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WO1994024306A1 - Technologie de liberation d'enzyme rapporteur: procedes de detection de la presence de proteases aspartiques et d'autres activites d'enzymes hydrolytiques - Google Patents

Technologie de liberation d'enzyme rapporteur: procedes de detection de la presence de proteases aspartiques et d'autres activites d'enzymes hydrolytiques Download PDF

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Publication number
WO1994024306A1
WO1994024306A1 PCT/US1994/004098 US9404098W WO9424306A1 WO 1994024306 A1 WO1994024306 A1 WO 1994024306A1 US 9404098 W US9404098 W US 9404098W WO 9424306 A1 WO9424306 A1 WO 9424306A1
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WO
WIPO (PCT)
Prior art keywords
sample
die
indicator
solid support
hydrolase
Prior art date
Application number
PCT/US1994/004098
Other languages
English (en)
Inventor
Paul J. Lawrence
Aulena Chauhuri
Terrence J. Andreasen
Original Assignee
Litmus Concepts, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/048,539 external-priority patent/US5418226A/en
Application filed by Litmus Concepts, Inc. filed Critical Litmus Concepts, Inc.
Priority to DE69424663T priority Critical patent/DE69424663T2/de
Priority to AT94914164T priority patent/ATE193330T1/de
Priority to PL94311140A priority patent/PL180526B1/pl
Priority to CA002160262A priority patent/CA2160262C/fr
Priority to EP94914164A priority patent/EP0694077B1/fr
Priority to BR9405963A priority patent/BR9405963A/pt
Priority to JP52344194A priority patent/JP3705441B2/ja
Priority to AU66338/94A priority patent/AU6633894A/en
Publication of WO1994024306A1 publication Critical patent/WO1994024306A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
    • G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida

Definitions

  • This invention relates generally to methods of assaying for the presence of hydrolase activity (i.e., hydrolytic enzymes) in a sample or specimen.
  • this invention relates to a method for detecting candidiasis by assaying for the presence of enzymatically active aspartic protease in a sample.
  • vulvovaginal 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.
  • 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 vulvovaginitis release this enzyme when they are subsequently grown in specifically defined culture medium.
  • Candida albicans acid proteinase antigen i.e., aspartic protease antigen
  • aspartic protease antigen has been detected immunologically in the vaginal fluid of all women from which vulvovaginal Candida albicans was isolated.
  • the concentration of Candida albicans acid proteinase antigen 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 ⁇ 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 ⁇ 18.5 ng/ml).
  • Aspartic proteases are one of the major classes of proteases. They contain one or more key aspartic acid residues which are required for activity.
  • Candida albicans aspartic protease has a broad protein substrate specificity which includes, for example, albumin, 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 albicans aspartic protease is strongly inhibited by pepstatin, but it is not inhibited by thiol reagents, chelators or serine protease inhibitors.
  • 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 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.
  • fluorogenic substrates have been described for aspartic proteases, but they, too, are of limited utility.
  • the substrate specificity of this particular class of enzymes requires the presence of several hydrophobic amino acids, rendering the substrates relatively insoluble.
  • the fluorogenic substrates are hydrolyzed very slowly and thus, fluorogenic assays are time-consuming.
  • many biological specimens contain fluorescent materials which can interfere with fluorogenic assays for aspartic proteases.
  • UV spectrophotometric assays are generally used to assay for the presence of this particular class of enzymes.
  • 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.
  • protein such as, for example, hemoglobin or albumin
  • 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.
  • this assay can be used for the detection of aspartic proteases, it is both time-consuming and laborious.
  • 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.
  • a sample e.g. , vaginal fluid
  • a 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.
  • 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.
  • 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.
  • proteases or (interchangeably) proteinases peptidases, lipases, nucleases, homo-oligosaccharidases, hetero-oligosaccharidases, homo- polysaccharidases, hetero-polysaccharidases, phosphatases, sulfatases, neuraminidases and esterases.
  • 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 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.
  • the lack of a detectable change in the indicator is an indication that the enzymatically active hydrolase is absent from the sample.
  • 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, Upases, 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.
  • 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 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.
  • the target hydrolase inhibitor In the event that the target hydrolase inhibitor is not present in the sample, the target hydrolase will 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.
  • test devices 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.
  • 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.
  • the sample 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.
  • 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.
  • 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.
  • 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.
  • 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 d e 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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 die 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.
  • 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.
  • FIG. 1 For water-based samples, the incorporation of a surface-active agent in the laminae immediately adjacent to die gap to be filled widi 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 ingredient in the lamina or combined in the lamina with test reagents.
  • both sides of the gap are lined with laminae bearing the surface-active agent.
  • a sample introduction port is included in die device to permit direct insertion of the sample into the chamber, and preferred embodiments include one or more vent holes in me chamber, spaced apart from the sample introduction port, to further facilitate the filling of the chamber.
  • FIG. 1 is a view in perspective of an illustrative test device in accordance with d e invention.
  • FIG. 2 is a side view in cutaway of a portion of the test device shown in FIG. 1.
  • a method of assaying for the presence of an enzymatically active hydrolase in a sample comprising: (a) contacting die sample with a solid support, the solid support having a reporter enzyme immobilized thereon in such a manner whereby me 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, die detectable change being an indication of d e presence of die enzymatically active hydrolase in me sample.
  • 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.
  • hydrolytic enzymes i.e., hydrolases
  • the method 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, acid proteases, and alkaline proteases.
  • the method of the present invention can be used to assay for d e presence of homo- or hetero-oligosaccharidases or homo- or hetero- polysaccharidases, including, but not limited to, chitinase, amylases, cellulase and lysozyme.
  • 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.
  • reporter enzymes include, but are not limited to, die following: peroxidases, phosphatases, oxidoreductases, dehydrogenases, transferases, isomerases, kinases, reductases, deaminases, catalases, urease and glucuronidase.
  • peroxidases phosphatases, oxidoreductases, dehydrogenases, transferases, isomerases, kinases, reductases, deaminases, catalases, urease and glucuronidase.
  • peroxidases such as, for example, horseradish peroxidase.
  • 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 die hydrolase whose presence is being assayed.
  • a solid support i.e., an insoluble polymeric material, inorganic or organic matrix, gel, aggregate, precipitate or resin
  • Preferred 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 and polymeric dialdehyde, starch, collagen, keratin, elastin, bovine hide powder, bacterial cell wall peptidoglycan or fragments tiiereof, nylon, polyethylene terephthalates, polycarbonates, and controlled pore glass. Immobilization of me reporter enzyme on the solid support is carried out using conventional methods and procedures known to and understood by ti ose skilled in die art.
  • the reporter enzyme can be attached directly to d e solid support.
  • the insoluble support serves directly as a substrate for the hydrolase.
  • the reporter or marker enzyme such as, for example, horseradish peroxidase
  • the reporter or marker enzyme can be attached directly to d e insoluble chitin. In die presence of chitinase, horseradish peroxidase will be released from the solid support.
  • die reporter enzyme e.g., horseradish peroxidase
  • die reporter enzyme e.g., horseradish peroxidase
  • lysozyme die hydrolase being detected
  • die reporter enzyme e.g. , horseradish peroxidase
  • bacterial cell wall peptidoglycan in d e presence of the hydrolase lysozyme, horseradish peroxidase will be released from the solid support.
  • die reporter enzyme can be immobilized on die solid support dirough the use of a linker molecule which is a hydrolyzable substrate for the hydrolase being detected.
  • 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 die selection in any given case will be readily apparent to those skilled in die art.
  • the term "indicator” is used herein to refer to any chemical species which undergoes a detectable change as a result of die reaction or as a result of the culmination of reactions occurring when the enzymatically active hydrolase is present in die 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 d e formation of color in an otherwise colorless material, upon action of die reporter or marker enzyme when it is released from the solid support by the enzymatically active hydrolase whose presence is being detected.
  • die reporter enzyme may be capable of catalyzing die 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 die action of the hydrolase.
  • die reporter enzyme may be capable of producing otiier visible or detectable signals, such as, for example, a clot, an agglutination, a precipitation, or a clearing zone.
  • the indicator would be the chemical species or substrate required by die reporter or marker enzyme in order to bring about die desired detectable change.
  • chromogenic indicators i.e., chromogens
  • otiier species having a similar effect may be used as visual indicators witii horseradish peroxidase as the reporter enzyme.
