjusted to pH 8.0, and the suspension
allowed
to stand overnight at room temperature. The beads were washed 10 times, using
centrifugation, with distilled water and washed sequentially with 50 mL of
each of the
following: 0.5 M potassium phosphate buffer, pH 7.5; 0.1 M potassium phosphate
buffer, pH 7.5; 3.5 ~ M sodium thiocyanate; and finally with large volumes of
phosphate
buffered saline (PBS) (0.01 M sodium phosphate, pH 7.2 containing 0.15 M
NaCI).
Step 2: Covalent Labeling with Horse Radish Peroxidase (ALDEHYDE METHOD)
Horse Radish Perozidase aldehyde (PREPARATION D) was used to label the above
myoglobin or albumin derivatized acrylic beads from step 1 above using
PROCEDURE
F.
R. COUPLING OF SIGMACELL~ 20 MYOGLOB1N TO HItPO WITH
GLUTARALDEHYDE
1. MATERIALS
a. Myoglobin covalently attached to cyanogen bromide activated Sigmacell~ 20
(PREPARATION E)
b. 0.5 M MES buffer, pH 5.0
c. 1 ~ (v/v) glutaraldehyde in water
d. HRPO-hydrazide (from Sigma Chemical Co., 200 units/mg)
e. 100 mM formaldehyde
f. 100 mM sodium cyanoborohydride
g. u.~ tv~ Nac:1
2. PROCEDURES
One mL of glutaraldehyde solution was added to 1 gram of myoglobin
conjugated Sigmacell~ 20, and the suspension rotated at room temperature for
30 minutes
and washed with water. Five mg HRPO hydrazide in 2 mL of 100 mM MES buffer
were added to the washed resin and the suspension rotated for 4 hours at room
temperature. Two hundred microliters of 100 mM formaldehyde solution were
added,
and the mizture allowed to incubate for 30 minutes at room temperature. The
resin was
washed with water, suspended in 5 mL of 100 mM sodium cyanoborohydride, and
SU85TITUTE SHEET (RULE 26)
WO 94/24306 2 ~. 6 p 2 ~ 2 PCT/US94/04098
S8
incubated overnight at 4°C. The resin was then washed with distilled
water and 0.5 M
NaCI and stored as a moist paste until used.
S. PREPARATION OF GUAIAC SOLUTION AND GUAIAC SHEETS
1. Guaiac, Hydrozypropyl Cellulose Solution (i. e. , Guaiac Ink)
150 grams of powdered guaiac are dissolved in 660 mL warm, stirred ethanol.
To the resulting solution 1330 mL of distilled water are added and the
resulting
suspension allowed to cool to room temperature. After two hours, the
supernatant is
decanted and the residual suspension retained.
250 mL of a 10 % (w/w) solution of hydrozypropyl cellulose in ethanol is mined
with an additional 250 mL of ethanol, and the resulting solution is added to
the guaiac
slurry. The mizture is stirred until the guaiac residue has completely
dissolved.
2. Guaiac Sheets
One mL of the above guaiac solution is pipetted onto the polyethylene side of
a 10
inch z 10 inch sheet of a Mylar/polyethylene laminate (7 mils Mylar/3 mL
polyethylene).
The guaiac solution is spread over the surface of the sheets by means of a
standard
wound-wire testing bar, and allowed to dry at room temperature.
T. PREPARATION OF SODIUM PERBORATE SUSPENSION (i.e., SODIUM
PERBORATE INK)
Twenty grams of finely ground solid sodium perborate is mined with a
sufficient
volume of 10 ~ (w/w) solution of hydrozypropyl cellulose solution in anhydrous
alcohol
to produce one liter of suspension.
II. ERPERIMENTS
EXPERIMENT I
This ezperiment involves the release of HRPO from [SEPHAROSE-CASEIN-
HRPO] and [SEPHAROSE-KAPPA-CASEIN-HRPO] by aspartic protease released into
Candida albicans culture.
A. MATERIALS: (CYANOGEN BROMIDE ACTIVATED SEPHAROSE) (HRPO
ALDEHYDE COUPLING METHOD)
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 ~ ~ ~ 0 2 6 2 PCT/U894/04098
59
1. Cyanogen bromide activated Sepharose 4B (PREPARATION A) first
derivatized with covalently bound casein or Kappa-Casein (PREPARATION P)
and subsequently labeled with HRPO aldehyde (PREPARATION D)
2. Aspartic Protease producing Candida albicans (ATCC 28366) culture
propagated and grown in liquid culture as described in Journal of General
Microbiology, (1983) 129:431 - 438.
3. Guaiac layered sheets (PREPARATION S)
4. Buffers and solutions
a. 20 mM hydrogen peroxide
b. 500 mM MES buffer, pH 6.0
B. PROCEDURES
10 mg (wet weight) [Sepharose-Casein-HRPO) conjugate (or the Kappa-Casein
equivalent) were suspended in 300 microliters of either Candida albicans
culture
containing active secreted aspartic protease, or 300 microliters of the same
Candida
albicans culture which had been boiled for 20 minutes to inactivate the
aspartic protease.
The mixture was rotated for 15 minutes at room temperature, and the suspension
centrifuged to sediment the solid conjugate and Candida albicans cells.
Eighty microliters of the clear supernatant were mixed with 10 microliters of
hydrogen peroxide solution and 10 microliters of MES buffer. Twenty
microliters of
each solution were added to the surface of a guaiac layered sheet, and the
sheet examined
for formation of a blue color.
SUBSTITUTE SHEET (RULE 26)
WO 94/2x306 _ PCT/US94/04098
COLOR SCORE: INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
1.5 BLUE COLOR BETWEEN 1.0 AND 2.0 IN INTENSITY
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
C. RESULTS
A dark blue color formed on the guaiac layered sheet in the area to which
Candida albicans treated Sepharose-protein-HRPO supernatant was added. No
color was
seen in the area to which boiled culture treated Sepharose-protein-HRPO
supernatant was
added.
ENZYME SOURCE COLOR SCORE
Candida albicans culture 1.5
Boiled culture 0
D. INTERPRETATION
Active aspartic protease secreted into the growth medium by Candida albicans
cells hydrolyzed Sepharose bound Casein or Kappa-Casein, releasing soluble,
active
HRPO. Centrifugation sedimented Sepharose bound HRPO, leaving only aspartic
protease-solubilized HRPO in solution. The soluble HRPO catalyzed the
oxidation of
guaiac by hydrogen peroxide, producing a blue color. Hence, blue color
formation on
the guaiac layered sheets provides a convenient method for detecting aspartic
protease.
Boiling the culture inactivated aspartic protease secreted into the growth
medium by Candida albicans cells. Hence, Sepharose bound casein or Kappa-
Casein
was not hydrolyzed, and soluble, active HRPO was not released from the
support.
SUBSTITUTE SHEET (RULE 26j
2.60262
WO 94/24306 - PCT/LTS94104098
61
Centrifugation sedimented Sepharose bound HRPO, leaving no aspartic protease-
solubilized HRPO in solution. The oxidation of guaiac by hydrogen peroxide was
not
catalyzed, and a barely detectable blue color formed. Release of HRPO from the
support
was not simply the result of non specific release of HRPO by salts or other
components
present in the growth medium.
EXPE;RnVIT~VT II
This experiment involves the release of HRPO from [EUPERGI'Tm C
ACRYLIC BEADS-CASEIN-HRPO] conjugates and [EUPERGI'I'm C ACRYLIC
BEADS-KAPPA-CASEIN-HRPO] conjugates by aspartic protease released into
Caruiida
albicans culture:
A. MATERIALS: (EUPERGIT~ C ACTIVATED ACRYLIC BEADS) (HRPO
ALDEHYDE COUPLING METHOD)
1. Ozirane acrylic beads (Eupergitm C) were treated with Casein or Kappa-
Casein
(PREPARATION M) and subsequently labeled with HRPO aldehyde
(PREPARATION D)
2. Aspartic Protease producing Candida albicans (ATCC 28366) culture
propagated and grown in liquid culture as described in Journal of General
MicrobioloQV, (1983) 129: 431 - 438.
