CZ266495A3 - Method of determining presence of enzymatically active hydrolase in a sample and apparatus for making the same - Google Patents

Abstract

Methods of assaying for the presence of enzymatically active hydrolases (i.e., hydrolytic enzymes) in a sample or specimen are disclosed. In particular, a method of detecting candidiasis by assaying for the presence of enzymatically active aspartic protease in a sample is provided. In these methods, a sample or specimen is contacted with a solid support. The solid support with which the sample is contacted has a reporter enzyme (i.e., a signal generating enzyme) immobilized thereon. The reporter enzyme is immobilized on the solid support in a manner such that it is released from the solid support upon action of the enzymatically active hydrolase if the enzymatically active hydrolase 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 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. Moreover, the preence of an enzymatically active hydrolase in a sample may indicate the presence of a particular pathogen or disease state such as, for example, candidiasis. In addition to the methods of assaying for the presence of enzymatically active aspartic protease and other hydrolytic enzymes, a dry, self-contained test device for assaying for the presence of an enzymatically active hydrolase in a sample is disclosed. In particular, a dry, self-contained test device for testing a sample for the presence of candidiasis by assaying for the presence of enzymatically active aspartic protease is also disclosed. These test devices combine a reporter enzyme immobilized on a solid support, an indicator, and all other reagents and components necessary to achieve a detectable indication of the presence or absence of the enzymatically active hydrolase whose presence is being detected in the sample, and preferred embodiments contain positive and negative controls as well.

Description

A method for detecting the presence of an enzymatically active hydrolase in a sample and apparatus for carrying out the method

Technical field

The invention relates to a method for detecting the presence of an enzymatically active hydrolase in a sample and to an apparatus for carrying out the method. It relates generally to methods for testing the presence of hydrolytic enzymes in a sample. In particular, the invention relates to a method for detecting candidiasis by testing for the presence of an enzymatically active aspartic protease in a sample.

BACKGROUND OF THE INVENTION

Candida albicans and other Candida species cause known medically significant infections. Oral candidiasis, for example, is well known in immunocompromised patients. Moreover, vulvovaginal candidiasis is the most common disease in obstetrics and gynecology. It is estimated that approximately three-quarters of all adult women suffer from at least one incidence of this disease (De Bernardis et al., J. Klíň. Microbiol. 27 (11), 2598-2603, 1989). Due to this widespread occurrence, extensive research has been devoted to understanding the etiology of candidiasis.

Research has shown that Candida albicans and other Candida species have an etiological involvement in human candidiasis, and it is now widely believed that candidiasis is mainly due to the presence of Candida albicans. Furthermore, the role of aspartic acid protease (interchangeable) acid proteinase as a virulence factor of Candida albicans is now largely evident. It is known that pure Candida albicans cultures secrete aspartic protease when grown under well-defined conditions. Similarly, pure Candida albicans strains isolated from women with symptoms of vulvovaginitis are known to release this enzyme when subsequently allowed to grow in a specifically defined culture environment.

Acidic proteinase Candida albicans, an aspartic acid protease antigen, was detected immunologically in the vaginal fluid of all women from whom vulvovaginal Candida albicans was isolated (De Bernardis et al., Abstract No. F-91 in the Abstracts of the Annual Meeting of the However, the concentration of Candida albicans acid proteinase antigen was significantly higher in patients with symptomatic vulvovaginal candidiasis than in asymptomatic carriers.The concentration of this antigen in the vaginal fluid of women with candidiasis is approximately 176 ± 15.2 ng / ml, while the concentration of this antigen in the vaginal fluid of women without isolation of Candida albicans, i.e. without clinical candidiasis, is less than 2 ng / ml Asymptomatic carriers of Candida albicans have mean antigen levels of 94 ± 18.5 ng / ml. suggest that an acidic proteinase (i.e. aspartic protease) is involved However, aspartic protease Candida albicans is known to be unstable at body temperature. Furthermore, the detection of aspartic protease antigen does not immunologically indicate whether the enzyme is present in an enzymatically active form.

Acidic proteinase Candida albicans is an extracellular protease of aspartic acid. Aspartic acid proteases are a major class of proteases. They contain one or more key aspartic acid residues that are required for activity. Asparagus protease Candida albicans has a wide specificity of a protein substrate that includes, for example, albumin, hemoglobin, casein, immunoglobin A and many other proteins. This enzyme functions optimally under acidic conditions (i.e., pH 2.5-5.5) and is rapidly inactivated at high pHs (i.e. pH 7.5). Asparagus protease Candida albicans is strongly inhibited by pepstatin but is not inhibited by thiol reagents, chelating agents or serine protease inhibitors.

Many pharmaceutical companies and leading academics are studying aspartic acid protease inhibitors for potential therapeutic use. However, their efforts are hampered by the lack of an appropriate aspartic acid protease enzyme assay. While simple colorimetric assays are available for some serine, thiol, metallic, acidic and basic proteases and peptidases, they are not available for aspartic acid proteases. The substrate specificity of this particular class of enzymes requires the presence of several hydrophobic amino acids. This property strongly hindered the search for simple synthetic chromogenic substrates, since the hydrophobic amino acids that serve as substrates for the aspartic protease are notoriously difficult to dissolve in water. As a result, chromogenic substrates for aspartic protease are not commercially available, difficult to synthesize and characterize, and poorly dissolve in water. The ultimate consequence of these drawbacks is that the enzymatic activity defined by these chromogenic substrates is extremely low and colorimetric assays for aspartic protease are therefore rather insensitive. Similarly, fluorogenic agents have been described for aspartic proteases, but they also have limited utility. First, as mentioned above, the substrate specificity of this particular class of enzymes requires the presence of several hydrophobic amino acids for which these substrates are relatively insoluble. Second, at low attainable concentrations of these substrates, the fluorogenic substances hydrolyze very slowly and the fluorogenic tests are therefore time consuming. In addition, many biological samples contain fluorescent materials that may interfere with fluorogenic assays for aspartic proteases.

Because of the lack of suitable colorimetric and fluorogenic assays to detect aspartic proteases, spectrophotometric assays in the ultraviolet (UV) region are typically used to test for the presence of this particular class of enzymes. In typical ultraviolet spectrophotometric assays, an aspartic protease is added to a protein solution (such as 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 frozen incubation mixture to precipitate unused protein, leaving ultraviolet absorbing peptides in solution. Finally, the precipitated unused protein is pelleted by centrifugation for approximately one hour at freezing temperatures, the supernatant is aspirated and its optical density at 280 nm is determined indicating the extent of protein hydrolysis. Although this assay can be used to detect aspartic proteases, this assay is both time consuming and laborious.

Thus, a convenient, simple, on-site test to detect the presence of enzymatically active aspartic proteases has not been developed. It is therefore an object of the present invention to provide a method of testing for the presence of enzymatically active aspartic proteases to address the problems and drawbacks of the prior art. The method of the invention is furthermore also suitable for testing the presence of other known hydrolytic enzymes, i.e. hydrolases.

SUMMARY OF THE INVENTION

The method for detecting the presence of an enzymatically active hydrolase in a sample is according to the invention in that:

(a) contacting the sample with a solid support containing the immobilized reporter enzyme released by the hydrolase;

(b) the sample, after contact with the solid support, is combined with an indicator which is sensitive to a detectable change by the reporting enzyme; and

(c) any detectable change in the indicator is observed, which is an indication of the presence of enzymatically active hydrolase in the sample.

Surprisingly, it has been found that the enzymatically active as paraguay protease Candida albicans is present in the vaginal fluid of women with vulvovaginal candidiasis. It has further been found that the presence of an enzymatically active aspartic protease in a sample may serve as an indicator for the detection and diagnosis of candidiasis. Based on these findings, a method for detecting candidiasis has now been developed by testing for the presence of an enzymatically active aspartic protease in a sample.

In this method, a sample, such as a vaginal fluid, is contacted with a solid carrier. The solid support with which the sample comes into contact has a reporter enzyme (i.e., a signal-inducing enzyme) which is immobilized thereon. The reporter enzyme is immobilized on a solid support such that it is released from it by the action of an enzymatically active aspartic protease, if the enzymatically active aspartic protease is actually present in the sample. Then, when the sample comes into contact with the solid support, it is combined with the indicator. The indicator is any chemical that is capable of visible or detectable changes (such as color change) due to the action of a reporter enzyme. If, after contact with the sample, the indicator undergoes a detectable change, the enzymatically active aspartic protease is present in the sample and can therefore be said to be candidiasis.

Prior to the present invention, there was no rapid and direct means to test for the presence of enzymatically active aspartic proteases or, more importantly, for the presence of candidiasis. Spectro-photometric, fluorogenic, and immunoassays have been used to test for the presence of aspartic protease activity, but these are time-consuming, laborious, and unsuitable for non-trained clinicians. Similarly, Candida albicans can be detected by growing the sample in a defined environment or by wet microscopy. The cultivation process is very time-consuming (it takes approximately 48 hours) and both procedures require expensive equipment and extensive training. In contrast to the aforementioned assays, the method for testing the presence of the enzymatically active aspartic protease of the invention and then candidiasis is rapid, accurate, cheap and easy to use.