  • Preferred chromogenic indicators 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 hydroperoxide and guaiac, a chromogen which is colorless in its reduced state and deep blue in its oxidized state.
  • 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 die reporter enzyme is capable of catalyzing or initiating, and die selection in any given case will be readily apparent to those skilled in d e art.
  • the visual indicator is a chromogenic indicator
  • a liquid chromogen system for detecting peroxidases such a system would comprise a solvent, a hydroperoxide and a chromogen capable of being oxidized by hydroperoxides in die presence of a peroxidase, such as, for example, horseradish peroxidase.
  • a solid chromogen system such a system would comprise a hydroperoxide, a chromogen capable of being oxidized by hydroperoxides in d e presence of a peroxidase, and a solid support onto which the chromogen has been impregnated or immobilized and dried.
  • d e chromogen can be impregnated onto a bibulous paper or support as is done witii Hemoccult* slides or, alternatively, die chromogen can be deposited onto a plastic or otiier sheet in the form of a tiiin layer.
  • the chromogen could 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 eitiier alone or in combination witii a water soluble or water insoluble polymer.
  • a polymeric material such as, for example, hydroxypropyl cellulose, ethyl cellulose, etc.
  • me hydroperoxide in die solid chromogen system for detecting peroxidases, me hydroperoxide can be provided eitiier in a solid form (such as, for example, titanium hydroperoxide) or it can be generated in situ.
  • Hydrogen peroxide for example, can be generated in situ with glucose, ambient oxygen and glucose oxidase.
  • hydrogen peroxide can be generated in situ through die use of a dried layer formed after depositing a suspension of sodium perborate in alcohol. At low pH, sodium perborate releases hydrogen peroxide spontaneously.
  • d e chromogen will be oxidized and a visually detectable change in color will result. This resulting change in color is an indication tiiat the enzymatically active hydrolase is present in the sample or specimen.
  • This metiiod as well as the other methods of me 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 support(s) 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.
  • the reactions conditions (such as, for example, die choice of me 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.
  • hydrolase specificity For example, it is known tiiat 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.
  • die pH of die assay one will be able to selectively detect die presence of a particular hydrolase.
  • tiiat specific enzyme inhibitors can also be used in die presently claimed methods to increase hydrolase activity and specificity and to differentiate between the different hydrolases.
  • a method for assaying for the presence of an enzymatically active hydrolase in a sample comprising: placing the sample in a device which contains first and second solid supports, the first solid support having a reporter enzyme immobilized tiiereon in such a manner whereby the reporter enzyme is released upon action of die hydrolase, die second solid support, which is not in contact with the first solid support, having immobilized tiiereon an indicator, the indicator being one which is susceptible to a detectable change upon action of die reporter enzyme, the sample being placed in the device in such a manner tiiat 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 me second solid support; and observing wheti er the indicator undergoes a detectable change, die detectable change being an indication of the presence of the enzymatically active hydrolase in die sample.
  • This metiiod of die 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, Upases, nucleases, hom or hetero-oligosaccharidases, homo- or hetero-polysaccharidases, phosphatases, sulfatases, neuraminidases and esterases.
  • 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 tiiis 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 die sample.
  • reporter enzymes include, but are not limited to, die 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 die enzymatically active hydrolase whose presence is being assayed.
  • the reporter enzyme can be attached directly to die 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.
  • linker molecules include, but are not limited to, d e following: proteins, carbohydrates, lipids, peptides, esters and nucleic acids.
  • linker molecule used to attach the reporter enzyme to the first solid support will depend on which hydrolase is being detected, and die selection in any given case will be readily apparent to those skilled in d e art. Immobilization of the reporter enzyme on the first solid support is carried out using conventional methods and procedures known to and understood by tiiose skilled in the art.
  • die indicator is attached to die second solid support which is not in contact with die first solid support.
  • Preferred first and second solid supports in accordance witii die present invention include, but are not limited to, die 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. Immobilization of the visual indicator on the second solid support is carried out using conventional metiiods and procedures known by those skilled in d e 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 die sample or specimen.
  • the resulting detectable change in die indicator is an indication tiiat d e enzymatically active hydrolase is present in die 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 die 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 die enzymatically active hydrolase whose presence is being detected.
  • chromogemc indicators i.e., chromogens
  • otiier species having a similar effect may be used as visual indicators.
  • Preferred chromogenic indicators for peroxidase-like reporter enzymes in accordance witii die 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 chromogemc 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 d e reaction or reactions which the reporter enzyme is capable of catalyzing or initiating, and die selection in any given case will be readily apparent to tiiose skilled in the art.
  • 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.
  • the chromogen can be impregnated onto a bibulous paper or support as is done witii Hemoccult ® slides or, alternatively, the chromogen can be deposited onto a plastic or other sheet in die form of a min layer.
  • the chromogen could 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 eitiier alone or in combination witii a water soluble or water insoluble polymer.
  • a polymeric material such as, for example, hydroxypropyl cellulose, ethyl cellulose, etc.
  • the hydroperoxide can be provided eitiier in a solid form (such as, for example, titanium hydroperoxide) or it can be generated in situ in the device.
  • a solid form such as, for example, titanium hydroperoxide
  • die chromogen will be oxidized and a visually detectable change in color will result. This resulting change in color is an indication of die presence of die enzymatically active hydrolase in die sample or specimen.
  • enzymatically active aspartic protease is the hydrolase being detected, die method comprising: placing the sample in a device which contains first and second solid supports, the first solid support being polyacrylate and having horseradish peroxidase immobilized tiiereon tiirough 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 hydroperoxide and guaiac, a chromogemc substrate which undergoes a color change upon action of horseradish peroxidase in die presence of a hydroperoxide, die sample being placed in die device in such a manner tiiat the sample contacts the first and second solid supports such that any horseradish peroxidase released by any enzymatically active aspartic protease present in the sample is permitted to diffuse
  • reaction conditions such as, for example, the choice of linker molecule, solid support, pH, buffer capacity, buffer identity, salts, etc.
  • specific inhibitors can be 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 die acid or alkaline proteases, one can selectively detect aspartic proteases.
  • a method for detecting candidiasis by assaying for die presence of enzymatically active aspartic protease in a sample comprising: (a) contacting the sample with a solid support, die solid support having a reporter enzyme immobilized tiiereon in such a manner whereby die reporter enzyme is released upon action of the aspartic protease; (b) combining the sample after having been contacted with die solid support with an indicator, die indicator being one which is susceptible to a detectable change upon action of die reporter enzyme; and (c) observing whether the indicator undergoes a detectable change, the detectable change being an indication of die presence of enzymatically active aspartic protease in the sample and tims, 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 otiier hydrolase activity present in the sample.
  • reporter enzymes include, but are not limited to, the following: peroxidases, phosphatases, oxidoreductases, dehydrogenases, transferases, isomerases, kinases, reductases, deaminases, catalases, urease and glucuronidase.
  • peroxidases phosphatases, oxidoreductases, dehydrogenases, transferases, isomerases, kinases, reductases, deaminases, catalases, urease and glucuronidase.
  • the preferred reporter enzymes are the peroxidases.
  • horseradish peroxidase is die 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.
  • 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 aspartic protease.
  • Solid supports in accordance witii the present invention include, but are not limited to, me 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.
  • Presently preferred solid supports in this method of die claimed invention include chitin, polyacrylate, cellulose, and tiieir 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 reporter enzyme can be immobilized on die solid support tiirough the use of a linker molecule which is a hydrolyzable substrate for the enzymatically active aspartic protease.
  • linker molecules include proteins and peptides, witii proteins being the preferred linker molecules.
  • preferred proteins include, but are not limited to, die following: azocasein, casein, xvcasein, immunoglobulins, hemoglobin, myoglobin, albumin, elastin, keratin and collagen.
  • die linker molecule is ⁇ -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 when enzymatically active aspartic 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 protease in the sample indicates die presence of candidiasis.
  • Preferred indicators are visual indicators and, in particular, chromogenic indicators, i.e., those in which die 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 die solid support by enzymatically active aspartic protease.
  • 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.
  • the reporter enzyme may be capable of producing otiier visible or detectable signals, such as, for example, a clot, an agglutination, a precipitation, or a clearing zone.