3. Guaiac layered sheets (PREPARATION S)
4. Buffers and solutions
a. 20 mM hydrogen peroxide
b. 500 mM MES buffer, pH 6.0
B. PROCEDURES
10 mg (wet weight) [Eupergit~ C-Casein-HRPO] conjugate (or the Kappa-
Casein equivalent) were suspended in 300 microliters of either Candida
albicans culture
containing secreted active aspartic protease, or 300 microliters of the same
Candida
albicans culture which had been boiled for 20 minutes to inactivate the
aspartic protease.
The mixture was rotated for 15 minutes at room temperature, and the suspension
centrifuged to sediment the solid conjugate and Candida albicarrs cells.
Eighty microliters of the clear supernatant were mixed with 10 microliters of
hydrogen peroxide solution and 10 microliters of MES buffer. Twenty
microliters of
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 2 ~ 6 0 2 6 ~ PCT/US94l04098
62
each solution were added to surface of a guaiac layered sheet, and the sheet
examined for
formation of a blue color.
COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
1.5 BLUE COLOR BETWEEN 1.0 AND 2.0 IN INTENSITY
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
C. RESULTS
A dark blue color formed on the guaiac layered sheet in the area to which
Candida albicans treated [Eupergit~ C-Protein-HRPO] supernatant was added. No
color
was seen in the area to which boiled culture treated [Eupergitm C-Protein-
HRPO]
supernatant was added.
ENZYME SOURCE COLOR SCORE
Candida albicans culture 1.5
Boiled culture 0
D. INTERPRETATION
Active aspartic protease secreted into the growth medium by Candida albicans
cells hydrolyzed Eupergit~ C bound Casein or Kappa-Casein, releasing soluble,
active
HRPO. Centrifugation sedimented Eupergit~ C bound HRPO, leaving only aspartic
protease-solubilized HRPO in solution. The soluble HRPO catalyzed the
oxidation of
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 216 0 2 6 ~ PCT/US94/04098
63
guaiac by hydrogen peroxide, producing a blue color. Hence, blue color
formation on
the guaiac layered sheets provides a convenient method for detecting aspartic
protease.
Boiling the culture inactivated aspartic protease secreted into the growth
medium by Candida albicans cells. Hence, Eupergit~ C bound casein or Kappa-
Casein
was not hydrolyzed, and soluble, active HRPO was not released from the
support.
Centrifugation sedimented Eupergit~ C bound HRPO, leaving no aspartic protease-
solubilized HRPO in solution. The oxidation of guaiac by hydrogen peroxide was
not
catalyzed, and a barely detectable blue color formed. Release of HRPO from the
support
was not simply the result of non specific release of HRPO by salts or other
components
present in the growth medium.
EXPERIMENT III
This experiment involves the release of HRPO from [SEPHAROSE-
HEMOGLOBIN-HRPO] or [SEPHAROSE=MYOGLOBIN-HRPO] by pepsin and aspartic
protease from Aspergillus saitoi.
A. MATERIALS: (CYANOGEN BROMIDE ACTIVATED SEPHAROSE) (HRPO
ALDEHYDE COUPLING METHOD)
1. Cyanogen bromide activated Sepharose 4B (PREPARATION A) first
derivatized with covalently bound hemoglobin or myoglobin (PREPARATION
E), and subsequently labeled with HRPO aldehyde (PREPARATIONS D and
F~. The conjugate was treated overnight with 100 mM sodium
cyanoborohydride at room temperature and washed with 0.5 M NaCI before
use.
2. Test Solutions: a. Commercially available Aspartic Protease (Type XIII)
from
Aspergillus saitoi (30 mg/mL,0.6 units/mg); b. pepsin (1 mg/mL, 2900
units/mg); and c. Bovine Serum Albumin (BSA) (2 mg/mL in water) (all
purchased from Sigma Chemical Co.)
3. Guaiac impregnated paper in the form of commercially available Hemoccult~
slides (from SmithKline Diagnostics)
4. Buffers and solutions
a. 0.02 ~ (v/v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0
b. 100 mM acetate buffer, pH 4.0
SUBSTITUTE SHEET (RULE 26)
WO 94/2x306 216 Q 2 G ~ PCT/US94/04098
64
B. PROCEDURES
40 mg (wet weight) [Sepharose-Hemoglobin-HRPO] (or the myoglobin
equivalent) conjugate were suspended in 75 microliters acetate buffer, pH 4.0
and 25
microliters of test solution were added. The suspension was incubated at room
temperature for 15 minutes and centrifuged to remove the solid phase
conjugate. Five
microliters of the supernatant were added to a guaiac slide, followed by five
microliters
of hydrogen peroxide solution.
COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE FAINT BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
C. RESULTS
A dark blue color formed on the guaiac impregnated sheet in the area to which
supernatant samples containing Aspartic Protease from Aspergillus saitoi or
pepsin were
added. Only a very faint color was seen in the area to which supernatant
aliquots
containing only BSA alone (2 mg/mL) or buffer were added.
ENZYME SOURCE COLOR SCORE
Aspartic Protease 2.0
Pepsin 2.0
~
BSA +/-
Buffer +/-
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 21 fi 0 2 6 2 PCT/IJS94/04098
D. INTERPRETATION
Active Aspergillus saitoi aspartic protease and pepsin, an aspartic protease
from porcine stomach, hydrolyzed Sepharose bound hemoglobin or myoglobin,
releasing
soluble, active HRPO. Centrifugation sedimented Sepharose bound HRPO, leaving
only
5 enzyme solubilized HRPO in solution. The soluble HRPO catalyzed the
oxidation of
guaiac by hydrogen peroxide, producing a blue color. Hence, blue color
formation on
the guaiac layered sheets provides a convenient method for detecting active
Aspergillus
aspartic protease or porcine pepsin.
Neither BSA nor buffer have aspartic protease activity. Hence, Sepharose
10 bound hemoglobin or myoglobin were not hydrolyzed, and soluble, active HRPO
was not
released from the support. Centrifugation sedimented Sepharose bound HRPO,
leaving
no aspartic protease-solubilized HRPO in solution. The oxidation of guaiac by
hydrogen
peroxide was not catalyzed, and a barely detectable blue color formed.
15 EXPE;~tllVIENT IV
This experiment involves the release of HRPO from [SEPHAROSE-CASEIN-
HRPO] or [SEPHAROSE-KAPPA-CASEIN-HRPO] by pepsin and aspartic protease from
Aspergillus saitoi:
A. MATERIALS: (CYANOGEN BROMIDE ACTIVATED SEPHAROSE) (HRPO
20 HYDRAZIDE COUPLING METHOD)
1. Cyanogen bromide activated Sepharose 4B (PREPARATION A) allowed to
react with Kappa-Casein-HRPO or Casein-HRPO (hydrazide method)
(PREPARATIONS B and C)
2. Test Solutions: a. Commercially available Aspartic Protease from
Aspergillus
25 saitoi (30 mg/mL,0.6 units/mg); b. pepsin (1 mg/mL,2900 units/mg); and c.
Bovine Serum Albumin (BSA)(2 mg/mL in water) (all purchased from Sigma
Chemical Co. )
3. Guaiac impregnated paper in the form of commercially available Hemoccult~
slides
30 4. Buffers and solutions
a. 0.02 ~ (v/v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0
b. 100 mM acetate buffer, pH 4.0
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 _ PCT/US94/04098
B. PROCEDURES
40 mg (wet weight) [Sepharose-Casein-HRPO] (or the Kappa-Casein
equivalent) conjugate were suspended in 75 microliters acetate buffer, pH 4.0
and 25
microliters of test solution were added. The suspension was incubated at room
temperature for 15 minutes and centrifuged to remove the solid phase
conjugate. Five
microliters of the supernatant were added to a guaiac slide, followed by five
microliters
of hydrogen perozide solution.
COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE FAINT BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
C. RESULTS
A dark blue color formed on the guaiac impregnated sheet in the area to which
supernatant samples containing Aspartic Protease from Aspergillus saitoi or
pepsin were
added. Only the faintest color detectable was seen in the area to which
supernatant
aliquots containing only BSA (2.0 mg/mL) or buffer were added.
SUBSTITUTE SHEET (RULE 26)
_ 216~2~2
WO 94/24306 PCT/US94104098
67
ENZYME SOURCE COLOR SCORE
Aspartic Protease2.0
Pepsin 2.0
BSA +/-
Buffer +/-
D. INTERPRETATION
Active Aspergillus saitoi aspartic protease and porcine pepsin hydrolyzed
Sepharose bound Casein or Kappa-Casein, releasing soluble, active HRPO.
Centrifugation sedimented Sepharose bound HRPO, leaving only enzyme
solubilized
HRPO in solution. The soluble HRPO catalyzed the oxidation of guaiac by
hydrogen
peroxide, producing a blue color. Hence, blue color formation on the guaiac
layered
sheets provides a convenient method for detecting aspartic protease or pepsin.
Neither BSA nor buffer have aspartic protease activity. Hence, Sepharose
bound casein or Kappa-Casein were not hydrolyzed, and soluble, active HRPO was
not
released from the support. Centrifugation sedimented Sepharose bound HRPO,
leaving
no aspartic protease-solubilized HRPO in solution. The oxidation of guaiac by
hydrogen
peroxide was not catalyzed, and a barely detectable blue color formed.
EXPE;RnVIENT V
This experiment involves the release of HRPO from [CHITIN-KAPPA-
CASEIN-HRPO] by pepsin and aspartic protease from Aspergillus saitoi.
A. MATERIALS: (CYANOGEN BROMIDE ACTIVATED CHITIN) (HRPO
HYDRAZIDE COUPLING METHOD)
1. Cyanogen bromide activated chitin (PREPARATION A) were allowed to react
with Kappa-Casein-HRPO (hydrazide method) (PREPARATION B and C)
2. Test Solutions: a. Commercially available Aspartic Protease from
Aspergillus
scritoi (30 mg/mL,0.6 units/mg); b. pepsin (1 mg/mL,2900 units/mg); and c.
Bovine Serum Albumin (BSA)(2 mg/mL in water) (all purchased from Sigma
Chemical Co. )
SUBSTITUTE SHEET (RULE 26)
WO 94/2.1306 _ ~ ~ PCT/US94/04098
68
3. Guaiac impregnated paper in the form of commercially available Hemoccult~
slides (from SmithKline Diagnostics) .
4. Buffers and solutions '
a. 0.02 ~ (v/v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0
S b. 100 mM acetate buffer, pH 4.0
B. PROCEDURES
40 mg (wet weight) [Chitin-Kappa-Casein-HRPO] conjugate were suspended in
75 microliters acetate buffer, pH 4.0 and 25 microliters of test solution were
added. The
suspension was incubated at room temperature for 15 minutes and centrifuged to
remove
the solid phase conjugate. Five ~l of the supernatant were added to a guaiac
slide,
followed by five microliters of hydrogen peroxide solution.
COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE FAINT BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
C. RESULTS
A dark blue color formed on the guaiac impregnated sheet in the area to which
supernatant samples containing Aspartic Protease from Aspergillus satoi or
pepsin were
added. Only a barely detectable blue color was seen in the area to which
supernatant
aliquots after BSA (2.0 mg/mL) or buffer were added.
SUBSTIME SHEET (RULE 26)
msozsz
WO 94/24306 PCTIUS94/04098
69
ENZYME SOURCE COLOR SCORE
Aspartic Protease 2.0
Pepsin 2.0
BSA +/-
Buffer +/-
D. INTERPRETATION
Active Aspergillus saitoi aspartic protease and porcine pepsin hydrolyzed
Sigmacellm 20 bound hemoglobin or myoglobin, releasing soluble, active HRPO.
Centrifugation sedimented Sigmacell~ 20 bound HRPO, leaving only enzyme
solubilized
HRPO in solution. The soluble HRPO catalyzed the oxidation of guaiac by
hydrogen
peroxide, producing a blue color. Hence, blue color formation on the guaiac
layered
sheets provides a convenient method for detecting aspartic protease or pepsin.
Neither BSA nor buffer have aspartic protease activity. Hence, Sigmacell~ 20
bound hemoglobin or myoglobin were not hydrolyzed, and soluble, active HRPO
was not
released from the support. Centrifugation sedimented Sigmacell~ 20 bound HRPO,
leaving no aspartic protease-solubilized HRPO in solution. The oxidation of
guaiac by
hydrogen peroxide was not catalyzed, and a barely detectable blue color
formed.
EXPER>aV»NT VI
This experiment involves the release of HRPO from [SIGMACELL~ 20-
HEMOGLOBIN-HRPO] or [SIGMACELLm 20-MYOGLOBIN-HRPO] by pepsin and
aspartic protease from Aspergillus saitoi.
A. MATERIALS: (CYANOGEN BROMIDE ACTIVATED SIGMACELL~ 20)
(HRPO ALDEHYDE COUPLING METHOD)
1. Cyanogen bromide activated Sigmacell~ 20 (PREPARATION A) first
derivatized with covalently bound hemoglobin or myoglobin (PREPARATION
E), and subsequently labeled with HRPO aldehyde (PREPARATIONS D-F~.
The conjugate was treated overnight with 100 mM sodium cyanoborohydride
at room temperature and washed with 0.5 M NaCI before use.
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 ~ ~ PCT/US94/04098
2. Test Solutions: a. Commercially available Aspartic Protease from
Aspergillus
saitoi (30 mg/mL,0.6 units/mg); b. pepsin (1 mg/mL,2900 units/mg); and c.
Bovine Serum Albumin (BSA)(2 mg/mL in water) (all purchased from Sigma
Chemical Co. )
5 3. Guaiac impregnated paper in the form of commercially available Hemoccult~
slides
4. Buffers and solutions
a. 0.02 ~ (v/v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0
b. 100 mM acetate buffer, pH 4.0
10 B. PROCEDURES
40 mg (wet weight) [Sigmacell~ 20-Hemoglobin-HRPO) (or the myoglobin
equivalent) conjugate were suspended in 75 microliters acetate buffer, pH 4.0
and 25
microliters of test solution were added. The suspension was incubated at room
temperature for 15 minutes and centrifuged to remove the solid phase
conjugate. Five
15 microliters of the supernatant were added to a guaiac slide, followed by
five microliters
of hydrogen peroxide solution.
COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE FAINT BLUE COLOR
20 0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
25 C. RESULTS
A dark blue color formed on the guaiac impregnated sheet in the area to which
supernatant samples containing Aspartic Protease from Aspergillus saitoi or
pepsin were
added. Only a very faint color was seen in the area to which supernatant
aliquots after
BSA (2.0 mg/mL) or buffer were added.
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 ~ . PCT/LTS94/04098
71
ENZYME SOURCE COLOR SCORE
Aspartic Protease2.0
Pepsin 2.0
BSA +/-
Buffer +/-
D. INTERPRETATION
Active Aspergillus saitoi aspartic protease and porcine pepsin hydrolyzed
Sigmacell~ 20 bound hemoglobin or myoglobin, releasing soluble, active HRPO.