The reporter enzyme release technology on which the aspartic protease assay is based can also be used to test for the presence of any active hydrolytic enzyme, including but not limited to: proteases (or interchangeably) proteinases, peptidases, lipases, nucleases, homo -oligosaccharidases, hetero-oligosaccharidases, homo-polysaccharidases, hetero-polysaccharidases, phosphatases, sulfatases, neuraminidases and esterases. Thus, methods for testing the presence of enzymatically active hydrolase in a sample have now been developed.

In these methods, the sample is contacted with a solid support. The solid support with which the sample comes into contact has a reporter enzyme (i.e., a signal-inducing enzyme) which is immobilized thereon. The reporter enzyme is immobilized on a solid support such that it is released from it by the action of hydrolase if the enzymatically active hydrolase is actually present in the sample. Then, when the sample comes into contact with the solid support, it is combined with the indicator. The indicator is any chemical that is capable of visible or detectable changes, usually color changes, due to the action of the reporter enzyme. A detectable change in the indicator is an indication that enzymatically active hydrolase is present in the sample. In contrast, the lack of detectable change in the indicator is an indication that the enzymatically active hydrolase is missing aspartic proteases in the sample and the presence of

Similar to the enzymatically active candidiasis assay, the methods of the invention are enzymatically active hydrolase in a sample rapid, accurate, cheap and easy to use.

In addition, the reporter enzyme release technology on which the methods of the invention are based can also be used to test for the presence of an inhibitor of any known hydrolytic enzyme, including, but not limited to: protease inhibitors (or interchangeably) proteases, peptidases, lipases, nucleases, homo-oligosaccharidases, hetero-oligosaccharidases, homo-polysaccharidases, hetero-polysaccharidases, phosphatases, sulfatases, neuraminidases, and esterases. Thus, a method for testing the presence of a hydrolase inhibitor in a sample has now been developed.

In these methods, the sample is contacted with the target hydrolase and a solid support. The solid support with which the sample comes into contact has a reporter enzyme (i.e., a signal-inducing enzyme) which is immobilized thereon. The reporter enzyme is immobilized on the solid support such that it is released from it by the action of the target hydrolase, unless the target hydrolase is inactivated due to the presence of the hydrolase inhibitor. Then, when the sample comes into contact with the target hydrolase and the reporter enzyme, it is combined with the indicator. The indicator is any chemical that is capable of visible or detectable changes, usually color changes, under the action of a reporter enzyme when the reporter enzyme is released from the solid support by the target hydrolase. If there is no target hydrolase inhibitor in the sample, the target hydrolase releases the enzyme from the carrier, causing a detectable change in the indicator. Conversely, if a hydrolase inhibitor is present in the sample, the target hydrolase is inhibited, the reporter enzyme is not released from the solid support, and no detectable change or response is produced in the indicator. Similar to the methods described above, methods for testing for the presence of a hydrolase inhibitor in a sample are fast, accurate, cheap, and easy to use.

In addition to methods for testing for the presence of enzymatically active aspartic protease and the activities of other hydrolytic enzymes, a dry, sealed test device for testing a sample for the presence of candidiasis is now being developed by testing for the presence of an enzymatically active aspartic protease. In addition, a dry sealed test device is also developed to test for the presence of enzymatically active hydrolase in a sample. These test devices are a combination of a reporter enzyme immobilized on a solid support, an indicator, and one or more dry-form reagents in a laminated plate with an inner chamber, the chamber being an empty sample receiving space. For convenience, portions of the plate and the location of the functional chemicals are described from the reference assembly, with the plate in a horizontal position, which is the position that the plate is most likely to occupy in use. When the plate is in this position, particularly preferred plates according to the invention, which are thin flat structures, the sample is inserted into the chamber through an opening at the top of the plate. For the laminates forming the plate, the uppermost laminate in this sample inserting position is called the top plate laminate, the bottom surface of the laminate forming the top surface of the chamber. Similarly, the bottom plate laminate is called the bottom plate laminate, wherein the bottom laminate forms the bottom surface of the chamber. The thin edges around the periphery of the top and bottom laminates are called the side edges of the plate, and the thin side ends of the chamber along the edges of its upper and lower surfaces are called the side walls of the chamber. Areas of any given surface facing each other in the same horizontal plane are called horizontally adjacent, while a laminate placed directly above the second laminates forming parallel horizontal planes is referred to as vertically adjacent.

Both the top and the bottom or both of the wafer laminates are made of a light transmissive material, preferably transparent. The solid-state reporting enzyme, indicator and other components and reagents required for the test are arranged in one or more laminates within the chamber, either as coatings on the top surface of the chamber, as coatings on the bottom surface of the chamber, or on both surfaces chambers. The indicator is any chemical that is capable of visible or detectable changes, usually color changes, due to the action of a reporter enzyme when released from a solid support by an enzymatically active hydrolase, the presence of which is detected. The laminate containing the indicator may be on the top or bottom surface of the chamber. In the same laminate as the indicator, there may be one or more reagents needed for the test, or may be in separate laminates on the same surface or on the opposite surface. According to some preferred embodiments of the invention, the indicator is contained in a laminate located directly below the light transmitting wall and the detector enzyme immobilized on the solid support is contained in the laminate placed on the opposite wall.

The reagents occupying the laminates may be selected such that a sample is sufficient to carry out the test and a minimum number of additional reagents, for example developers, according to particularly preferred embodiments, the laminates contain all the necessary reagents except the sample, so sample insertion.

All laminates are solid layers prior to contact with the sample, and the laminate containing the indicator preferably has a composition that is insoluble in the liquid sample for which the test is intended, so that the indicator remains in the laminate for the duration of the test. For samples in an aqueous or water-soluble environment, therefore, the preferred indicator laminate is either a water insoluble indicator or an indicator contained in a water-insoluble matrix. Thus, if the indicator is contained in a thin concentrated laminate directly below the translucent wall, the indicator changes, which is detectable through the translucent wall within a short period of time, resulting in both high sensitivity and a rapid result.

The method of the invention can be adapted and used to test a wide variety of hydrolases in test samples from a variety of sources. In addition, the process of the invention can be modified and used to test a wide variety of hydrolase inhibitors. The test may include 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 vary from one test to another depending on which hydrolase is being detected. In some cases, best results are obtained when the reagents are distributed between the top and bottom surfaces of the chamber prior to application, separated by a gap until the gap is filled with the test sample. In other cases, the reactants may be placed in a common layer or in two or more separate but vertically adjacent layers on the top or bottom surface of the chamber, without loss of test reliability. In all cases, however, the layers are formed and arranged such that the reaction peaking in a detectable indicator change occurs only when the chamber is filled with the test sample and so that when the indicator change occurs, it is at least concentrated and preferably limited to the layer immediately adjacent the translucent wall.

According to a preferred embodiment of the invention, the test device comprises a built-in positive control and / or a built-in negative control, which are activated by adding a single sample. Activation of these controls occurs concurrently with the test being performed and a detectable indication (such as a color change or missing color change) representing both controls, and the test is accomplished by a single loading of the sample into the test apparatus and is detectable by a translucent wall. The controls are located in the test device at positions horizontally adjacent to the test area with respective indications on the upper and lower surface of the device, preferably on the upper surface of the device, to identify the controls and to distinguish them from the test. Controls themselves generally consist of additional layers containing reagents or other suitable substances that either cause a detectable change in the indicator on its own or prevent the change from occurring and do so only when the sample is present and yet independent of the presence or absence of the suspected hydrolase in the sample. Again, the choice of these controls and the chemical mechanism by which they operate, as well as the choice of placing these layers on the same chamber surface as the indicator or on the opposite surface, will vary from test to test.

Other preferred embodiments of the invention are based on additional features to improve the performance of the assay. For water-based samples, the incorporation of a surfactant into the layers immediately facing the sample-filled gap will promote wetting of the layers with the sample and rapid and uniform filling of the chamber. The surfactant may be the functional ingredient alone in the layer or in combination with the test reagent in the layer. Preferably, both sides of the gap are coated with layers containing a surfactant. In the test device there is an inlet opening for sample introduction to allow direct introduction of the sample into the chamber, and preferably there are several vents in the chamber spaced apart from the inlet opening to further facilitate filling of the chamber.

Other features and advantages of the invention and its preferred embodiments will become apparent from the following description with the accompanying drawings.

Fig. 1 is a perspective view of a test device according to the invention.

Figure 2 is a cross-sectional side view of the device of Figure 1;

The invention relates to a method for testing for the presence of an enzymatically active hydrolase in a sample, wherein:

(a) contacting the sample with a solid support containing the immobilized reporter enzyme released by the hydrolase;

(b) the sample, after contact with the solid support, is combined with an indicator which is sensitive to a detectable change by the reporting enzyme; and

(c) any detectable change in the indicator is observed, which is an indication of the presence of enzymatically active hydrolase in the sample.

The term hydrolase as used herein refers to an enzyme that catalyzes hydrolytic reactions. The method of the invention can be used to test for the presence of any known hydrolytic enzyme. Such hydrolytic enzymes, i.e. hydrolases, are without limitation: proteases (or interchangeably) proteinases, peptidases, lipases, nucleases, homo-oligosaccharidases, heterooligosaccharidases, homo-polysaccharidases, hetero-polysaccharidases, phosphatases, sulfatases, neuraminidases, and esterases. According to a preferred embodiment of the invention, the method of the invention can be used to test for the presence of proteases including, but not limited to: aspartic acid proteases, serine proteases, thiol proteases, metalloproteases, acid proteases and alkaline proteases. In another preferred embodiment, the method of the invention may be used to test for the presence of homo-oligosaccharidases or hetero-oligosaccharides, or homo-polysaccharidases or hetero-polysaccharidases, including, but not limited to, chitinases, amylases, cellulases, and lysozyme.