  • the indicator would be the chemical species or substrate required by die reporter enzyme in order to bring about the desired detectable change.
  • chromogemc indicators i.e., chromogens
  • other species having a similar effect may be used as visual indicators when peroxidases are used as d e reporter or marker enzymes.
  • Preferred chromogenic indicators in accordance witii 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 hydroperoxide and guaiac, a chromogen which is colorless in its reduced state and deep blue in its oxidized state.
  • 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 die reaction or reactions which die reporter enzyme is capable of catalyzing or initiating, and the selection in any given case will be readily apparent to tiiose skilled in the art. As previously described, if die visual indicator is a chromogenic indicator, it is possible to employ either a liquid chromogen system or a solid chromogen system.
  • a method for detecting Tnchomonas vaginalis by assaying for the presence of enzymatically active thiol protease in a sample comprising: (a) contacting the sample with a solid support, die solid support having a reporter enzyme immobilized tiiereon 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 witii the solid support witii an indicator, d e 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, die detectable change being an indication of d e presence of enzymatically active thiol protease in the sample and tiius, T chomonas vaginalis.
  • the reporter enzyme or marker enzyme used in tiiis 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 die sample, including inactivating hydrolysis by any hydrolase activity present in the sample.
  • reporter enzymes include, but are not limited to, die following: peroxidases, phosphatases, oxidoreductases, dehydrogenases, transferases, isomerases, kinases, reductases, deaminases, catalases, urease and glucuronidase.
  • die 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 enzymatically active thiol protease.
  • 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.
  • Presently preferred solid supports in this metiiod of the claimed invention include chitin, polyacrylate, cellulose, and tiieir 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 die art.
  • the reporter enzyme can be immobilized on die solid support through the use of a linker molecule which is a hydrolyzable substrate for the enzymatically active thiol protease.
  • linker molecules include proteins and peptides, with proteins being the preferred linker molecules.
  • linker molecule is a protein
  • preferred proteins include, but are not limited to, die following: azocasein, casein, / -casein, immunoglobulins, hemoglobin, myoglobin, albumin, elastin, keratin and collagen.
  • the linker molecule is ⁇ -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 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 Tnchomonas vaginalis.
  • Preferred indicators are visual indicators and, in particular, chromogenic indicators, i.e., those in which the detectable change is a change in color, including die 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 die enzymatically active aspartic protease.
  • 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.
  • die reporter enzyme may be capable of producing otiier visible or detectable signals, such as, for example, a clot, an agglutination, a precipitation, or a clearing zone.
  • the indicator would be the chemical species or substrate required by the reporter enzyme in order to bring about the desired detectable change.
  • chromogenic indicators i.e., chromogens
  • otiier species having a similar effect may be used as visual indicators when peroxidases are used as the reporter or marker enzymes.
  • Preferred chromogemc indicators in accordance witii die present invention comprise a hydroperoxide and a chromogen including, but not limited to, one of die 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 chromogemc 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 chromogemc indicator for any given reporter enzyme will depend on die reaction or reactions which die reporter enzyme is capable of catalyzing or initiating, and die selection in any given case will be readily apparent to those skilled in the art.
  • the visual indicator is a chromogenic indicator, it is possible to employ eitiier a liquid chromogen system or a solid chromogen system.
  • certain reaction conditions and specific inhibitors can be used to increase thiol protease activity and specificity.
  • 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
  • pepstatin to inhibit aspartic proteases
  • soybean trypsin inhibitor to inhibit trypsin
  • EDTA or other chelators to inhibit metallo-proteases
  • any of the many known naturally occurring inhibitors of non-thiol protein proteases 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
  • the reporter enzyme release technology of the present invention can also be employed to detect d e 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.
  • 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 die blood concentration of a hydrolase- inhibiting therapeutic drug or to determine die presence of a potential pesticide hydrolase inhibitor contamination in produce, etc. In these cases, it is, therefore, necessary to detect die inhibitor of a hydrolase, ratiier than the hydrolase itself.
  • a method for assaying for die presence of an inhibitor of a target hydrolase in a sample comprising: (a) contacting the sample with the target hydrolase and a solid support, die solid support having a reporter enzyme immobilized thereon in such a manner whereby the reporter enzyme is released upon action of die target hydrolase if die target hydrolase is not inactivated by the presence of the inhibitor; (b) combining the sample after having been contacted widi die target hydrolase and d e 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 whetiier the indicator undergoes a detectable change, d e detectable change being an indication of the absence of the inhibitor of the target hydrolase in die sample.
  • test performance involves detecting die ability of the sample to inhibit the target hydrolase.
  • the target hydrolase will release the reporter enzyme from the solid support, tiiereby producing a detectable change in the indicator.
  • the target hydrolase inhibitor is present in the sample, the target hydrolase will be inhibited, die reporter enzyme will not be released from the solid support, and a detectable change or response will not be produced in die indicator. It is not essential tiiat the inhibitor in the sample completely inhibit die target hydrolase added to the test system.
  • This method of die 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, die following: inhibitors of the proteases or (interchangeably) proteinases, peptidases, Upases, nucleases, homo- or hetero-oligosaccharidases, homo- or hetero- polysaccharidases, phosphatases, sulfatases, neuramimdases and esterases.
  • this metiiod 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.
  • mis metiiod 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 die literature.
  • pepstatin is the aspartic protease inhibitor whose presence is being detected.
  • target hydrolases include, but are not limited to, the following: proteases, proteinases, peptidases, Upases, nucleases, homo- or hetero-oligosaccharidases, homo- or hetero-polysaccharidases, phosphatases, sulfatases, neuramimdases and esterases.
  • the particular target hydrolase used in die above method will depend upon which inhibitor is being detected, and die selection in any given case will be readily apparent to tiiose skilled in die art. If, for example, pepstatin is die inhibitor being detected, ti en aspartic protease would be the target hydrolase used in die test system described above.
  • the reporter enzyme or marker enzyme used in tiiis method of die 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 die target hydrolase which is incorporated into d e test system
  • Such reporter enzymes include, but are not limited to, die following: peroxidases, phosphatases, oxidoreductases, dehydrogenases, transferases, isomerases, kinases, reductases, deaminases, catalases, urease and glucuronidase.
  • the preferred reporter enzymes are die 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 die presence of the inhibitor.
  • Solid supports in accordance with the present invention include, but are not limited to, die 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 die solid support through die use of a linker molecule which is a hydrolyzable substrate for the enzymatically active target hydrolase.
  • linker molecules include, but are not limited to, die 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 die test system, and die 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 die reaction or as a result of die culmination of reactions occurring if the enzymatically active target hydrolase is not inactivated by die presence of the inhibitor in the sample or specimen.
  • Preferred indicators are visual indicators and, in particular, chromogemc indicators, i.e., those in which the visible change is a change in color, including die 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 die enzymatically active target hydrolase provided it is not inactivated by the presence of the inhibitor in the sample or specimen.
  • chromogemc indicators i.e., chromogens
  • otiier species having a similar effect may be used as visual indicators.
  • Preferred chromogemc indicators for peroxidase-like reporter enzymes in accordance witii die 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 chromogemc indicator is comprised of a hydroperoxide and guaiac, a chromogen which is colorless in its reduced state and deep blue in its oxidized state.
  • die visual indicator is a chromogemc indicator, it is possible to employ either a liquid chromogen system or a solid chromogen system.
  • a test device for assaying for the presence of an enzymatically active hydrolase in a sample 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 hydrolase; an indicator contained on the interior-facing surface of one of the first and second walls, d e 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.
  • the receptacle is preferably flat and tiiin and of a size which can be easily held by hand.
  • die chamber is preferably flat and shallow as well, with a width and lengtii much greater than its deptii, die deptii being substantially constant.
  • the chamber is preferably shallow enough to promote spontaneous wetting of die chamber walls with die specimen to achieve the maximum contact between the specimen and die 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 d e reagents on the surface opposite that to which the visual indicator has been applied will need to diffuse in order to reach the indicator.
  • die chamber deptii 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 deptii, preferably from about 5 mil to about 15 mil (0.005-0.0.015 inch; 0.0127-0.0381 cm), will give the best results. For any given deptii, die 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 die size and shape of the visible test area on the outer surface of the device.
  • the lateral dimensions should tiius 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 die 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 cm 2 to about 10 cm 2 , or preferably from about 0.3 cm 2 to about 3 cm 2 .