Centrifugation sedimented Sigmacell~ 20 bound HRPO, leaving only enzyme
solubilized
HRPO in solution. The soluble HRPO catalyzed the oxidation of guaiac by
hydrogen
peroxide, producing a blue color. Hence, blue color formation on the guaiac
layered
sheets provides a convenient method for detecting aspartic protease or pepsin.
Neither BSA nor buffer have aspartic protease activity. Hence, Sigmacell~ 20
bound hemoglobin or myoglobin were not hydrolyzed, and soluble, active HRPO
was not
released from the support. Centrifugation sedimented Sigmacell~ 20 bound HRPO,
leaving no aspartic protease-solubilized HRPO in solution. The oxidation of
guaiac by
hydrogen peroxide was not catalyzed, and a barely detectable blue color
formed.
EXPE;RIIVVIENT VII
This experiment is an example of the release of HRPO from [SIGMACELL~
20-KAPPA-CASEIN-HRPO] by pepsin and aspartic protease from Aspergillus saitoi.
A. MATERIALS: (CYANOGEN BROMIDE ACTIVATED SIGMACELL~ 20)
(HRPO HYDRAZIDE COUPLING METHOD)
1. Cyanogen bromide activated Sigmacell~ 20 (PREPARATION A) allowed to
react with Kappa-Casein-HRPO (hydrazide method) (PREPARATIONS B and
C)
2. Test Solutions: a. Commercially available Aspartic Protease from
Aspergillus
saitoi (30 mg/mL,0.6 units/mg); b. pepsin (1 mg/mL,2900 units/mg); and c.
Bovine Serum Albumin (BSA)(2 mg/mL in water) (all purchased from Sigma
f hPmiral ('_n_1
SUBSTITUTE SHEET (RULE 2S)
WO 94/24306 216 ~ ~ ~ ~ PCT/iJS94/04098
72
3. Guaiac impregnated paper in the form of commercially available Hemoccult~
slides
4. Buffers and solutions
a. 0.02 ~O (v/v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0
b. 100 mM acetate buffer, pH 4.0
B. PROCEDURES
40 mg (wet weight) [Sigmacell~ 20-Kappa-Casein-HRPO] conjugate were
suspended in 75 microliters acetate buffer, pH 4.0 and 25 microliters of test
solution
were added. The suspension was incubated at room temperature for 15 minutes
and
centrifuged to remove the solid phase conjugate. Five microliters of the
supernatant were
added to a guaiac slide, followed by five microliters of hydrogen peroxide
solution.
COLOR SCORE INTERPRETATION
'
i
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE FAINT BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
C. RESULTS
A dark blue color formed on the guaiac impregnated sheet in the area to which
supernatant samples containing Aspartic Protease from Aspergillus saitoi or
pepsin were
added. Only a very faint color was seen in the area to which supernatant
aliquots after
BSA (2.0 mg/mL) or buffer were added.
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 _ ~ PCT/US94104098
73
ENZYME SOURCE COLOR SCORE
Aspartic Protease2.0
Pepsin 2.0
BSA +/-
Buffer +/-
D. INTERPRETATION
Active Aspergillus saitoi aspartic protease and porcine pepsin hydrolyzed
Sigmacellm 20 bound Kappa-Casein, releasing soluble, active HRPO.
Centrifugation
sedimented Sigmacell~ 20 bound HRPO, leaving only enzyme solubilized HRPO in
solution. The soluble HRPO catalyzed the oxidation of guaiac by hydrogen
peroxide,
producing a blue color. Hence, blue color formation on the guaiac layered
sheets
provides a convenient method for detecting aspartic protease or pepsin.
Neither BSA nor buffer have aspartic protease activity. Hence, Sigmacell~ 20
bound Kappa-Casein were not hydrolyzed, and soluble, active HRPO was not
released
from the support. Centrifugation sedimented Sigmacell~ 20 bound HRPO, leaving
no
aspartic protease-solubilized HRPO in solution. The oxidation of guaiac by
hydrogen
peroxide was not catalyzed, and a barely detectable blue color formed.
ERPERIMENT VIII
This example is an example of the release of HRPO from [SIGMACELL~ 20-
KAPPA-CASEIN-HRPO] (HYDRAZIDE METHOD) or [SIGMACELL~ 20-
MYOGLOBIN/HEMOGLOBIN-HRPO] (ALDEHYDE METHOD) by aspartic protease
from Aspergillus saitoi or Pepsin.
A. MATERIALS: (CYANOGEN BROMIDE ACTIVATED SIGMACELL~ 20)
1. Cyanogen bromide activated Sigmacellm 20 (PREPARATION A) were
derivatized with Kappa-Casein and HRPO (hydrazide method)
(PREPARATION B). Alternatively, cyanogen bromide activated Sigmacell~
20 were derivatized with hemoglobin or myoglobin (PREPARATION E) and
then coupled to HRPO aldehyde (PREPARATION D)
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 216 0 2 6 ~ pCT/iJS94/04098
74
2. Test Solutions: a. Commercially available Aspartic Protease from
Aspergillus
saitoi (30 mg/mL,0.6 units/mg); b. pepsin (1 mg/mL,2900 units/mg); and c.
Bovine Serum Albumin (BSA)(2 mg/mL in water) (all purchased from Sigma
Chemical Co.)
3. Guaiac impregnated paper in the form of commercially available Hemoccult~
slides
4. Buffers and solutions
a. 0.02 % (v/v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0
b. 100 mM acetate buffer, pH 4.0
B. PROCEDURES
40 mg (wet weight) [Sigmacell~ 20-Kappa-Casein-HRPO] conjugate were
suspended in 75 microliters acetate buffer, pH 4.0 and 25 microliters of test
solution
were added. The suspension was incubated at room temperature for 15 minutes
and
centrifuged to remove the solid phase conjugate. Five microliters of the
supernatant were
added to a guaiac slide, followed by five microliters of hydrogen peroxide
solution.
COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE FAINT BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
C. RESULTS
A dark blue color formed on the guaiac impregnated sheet in the area to which
supernatant samples containing aspartic protease from Aspergillus saitoi or
pepsin were
added. Only a very faint color was seen in the area to which supernatant
aliquots
containing BSA (2.0 mg/mL) or buffer were added.
SUBSTITUTE SHEET (RULE 26)
2160262
WO 94/24306 ' PCT/US94/04098
7S
ENZYME SOURCE COLOR SCORE
Aspartic Protease 2.0
Pepsin ~ 2.0
BSA +/-
Buffer +/-
D. INTERPRETATION
Active Aspergillus saitoi aspartic protease and porcine pepsin hydrolyzed
Sigmacell~ 20 bound Kappa-Casein, releasing soluble, active HRPO.
Centrifugation
sedimented Sigmacell~ 20 bound I~RPO, leaving only enzyme solubilized HRPO in
solution. The soluble HRPO catalyzed the oxidation of guaiac by hydrogen
peroxide,
producing a blue color. Hence, blue color formation on the guaiac layered
sheets
provides a convenient method for detecting aspartic protease or pepsin.
Neither BSA nor buffer have aspartic protease activity. Hence, Sigmacell~ 20
bound Kappa-Casein were not hydrolyzed, and soluble, active HRPO was not
released
from the support. Centrifugation sedimented Sigmacell~ 20 bound HRPO, leaving
no
aspartic protease-solubilized HRPO in solution: The oxidation of guaiac by
hydrogen
peroxide was not catalyzed, and a barely detectable blue color formed.
.
EXPERIMENT IR
This experiment is an example of the release of HRPO from [SIGMACELL~
20-HEMOGLOBIN-HRPO) or [SIGMACELL~ 20-MYOGLOBIN-HRPO] by aspartic
protease released into Candida albicans culture.