The term "reporter enzyme" or (interchangeably) "signal enzyme" is used herein to refer to a signal generating enzyme, that is, an enzyme whose activity causes a detectable change. Such enzymes include, but are not limited to: peroxidase, phosphatase, oxidoreductase, dehydrogenase, transferase, isomerase, kinase, reductase, deaminase, catalase, urease, and glucuronidase. When selecting a reporter enzyme for use in the method of the invention, it is critical that the reporter enzyme is not subject to inactivation by any agent in the sample, including inactivating hydrolysis, by any hydrolase activity in the sample. The selection of a suitable reporter or signal enzyme is known to those skilled in the art. At the same time, peroxidases such as horseradish peroxidase are suitable reporter enzymes.

The reporter enzyme is immobilized on a solid support, i.e., on an insoluble polymeric material, inorganic or organic matrix, gel, aggregate, precipitate or resin, in such a way that the reporter enzyme is released by the hydrolase being tested. Suitable carriers of the invention include, but are not limited to, cellulose, agarose, dextran, polyacrylate, polyacrylamide or derivatives thereof, chitin, sepharose, oxirane, acrylic beads and polymer dialdehyde, starch, collagen, keratin, elastin, cowhide powder, bacterial peptidoglycan cell wall or fragments thereof, nylon, polyethylene terephthalates, polycarbonates and controlled pore glass. The immobilization of the reporter enzyme on a solid support is accomplished by conventional methods known in the art.

The reporting enzyme may be attached directly to the solid support. In this case, the insoluble carrier serves directly as a substrate for the hydrolase. If, for example, chitinase is tested as a hydrolase, a reporter or labeled enzyme (such as horseradish peroxidase) can be attached directly to the insoluble chitin. In the presence of chitinase, horseradish peroxidase is released from the solid support. Similarly, if cellulase is to be detected as the hydrolase, the reporter or labeled enzyme (such as horseradish peroxidase) can be attached directly to cellulose and horseradish peroxidase is released from the solid support in the presence of cellulase hydrolase. Finally, if lysozyme is to be detected as a hydrolase, the reporter or labeled enzyme, for example horseradish peridase, can be attached directly to the peptidoglycan of the cell wall of the bacteria and, in the presence of lysozyme hydrolase, horseradish peroxidase is released from the solid support.

Alternatively, the reporter enzyme may be immobilized on a solid support using a linker molecule, which is a hydrolyzable substrate in detecting the hydrolase. Such linker molecules include, but are not limited to: proteins, hydrocarbons, lipids, peptides, esters, and nucleic acids. The particular linker molecule used to attach the reporter enzyme to the solid support depends on which hydrolase is detected and the choice is known to those skilled in the art.

As used herein, the term "indicator" refers to any chemical that undergoes a detectable change due to the reaction or as a result of the culmination of reactions occurring when an enzymatically active hydrolase is present in the sample. The resulting detectable change is an indication that the enzymatically active hydrolase is contained in the sample.

Preferred indicators are visual indicators and in particular chromogenic indicators, i.e. those in which the color change, including the coloring of otherwise colorless material by the reporter or signaling enzyme, is released from the solid support by the enzymatically active hydrolase to be detected. Alternatively, the reporter enzyme may be able to catalyze the generation of a fluorescent, phosphorescent, bioluminescent, chemiluminescent, or electrochemical signal by its release from the solid support by hydrolase. Additionally, the reporter enzyme may be capable of generating other visible or detectable signals, a debris zone, agglutination, precipitation or clarification. In such cases, the indicator would be the chemicals or substrates required by the reporter or signaling enzyme to produce the desired detectable change.

A variety of chromogenic indicators (i.e., chromogens) and other species having a similar effect with horseradish peroxidase as the reporter enzyme may be used as visual indicators. Preferred chromogenic indicators of the invention include hydrogen peroxide and chromogen, including, but not limited to, one of the following: guaiac, 2-2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid, tetramethylbenzidine, phenol, 4-ol); A particularly preferred chromogenic indicator consists of hydrogen peroxide and guaiac, which is a chromogen colorless in a reduced state and dark blue in an oxidized state, and the guaiac may optionally be purified prior to use, for example, by use of an aminoantipyrine and 4,5-dihydroxynaphthalene-2,7-disulfonic acid. The most aqueous chromogenic indicator for each given reporter enzyme depends on the reaction or reactions that the reporter enzyme is able to catalyze or initiate, and the choice in this case is known to those skilled in the art.

If the visual indicator is a chromogenic indicator, either a liquid chromogenic or solid chromogenic system may be used. When a liquid chromogen system is used to detect peroxidases, such a system will consist of a solvent, hydrogen peroxides and chromogens capable of oxidation by hydrogen peroxide in the presence of a peroxidase such as horseradish peroxidase. If an alternatively solid chromogenic system is used, such a system will consist of hydrogen peroxides and a chromogen capable of oxidizing with hydrogen peroxide in the presence of peroxidase, and a solid carrier through which the chromogen is impregnated or immobilized and dried. In this system, the chromogen may be impregnated on a suction paper or on a carrier formed by Hemoccult ^R strips, or alternatively, the chromogen may be deposited on a plastic or other thin-film sheet. According to this last embodiment, the chromogen may be layered as a solution containing a polymeric material (such as hydroxypropyl cellulose and ethyl cellulose). If the chromogen itself is water-soluble, it may be trapped in a matrix of water-insoluble material. Alternatively, if the chromogen itself is not water-soluble, it may be layered as a solution in an organic solvent either alone or in combination with a water-soluble or water-insoluble polymer.

In a solid chromogenic peroxidase detection system, the hydrogen peroxide may either be in a solid shape (such as titanium hydroperoxide) or may be formed in situ. In situ, the hydrogen peroxide may be formed, for example, by glucose, ambient oxygen and glucose oxidase. Alternatively, hydrogen peroxide may be formed in situ using a dried layer formed by depositing a sodium perborate suspension in alcohol. At low pH, sodium perborate releases hydrogen peroxide spontaneously. In the presence of hydrogen peroxide and peroxidase, when released from the solid support by the hydrolase, the chromogen oxidizes and results in a visually detectable color change. This resulting color change is an indication of the presence of enzymatically active hydrolase in the sample.

This method, like other methods of the invention, can be used to simultaneously test the presence of two or more active hydrolase in a sample. Such a method uses mixtures of two or more reporter enzymes immobilized on the solid support (s) by substrate bonds that are sensitive to specific hydrolases and two or more indicators and reaction systems to generate a detectable response to each reporter enzyme. For example, it would be possible to use the method of the invention to simultaneously remove a mixture of proteases, lipases, and polysaccharidases, thereby obtaining a hydrolytic profile of a given pathogenic or disease process.

In addition, it will be apparent to those skilled in the art that reaction conditions (such as choice of linker molecule or bridge, solid supports, pH, buffer capacity, buffer identity, salts) of the process of the invention can be modified and controlled to increase the specificity of hydrolase and in the sample. For example, it is known that certain hydrolases function at low pH and are inhibited at high pH. In contrast, other hydrolases function at high pH and are inhibited at low pH. By controlling the pH, the presence of the hydrolase can be selectively detected. In addition, it is clear to those skilled in the art that specific enzyme inhibitors can also be used in the method of the invention to increase hydrolase activity and specificity and to distinguish between different hydrolase.

The invention also relates to a method for testing for the presence of an enzymatically active hydrolase in a sample, the method comprising placing the sample in a test apparatus comprising a first and a second solid support, the first solid support having the reporter enzyme immobilized therewith in such a way that the reporter enzyme is released. hydrolase and a second solid support not in contact with the first solid support having an immobilized indicator thereon that is sensitive to a detectable change due to the reporter enzyme action, wherein the sample is placed in a test apparatus so that the sample contacts the first and a second solid support such that each reporter enzyme released by the activity of the hydrolase present in the sample is allowed to diffuse through the sample to the second solid support; it is observed whether the indicator undergoes a detectable change, the detectable change being an indication of the presence of an enzymatically active hydrolase in the sample.

The method of the invention may also be used to test for the presence of any known hydrolytic enzyme. Such hydrolytic enzymes, i.e. hydrolases, include, but are not limited to: proteases (or interchangeably) proteinases, peptidases, lipases, nucleases, homo-oligosaccharidases, hetero-oligosaccharidases, homo-polysaccharidases, hetero-polysaccharidases, phosphatases, sulfatases, neuraminidases and esterases. According to a preferred embodiment of the invention, the method of the invention can be used to test for the presence of proteases including but not limited to: aspartic acid proteases, serine proteases, thiol proteases, metalloproteases, acid proteases and alkaline proteases.

The reporting or signaling enzyme of the method may be any signal generating enzyme not subject to inactivation by any agent in the sample, including inactivating hydrolysis by the hydrolase activity present in the sample. Such enzymes include, but are not limited to: peroxidase, phosphatase, oxidoreductase, dehydrogenase, transferase, isomerase, kinase, reductase, deaminase, catalase, urease, and glucuronidase. At the same time, peroxidases such as horseradish peroxidase are suitable reporter enzymes.