  • 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 ⁇ L will be the most appropriate and convenient.
  • the test device of die present invention can be used to assay for the presence of any known hydrolytic enzyme including, but not limited to, die following: proteases or (interchangeably) proteinases, peptidases, Upases, nucleases, homo- or hetero- oligosaccharidases, homo- or hetero-polysaccharidases, phosphatases, sulfatases, neuraminidases and esterases.
  • me test device of the present invention can be used to assay for the presence of proteases, including, but not limited to, die following: aspartic proteases, serine proteases, thiol proteases, metallo proteases, and acid or alkaline proteases.
  • the test device of die 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 die 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.
  • reporter enzymes include, but are not limited to, die following: peroxidases, phosphatases, oxidoreductases, dehydrogenases, transferases, isomerases, kinases, reductases, deaminases, catalases, urease and glucuronidase.
  • peroxidases phosphatases, oxidoreductases, dehydrogenases, transferases, isomerases, kinases, reductases, deaminases, catalases, urease and glucuronidase.
  • me 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 me solid support upon action of die hydrolase whose presence is being detected.
  • a first solid support i.e., an insoluble matrix, gel or resin
  • Preferred solid supports in accordance witii the present invention include, but are not limited to, die 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. Immobilization of the reporter enzyme on the first solid support is carried out using conventional methods and procedures known to and understood by tiiose skilled in d e art.
  • the reporter enzyme can be attached directly to die first solid support or, alternatively, the reporter enzyme can be immobilized on d e first solid support tiirough die use of a linker molecule having a hydrolyzable linkage which is a substrate for the enzymatically active hydrolase being detected.
  • linker molecules include, but are not limited to, die 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 d e selection in any given case will be readily apparent to tiiose skilled in the art.
  • the indicator is immobilized on a second solid support which is not in contact witii the first solid support.
  • Preferred solid supports in accordance witii 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 d e 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 d e sample or specimen.
  • This detectable change is, tiierefore, an indication that d e enzymatically active hydrolase being assayed is present in die 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 d e 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 die hydrolase whose presence is being detected.
  • 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 die action of the hydrolase.
  • the reporter enzyme may be capable of producing otiier 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 d e chemical species or substrate required by the reporter or marker enzyme in order to bring about the desired detectable change.
  • chromogemc indicators i.e., chromogens
  • otiier species having a similar effect may be used as visual indicators.
  • preferred chromogemc indicators comprise a hydroperoxide and a chromogen including, but not limited to, one of die 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, a chromogen which is colorless in its reduced state and deep blue in its oxidized state.
  • die guaiac may be purified prior to use, e.g. , by solvent extraction.
  • the most appropriate chromogemc indicator for any given reporter enzyme will depend on die reaction or reactions which die reporter enzyme is capable of catalyzing or initiating, and die selection in any given case will be readily apparent to those skilled in die art. If the visual indicator is a chromogemc indicator, a solid chromogen system is employed.
  • This solid chromogen system comprises a hydroperoxide, a chromogen capable of being oxidized by hydroperoxides in die presence of a peroxidase, and a solid support onto which die chromogen has been impregnated or immobilized and dried.
  • the chromogen can be impregnated onto a bibulous paper or support as is done witii Hemoccult* slides or, alternatively, the chromogen can be deposited onto a plastic or other sheet in the form of a thin layer.
  • the chromogen could 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 eitiier alone or in combination with a water soluble or water insoluble polymer. In this particular chromogen system, the hydroperoxide can be provided eitiier in a solid form (such as, for example, titanium hydroperoxide) or it can be generated in situ in d e device.
  • a polymeric material such as, for example, hydroxypropyl cellulose, ethyl cellulose, etc.
  • Hydrogen peroxide for example, can be generated in situ by layering dried glucose and glucose oxidase on the interior-facing surfaces of the test device.
  • 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.
  • sodium perborate releases hydrogen peroxide spontaneously.
  • die peroxidase e.g. , horseradish peroxidase
  • die hydrolase upon its release from the first solid support by die hydrolase, die chromogen will be oxidized and a visually detectable change in color will result. As previously mentioned, this resulting change in color is an indication mat the enzymatically active hydrolase is present in die sample or specimen.
  • die reactions conditions (such as, for example, the selection of the solid support and linker molecule, pH, buffer capacity, buffer identity, salts, etc.) of the presentiy 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.
  • 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 witii a sample introduction port by which die specimen is placed in die chamber.
  • the port is preferably in the same wall through which changes in d e visual indicator are observed, i.e., the light-transmitting wall.
  • the port will be shaped to accommodate d e transfer device which is used to convey the sample from its source, and die port may thus be varied to suit any of die 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 die art.
  • a circular port is generally adequate, altiiough for transfer devices such as swabs, d e 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 die fluid migration needed to fill d e chamber and tiiereby place all reagents in contact with the specimen.
  • One such feature is the inclusion of one or more vent holes in the chamber to permit the escape of air.
  • the vent holes will be adequately distanced from me sample introduction port to maximize the surface area wetted by die specimen.
  • the vent holes will be arranged to assure that the specimen reaches both controls and fills them to avoid any false or ambiguous readings.
  • one preferred arrangement of die device is die 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 witii die test area.
  • the sample 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 invention is die placement of a surface-active agent along the interior surface of the chamber.
  • the agent may be along one or the otiier 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 die surface.
  • the lamina will also contain one or more reagents taking part in the test reactions.
  • 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 emulsifiers, and will vary widely in chemical structure and electronic character, including anionic, cationic, zwitterionic and nonionic substances.
  • alkyl-alkoxy sulfates examples are alkyl-alkoxy sulfates, alkyl aryl sulfonates, glycerol fatty acid esters, lanolin-based derivatives, polyoxyethylene alkyl phenols, polyoxyethylene amines, polyoxyethylene fatty acids and esters, polyoxyethylene fatty alcohols and ethers, poly(ethylene glycol) fatty acids and esters, polyoxyethylene fatty esters and oils, polyoxypropylene/polyoxyethylene condensates and block polymers, sorbitan fatty acid esters, sulfo derivatives of succinates, alkyl glucosides, and cholic acid derivatives.
  • Trade names of products falling within some of these classes are Lubrol, Brij, Tween, Tergitol, Igepal, Triton, Teepol and many otiiers.
  • Formation of the solid laminae may be done by applying d e lamina material in liquid form followed by drying or otiier solidification.
  • the liquid form of the substance may be, for example, a solution, a suspension, or an uncured liquid state of the substance, and die 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 die 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 die solubility of the layer witii solvents used in layers to be applied over it or to make die layer soluble in solvents which do not dissolve layers applied underneatii, or all of these at die 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.
  • die 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.
  • one or more additional reagents will be included for each control.
  • These additional reagents will eitiier be incorporated witiiin one of the existing laminae in a horizontally defined portion of tiiat lamina or applied as a separate, vertically adjacent lamina over a horizontally defined portion of the existing lamina.
  • 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 me overall test is directed, die type of visual indicator used to detect die presence of d e hydrolase, and wheti er 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 die 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 d e 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 tiiereby prevents the detectable change from occurring regardless of whetiier or not the enzymatically active hydrolase is present in the sample or specimen. Both controls are activated when die specimen is applied to the test device.
  • control reagents in laminae on the same surface as the lamina(e) containing the other reagent(s). In others, best results are achieved when die control reagents are placed in laminae on the chamber surface opposite that which bears the other reagent(s), such that the control reagent and die remaining reagent(s) are separated by die air gap.
  • the control areas of the device will contain all components and reagents used in die test area with the addition of die control reagents, either incorporated in horizontally delineated sections of one or more of the same laminae used in die test area or applied as separate laminae over such horizontally delineated sections.
  • discontinuities in the laminae may be in laminae along the upper surface, the lower surface, or both.
  • the controls are preferably activated by die 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 die same chamber in the test device, witii unobstructed fluid communication between the various areas.
  • die control areas are isolated from each otiier by die test area which is positioned in between the two. Filling of all areas with a single application can be accomplished witii the arrangement of the sample introduction port and vent holes described above. Since the detectable changes, or absence thereof, are detectable tiirough die light-transmitting wall of the device, d e identification of areas as positive and negative controls is conveniently achieved by placing appropriate indicia on the outer surface of the device.