A. MATERIALS: (ALDEHYDE ACTIVATED SIGMACELL~ 20) (HRPO
ALDEHYDE COUPLING METHOD)
1. Aldehyde activated Sigmacellm 20 (PREPARATION G) was first derivatized
with covalently bound hemoglobin or myoglobin (PREPARATION H) and
subsequently labeled with HRPO aldehyde (PREPARATIONS D AND F~
SUBSTITUTE SHEET (RULE 26j
PCT/US94104098
WO 94124306 216 0 2 6 2
76
2. Aspartic Protease producing Candida albicans (ATCC 28366) culture
propagated and grown in liquid culture as described in Journal of General
Microbiology, (1983) 129: 431 - 438.
3. Guaiac layered sheets (PREPARATION S)
4. Buffers and solutions
a. 20 mM hydrogen peroxide
b. 500 mM MES buffer, pH 6.0
B. PROCEDURES
mg (wet weight) [Sigmacell~ 20-Protein-HRPO] conjugate were suspended
10 in 300 microliters of either Candida albicans culture containing secreted
aspartic
protease, or 300 microliters of the same Candida albicans culture which had
been boiled
for 20 minutes to inactivate the aspartic protease. The mixture was rotated
for 15
minutes at room temperature, and the suspension centrifuged to sediment the
solid
conjugate and Candida albicans cells.
Eighty microliters of the clear supernatant were mixed with 10 microliters of
hydrogen peroxide solution and 10 microliters of MES buffer. Twenty
microliters of
each solution were added to surface of a guaiac layered sheet, and the sheet
examined for
formation of a blue color.
COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/_ POSSIBLE FAINT BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
1.5 BLUE COLOR BETWEEN 1.0 AND 2.0 IN INTENSITY
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
SUBSTITUTE SHEET (RULE 26)
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C. RESULTS
A dark blue color formed on the guaiac layered sheet in the area to which
Candida albicans treated Sepharose-protein-HRPO was added. No color was seen
in the
area to which boiled-culture treated Sepharose-protein-HRPO was added.
ENZYME SOURCE COLOR SCORE
Candida Albicarrs culture 1.5
Boiled culture 0
D. INTERPRETATION
Aspartic protease secreted into the growth medium by Candida albicans cells
hydrolyzed Sigmacell~ 20 bound protein, releasing soluble, active HRPO.
Centrifugation
sedimented Sepharose bound HRPO, leaving only aspartic protease-solubilized
HRPO in
solution. The soluble HRPO catalyzed the oxidation of guaiac by hydrogen
peroxide,
producing a blue color. Hence, blue color formation on the guaiac layered
sheets
provides a convenient method for detecting aspartic protease.
Boiling the culture inactivated aspartic protease secreted into the growth
medium by Candida albicans cells. Hence, Sigmacell~ 20 bound protein was not
hydrolyzed, and soluble, active HRPO was not released from the support.
Centrifugation
sedimented Sigmacellm bound HRPO, leaving no aspartic protease-solubilized
HRPO in
solution. The oxidation of guaiac by hydrogen peroxide was not catalyzed, and
a barely
detectable blue color formed. Release of HRPO from the support was not simply
the
result of non-specific release of HRPO by salts or other components present in
the
growth medium.
ERPEItnVIENT R
This example is an example of the release of HRPO from [POLYMERIC
DIALDEHYDE-KAPPA-CASEIN-HRPO) by pepsin and aspartic protease from
Aspergillus saitoi.
A. MATERIALS: (POLYMERIC DIALDEHYDE) (HRPO HYDRAZIDE
COUPLING METHOD)
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78
1. Commercial polymeric dialdehyde from Sigma Chemical Co. allowed to react
with Kappa-Casein-HRPO (hydrazide method) (PREPARATIONS B and L)
2. Test Solutions: a. Commercially available Aspartic Protease from
Aspergillus
saitoi (30 mg/mL, 0.6 units/mg); b. pepsin (1 mg/mL, 2900 units/mg); and c.
Bovine Serum Albumin (BSA)(2 mg/mL in water) (all purchased from Sigma
Chemical Co. )
3. Guaiac impregnated paper in the form of commercially available Hemoccult~
slides
4. Buffers and solutions
a. 0.02 °b (v/v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0
b. 100 mM acetate buffer, pH 4.0
B. PROCEDURES
40 mg (wet weight) [POLYMERIC DIALDEHYDE-KAPPA-CASEIN-HRPO]
conjugate were suspended in 75 microliters acetate buffer, pH 4.0 and 25
microliters of
test solution were added. The suspension was incubated at room temperature for
15
minutes and centrifuged to remove the solid phase conjugate. Five microliters
of the
supernatant were added to a guaiac slide, followed by five microliters of
hydrogen
peroxide solution.
COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE FAINT BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
1.5 BLUE COLOR BETWEEN 1.0 AND 2.0 IN INTENSITY
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
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C. RESULTS
A dark blue color formed on the guaiac impregnated sheet in the area to which
supernatant samples containing Aspartic Protease from Aspergillus saitoi or
pepsin were
added. Only a very faint color was seen in the area to which supernatant
aliquots after
S BSA (2.0 mg/mL) or buffer were added.
ENZYME SOURCE COLOR SCORE
Aspartic Protease 2.0
Pepsin 2.0
BSA +/-
Buffer +/_
D. INTERPRETATION
Active Aspergillus saitoi aspartic protease and porcine pepsin hydrolyzed
polymeric dialdehyde bound Kappa-Casein, releasing soluble, active HRPO.
Centrifugation sedimented polymeric dialdehyde bound HRPO, leaving only enzyme
solubilized HRPO in solution. The soluble HRPO catalyzed the oxidation of
guaiac by
hydrogen peroxide, producing a blue color. Hence, blue color formation on the
guaiac
layered sheets provides a convenient method for detecting aspartic protease or
pepsin.
Neither BSA nor buffer have aspartic protease activity. Hence, polymeric
dialdehyde bound Kappa-Casein were not hydrolyzed, and soluble, active HRPO
was not
released from the support. Centrifugation sedimented polymeric dialdehyde
bound
HRPO, leaving no aspartic protease-solubilized HRPO in solution. The oxidation
of
guaiac by hydrogen peroxide was not catalyzed, and a barely detectable blue
color
formed.
EXPF;R)(MT.NT XI
This experiment involves the release of HRPO from [POLYMERIC
DIALDEHYDE-MYOGLOBIN-HRPO] by aspartic protease from Candida albicans.
A. MATERIALS: (POLYMERIC DIALDEHYDE)(HRPO ALDEHYDE
COUPLING METHOD)
SUB~fITUTE SHEET (RULE 26)
PCT/US94/04098
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1. Commercial polymeric dialdehyde from Sigma Chemical Co. allowed to react
with myoglobin (PREPARATION N) and subsequently with HRPO aldehyde
(PREPARATION N):
2. Aspartic Protease producing Candida albicans (ATCC 28366) culture
5 propagated and grown in liquid culture as described in Journal of General
Microbiology, (1983) 129: 431 - 438.
3. Guaiac layered sheets (PREPARATION S)
4. Buffers and solutions
1. 20 mM hydrogen peroxide
10 2. 500 mM MES buffer, pH 6.0
B. PROCEDURES
10 mg (wet weight) [Polymeric Dialdehyde-Myoglobin-HRI'O] conjugate were
suspended in 300 microliters of either Candida albicans culture containing
secreted
aspartic protease, or 300 microliters of the 'same Candida albicans culture
which had
15 been boiled for 20 minutes to inactivate the aspartic protease. The mixture
was rotated
for 20 minutes at room temperature, and the suspension centrifuged to sediment
the solid
conjugate and Candida albicans cells.
Eighty microliters of the clear supernatant were mixed with 10 microliters of
hydrogen peroxide solution and 10 microliters of MES buffer. Twenty
microliters of
20 each solution were added to surface of a guaiac layered sheet, and the
sheet examined for
formation of a blue color.