The reporter enzyme is immobilized on the first solid support, i.e., on an insoluble polymeric material, i.e., an inorganic or organic matrix, gel, or resin, in such a way that the reporter enzyme is released from the solid support by the enzymatically active hydrolase to be tested. The reporter enzyme may be attached directly to the first solid support if the solid support is a hydrolase substrate, or alternatively, the reporter enzyme may be immobilized on the first solid support using a linker molecule, which is the substrate for the enzymatically active hydrolase being detected. Such linker molecules include, but are not limited to: proteins, hydrocarbons, lipids, peptides, esters, and nucleic acids. The particular linker molecule used to attach the reporter enzyme to the solid support depends on which hydrolase is detected and the selection in the present case does not cause difficulties to those skilled in the art. The immobilization of the reporter enzyme on the first solid support is accomplished by conventional methods known to those skilled in the art.

In this method of the invention, the indicator is attached to a second solid support that is not in contact with the first solid support. Cellulose, agarose, dextran, poly17 acrylate, polyacrylamide or derivatives thereof, chitin, sepharose, oxirane, acrylic beads, polymeric dialdehyde, starch, collagen, keratin, elastin, bovine powder are suitable first and second carriers of the invention without limitation skin, bacterial cell wall peptidoglycan or fragments thereof, nylon, polyethylene terephthalates, polycarbonates and controlled pore glass. The immobilization of the visual indicator on the second solid support is accomplished by conventional methods known in the art.

The indicator may be any chemical that undergoes a detectable change due to the reaction or as a result of the culmination of reactions occurring when an enzymatically active hydrolase is present in the sample. The resulting detectable change in the indicator is an indication that the enzymatically active hydrolase is present in the sample. Preferred indicators are visual indicators, and in particular chromogenic indicators, i.e. those in which the color change is a visual change, including the coloring of otherwise colorless material under the action of a reporter or signaling enzyme when released from the solid support by an enzymatically active hydrolase.

As visual indicators, chromogenic indicators (i.e., chromogens) and other substances having a similar effect may be used. Preferred chromogenic indicators for the peroxidase-like reporter systems of the invention are hydrogen peroxide and a chromogen including, but not limited to, one of the following: guaiac, 2-2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid, tetramethylbenzidine, phenol, 4-aminoantipyrine and 4,5-dihydroxynaphthalene-2,7-disulfonic acid A particularly preferred chromogenic indicator consists of hydrogen peroxide and guaiac, a chromogen colorless in a reduced state and dark blue in an oxidized state. the enzyme will depend on the reaction or reactions that the reporter enzyme is able to catalyze or initiate and the choice in the present case will be apparent to those skilled in the art.

When the visual indicator is a chromogenic indicator for peroxide reactions, a solid chromogenic system is used and such a system consists of hydrogen peroxide and chromogens capable of oxidizing with hydrogen peroxides in the presence of peroxidase and a solid carrier onto which the chromogen is impregnated or immobilized and dried. In this system, the chromogen may be impregnated on a suction paper or on a carrier formed by Hemoccult ^R strips, or alternatively, the chromogen may be deposited on a plastic or other thin-film sheet. According to this last embodiment, the chromogen may be layered as a solution containing a polymeric material (such as hydroxypropyl cellulose and ethyl cellulose). If the chromogen itself is water-soluble, it may be trapped in a matrix of water-insoluble material. Alternatively, if the chromogen itself is not water-soluble, it may be layered as a solution in an organic solvent either alone or in combination with a water-soluble or water-insoluble polymer. In a solid chromogenic system, hydrogen peroxide may either be in a solid state (such as titanium hydroperoxide) or may be formed in situ in a test apparatus. In the presence of hydrogen peroxide and peroxidase, when released from the solid support by the hydrolase, the chromogen oxidizes and results in a visually detectable color change. This resulting color change is an indication of the presence of enzymatically active hydrolase in the sample.

In a preferred embodiment of the method of the invention, the hydrolase to be detected is an enzymatically active aspartic protease, the method comprising placing a sample in a test apparatus comprising first and second solid supports, the first solid support being a polyacrylate having horseradish peroxidase immobilized thereon. the myoglobin molecule, which is a substrate for the aspartic protease and the second solid carrier not in contact with the first solid carrier, is a cellulose derivative on which hydrogen peroxide and guaiac are immobilized, a chromogenic substrate that is subject to horseradish peroxidase color change in the presence of hydrogen peroxide, wherein the sample is placed in a test device in such a way that the sample is contacted with both the first and second solid supports such that any horseradish peroxidase released by the enzymatically active aspartic protease agate present in the sample is allowed to diffuse through the sample to a second solid support; and observing whether the guaiac changes color, wherein the color change is an indication of the presence of an enzymatically active protease in the sample.

As noted above, those skilled in the art will appreciate that specific reaction conditions (such as linker molecule, solid carrier, pH, buffer capacity, buffer identity, salt) and specific inhibitors may be used to increase the specificity of aspartic protease. For example, to test aspartic protease at a pH of about 2.5 to 5.0, aspartic proteases can be selectively detected among numerous thiol, serine, metallo- and alkaline proteases. Furthermore, by adding metallo-protease inhibitors, thiol, serine and acidic or alkaline proteases, aspartic acid proteases can be selectively detected.

The invention also relates to a method for detecting candidiasis by testing for the presence of an enzymatically active aspartic protease in a sample, wherein:

(a) contacting the sample with a solid carrier containing an immobilized reporter enzyme released by the action of aspartic protease;

(b) the sample, after contact with the solid support, is combined with an indicator which is sensitive to a detectable change by the reporting enzyme; and

(c) any detectable change in the indicator is observed, which is an indication of the presence of an enzymatically active aspartic protease in the sample and hence candidiasis.

The reporting enzyme or labeled enzyme used in the method of the invention may be a signal inducing enzyme, i.e. an enzyme whose activity induces a visible or detectable change not subject to inactivation by any agent in the sample, including inactivating hydrolysis by any aspartic protease or any other hydrolase activity present in the sample. Such enzymes include, but are not limited to: peroxidase, phosphatase, oxidoreductase, dehydrogenase, transferase, isomerase, kinase, reductase, deaminase, catalase, urease, and glucuronidase. The selection of the appropriate reporter or marker enzyme is readily apparent to those skilled in the art. Peroxidases are suitable reporter enzymes in this method of the invention. In particular, horseradish peroxidase is a suitable reporter enzyme since horseradish peroxidase is not readily hydrolyzed by aspartic protease or many other known proteases that may be present in the sample.

The reporter enzyme is immobilized on a solid support, i.e., on an insoluble matrix, gel or resin, in such a way that the reporter enzyme is released by the action of aspartic protease. Suitable solid carriers according to the invention include, but are not limited to, cellulose, agarose, dextran, polyacrylate, polyacrylamide or derivatives thereof, chitin, sepharose, oxirane, acrylic beads, polymeric dialdehyde, starch, collagen, keratin, elastin, cowhide powder, peptidoglycan bacterial cell wall or fragments thereof, nylon, polyethylene phthalate, polycarbonate, and controlled pore glass. Suitable solid carriers according to the invention are: chitin, polyacrylate, cellulose and derivatives thereof and sepharose. The immobilization of the reporter enzyme on a solid support is accomplished by conventional methods known in the art.

The reporter enzyme can be immobilized on a solid support using a linker molecule, which is a hydrolysable substrate for an enzymatically active aspartic protease. Such linker molecules of the invention include, but are not limited to, proteins and peptides, with suitable linker molecules being proteins. When the linker molecule is a protein, preferred proteins include, but are not limited to: azocasin, casein, K-casein, immunoglobulins, hemoglobin, myoglobin, albumin, elastin, keratin, and collagen. According to a preferred embodiment of the invention, the linker molecule is K-casein, casein, hemoglobin or myoglobin.

The indicator may be any chemical that undergoes a detectable change due to the reaction or as a result of the culmination of reactions occurring when an enzymatically active aspartic protease is present in the sample. The resulting detectable change is an indication that an enzymatically active aspartic protease is present in the sample and the presence of an enzymatically active aspartic protease again indicates the presence of candidiasis.

Preferred indicators are visual indicators, and in particular chromogenic indicators, i.e. those in which the color change is a visual change, including the coloring of otherwise colorless material by a reporter or signaling enzyme when released from the solid support by an enzymatically active aspartic protease. Alternatively, the reporter enzyme may be able to catalyze the generation of a fluorescent, phosphorescent, bioluminescent, chemiluminescent, or electrochemical signal by its release from the solid support under the action of aspartic protease. Additionally, the reporter enzyme may be capable of generating other visible or detectable signals, a debris zone, agglutination, precipitation or clarification. In such cases, the indicator would be the chemicals or substrates required by the reporter or signaling enzyme to produce the desired detectable change.

As visual indicators! a variety of chromogenic indicators (i.e., chromogens) and other species having a similar effect may be used when a peroxidase is used as a reporter or signaling enzyme. Preferred chromogenic indicators of the invention include hydrogen peroxide and chromogen, including, but not limited to, one of the following: guaiac, 2-2'azino-bis (3-ethylbenzthiazol-6-sulfonic acid, tetramethylbenzidine, phenol, 4- A particularly preferred chromogenic indicator consists of hydrogen peroxide and guaiac, which is a chromogen colorless in a reduced state and dark blue in an oxidized state, and the guaiac may optionally be purified prior to use, for example by solvent extraction, for example, aminoantipyrine and 4,5-dihydroxynaphthalene-2,7-disulfonic acid. The most aqueous chromogenic indicator for each given reporter enzyme depends on the reaction or reactions that the reporter enzyme is able to catalyze or initiate and the choice in the case is obvious to those skilled in the art. either liquid chromogen or solid chromogen system.