  • the test device may be formed in a variety of ways. Sheets of polymeric material may be laminated together, with appropriate cutouts to define die shape of the chamber and holes for the sample introduction port and die vent holes. The deptii of the chamber as well as its shape and lateral dimensions will then be defined by die thickness of the central sheet, while the placement of the holes will be controlled by die top sheet.
  • the indicator and reagent coatings may be applied to die top sheet, bottom sheet or both, as required, before the sheets are assembled into die laminate. The sheets may then be secured togedier 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 otiierwise light-transmitting polymeric material, witii a section of the sheet embossed or otiierwise processed, mechanically or chemically, to contain a depression or indentation of constant deptii in the inner surface of the chamber.
  • the depression is located on one half of die sheet, witii die holes for sample introduction and venting on die otiier half.
  • the indicator and reagent coatings are applied at appropriate locations on the sheet, and the half containing the holes is then folded over die otiier 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 die two halves together is tiirough 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.
  • appropriate adhesives will be tiiose which are transparent, inert, wettable by, and otiierwise compatible with die layers to be applied over it.
  • a test device for testing a sample for the presence of candidiasis by assaying for the presence of enzymatically active aspartic protease comprising: a receptacle defined at least in part by first and second opposing walls having interior-facing surfaces with a gap dierebetween, the first, second, or both walls being of light-transmitting material; a reporter enzyme immobilized on a solid support on die interior-facing surface of one of the first and second walls in such a manner whereby the reporter enzyme is released upon action of die aspartic protease; an indicator immobilized on die 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.
  • the reporter enzyme or marker enzyme used in tiiis 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.
  • reporter enzymes include, but are not limited to, the following: peroxidases, phosphatases, oxidoreductases, dehydrogenases, transferases, isomerases, kinases, reductases, deaminases, catalases, urease and glucuronidase.
  • Presentiy preferred reporter 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 upon action of aspartic protease.
  • the reporter enzyme can be immobilized on die solid support tiirough the use of a linker molecule having a hydrolyzable linkage which is a substrate for aspartic protease.
  • linker molecules include proteins and peptides, witii proteins being the preferred linker molecules.
  • linker molecule is a protein
  • preferred proteins include, but are not limited to, d e following: azocasein, casein, / -casein, immunoglobulins, hemoglobin, myoglobin, albumin, elastin, keratin and collagen.
  • die linker molecule is /c-casein, casein, hemoglobin, or myoglobin.
  • the indicator is immobilized on a second solid support which is not in contact with die first solid support.
  • Preferred solid supports in accordance witii die present invention include, but are not limited to, die 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 d e second solid support is carried out using conventional methods and procedures known by those skilled in die 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 die sample or specimen.
  • the resulting detectable change is an indication of the presence of enzymatically active aspartic protease in the sample and, in turn, die presence of enzymatically active aspartic protease in the sample indicates die presence of candidiasis.
  • 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 die reporter or marker enzyme when it is released from me solid support by the enzymatically active aspartic protease whose presence is being detected.
  • chromogemc indicators i.e., chromogens
  • otiier species having a similar effect may be used as visual indicators.
  • Preferred chromogemc indicators in accordance witii the present invention when peroxidase is die 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 chromogemc indicator is comprised of a hydroperoxide and guaiac, which is colorless in its reduced state and deep blue in its oxidized state.
  • a solid chromogen system is employed, and such a system comprises a hydroperoxide, a chromogen capable of being oxidized by hydroperoxides in die presence of a peroxidase, and a solid support onto which the chromogen has been impregnated or immobilized and dried.
  • the chromogen can be impregnated onto a bibulous paper or support as is done witii Hemoccult ® slides or, alternatively, the chromogen can be deposited onto a plastic or otiier sheet in the form of a thin layer.
  • 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 die 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.
  • a polymeric material such as, for example, hydroxypropyl cellulose, ethyl cellulose, etc.
  • the hydroperoxide can be provided eitiier in a solid form (such as, for example, titanium hydroperoxide) or it can be generated in situ in the test device.
  • a solid form such as, for example, titanium hydroperoxide
  • 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 enzymatically active aspartic protease in the sample or specimen and, in mm, the presence of enzymatically active aspartic protease indicates d e presence of candidiasis.
  • a test device for testing a sample for the presence of an inhibitor of a target hydrolase 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 die interior-facing surface of one of die 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 die interior-facing surface of one of the first and second walls in such a manner whereby die reporter enzyme is released upon action of the target hydrolase if the target hydrolase is not inactivated by d e presence of the inhibitor; an indicator contained on die interior- facing surface of one of die 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 d e receptacle for introduction of die sample.
  • test performance involves detecting the ability of the sample to inhibit die target hydrolase.
  • the target hydrolase In die event tiiat die target hydrolase inhibitor is not present in the sample, the target hydrolase will release the reporter enzyme from the solid support, tiiereby producing a detectable response in the indicator. Conversely, if the target hydrolase inhibitor is present in die 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 die indicator.
  • die inhibitor in the sample completely inhibit the target hydrolase added to die 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 hydrolase 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 die test device.
  • exposure time of the target hydrolase to any inhibitor in the sample can be regulated by physically or chemically separating die target hydrolase from the immobilized reporter enzyme.
  • 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.
  • 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.
  • the target hydrolase 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, die 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, die following: inhibitors of the proteases or (interchangeably) proteinases, peptidases, Upases, nucleases, homo- or hetero-oligosaccharidases, homo- or hetero- polysaccharidases, phosphatases, sulfatases, neuramimdases and esterases.
  • inhibitors of the proteases or (interchangeably) proteinases including, but not limited to, die following: inhibitors of the proteases or (interchangeably) proteinases, peptidases, Upases, nucleases, homo- or hetero-oligosaccharidases, homo- or hetero- polysaccharidases, phosphatases, sulfatases, neuramimdases and esterases.
  • tiiis 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.
  • 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.
  • pepstatin is the aspartic protease inhibitor whose presence is being detected.
  • target hydrolases include, but are not limited to, die following: proteases, proteinases, peptidases, Upases, 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 die art. If, for example, pepstatin is the inhibitor being detected, tiien aspartic protease would be the target hydrolase used in die test device described above.
  • die 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 die test device.
  • reporter enzymes include, but are not limited to, die following: peroxidases, phosphatases, oxidoreductases, dehydrogenases, transferases, isomerases, kinases, reductases, deaminases, catalases, urease and glucuronidase.
  • peroxidases phosphatases, oxidoreductases, dehydrogenases, transferases, isomerases, kinases, reductases, deaminases, catalases, urease and glucuronidase.
  • 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 d e presence of the inhibitor in the sample.
  • Solid supports in accordance witii 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.
  • die reporter enzyme can be immobilized on the solid support tiirough die use of a linker molecule which is a hydrolyzable substrate for the enzymatically active target hydrolase.
  • linker molecules include, but are not limited to, die 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 die test device, and d e 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 die presence of the inhibitor in the sample or specimen.
  • Preferred indicators are visual indicators and, in particular, chromogemc indicators, i.e., those in which the visible change is a change in color, including die formation of color in an otherwise colorless material, upon action of the reporter or marker enzyme when it is released from me solid support by die enzymatically active target hydrolase provided it is not inactivated by d e presence of the inhibitor in the sample or specimen.
  • chromogenic indicators i.e., chromogens
  • otiier species having a similar effect may be used as visual indicators.
  • Preferred chromogemc 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 die 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 chromogemc indicator is comprised of a hydroperoxide and guaiac, a chromogen which is colorless in its reduced state and deep blue in its oxidized state.
  • die visual indicator is a chromogemc indicator, it is possible to employ either a liquid chromogen system or a solid chromogen system.
  • 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 die test device for testing a sample for the presence of an inhibitor of a target hydrolase.
  • die invention is not intended to be limited to any particular construction of a test device, die attached Figures, which are not drawn to scale, illustrate how one such device may be constructed.
  • FIG. 1 depicts die support structure of the device in a perspective view, prior to the indicator and reagents being applied and die chamber being enclosed.
  • the support structure consists of a single sheet 11 of relatively stiff, transparent, chemically inert plastic material, with a score line 12 defining a fold separating die sheet into two halves 13, 14, each having the same length and widti .