SUBSTITUTE SHEET (RULE 26)
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COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE FAINT BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
1.5 BLUE COLOR BETWEEN 1.0 AND 2.0 IN INTENSITY
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
C. RESULTS
A dark blue color formed on the guaiac layered sheet in the area to which
Candida albicans treated polymeric dialdehyde-myoglobin-HRPO was added. No
color
was seen in the area to which boiled culture ueated dialdehyde-myoglobin-HRPO
was
added.
ENZYME SOURCE COLOR SCORE
Candida albicans culture 1.5
Boiled culture 0
D. INTERPRETATION
Active aspartic protease secreted into the growth medium by Candida albicans
cells hydrolyzed polymeric dialdehyde-myoglobin, releasing soluble, active
HRPO.
Centrifugation sedimented polymeric dialdehyde bound HRPO, leaving only
aspartic
protease-solubilized HRPO in solution. The soluble HRPO catalyzed the
oxidation of
guaiac by hydrogen peroxide, producing a blue color. Hence, blue color
formation on
the guaiac layered sheets provides a convenient method for detecting aspartic
protease.
Boiling the culture inactivated aspartic protease secreted into the growth
medium by Candida albicans cells. Hence, polymeric dialdehyde bound myoglobin
was
SUBSTITUTE SHEET (RULE 26j
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82
not hydrolyzed, and soluble, active HRPO was not released fmm the support.
Centrifugation sedimented polymeric dialdehyde bound HRPO, leaving no aspartic
protease-solubilized HRPO in solution. The oxidation of guaiac by hydrogen
peroxide
was not catalyzed, and a barely detectable blue color formed. Release of HRPO
from the
support was not simply the result of non specific release of HRPO by salts or
other
components present in the growth medium.
EXPERnViENT XII
This experiment involves the release of HLtPO from [SEPHAROSE 6 MB-
KAPPA-CASEIN-HRPO] by vaginal fluid specimens:
1. Normal vaginal fluid
2. Normal vaginal fluid to which Candida albicans culture had been added
3. Vaginal fluid from women with clinically diagnosed wlvovaginal
c~ndidiasis
A. MATERIALS: (COMMERCIAL CYANOGEN BROMIDE ACTIVATED
SEPHAROSE 6 MB) (HRPO HYDRAZIDE COUPLING METHOD)
1. Sepharose 6 MB-Kappa-Casein-HRPO conjugate (PREPARATION C)
2. Vaginal fluid samples obtained on standard Dacron swabs. The specimens
were frozen until immediately prior to use. The specimens were thawed, and
centrifuged in specially adapted tubes to permit extraction of undiluted
vaginal
fluid from the swab. The entire specimen from each swab was tested. Where
necxssary, distilled water was added to the specimen to produce a final volume
of 75 microliters.
Where indicated, 25 microliters of a C~rdida albicans culture (See, e.g.,
Experiment I) were added to vaginal fluid specimens obtained from women
without clinical vulvovaginal candidiasis.
3. Guaiac impregnated filter paper (commercial Hemoccultm test slides)
4. Buffers and solutions
a. 6 mM hydrogen peroxide
b. 250 mM glycylglycine buffer, pH 3.0
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83
B. PROCEDURES
30 mg (wet weight) [Sepharose-Kappa-Casein-HRPO] conjugate were
suspended in 75 microliters of treated or untreated vaginal fluid. The
suspensions were
incubated at room temperature for 15 minutes, and the suspension centrifuged
to
sediment the solid conjugate and other debris.
Five microliters of the clear supernatants were added to the guaiac-
impregnated paper, followed by 5 microliters of hydrogen peroxide solution,
and the
sheets were examined for formation of a blue color.
COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE FAINT BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
C. RESULTS
A dark blue color formed on the guaiac impregnated paper in the area to
which vaginal fluid supernatant from women infected with wlvovaginal
candidiasis was
added. No color was formed in the areas of the guaiac impregnated paper to
which
vaginal fluid supernatant from control women was added. A strong blue color
also
formed on the guaiac impregnated paper in the area to which vaginal fluid
supernatant
from normal women which had been supplemented with medium from a growing
Candida albicar~.r culture was added.
SUBSTIME SHEET (RULE 26)
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84
SPECIMEN NO. DIAGNOSIS COLOR SCORE
7757 CANDIDIASIS 2.0
7701 CANDIDIASIS 2.0
7749 CANDIDIASIS 2.0
7760 CANDIDIASIS 2.0
7753 CANDIDIASIS 2.0
7795 CANDIDIASIS 2.0
7779 CANDIDIASIS 2.0
7799 CANDIDIASIS 2.0
7768 NORMAL 1.0
7770 NORMAL 0.0
7744 NORMAL 0.0
7745 NORMAL 0.0
7750 NORMAL 0.0
7761 NORMAL 0.0
0001 NORMAL 0.0
0001 NORMAL + Candida culture2.0
7747 NORMAL + Candida culture2.0
7748 NORMAL + Candida culture1.0
7750 NORMAL + Candida culture2.0
7756 NORMAL + Candida culture2.0
SUBSTITUTE SHEET (RULE 26)
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8S
D. INTERPRETATION
Vaginal fluid from women with proven clinical wlvovaginal candidiasis
contained aspartic protease which hydrolyzed Sepharose 6 MB-bound Kappa-
Casein, at
pH 3.0 in 15 minutes at room temperature, releasing soluble, active HRPO.
Centrifugation sedimented Sepharose 6 MB-bound HRPO, leaving only released,
solubilized HRPO in solution. The soluble HRPO catalyzed oxidation of the
commercial
Hemoccult~ slides, producing a blue color. Hence, blue color formation on the
Hemoccult~ slides provides a rapid and convenient method for detecting Candida-
derived
aspartic protease in vaginal fluid, and hence, wlvovaginal candidiasis.
Vaginal fluid from normal women without wlvovaginal candidiasis did not
contain aspartic protease, and failed to hydrolyze Sepharose 6 MB-bound Kappa-
casein,
at pH 3.0 in 15 minutes at room temperature, and failed to release soluble,
active HRPO.
Centrifugation sedimented Sepharose 6 MB-bound HRPO, leaving no released,
solubilized HRPO in solution. Lacking the HRPO catalyst, no oxidation of the
commercial Hemoccultm slides occurred and no blue color was produced. Hence,
lack of
a blue color formation on the Hemoccultm slides provides a rapid and
convenient method
for detecting normal women lacking Candida-derived aspartic protease in
vaginal fluid
and, hence, women who did not have wlvovaginal candidiasis.
Finally, when medium from a Candida albicans culture was added to vaginal
fluid from normal women without wlvovaginal candidiasis hydrolysis of
Sepharose 6
MB-bound Kappa-Casein occurred at pH 3.0 in 15 minutes at room temperature,
and
soluble, active HRPO was released. Centrifugation sedimented Sepharose 6 MB-
bound
HRPO, leaving released, solubilized HRPO in solution. The soluble HRPO
catalyzed
oxidation of commercial Hemoccult~ slides by hydrogen peroxide and produced a
blue
color. Hence, blue color formation on the Hemoccultm slides demonstrates that
aspartic
protease released into growth medium by Candida albicans cells can readily be
detected
rapidly and conveniently even when added to vaginal fluid from normal women.
ERPERIMENT RI1Q
This experiment involves the release of HRPO from [EUPERGIT'm C-
MYOGLOBIN-HRPO] and [SIGMACELL~ 20-MYOGLOBIN-HRPO] by vaginal fluid
specimens.