The invention also relates to a method for detecting Trichomonas vaginalis by testing for the presence of an enzymatically active thiol protease in a sample, wherein:

(a) contacting the sample with a solid carrier comprising an immobilized reporter enzyme released by the action of an enzymatically active thiol protease;

(b) the sample, after contact with the solid support, is combined with an indicator which is sensitive to a detectable change by the reporting enzyme; and

(c) any detectable change in the indicator is observed, which is an indication of the presence of an enzymatically active thiol protease in the sample and therefore Trichomonas vaginalis.

As with the methods described above, the reporting enzyme or signaling enzyme used in the method of the invention may be a signal-inducing enzyme, i.e. an enzyme whose activity induces a visible or detectable change not subject to inactivation by any agent in the sample, including inactivating hydrolysis by any hydrolase activity present. in the sample. Such reporter enzymes include, but are not limited to, peroxidase, phosphatase, oxidoreductase, debydrogenase, transferase, isomerase, kinase, reductase, deaminase, catalase, urease, and glucuronidase. The selection of the appropriate reporter or marker enzyme is readily apparent to those skilled in the art. Peroxidases, in particular horseradish peroxidase, are suitable reporter enzymes in this method of the invention.

The reporter enzyme is immobilized on a solid support, i.e., on an insoluble matrix, gel or resin, in such a way that the reporter enzyme is released from the solid support by the action of an enzymatically active thiol protease. Suitable solid carriers according to the invention include, but are not limited to, cellulose, agarose, dextran, polyacrylate, polyacrylamide or derivatives thereof, chitin, sepharose, oxirane, acrylic beads, polymeric dialdehyde, starch, collagen, keratin, elastin, cowhide powder, peptidoglycan bacterial cell walls or fragments thereof, nylon, polyethylene terephthalates, polycarbonates and controlled pore glass. Suitable solid carriers according to the invention are: chitin, polyacrylate, cellulose and derivatives thereof and sepharose. The immobilization of the reporter enzyme on a solid support is accomplished by conventional methods known in the art.

The reporter enzyme may be immobilized on a solid support using a linker molecule, which is a hydrolyzable substrate for an enzymatically active thiol protease. Such linker molecules of the invention include, but are not limited to, proteins and peptides, with suitable linker molecules being proteins. When the linker molecule is a protein, preferred proteins include, but are not limited to: azocasin, casein, K-casein, immunoglobulins, hemoglobin, myoglobin, albumin, elastin, keratin, and collagen. In a preferred embodiment of the invention, the linker molecule is K-casein, casein, hemoglobin or myoglobin.

The indicator may be any chemical that undergoes a detectable change due to the reaction or as a result of the culmination of reactions occurring when an enzymatically active thiol protease is present in the sample. The resulting detectable change is an indication that an enzymatically active aspartic acid protease is present in the sample and the presence of an enzymatically active acid thiol protease again indicates the presence of Trichomonas vaginalis.

Preferred indicators are visual indicators, and in particular chromogenic indicators, i.e. those in which the color change is a visual change, including the coloring of otherwise colorless material by the reporter or signaling enzyme when released from the solid support by the enzymatically active aspartic protease. Alternatively, the reporter enzyme may be able to catalyze the generation of a fluorescent, phosphorescent, bioluminescent, chemiluminescent, or electrochemical signal by its release from the solid support by a thiol protease. Additionally, the reporter enzyme may be capable of generating other visible or detectable signals, a clotting zone, agglomeration, precipitation or clarification. In such cases, the indicator would be the chemicals or substrates required by the reporter or signaling enzyme to produce the desired detectable change.

A variety of chromogenic indicators (i.e., chromogens) and other substances having a similar effect can be used as visual indicators when peroxidases are used as a reporter or signaling enzyme. Preferred chromogenic indicators of the invention include hydrogen peroxide and chromogen, including, but not limited to, one of the following: guaiac, 2-2'azino-bis (3-ethylbenzthiazoline-6-sulfonic acid, tetramethylbenzidine, phenol, 4-aminoantipyrine and A particularly preferred chromogenic indicator consists of hydrogen peroxide and guaiac, which is a chromogen colorless in a reduced state and dark blue in an oxidized state, and the guaiac may optionally be purified before use, for example by solvent extraction. the indicator for each given reporter enzyme depends on the reaction or reactions that the reporter enzyme is able to catalyze or initiate and the choice in the present case is obvious to the person skilled in the art.As mentioned above, if the visual indicator is a chromogenic indicator, either liquid chrora or solid chromogen system.

Similar to aspartic protease assays, some reaction conditions and specific inhibitors may be used to increase the activity and specificity of thi protease. For example, inhibitors (such as pepstatin to inhibit aspartic acid proteases, soybean trypsin to try tripsin, ethylenediaminetetraacetic acid or other chelating agents to inhibit metal10-proteases, or any of the known naturally occurring inhibitors of non-thiol protein proteases) may be added to test system so that only thiol proteases function and can therefore be detected. In addition, by testing at a pH of about 7.4, many proteases will be active, but aspartic proteases will be inactivated.

It is important to note that the reporting enzyme release technology of the invention can also be used to detect the presence or absence of a hydrolase inhibitor in a sample. Numerous biological processes, including, for example, blood pressure regulation, blood clotting, bacterial replication involve the use of very specific, carefully modulated hydrolases. In addition, it is known that, for example, numerous drugs, pesticides and herbicides function by inhibiting specific hydrolases. Under certain circumstances, it is highly desirable to determine, for example, the concentration of a therapeutic hydrolase inhibiting drug in the blood or to determine the presence of potential contamination of a hydrolase inhibitor by pesticides in production. In these cases it is therefore necessary to detect the hydrolase inhibitor rather than the hydrolase itself.

The invention therefore also relates to a method for testing for the presence of a target hydrolase inhibitor in a sample, (a) contacting the sample with a target hydrolase and a solid support, wherein the reporter enzyme is released immovably on the support when released by the target hydrolase. hydrolase inactivated by the presence of inhibitor, (b) combining the sample with the indicator upon contact with the target hydrolase and the solid support with the indicator, the indicator being sensitive to a detectable change by a reporter enzyme, (c) observing whether the indicator is undergoing a detectable change; this detectable change being an indication of the absence of a target hydrolase inhibitor in the sample.

To detect the presence of the hydrolase inhibitor in the sample, a defined amount of target hydrolase is included in the assay system, and the assay involves determining the ability of the sample to inhibit the target hydrolase. In the absence of a target hydrolase inhibitor in the sample, the target hydrolase releases the reporter enzyme from the solid support, causing a detectable change in the indicator. Conversely, if a target hydrolase inhibitor is present in the sample, the target hydrolase is inhibited, the reporter enzyme is not released from the solid support, and no detectable change or response is generated in the indicator. There is no need for the inhibitor in the sample to completely inhibit the added hydrolase added to the system. It is only required that there is a sufficient degree of inhibition of the target hydrolase to produce a noticeable difference in the expected detectable response.

The method of the invention can be used to test for the presence or absence of any known hydrolytic enzyme inhibitor including, but not limited to: protease inhibitors (or interchangeably) proteases, peptidases, lipases, nucleases, homo-oligosaccharidases, hetero-oligosaccharidases, homo-polysaccharidases , hetero-polysaccharidases, phosphatases, sulfatases, neuraminidases and esterases. According to a preferred embodiment of the invention, the method of the invention can be used to test for the presence of protease inhibitors including, but not limited to: aspartic protease inhibitors, serine protease inhibitors, thiol protease inhibitors, metalloprotease inhibitors, acid protease inhibitors and alkaline protease inhibitors. In another preferred embodiment, the method of the invention can be used to test for the presence of aspartic acid protease inhibitors, such as pepstatin ovomacroglobulin, haloperidol, transit state mimetics, U-81749, H-261, MV7-101, A-75925, A-76928 and A-7OO3. U-81749, H-261, MV7-101, A-75925, A-76928 and A-7OO3 are experimental drugs recently described in the literature. According to yet another preferred embodiment of the method of the invention, the presence of pepsatine as an aspartic protease inhibitor is detected.

According to the method of the invention, the target hydrolase includes, but is not limited to: proteases, proteinases, peptidases, lipases, nucleases, homo-oligosaccharidases, hetero-oligosaccharidases, homo-polysaccharidases, hetero-polysaccharidases, phosphatases, sulfatases, neuraminidases and the like. esterases. The particular target hydrolase used in the process depends on which inhibitor is being detected and the choice in the present case is readily apparent to those skilled in the art. For example, if the inhibitor of interest is pepstatin, the target hydrolase used in the above assay system is aspartic acid protease.