  • the lower half 13 contains an indentation of a composite shape consisting of a circle 15 at the center with two 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 tiiat when the plastic is folded at die score line 12 and die top half 14 is placed in contact with die bottom half 13, the sample introduction port 18 is above the center of the circular part 15 of the indentation, and die vent holes 19, 20 are above die 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.
  • the two halves 13, 14 may be of differing lengti s, widtiis or both for various reasons.
  • the only critical feature is that the indentations in die lower half and die holes in die 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 d e score line.
  • 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 d e advantage of preventing user confusion as to where to introduce die sample.
  • FIG. 2 is a side cutaway view of the device of FIG. 1, showing die chamber 31 in cutaway after the coatings have been applied and die two halves folded over and sealed to one another.
  • the inner surfaces of each of the two halves 13, 14 of the transparent polymer are coated witii an adhesive 32, 33, respectively.
  • the upper adhesive layer 33 is the layer containing the visual indicator 34, and beneadi the latter is a layer of reagent 35.
  • tiiat both the visual indicator layer 34 and die reagent layer 35 can extend die full length and widtii of the chamber, surrounding die sample introduction port 18 and extending into all areas of the chamber.
  • test and control areas of the chamber are defined by die 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 die otiier rectangular extension 17.
  • reagents for the controls may be placed on die 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 central circular portion 15 of the chamber is coated with a layer 38 which may either contain an additional reagent used in the test reaction or no reagent at all.
  • die circular test area 41 is flanked by a rectangular negative control area 42 and a rectangular positive control area 43.
  • the three segments 36, 37, 38 can be separated by gaps or discontinuities 44, 45 to retard or minimize diffusion between, or contact of, the contents of these segments.
  • Similar discontinuities may also be placed in eitiier or both of the visual indicator and reagent layers 34, 35, directly above the discontinuities 44, 45 in the lower layer. The discontinuities in die visual indicator and reagent layers will further prevent diffusion of control components or other reagents from the control areas into the test area.
  • die layers 35, 36, 37, 38 may contain, in addition to any reagents present, 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.
  • the reagents tend to deteriorate upon prolonged exposure to air or to air-borne moisture.
  • this is prevented by a tiiin 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 use, whereupon the sheet is readily peeled off.
  • a moisture- and air-impermeable sheet on the bottom of the device, die sheet being either permanently attached or capable of being peeled off. Further protection against moisture and air can be achieved by placing die device in a pouch which completely surrounds die device.
  • each of the dimensions of the device shown in Figures 1 and 2 may vary, as may their arrangements and shapes.
  • a typical example is one in which the support sheet is Mylar 5 mil in thickness (0.005 inch, 0.0127 cm), and die adhesive layer is low density polyethylene 2 mil in thickness (0.002 inch, 0.0051 cm), the gap widtii 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 2 ), and d e negative and positive control areas each measure 1/8 inch x 1/16 inch (area: 0.0078 square inch, 0.0504 cm 2 ).
  • the air vents in this example are each circular, and tiiey and die sample introduction port are each 1/8 inch (0.32 cm) in diameter.
  • the chamber volume is approximately 12 ⁇ L.
  • 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 otiierwise.
  • 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.
  • MATERIALS a. Solid, insoluble supports (Sepharose 4B, chitin, and Sigmacell* 20 [purchased from Sigma Chemical Co.]) b. Distilled water and ice made witii distilled water c. 4.0 M NaOH d. Solid CNBr e. Coupling buffer (0.1 M NaHCO 3 containing 0.5 M NaCl) f. Magnetic stirring motor and stirring bar; pH meter; chemical fume hood 2.
  • the filtrate was collected in a suction flask containing solid ferrous sulfate which inactivated residual CNBr and cyanide remaining in d e reaction mixmre.
  • the solid support was washed witii 1 liter cold distilled water and 1 liter coupling buffer under suction and stored as a moist paste at 4°C.
  • 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 NaCl iii. 100 mM Sodium borate, pH 9.0 containing 0.5 M NaCl d. Solid sodium periodate e. 100 mM Formaldehyde
  • HRPO hydrazide approximately 175 Units/mg protein
  • HRPO hydrazide approximately 175 Units/mg protein
  • Two hundred microliters of 100 mM formaldehyde were added to d e mixmre, and incubation continued at room temperamre for and additional 30 minutes.
  • Two mL 1 M cold acetate buffer, pH 4.0 were added, and die conjugate allowed to precipitate for 30 minutes at 4°C.
  • MATERIALS a. [Kappa-Casein-HRPO] conjugates (PREPARATION B) b. Distilled water c. Buffers: i. Coupling Buffer (0.1 M NaHCO 3 containing 0.5 M NaCl) ii. 1.0 M Tris buffer, pH 8.0 iii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCl iv. 100 mM Sodium borate buffer, pH 9.0, containing 0.5 M NaCl d. Cyanogen bromide activated Sepharose 4B and Sigmacell ® 20 (PREPARATION A) e. Cyanogen bromide activated Sepharose 6 MB (from Sigma Chemical Co.)
  • Two milliliters [Kappa-Casein-HRPO] conjugate prepared from the hydrazide of HRPO as described in PREPARATION B were diluted witii 3 mL coupling buffer and added to one gram moist cyanogen bromide-activated Sepharose 4B or Sigmacell ® .
  • the suspension was mixed end-over-end at room temperamre for two hours.
  • the solid support was washed witii 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 remaimng active sites on the solid support.
  • MATERIALS a. HRPO, Type II, 200 Units/mg (from Sigma Chemical Co.) b. 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 5.0 c. Solid sodium periodate
  • 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 NaHCO 3 containing 0.5 M NaCl) ii. 1.0 M Tris buffer, pH 8.0 iii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCl iv. 100 mM Sodium borate buffer, pH 8.0 containing 0.5 M NaCl v. 0.1 Percent (v/v) glutaraldehyde solution in water 2.
  • Coupling Buffer 0.1 M NaHCO 3 containing 0.5 M NaCl
  • 1.0 M Tris buffer pH 8.0 iii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCl iv. 100 mM Sodium borate
  • 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 NaCl e. distilled water
  • Solid sodium periodate (428 mg) was added to one gram Sigmacell ® suspended in 10 mL distilled water. The mixmre was rotated continuously at room temperamre 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.
  • 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 NaCl iv. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCl v. 20 mM MES buffer, pH 5.0 2.
  • PROCEDURE 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. Buffer
  • 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 NaCl ii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCl iii. 20 mM MES buffer, pH 5.0 e. Aldehyde-Activated HRPO (PREPARATION D)
  • 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 mixmre was allowed to incubate without agitation for 72 hours at room temperamre. The support was washed tiiree times with 1.0 M NaCl and five times with distilled water. The support was mixed witii 2.5 mL mercaptoethanol previously adjusted to pH 8.0, and die suspension allowed to stand overnight at room temperamre.
  • 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 phosphate buffer pH 7.5; 3.5 M sodium thiocyanate; and finally witii large volumes of phosphate buffered saline (PBS) (0.01 M sodium phosphate, pH 7.2 containing 0.15 M NaCl).
  • PBS phosphate buffered saline
  • the derivatized beads were treated witii 0.1 % (v/v) glutaraldehyde witii rotation for 2 hours at room temperamre, overnight at 4°C, and tiien washed witii distilled water.
  • MATERIALS a. HRPO aldehyde (PREPARATION D) b. Hemoglobin derivatized Eupergit ® C (PREPARATION J) c. 100 mM Sodium cyanoborohydride d. 1.0 M NaCl e. Distilled water 2.
  • MATERIALS a. 25 mg Kappa-Casein-HRPO conjugate prepared by die 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 NaCl iii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCl iv. 50 mM MES buffer, pH 6.0 d. 100 mM Formaldehyde e. 100 mM Sodium cyanoborohydride 2.
  • PROCEDURE 25 mg Kappa-Casein-HRPO conjugate prepared by die hydrazide method (PREPARATION B) dissolved in 2 mL 100 mM borate buffer, pH 9.0
  • This solution was mixed with three mL MES buffer, and die solution added to 1 gram polymeric dialdehyde.
  • the suspension was rotated at room temperamre for 6 hours, and incubated overnight at 4°C.
  • Two hundred microliters of 100 mM formaldehyde were added to die suspension and die mixmre incubated for 1 hour at room temperamre.