1. Normal vaginal fluid
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86
2. Vaginal fluid from women with clinically diagnosed vulvovaginal candidiasis
A. MATERIALS: (CYANOGEN BROMIDE ACTIVATED SIGMACELL~ 20-
MYOGLOBIN-HRPO--PREPARATIONS A AND I) AND (EUPERGI'T~ C-
MYOGLOBIN-HRPO--PREPARATION Q) (HRPO ALDEHYDE COUPLING
METHOD)
1. Finely ground oxirane acrylic beads coupled to Myoglobin-HRPO Aldehyde
(PREPARATION Q)
2. Sigmacell~ 20 (cyanogen bromide activated, PREPARATION A) coupled to
Myoglobin-HRPO Aldehyde (PREPARATION I)
3. Vaginal fluid samples obtained on standard Dacron swabs. The specimens
were frozen until immediately prior to use. The specimens were thawed, and
centrifuged in specially adapted tubes to permit extraction of undiluted
vaginal
fluid from the swab. The entire specimen from each swab was tested.
4. Guaiac impregnated filter paper (commercial Hemoccult~ test slides)
5. Buffers and solutions
a. 6 mM hydrogen peroxide
b. 250 mM glycylglycine buffer, pH 3.0
c. 1.0 M acetate buffer, pH 4.0
B. PROCEDURES
20 mg (wet weight) of Eupergit~-bound myoglobin-HRPO conjugate were
suspended in the undiluted vaginal fluid and 10 microliters of 1 M acetate
buffer. The
suspensions were incubated at room temperature for 10 minutes, and the
suspension
centrifuged to sediment the solid conjugate and other debris.
Five microliters of the clear supernatant were added to the guaiac-impregnated
paper, followed by 5 microliters of hydrogen peroxide solution, and the sheets
were
examined for formation of a blue color.
20 mg (wet weight) of Sigmacell~ 20-bound myoglobin-HRPO conjugate were
suspended in undiluted vaginal fluid and 10 microliters of 250 mM acetate
buffer. The
suspensions were incubated at room temperature for 15 minutes, and the
suspension
centrifuged to sediment the solid conjugate and other debris.
SUBSTITUTE SHEET (RULE 26)
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WO 94/24306 PCT/US94/04098
g7
Five microliters of the clear supernatant were added to the guaiac-impregnated
paper, followed by 5 microliters of hydrogen peroxide solution, and the sheets
were
examined for formation of a blue color.
COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE FAINT BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
C. RESULTS
A dark blue color formed on the guaiac impregnated paper in the area to
which vaginal fluid from women infected with wlvovaginal candidiasis was
added. No
color was formed in the areas of the guaiac impregnated paper to which vaginal
fluid
from control was added.
SUBSTITUTE SHEET (RULE 26)
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88
SPECIMEN NO. SCORE DIAGNOSIS SUBSTRATE COLOR
7823 CANDIDIASIS Sigmacell~ 2.0
7824 CANDIDIASIS Sigmacell~ 2.0
7844 CANDIDIASIS Sigmacell~ 2.0
7848 CANDIDIASIS Sigmacell~ 2.0
7820 CONTROL Sigmacell~ 0.0
7821 CONTROL Sigmacell~ 0.0
7826 CONTROL Sigmacell~ 0.0
. 7831 CONTROL Sigmacell~ 0.0
7891 CANDIDIASIS EUPERGIT~ C 2.0
7921 CANDIDIASIS EUPERGI'I'm 2.0
C
7914 CONTROL EUPERGI'I'm +/-
C
7915 CONTROL EUPERGI'I'~ 0.0
C
D. INTERPRETATION
Active aspartic protease secreted into the vaginal fluid of women with
clinical
candidiasis by Candida albicans cells hydrolyzed both Sigmacell~ 20-bound
protein and
Eupergitm-bound protein, releasing soluble, active HRPO. Centrifugation
sedimented
polymer-bound HRPO, leaving only aspartic protease-solubilized HRPO in
solution.
The soluble HRPO catalyzed the oxidation of guaiac by hydrogen peroxide,
producing a
blue color. Hence, blue color formation on the guaiac-impregnated paper
provides a
convenient and effective method for detecting active aspartic protease in
vaginal fluid,
and thereby, wlvovaginal candidiasis.
Active aspartic protease is not found in the vaginal fluid of control women,
i.e., those without clinical candidiasis. Hence, vaginal fluid specimens from
control
women failed to hydrolyze polymer-bound protein, and failed to release
soluble, active
HRPn from either suuvort. Centrifugation sedimented polymer bound HRPO,
leaving no
SUBSTITUTE SHEET (RULE 26)
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aspartic protease-solubilized HRPO in solution. Lacking HRPO, the supernatant
from
control specimens failed to catalyze the oxidation of guaiac by hydrogen
peroxide, and
failed to produce a blue color. Hence, failure to form a blue color on the
guaiac- '
impregnated paper provides a convenient method for identifying women who were
not
infected with wlvovaginal Candida albicans.
ERPE~RnVIENT XIV
This experiment involves the differential hydrolytic activity of several
microbial proteases on [Sigmacellm-Myoglobin-HRPO].
A. MATERIALS
1. [Sigmacell~-Myoglobin-HRPO] (PREPARATIONS E and F).
2. Aspartic protease producing Candida albicans (ATCC 28366) culture.
3. Trichomonas vaginalis (ATCC 3001) culture.
4. Mobiluncus curtisii cell suspension (ATCC 35241) in saline solution.
5. Buffers and solutions.
a. 0.02 ~ hydrogen peroxide solution
b. Potassium phosphate buffer pH 7.5, 300 mM
c. Sodium acetate buffer pH 4.0, 100 mM.
6. Guaiac impregnated paper (commercial Hemoccult~ slides).
B. PROCEDURE.
Twenty milligrams of substrate [Sigmacellm-Myoglobin-HRPO] were
suspended in 75 microliters of the appropriate buffer (see table below),
followed by
addition of 25 microliters of cell culture/suspension. The reaction was
incubated between
10-30 mins after which the sample was centrifuged to remove solid phase
conjugated
substrate and cell debris. Five microliters of the reaction supernatant were
added to the
guaiac slide and developed with 5 microliters of hydrogen peroxide solution.
The reaction
conditions and color development are reported in the table that follows.
SUBSTITUTE SHEET (RULE 26)
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COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE FAINT BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
C. RESULTS
No color formed when the reaction supernatant from tubes incubated with
buffer at pH 4.0 or pH 7.5 for 30 minutes was added to guaiac slides and
developed with
hydrogen perozide. A similar result was seen (i.e., no color formation) with
boiled
cultures from Candida albicans (pH 4.0 and 7.5), Trichomonas vaginalis (pH 4.0
and
7.5) or Mobiluncus curtisii (pH 4.0 and 7.5). Candida albicans culture
produced a
strong blue color after a 10 minute incubation at pH 4.0, but not at pH 7.5
even after a
30 minute incubation. Trichomonas vaginalis produced a strong blue color after
a 30
minute incubation at pH 7.5, but only a barely detectable color after a 30
minute
incubation at pH 4Ø Mobiluncus Curtisii culture produced no blue color in 30
minutes
at either pH 4.0 or 7.5.