The reporting enzyme or signaling enzyme used in the method of the invention may be any signal-inducing enzyme, i.e. an enzyme whose activity induces a visible or detectable change that is not inactivated by any agent in the sample, including inactivating hydrolysis by the target hydrolase incorporated into the assay system. . Such enzymes include, but are not limited to: peroxidase, phosphatase, oxidoreductase, dehydrogenase, transferase, isomerase, kinase, reductase, de27 aminase, catalase, urease, and glucuronidase. The selection of the appropriate reporter or signaling enzyme will be readily apparent to those skilled in the art. Peroxidases and, in particular, horseradish peroxidase are suitable reporter enzymes in this method of the invention.

The reporter enzyme is immobilized on a solid support, i.e., on an insoluble matrix, gel or resin, in such a way that the reporter enzyme is released from the solid support by the target hydrolase, unless the target hydrolase is inactivated by the presence of the inhibitor. Suitable solid carriers according to the invention include, but are not limited to, cellulose, agarose, dextran, polyacrylate, polyacrylamide or derivatives thereof, chitin, sepharose, oxirane, acrylic beads, polymeric dialdehyde, starch, collagen, keratin, elastin, cowhide powder, peptidoglycan bacterial cell wall or fragments thereof, nylon, polyethylene terephthalates, polycarbonates and controlled pore glass.

The reporter enzyme may be immobilized on a solid support using a linker molecule, which is a hydrolyzable substrate for an enzymatically active target protease. Such linker molecules of the invention include, but are not limited to: proteins, carbohydrates, lipids, peptides, esters, and nucleic acids. The particular linker molecule used to attach the reporter enzyme to the solid support depends on which target hydrolase has been added to the assay system and the choice in each particular case is obvious to those skilled in the art.

The indicator may be any chemical that undergoes a detectable change due to the reaction or as a result of the culmination of reactions taking place if the enzymatically active target hydrolase is not inactivated by the presence of the inhibitor in the sample. Preferred indicators are visual indicators, and in particular chromogenic indicators, i.e. those in which the change in color is a visual change, including the coloring of otherwise colorless material by the reporter or signaling enzyme when released from the solid support by the enzymatically active target hydrolase. the target hydrolase is not inactivated by the presence of inhibitor in the sample.

A variety of chromogenic indicators (i.e. chromogens) and other substances having a similar effect may be used as visual indicators. Preferred chromogenic indicators of the invention for reporter enzyme-like peroxidases include hydrogen peroxide and chromogen, including, but not limited to, one of the following: guaiac, 2'-2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid, tetramethylbenz) an especially preferred chromogenic indicator consists of hydrogen peroxide and guaiac, which is a chromogen colorless in a reduced state and dark blue in an oxidized state, as described above, such as phenol, 4-aminoantipyrine and 4,5-dihydroxynaphthalene-2,7-disulfonic acid. described, if the visual indicator is a chromogenic indicator, either a liquid chromogenic system or a solid chromogenic system may be used.

An apparatus for carrying out a method for detecting the presence of an enzymatically active hydrolase in a sample according to the invention consists of a box defined at least in part by a first and a second opposing wall with surfaces inwardly spaced therefrom, the first wall and / or the second wall being translucent. material; from a reporter enzyme immobilized on a solid support on an inwardly facing surface of the first or second wall such that the reporter enzyme is released by the action of hydrolase;

an indicator contained on an inwardly facing surface of the first and second walls, wherein the indicator is a substance susceptible to the change detected by the reporting system;

and a hole in the sample insertion box.

According to the invention, the box is preferably flat and large enough to be easily held by hand. The chamber in the box is preferably flat and at the same time shallow, its width and length being considerably greater than its depth, which is substantially constant. Preferably, the chamber is shallow enough to allow spontaneous wetting of the chamber walls with the sample to achieve maximum contact between the sample and the dry reagent coatings on the top and bottom surfaces. This is particularly important when the reagent coatings are on the top and bottom surfaces of the chamber. In such cases, a small constant distance between these surfaces also minimizes the diffusion distance diffused by the reagents on the opposite surface on which the visual indicator is applied to arrive at the indicator.

Within these considerations, the depth of the chamber is not critical to the invention and may vary. In most cases, a chamber with a depth of 0.076 to 1.27 mm, preferably 0.127 to 0.381 mm, will give the best results. For each given depth, the lateral dimensions of the chamber (i.e., the distance between the side walls) will define the sample size that the test apparatus can accommodate and are not otherwise important, except that they define the shape and size of the visible test surface on the test surface. Thus, the lateral dimensions are intended to provide a test area large enough to be visible and yet small enough to allow the chamber to be completely filled with a sample of reasonable size. The sample size will vary depending on the type of sample and its source and the sampling method. In typical designs, a side area of the chamber of about 0.1 to 10 cm ² , or preferably about 0.3 to 3 cm ^{2, is considered} . The chamber's internal volume for typical designs will vary similarly, and for most samples approximately 3 μΐ to 300 μΐ will be most convenient and comfortable.

The assay device of the invention can be used to test for the presence of any known hydrolytic enzyme, without limitation: proteases, proteinases, peptidases, lipases, nucleases, homo-oligosaccharidases, hetero-oligosaccharidases, homo-polysaccharidases, hetero -polysaccharidases, phosphatases, sulfatases, neuraminidases and esterases. According to a preferred embodiment of the invention, the assay device can be used to test for the presence of proteases including, but not limited to: aspartic protease, serine protease, thiol protease, metalloprotease, acid protease, and alkaline protease. In another preferred embodiment, the assay device may be used to test for the presence of homo-oligosaccharidases, hetero-oligosaccharidases, homo-polysaccharidases, hetero-polysaccharidases including, but not limited to, chitinases, amy30 baths, cellulases, and lysozyme.

The reporting or labeling enzyme according to the method may be any signal generating enzyme, that is, an enzyme whose activity yields a detectable change not subject to inactivation by any agent in the sample, including inactivating hydrolysis by the hydrolase activity present in the sample. Such enzymes include, but are not limited to, peroxidase, phosphatase, oxidoreductase, dehydrogenase, transferase, isomerase, kinase, reductase, deaminase, catalase, urease, and glucuronidase. At the same time, peroxidases such as horseradish peroxidase are suitable reporter enzymes.

The reporter enzyme is immobilized on the first solid support, i.e. on an insoluble polymeric material, i.e., an inorganic or organic matrix, gel, or resin, in such a way that the reporter enzyme is released from the solid support by the enzymatically active hydrolase to be tested. Suitable solid carriers according to the invention include, but are not limited to, cellulose, agarose, dextran, polyacrylate, polyacrylamide or derivatives thereof, chitin, sepharose, oxirane, acrylic beads, polymeric dialdehyde, starch, collagen, keratin, elastin, cowhide powder, pepti bacterial cell wall doglycan or fragments thereof, nylon, polyethylene terephthalates, polycarbonates, and controlled pore glass. The immobilization of the visual indicator on the first solid support is accomplished by conventional methods known in the art.

The reporter enzyme may be attached directly to the first solid support, or alternatively, the reporter enzyme may be immobilized on the first solid support using a linker molecule that is a hydrolyzable substrate for the enzymatically active target hydrolase. Such linker molecules of the invention include, but are not limited to: proteins, carbohydrates, lipids, peptides, esters, and nucleic acids. The particular linker molecule used to attach the reporter enzyme to the first solid support depends on which target hydrolase is detected and the choice in each particular case is obvious to those skilled in the art.

In the test device of the invention, the indicator is immobilized on a second solid support that is not in direct contact with the first solid support. Cellulose, agarose, dextran, polyacrylate, polyacrylamide or derivatives thereof, chitin, sepharose, oxirane, acrylic beads, polymer dialdehyde, starch, collagen, keratin, elastin, cowhide powder include, but are not limited to, suitable solid carriers according to the invention. , bacterial cell wall peptidoglycan or fragments thereof, nylon, polyethylene terephthalates, polycarbonates and controlled pore glass. The immobilization of the visual indicator on the first solid support is accomplished by conventional methods known in the art.

The indicator may be any chemical that undergoes a detectable change due to the reaction or as a result of the culmination of reactions occurring when an enzymatically active hydrolase is present in the sample. This detectable change is an indication that the enzymatically active hydrolase is present in the sample. Preferred indicators are visual indicators, and in particular chromogenic indicators, i.e. those in which the color change, including the coloring of otherwise colorless material under the action of a reporter or marker enzyme, is released from the solid support by the enzymatically active hydrolase to be detected. Alternatively, the reporter enzyme may be able to catalyze the generation of a fluorescent, phosphorescent, bioluminescent, chemiluminescent, or electrochemical signal by its release from the solid support by the action of aspartic protease. Additionally, the reporter enzyme may be capable of generating other visible or detectable signals, a clotting zone, agglomeration, precipitation or clarification. In such cases, the indicator would be the chemicals or substrates required by the reporter or marker enzyme to produce the desired detectable change.

A variety of chromogenic indicators (i.e., chromogens) and other substances having a similar effect can be used as visual indicators. Preferred chromogenic indicators for the peroxidase reporter systems of the invention are hydrogen peroxide and a chromogen including, but not limited to, one of the following: guaiac, 2-2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid, tetramethylbenzidine, phenol, 4- aminoantipyrine and 4,5-di-hydroxynaphthalene-2,7-disulfonic acid A particularly preferred chromogenic indicator consists of hydrogen peroxide and guaiac, which is the chromogenic in the reduced state and the dark blue in the oxidized state. it will depend on the reaction or reactions that the reporter enzyme is able to catalyze or initiate and the choice in the present case will be apparent to those skilled in the art.