  • the resin was removed by centrifugation, washed witii water, and resuspended in 3 mL 100 mM ethanolamine, pH 8.5. After a two hour incubation with rotation at room temperamre, 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 4°C. A final wash was done witii distilled water, and the moist suspension stored at 4°C until used.
  • 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 NaCl ii. 100 mM Sodium acetate buffer pH 4.0 containing 0.5 M NaCl iii. 100 mM Tris buffer pH 8.0, containing 0.5 M NaCl 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
  • 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 NaCl were added to 1 mL of Eupergit ® C beads suspended in water. The mixmre was incubated with rotation for 48 hours at room temperamre. 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.
  • 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 NaCl iii. 100 mM Sodium acetate buffer, pH 4.0 containing 0.5 M NaCl 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
  • 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 witii HRPO Aldehyde
  • Step 1 Covalent Attachment of Myoglobin and Bovine Serum Albumin to Eupergit ® C
  • Acrylic Beads (Eupergit ® C) 1.
  • MATERIALS a. Oxirane acrylic beads (Eupergit ® C, 150 micron beads from Sigma Chemical Co.)
  • b Horse heart myoglobin (type IV) or bovine serum albumin (from Sigma)
  • 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 mixmre was allowed to incubate without agitation for 72 hours at room temperamre.
  • the support was washed tiiree times with 1.0 M NaCl and five times with 20 mL distilled water.
  • the support was mixed witii 2.5 mL mercaptoethanol previously adjusted to pH 8.0, and die suspension allowed to stand overnight at room temperamre.
  • 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 NaCl).
  • PBS phosphate buffered saline
  • Step 2 Covalent Labeling with Horse Radish Peroxidase (ALDEHYDE METHOD) Horse Radish Peroxidase aldehyde (PREPARATION D) was used to label the above myoglobin or albumin derivatized acrylic beads from step 1 above using PROCEDURE F.
  • MATERIALS a. Myoglobin covalentiy 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. 0.5 M NaCl 2.
  • Guaiac Sheets One mL of the above guaiac solution is pipetted onto the polyethylene side of a 10 inch x 10 inch sheet of a Mylar/polyethylene laminate (7 mils Mylar/3 mL polyethylene). The guaiac solution is spread over die surface of the sheets by means of a standard wound-wire testing bar, and allowed to dry at room temperamre.
  • This experiment involves the release of HRPO from [SEPHAROSE-CASEIN- HRPO] and [SEPHAROSE-KAPPA-CASEIN-HRPO] by aspartic protease released into Candida albicans culture.
  • MATERIALS (CYANOGEN BROMIDE ACTIVATED SEPHAROSE) (HRPO ALDEHYDE COUPLING METHOD) 1. Cyanogen bromide activated Sepharose 4B (PREPARATION A) first derivatized witii covalently bound casein or Kappa-Casein (PREPARATION P) and subsequently labeled witii HRPO aldehyde (PREPARATION D)
  • MATERIALS (EUPERGIT ® C ACTIVATED ACRYLIC BEADS) (HRPO ALDEHYDE COUPLING METHOD)
  • Oxirane acrylic beads (Eupergit ® C) were treated with Casein or Kappa-Casein (PREPARATION M) and subsequently labeled witii HRPO aldehyde
  • Casein equivalent were suspended in 300 microliters of either Candida albicans culmre containing secreted active aspartic protease, or 300 microliters of the same Candida albicans culmre which had been boiled for 20 minutes to inactivate the aspartic protease.
  • the mixmre was rotated for 15 minutes at room temperamre, and d e suspension centrifuged to sediment the solid conjugate and Candida albicans cells.
  • This experiment involves the release of HRPO from [SEPHAROSE- HEMOGLOBIN-HRPO] or [SEPHAROSE-MYOGLOBIN-HRPO] by pepsin and aspartic protease from Aspergillus saitoi.
  • MATERIALS (CYANOGEN BROMIDE ACTIVATED SEPHAROSE) (HRPO ALDEHYDE COUPLING METHOD)
  • the conjugate was treated overnight with 100 mM sodium cyanoborohydride at room temperamre and washed witii 0.5 M NaCl before use.
  • 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.)
  • Aspartic Protease Type XIII
  • pepsin (1 mg/mL, 2900 units/mg
  • BSA Bovine Serum Albumin
  • MATERIALS (CYANOGEN BROMIDE ACTIVATED SEPHAROSE) (HRPO HYDRAZIDE COUPLING METHOD)
  • 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.)
  • Centrifugation sedimented Sepharose bound HRPO leaving only enzyme solubilized HRPO in solution.
  • the soluble HRPO catalyzed die oxidation of guaiac by hydrogen peroxide, producing a blue color.
  • blue color formation on the guaiac layered sheets provides a convenient method for detecting aspartic protease or pepsin.
  • BSA nor buffer have aspartic protease activity.
  • 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.
  • MATERIALS (CYANOGEN BROMIDE ACTIVATED CHITIN) (HRPO HYDRAZIDE COUPLING METHOD)
  • 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
  • This experiment involves the release of HRPO from [SIGMACELL* 20- HEMOGLOBIN-HRPO] or [SIGMACELL* 20-MYOGLOBIN-HRPO] by pepsin and aspartic protease from Aspergillus saitoi.
  • MATERIALS (CYANOGEN BROMIDE ACTIVATED SIGMACELL* 20) (HRPO ALDEHYDE COUPLING METHOD)
  • 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 3. Guaiac impregnated paper in the form of commercially available Hemoccult ® slides
  • 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 temperamre 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.
  • EXPERIMENT VHI 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.
  • 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 temperamre for 15 minutes and centrifuged to remove the solid phase conjugate. Five microliters of die supernatant were added to a guaiac slide, followed by five microliters of hydrogen peroxide solution.
  • EXPERIMENT X This example is an example of the release of HRPO from [POLYMERIC DIALDEHYDE-KAPPA-CASEIN-HRPO] by pepsin and aspartic protease from Aspergillus saitoi.
  • MATERIALS (POLYMERIC DIALDEHYDE) (HRPO HYDRAZIDE
  • 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.)
  • BSA Bovine Serum Albumin
  • 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 d e 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.
  • MATERIALS (COMMERCIAL CYANOGEN BROMIDE ACTIVATED SEPHAROSE 6 MB) (HRPO HYDRAZIDE COUPLING METHOD)
  • Vaginal fluid from women with proven clinical vulvovaginal candidiasis contained aspartic protease which hydrolyzed Sepharose 6 MB-bound Kappa-Casein, at pH 3.0 in 15 minutes at room temperamre, 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.
  • vaginal fluid from women with clinically diagnosed vulvovaginal candidiasis
  • MATERIALS (CYANOGEN BROMIDE ACTIVATED SIGMACELL ® 20- MYOGLOBIN-HRPO--PREPARATIONS A AND I) AND (EUPERGIT ® C- MYOGLOBIN-HRPO--PREPARATION Q) (HRPO ALDEHYDE COUPLING
  • vaginal fluid specimens from control women failed to hydrolyze polymer-bound protein, and failed to release soluble, active HRPO from either suDDOrt. Centrifugation sedimented polymer bound HRPO, leaving no 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 witii vulvovaginal Candida albicans.
  • Tnchomonas vaginalis (ATCC 3001) culmre.
  • 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 4.0.
  • Mobiluncus Curtisii culmre produced no blue color in 30 minutes at either pH 4.0 or 7.5.
  • EXPERIMENT XV This experiment involves the activity of various proteases and tiieir inhibitors on the substrate [Eupergit ® C-Myoglobin-HRPO].
  • Pepstatin A from a microbial source obtained from Sigma Chemical Co., 2mg/ml in ethanol.
  • 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 enzymes 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.
  • the enzymes are preincubated witii their respective inhibitors and appropriate buffers for 15 mins. These are then added to die substrate and incubated at room temp for further 15 mins.
  • reaction mixmre is centrifuged to remove solid phase conjugate.
  • the supernatant (5 ⁇ l) is added to Hemoccult ® slides and developed with 5 ⁇ l of hydrogen peroxide.
  • 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 Can ⁇ da albicans aspartic protease.
  • 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 tiiiol protease).
  • Pepstatin a known inhibitor of aspartic proteases inhibits color formation by the Can ⁇ da albicans aspartic protease.