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Enryme Source
(Cell Cultures) pH of IncubationTime of incubationColor Score
Candida albicans 4.0 10 min 2.0
Candida albicarts 7.5 30 min 0.0
Boiled Candida albicans4.0 30 min 0.0
Boiled Candida albicarrs7.5 30 min 0.0
Mobiluncus curtisii 4.0 30 min 0.0
Mobiluncus curtisii 7.5 30 min 0.0
Boiled Mobiluncus 4.0 30 min 0.0
curtisii
Boiled Mobiluncus 7.5 30 min 0.0
cunisii
Trichomonos vaginalis4.0 30 min 0.25
Trichomonus vaginalis7.5 30 min 2.0
Boiled T. vaginalis 4.0 30 min 0.0
Boiled T. vaginalis 7.5 30 min 0.0
Buffer 4.0 30 min 0.0
Buffer 7.5 30 min 0.0
D. INTERPRETATION
HRPO is not released from the solid support by 300 mM phosphate buffer at
pH 7.5 or by 100 mM acetate buffer at pH 4.0 over a 30 minute incubation
interval at
room temperature. Similarly, Boiled suspensions of Candida albicans,
Tric)wmonas
vaginalis or Mobiluncus curtisii do not release HRPO at pH 7.5 or 4.0 over a
30 minute
incubation at room temperature. A Caruiida albicans culture, however releases
HRPO
from the solid support at pH 4.0 in 10 minutes, but fails to release HRPO at
pH 7.5,
even over a 30 minute incubation period. This is consistent with the
established pH
profile of the Candida albicans aspartic protease, which is active at low pH,
but poorly
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 ~ ~ PCT/US94/04098
active or inactive at high pH. Conversely, a T. vaginalis culture readily
releases HRPO
from the solid support at pH 7.4 over a 30 minute incubation interval, but
releases barely
detectable quantities ~of HRPO at pH 4Ø This behavior is also consistent
with the
known pH profile of the T. vaginalis thiol proteases (i. e. , active at high
pH, but much
less active or inactive at low pH). Finally, Mobiluncus curtisii, which is not
known to
excrete proteases, fails to release HRPO at either pH 4.0 or pH 7.5 even over
a 30
minute incubation interval.
Hence, by performing the tests under different pH conditions, it is possible
to
differentiate between the three microbes: only Candida albicans will cause
color
formation in 10 minutes at room temperature at pH 4.0; only Trichomonas
vaginalis will
cause color formation at pH 7.5 in 30 minutes, and; Mobiluncus curtisii will
not cause
color formation in 30 minutes at either pH 7.5 or 4:0.
ERPE;~e~ViENT XV
This experiment involves the activity of various proteases and their
inhibitors on
the substrate [Eupergitm C-Myoglobin-HRPO].
A. MATERIALS
1. Pulverized [Eupergitm C-Myoglobin-HRPO] (PREPARATION ~.
2. Commercially available aspartic protease from Sigma Chemical Co. (Type
XIII, from Aspergillus saitoi, 0.6 units/mg activity) 40 mg/ml.
3. Trypsin (serine protease) from bovine. pancreas (2900 units/mg activity),
obtained from U.S. Biochemicals, 2 mg/ml.
4. Papain (thiol protease) from papaya latex (12 units/mg activity) from Sigma
Chemical Co., 2mg/ml.
5. Aspartic protease producing Candida albicans culture (ATCC 28366)
6. Tosyl lysine chloromethyl ketone (TLCK) hydrochloride 50 mM in ethanol
from Sigma Chemical Co..
7. Pepstatin A from a microbial source, obtained from Sigma Chemical Co.,
2mg/ml
in ethanol.
8. Buffers and solutions.
a. Potassium phosphate buffer pH 7.0, 200 mM.
b. Potassium phosphate buffer pH 7.4, 100 mM.
SUBSTITUTE SHEET (RULE 26)
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c. Sodium acetate buffer pH 4.0, 100 mM.
d. 0.02 ~ Hydrogen peroxide solution.
e. Absolute ethanol.
9. Guaiac impregnated paper (commercial Hemoccult~ slides).
B. PROCEDURE.
This assay is set up in two parts. The first is to determine the activity of
the
different proteases on the substrate [Eupergit~ C-Myoglobin-HRPO]. The enrymes
and
controls (enzymes boiled for 15 min) are incubated with 20 mg of substrate and
buffers
(see quantities in table below) for 15 minutes prior to assay.
In the second part, the enzymes are preincubated with their respective
inhibitors and appropriate buffers for 15 mins. These are then added to the
substrate and
incubated at room temp for further 15 mins.
In both cases, the reaction mixture is centrifuged to remove solid phase
conjugate. The supernatant (5 ~cl) is added ~to Hemoccult~ slides and
developed with 5 ~1
of hydrogen peroxide.
COLOR SCORE INTERPRETATION
0 NO VISIBLE BLUE COLOR FORMATION
+/- POSSIBLE FAINT BLUE COLOR
0.25 FAINTEST BLUE COLOR DETECTABLE VISUALLY
0.5 DISTINCT BLUE COLOR
1.0 DARK BLUE COLOR
2.0 DARKEST BLUE COLOR POSSIBLE IN TEST SYSTEM
C. RESULTS
1. PART I: The reaction supernatants from tubes containing Buffer alone at
either pH 4.0 or pH 7.5 produced the only faintest color detectable when added
to guaiac
slides and developed with hydrogen peroxide developer. The same result was
seen with
reaction supernatants from tubes containing boiled trypsin at pH 7.4, boiled
Candida
culture at pH 4.0, and boiled papain at pH 7.4. A strong blue color was formed
with
SUBSTITUTE SHEET (RULE 26)
WO 94/24306 ~ PCT/US94/04098
unboiled trypsin at pH 7.4, unboiled Candida culture at pH 4.0, and unboiled
papain at
pH 7Ø
2. PART II: TLCK, a protease inhibitor capable of inhibiting both serine and
thiol type proteases inhibits color formation by both trypsin (a serine
protease) and papain
(a thiol protease). Pepstatin, a known inhibitor of aspartic proteases
inhibits color
formation by the Candida albicans aspartic protease.
TABLE - PART 1
Enzyme Source (vol. = 25 ~cl) Buffer (vol. = 75 Color score
~cl)
Trypsin pH 7.4 2.0
Trypsin (boiled) pH 7.4 0.25
Candida culture pH 4.0 1.5
Candida boiled pH 4.0 0.25
Papain pH 7.0 1.0
Papain (boiled) pH 7.4 0.25
buffers only 0.25
TABLE - PART 2
Inhibitor Ethanol
Enzyme Solution (Control)Buffer pH, Color Score
Vol.
Trypsin TLCK(10~,1) 7.4, 65 ~,1 1.0
Trypsin - - 7.4, 75 ~1 2.0
Papain TLCK(25~1) 251 7.0, 50 ~,1 0.5
Papain , - 7.0, 50 ~.l 1.0
Aspartic proteasePepstatin (25,1) 4.0, 50 ~,1 0.5
Aspartic protease- 25~c1 4.0, 50 ~,1 1.5
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WO 94/24306 PCTlUS94/04098
D. INTERPRETATION
Trypsin, a serine protease hydrolyzed the solid phase substrate at pH 7.4,
releasing soluble HRPO which catalyzed blue color formation on developed
guaiac slides.
The same was true for the Candida albicans aspartic protease at pH 4.0, and
papain, a
5 thiol protease at pH 7Ø Boiling prevented HRPO release by each enzyme.
Hence, each
of the three different enzyme types was capable of hydrolytic release of
soluble HRPO
from the support under the appropriate reaction conditions. Neither buffers
nor heat
inactivated enzymes released soluble HRPO, indicating that HRPO release was
not simply
a non specific release caused by salts, etc. in the growth medium or
incubation mixture.
10 TLCK, a protease inhibitor capable of inhibiting both serine and thiol type
proteases inhibits color formation by both trypsin (a serine protease) and
papain (a thiol
protease). Pepstatin, a known inhibitor of aspartic proteases inhibits color
formation by
the Candida albicans aspartic protease. Hence, by performing incubations in
the
presence of known specific enzyme inhibitors or inhibitors of specific classes
of enzymes,
15 specificity of hydrolase detection can be attained.
The foregoing is offered for purposes of illustration. It will be readily
apparent to those skilled in the art that the operating conditions, materials,
procedural
20 steps and other parameters of the methods and test devices described herein
may be
further modified or substituted in ways without departing from the spirit and
scope of the
invention.
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