When the visual indicator is a chromogenic indicator for peroxide reactions, a solid chromogenic system is used, and such a system consists of hydrogen peroxide and a chromogen capable of oxidizing hydrogen peroxides in the presence of peroxidase and a solid carrier to which the chromogen is impregnated or immobilized and dried. In this system, the chromogen may be impregnated on a suction paper or on a carrier formed by Hemoccult ^R strips, or alternatively, the chromogen may be deposited on a plastic or other thin-film sheet. According to this last embodiment, the chromogen may be layered as a solution containing a polymeric material (such as hydroxypropyl cellulose or ethyl cellulose). If the chromogen itself is water-soluble, it may be trapped in a matrix of water-insoluble material. Alternatively, if the chromogen itself is not water-soluble, it may be layered as a solution in an organic solvent either alone or in combination with a water-soluble or water-insoluble polymer.

In this particular chromogen system, the hydrogen peroxide may either be in a solid state (such as titanium hydroperoxide) or may be formed in situ in a test apparatus. Alternatively, hydrogen peroxide can be generated in situ by superimposing a sodium perborate suspension in alcohol on the inner face surfaces of the test device. At low pH values, sodium perborate releases hydrogen peroxide spontaneously. In the presence of hydrogen peroxide and peroxidase (e.g. horseradish peroxidase), when released from the first solid support by hydrolase, the chromogen is oxidized and results in a visually detectable color change. As mentioned above, this resulting change in color is an indication of the presence of enzymatically active hydrolase in the sample.

It will be apparent to those skilled in the art that reaction conditions (such as choice of linker molecule or bridge, solid supports, pH, buffer capacity, buffer identity, salts) of the assay device of the invention can be modified and controlled to increase the specificity of the hydrolase and which may be present in the sample. In addition, those skilled in the art will recognize that specific enzyme inhibitors may also be added to the claimed assay devices to increase hydrolase activity and specificity and to distinguish between different hydrolase in the sample.

The test device is provided with an inlet for introducing the sample through which the sample is inserted into the chamber. Preferably, the entrance is in the same wall by which changes in the visual indicator are observed, i.e. in the translucent wall. The inlet has a shape adapted to the transfer device that is used to transport the sample from its source, and the inlet can therefore be varied to suit the different types of transfer devices that can be used. Examples of transfer devices are syringes, pipettes, swabs and examination mirrors. Other devices are known to those skilled in the art. In general, a circular inlet is appropriate, although for transfer devices such as swabs, the inlet may have a sharp edge against which the transfer device rubs to facilitate scraping of the sample.

Preferred embodiments of the test device include additional measures that further promote liquid penetration to fill the chamber and thereby bring all reagents into contact with the sample. One such measure is the vents in the chamber to allow air to escape. The vents are located at reasonable distances from the inlet to achieve the maximum surface area of the sample wetted surface. For test equipment incorporating positive and negative controls within the chamber at positions horizontally facing the test area, the vents shall be arranged to ensure that the sample reaches both controls and fills them to prevent any false or erroneous readings. As will be discussed further below, one preferred arrangement is the location of the test area between the control areas such that the positive and negative control areas do not meet at the common interface although they have a common interface with the test area. In this arrangement, the inlet opening is most preferably located in the wall directly above the test area and one vent is above each of the two control areas or near their outer perimeter, thereby ensuring that the test area is filled first and then both control areas.

Another measure to promote fluid migration in preferred embodiments of the invention is to position surfactants along the inner surface of the chamber. The agent may be on the top and / or bottom surface of the chamber and may be incorporated in the form of a dried solution in a carrier matrix comprising mostly a laminate or coating on the surface. In some cases, the laminate also contains one or more reagents involved in the reactions. In other cases, the surfactant is only a functional component of the laminate.

Surfactants are useful in water-based samples, as biological samples tend to be. Suitable surfactants are those which can be obtained in solid form and a wide variety of surfactant agents can be used. These substances are generally detergents, wetting or emulsifying agents and differ widely in their chemical structure and electronic nature, including anionic, cationic, zwitterionic and nonionic. Examples are alkylalkoxysulfates, alkylaryl sulfonates, glycerol fatty acid esters, lanolin-based derivatives, polyoxyethylene alkylphenols, polyoxyethylene amines, polyoxyethylene fatty acids and esters, polyoxyethylene fatty alcohols and esters, poly (ethylene glycol 1) fatty acids and esters, polyoxyethylene fatty acids and esters, polyoxyethylene fatty acids and esters, / polyoxyethylene condensates and block polymers, sorbitan fatty acid esters, sulfodates and succinates, alkyl glucosides and cholic acid derivatives. Trade names of some of these are, for example, Lubrol, Brij, Tween, Tergitol, Igepal, Triton and Teepol.

The formation of a solid laminate of both the indicator and the reagents is possible by depositing the laminate in liquid form followed by drying or otherwise converting it into a solid shape. For example, the liquid form of the substance may be a solution, suspension, or uncured state of the substance, and the solid state operation may thus be solvent evaporation or cure of the substance. The substance may be combined with additive materials for one or more purposes, such as:

(1) to facilitate the application of liquid to the surface by modifying the viscosity of the liquid; (2) to help create a continuous smooth solid layer that remains uniform and does not disintegrate or granulate over time due to the application of additional layers; they are to be applied to it or to form a solvent-soluble layer which does not dissolve the layers applied underneath or all of these objectives simultaneously.

Suitable additives to serve one or all of these purposes are soluble polymeric materials. Examples are cellulose and various cellulose derivatives with substituents appropriately selected to achieve the desired solubility. For test devices intended for aqueous or water-based samples, the indicator laminate preferably comprises an indicator trapped in a matrix of solid material that is insoluble in water. This prevents the indicator from migrating from the laminate and away from the translucent surface. Or, however, the indicator itself is insoluble in water and itself constitutes a continuous laminate that remains intact.

In embodiments of the invention where both the positive and negative control indicators are present in the test apparatus, one or more additional reagents are included in each control. These additional agents are either incorporated into one of the existing laminates in the horizontally defined portion of the laminate or applied as a separate vertically adjacent laminate above the horizontally defined portion of the existing laminate. Due to their position in the chamber, these additional reagents define control areas which are horizontally separated from each other and from the test area.

The choice of a suitable positive and negative control reagent depends on the hydrolase targeted by the overall assay, the type of visual indicator used to detect the hydrolase, and whether the reagent is to serve as a positive or negative control. Using known chemicals, the choice of a suitable reagent will in most cases be obvious to those skilled in the art. For example, the negative control reagent may be a sample of hydrolase, hydrolase analogue, or any other species with a concurrent effect that initiates and induces a reaction or a sequence of reactions that culminate in a detectable change in the indicator. The laminate containing this reagent will be either on the upper or lower surface of the chamber, provided that the reagent does not initiate or induce a detectable change while the sample is present, but will do this independently of the presence or absence of enzymatically active hydrolase in the sample. The negative control reagent may be a similarly inhibitory agent as a denaturing, inhibiting or otherwise inactivating agent that prevents or blocks the reaction or sequence of reactions, thereby preventing a detectable change from occurring regardless of whether or not the enzymatically active hydrolase is present in the sample. .

Both controls are activated when the sample is introduced into the test equipment. In some cases, this is most effectively achieved by placing control reagents in the laminate on the same surface as the laminate (s) containing the second reagent (s). Otherwise, best results are obtained when the control reagents are placed in a laminate on the opposite side of the chamber to the second reagent (s), so that the control reagent and the other reagents are separated by an air gap. In preferred embodiments, test area control areas include all components and reagents used in the test area with the addition of control reagents either incorporated into the horizontally aligned sections of one or more test laminates used in the test area or deposited as separate laminates above such horizontally aligned sections. To achieve sharp interfaces for control areas and to prevent activation or deactivation of the test area by control reagents, it is often advantageous to create imperfections in the laminate at the interface to separate the control areas from the test area to minimize or eliminate lateral diffusion of control reagents from their respective control agents. areas. These impurities may be in the laminate along the upper and / or lower surface.

As mentioned, the controls are preferably activated by the sample itself. This can conveniently be achieved by arranging the control areas in the extension of the test area, which occupy a position in the same chamber of the test facility where communication between the individual areas is enabled without hindrance. According to a preferred embodiment, where both positive and negative control areas are incorporated, the control areas are separated from each other by the test area between them. Filling all areas with a single application is made possible by arranging the sample inlet port and vents as described above. Since the detectable changes or their absence are observable through the translucent wall of the test device, identification of the positive and negative areas is conveniently achieved by marking on the outside of the test device.

The translucent wall may be of a material that is inert and sufficiently rigid to hold the indicator laminate and yet translucent enough to detect a change in the indicator when it occurs. Translucent or transparent, preferably non-absorbent materials may be used: transparent materials are more preferred. Examples of polymeric materials suitable for use herein are polyethylene terephthalates (such as Mylar ^R ) and polycarbonates (such as Lexan ^R ). The opposite (i.e., bottom) wall of the device may similarly be made of a translucent or transparent material, although it may also be of a matt material, since viewing both test results and positive and negative controls is required from only one side of the test device. If the bottom wall is translucent, the detection of changes in the test area and both control areas can be facilitated by printing or coating any side of the bottom wall with color or reflective material to enhance color contrast.