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Abstract

Procédés de dosage permettant de détecter la présence d'hydrolases à activité enzymatique (en d'autres termes, d'enzymes hydrolytiques) dans un échantillon ou une éprouvette. L'invention se rapporte plus particulièrement à un procédé de détection de la candidose, consistant à détecter la présence de la protéase aspartique à activité enzymatique dans un échantillon. Selon ces procédés, un échantillon ou une éprouvette est mis en contact avec un support solide. Le support solide avec lequel l'échantillon est mis en contact comprend une enzyme rapporteur (en d'autres termes, une enzyme de génération de signaux) qui y est immobilisée. L'enzyme rapporteur est immobilisée sur le support solide de manière à être libérée de ce dernier par l'action de l'hydrolase à activité enzymatique si cette dernière est présente dans l'échantillon. Celui-ci, après avoir été mis en contact avec le support solide, est combiné avec un indicateur. L'indicateur se compose de n'importe quelle espèce chimique susceptible de présenter une modification détectable, généralement une variation de couleur, losqu'elle est soumise à l'action de l'enzyme rapporteur. Une modification décelable de l'indicateur révèle la présence de l'hydrolase à activité enzymatique dans l'échantillon. En outre, la présence d'une telle hydrolase dans un échantillon peut indiquer la présence d'un pathogène ou d'un état pathologique particulier tel que la candidose, par exemple. Outre les procédés de détection de la présence d'une protéase aspartique à activité enzymatique et d'autre enzymes hydrolytiques, l'invention se rapporte également à un dispositif d'essai autonome, à sec, permettant de détecter la présence d'une hydrolase à activité enzymatique dans un échantillon, plus particulièrement à un tel dispositif d'essai qui permettrait de tester un échantillon afin d'y détecter la présence de la candidose par l'intermédiaire de la détection d'une hydrolase à activité enzymatique. Ces dispositifs d'essai comprenennt, en combinaison, une enzyme rapporteur immobilisée sur support solide, un indicateur, et tous les autres réactifs et constituants nécessaires pour indiquer de manière détectable la présence ou l'absence d'une telle hydrolase dans l'échantillon, les modes préférés de réalisation contenant également des témoins positifs et négatifs.
PCT/US1994/004098 1993-04-14 1994-04-13 Technologie de liberation d'enzyme rapporteur: procedes de detection de la presence de proteases aspartiques et d'autres activites d'enzymes hydrolytiques WO1994024306A1 (fr)

Priority Applications (8)

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DE69424663T DE69424663T2 (de) 1993-04-14 1994-04-13 Reporterenzym-freisetzungstechnologie: verfahren zum nachweis der gegenwart von aspartat-proteasen und anderen aktivitaeten von hydrolytischen enzymen
AT94914164T ATE193330T1 (de) 1993-04-14 1994-04-13 Reporterenzym-freisetzungstechnologie: verfahren zum nachweis der gegenwart von aspartat-proteasen und anderen aktivitaeten von hydrolytischen enzymen
PL94311140A PL180526B1 (pl) 1993-04-14 1994-04-13 Sposób oznaczania obecnosci enzymatycznie aktywnej hydrolazy w próbce i urzadzenie testowe do badania obecnosci enzymatycznie aktywnej hydrolazy w próbce PL PL PL PL PL
CA002160262A CA2160262C (fr) 1993-04-14 1994-04-13 Technique de liberation d'enzyme marqueur; methodes d'essai pour deceler la presence de proteases aspartiques et diverses activites enzymatiques hydrolytiques
EP94914164A EP0694077B1 (fr) 1993-04-14 1994-04-13 Technologie de liberation d'enzyme rapporteur: procedes de detection de la presence de proteases aspartiques et d'autres activites d'enzymes hydrolytiques
BR9405963A BR9405963A (pt) 1993-04-14 1994-04-13 Processo para testar a presença de hidrolase enzimaticamente ativa e de um inibidor de uma hidrolase alvo em uma amostra e para detectar candidíase e trichomonas vaginalis e dispositivos de teste para testar em uma amostra a presença de uma hidrolase enzimaticamente ativa de candidíase de protease aspártica enzimaticamente ativa e de um inibidor de uma hidrolase alvo
JP52344194A JP3705441B2 (ja) 1993-04-14 1994-04-13 リポーター酵素放出技術：アスパラギン酸プロテアーゼ及びその他の加水分解酵素活性の存在についての検定方法
AU66338/94A AU6633894A (en) 1993-04-14 1994-04-13 Reporter enzyme release technology: methods of assaying for the presence of aspartic proteases and other hydrolytic enzyme activities

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US08/048,539 US5418226A (en) 1992-04-22 1993-04-14 Monoquaternary 2,16-bispiperidinylandrostane derivatives
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US5571684A (en) * 1994-11-07 1996-11-05 Litmus Concepts, Inc. Assay for proline iminopeptidase and other hydrolytic activities
GB2378246A (en) * 2001-05-16 2003-02-05 Univ Sheffield Detection of enzymatically active hydrolases
WO2007009047A2 (fr) * 2005-07-13 2007-01-18 Expressive Constructs, Inc. Substrats, detecteurs et methodes servant a evaluer des etats chez la femelle
WO2007141030A2 (fr) * 2006-06-09 2007-12-13 Bio Pur Ag Procédé pour la détection de réactions enzymatiques
US7878644B2 (en) 2005-11-16 2011-02-01 Gerber Scientific International, Inc. Light cure of cationic ink on acidic substrates
US9017963B2 (en) 2002-01-31 2015-04-28 Woundchek Laboratories (Us), Inc. Method for detecting microorganisms
CN116970479A (zh) * 2023-09-25 2023-10-31 哈尔滨瀚邦医疗科技有限公司 一种胃蛋白酶测定试剂盒及其应用

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AT509355B1 (de) * 2010-02-10 2012-04-15 Univ Graz Tech Testanordnung

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WO1996015255A2 (fr) * 1994-11-07 1996-05-23 Litmus Concepts, Inc. Test pour la proline iminopeptidase et pour d'autres activites hydrolytiques
WO1996015255A3 (fr) * 1994-11-07 1996-08-08 Litmus Concepts Inc Test pour la proline iminopeptidase et pour d'autres activites hydrolytiques
US5571684A (en) * 1994-11-07 1996-11-05 Litmus Concepts, Inc. Assay for proline iminopeptidase and other hydrolytic activities
GB2378246A (en) * 2001-05-16 2003-02-05 Univ Sheffield Detection of enzymatically active hydrolases
US9017963B2 (en) 2002-01-31 2015-04-28 Woundchek Laboratories (Us), Inc. Method for detecting microorganisms
WO2007009047A2 (fr) * 2005-07-13 2007-01-18 Expressive Constructs, Inc. Substrats, detecteurs et methodes servant a evaluer des etats chez la femelle
WO2007009047A3 (fr) * 2005-07-13 2007-04-19 Expressive Constructs Inc Substrats, detecteurs et methodes servant a evaluer des etats chez la femelle
US7896485B2 (en) 2005-11-16 2011-03-01 Gerber Scientific International, Inc. Light cure of cationic ink on acidic substrates
US7878644B2 (en) 2005-11-16 2011-02-01 Gerber Scientific International, Inc. Light cure of cationic ink on acidic substrates
WO2007141030A3 (fr) * 2006-06-09 2008-04-10 Bio Pur Ag Procédé pour la détection de réactions enzymatiques
WO2007141030A2 (fr) * 2006-06-09 2007-12-13 Bio Pur Ag Procédé pour la détection de réactions enzymatiques
CN116970479A (zh) * 2023-09-25 2023-10-31 哈尔滨瀚邦医疗科技有限公司 一种胃蛋白酶测定试剂盒及其应用
CN116970479B (zh) * 2023-09-25 2023-12-19 哈尔滨瀚邦医疗科技有限公司 一种胃蛋白酶测定试剂盒及其应用

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JPH08509124A (ja) 1996-10-01
CA2160262C (fr) 2002-03-26
PL311140A1 (en) 1996-02-05
CA2160262A1 (fr) 1994-10-27
JP3705441B2 (ja) 2005-10-12
CZ289902B6 (cs) 2002-04-17
CZ266495A3 (en) 1996-05-15
HUT73393A (en) 1996-07-29
BR9405963A (pt) 1996-01-30
PL180526B1 (pl) 2001-02-28
AU6633894A (en) 1994-11-08

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