The test device (box) can be made in many ways. The sheets of polymeric material may be laminated together with respective recesses to define the shape of the chamber and the sample introduction opening and vents. The depth of the chamber as well as its shape and lateral dimensions are then defined by the thickness of the central sheet, while the location of the openings is determined by the top sheet. The indicator and reagent coatings may be applied to the topsheet and / or the backsheet as desired prior to assembly of the sheets into the laminate. The sheets can then be joined together by conventional means, such as by heat setting or by means of an adhesive.

A particularly preferred method of molding the test device is to use a single sheet of transparent or otherwise translucent polymer material, extruded mechanically or chemically to produce a crimp or recess of constant depth in the inner surface of the chamber. The insert is in one half of the sheet, while the sample introduction and wetting holes are in the other half. The indicator and reagent coatings are deposited at the respective locations of the sheet, and the half with the holes is then folded over the other half to form a closed chamber and to achieve proper alignment of the areas representing the top and bottom surfaces of the chamber. The front surfaces of the sheet are joined as in the laminate of the previous paragraph.

A preferred method of joining the two halves together is by means of heat or pressure sensitive water or solvent based adhesives. The adhesive may be confined to the peripheral areas of the chamber to prevent contact with the test reagents, or may cover the entire inner surface of the chamber and then be applied prior to application of the indicator coat and reagents. In the latter case, suitable adhesives are translucent, inert, wettable and otherwise compatible with the layers applied thereto. There are many types of suitable adhesives for this application, and the most appropriate choice varies between systems depending on the layers to be applied.

The invention also relates to a test device for testing the presence of candidiasis by testing for the presence of an enzymatically active aspartic protease in the sample, the test device comprising a box defined at least in part by a first and a second opposed wall with surfaces therebetween. and / or the second wall is of a translucent material; from a reporter enzyme immobilized on a solid support on an inwardly facing surface of the first or second wall such that the reporter enzyme is released by the action of hydrolase;

an indicator contained on an inwardly facing surface of the first and second walls, wherein the indicator is a substance susceptible to the detected change induced by the reporting system;

and a hole in the sample insertion box.

The reporting or signaling enzyme of this embodiment may be any signal generating enzyme not subject to inactivation by any agent in the sample, including inactivating hydrolysis by the hydrolase activity present in the sample. Such enzymes include, but are not limited to: peroxidase, phosphatase, oxidoreductase, dehydrogenase, transferase, isomerase, kinase, reductase, deaminase, catalase, urease, and glucuronidase. At the same time, peroxidases such as horseradish peroxidase are suitable reporter enzymes.

The reporter enzyme is immobilized on the first solid support, i.e., on an insoluble matrix, gel, or resin, in such a way that the reporter enzyme is released from the solid support by the action of aspartic acid protease. The reporter enzyme can be immobilized on a solid support using a linker molecule, which is a hydrolyzable link, which is a substrate for aspartic acid protease. In this assay device, such linker molecules are proteins and peptides, with suitable linker molecules being proteins. When the linker molecule is a protein, preferred proteins include, but are not limited to: azocasin, casein, K-casein, immunoglobulin, hemoglobin, myoglobin, albumin, elastin, keratin, and collagen. In a preferred embodiment of the invention, the linker molecule is K-casein, casein, hemoglobin or myoglobin. In the test device of the invention, the indicator is immobilized on a second solid support that is not in direct contact with the first solid support. Suitable solid carriers according to the invention include, but are not limited to, cellulose, agarose, dextran, polyacrylate, polyacrylamide or derivatives thereof, chitin, sepharose, oxirane, acrylic beads, polymeric dialdehyde, starch, collagen, keratin, elastin, cowhide powder, peptidoglycan bacterial cell wall or fragments thereof, nylon, polyethylene phthalate, polycarbonate, and controlled pore glass. The immobilization of the visual indicator on the first solid support is accomplished by conventional methods known in the art.

The indicator may be any chemical that undergoes a detectable change due to the reaction or as a result of the culmination of reactions occurring when an enzymatically active aspartic protease is present in the sample and the presence of an enzymatically active aspartic protease then indicates the presence of candidiasis. Preferred indicators are visual indicators, and in particular chroraogenic indicators, i.e. those in which the color change is a visual change, including the coloring of otherwise colorless material by the reporter or marker enzyme when released from the solid support by the enzymatically active aspartic protease detected. .

A variety of chromogenic indicators (i.e., chromogens) and other substances having a similar effect can be used as visual indicators. Preferred chromogenic indicators according to the invention, when the releasable reporter enzyme used is peroxidase, are hydrogen peroxide and a chromogen including, but not limited to, one of the following: guaiac, 2-2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid, tetramethylbenzidine) , a phenol, 4-aminoantipyrine and 4,5-dihydroxy-naphthalene-2,7-disulfonic acid A particularly preferred chromogenic indicator consists of hydrogen peroxide and guaiac, which is a chromogen in the reduced state and dark blue in the oxidized state.

When the visual indicator is a chromogenic indicator for peroxidases or pseudoperoxidases, a solid chromogenic system is used and such system consists of hydrogen peroxide and chromogens capable of oxidizing hydrogen peroxides in the presence of peroxidase and a solid carrier on which the chromogen is impregnated or immobilized and dried. In this system, the chromogen may be impregnated on a suction paper or on a carrier formed by Hemoccult ^R strips, or alternatively the chromogen may be deposited on a plastic or other thin-film sheet. In this latter embodiment, the chromogen may be layered as a solution containing a polymeric material (such as hydroxypropyl cellulose or ethyl cellulose). If the chromogen itself is water-soluble, it can be bound in a matrix of water-insoluble material. or, if the chromogen itself is not water-soluble, it may be layered as a solution in an organic solvent either alone or in combination with a water-soluble or water-insoluble polymer.

In this solid chromogenic system, hydrogen peroxide may either be in a solid state (such as titanium hydroperoxide) or may be formed in situ in a test apparatus. In the presence of hydrogen peroxide and peroxidase, when released from the first solid support by an enzymatically active aspartic protease, the chromogen is oxidized, resulting in a visually detectable color change. As noted above, this resulting color change is an indication of the presence of an enzymatically active aspartic protease in the sample, and the presence of an enzymatically active aspartic protease indicates the presence of candidiasis.

The invention also relates to a test device for testing a sample for the presence of candidiasis by testing for the presence of a target hydrolase inhibitor in the sample, the test device comprising a box defined at least in part by a first and a second opposed wall with surfaces therebetween. the second wall being of a translucent material; from a target hydrolase susceptible to inactivation by the presence of an inhibitor; from a reporter enzyme immobilized on a solid support on an inwardly facing surface of the first or second wall such that it is released by the target hydrolase unless inhibited by the presence of an inhibitor; the indicator contained on the inwardly directed detector enzyme is a target hydrolase of the surface of the first and second walls, the indicator being a substance sensitive to the detected change induced by the reporting system; and a hole in the sample insertion box.

To detect the presence of the hydrolase inhibitor in the sample, a defined amount of target hydrolase is incorporated into the assay device either immediately prior to the assay or more preferably in the manufacture of the assay device. Performing the assay involves determining the ability of the sample to inhibit the target hydrolase. In the absence of a target hydrolase inhibitor in the sample, the target hydrolase releases the reporter enzyme from the solid support, causing a detectable change in the indicator. Conversely, if a target hydrolase inhibitor is present in the sample, the target hydrolase is inhaled, the reporter enzyme is not released from the solid support, and no detectable change or response is produced in the indicator. There is no need for the inhibitor in the sample to completely inhibit the added hydrolase added to the system. It is only required that there is a sufficient degree of inhibition of the target hydrolase to produce a noticeable difference in the expected detectable response. Positive and negative control elements of the assay device illustrate the occurrence of completely inhaled target hydrolase and free inhibition target hydrolase.

The sensitivity of this enzyme release technology can be adjusted as desired by introducing defined amounts of the target hydrolase into the assay device. Similarly, if desired, the exposure time of the target hydrolase to the inhibitor in the sample can be controlled physically or chemically by separating the target hydrolase from the immobilized reporter enzyme. For example, it is possible to control the access time of the target hydrolase to the immobilized reporter enzyme by coating the reporter enzyme in the time release matrix with a coating of 1 pH or other controlled release material. Alternatively, the target hydrolase may be present at a site of a chemical form that is readily accessible to any inhibitor present in the sample. Thus, before it reaches the immobilized reporter enzyme, the target hydrolase may be exposed to the inhibitor for a sufficient period of time to inhibit it. Since accessibility to the immobilized reporter enzyme can be controlled over time, test results may indicate the presence of slow-acting inhibitors.

The assay device of the invention may be used to test for the presence or absence of any known hydrolytic enzyme inhibitor including, but not limited to: protease inhibitors (or interchangeably) proteases, peptidases, lipases, nucleases, homo-oligosaccharidases, hetero-oligosaccharaseases, homo-polysaccharidases, hetero-polysaccharidases, phosphatases, sulfatases, neuraminidases and esterases. According to a preferred embodiment of the invention, the assay device of the invention is used to test for the presence of protease inhibitors including, but not limited to: aspartic protease inhibitors, serine protease inhibitors, thiol protease inhibitors, metallo protease inhibitors, acid protease inhibitors and alkaline protease inhibitors. According to another preferred embodiment, the assay device of the invention is used to test for the presence of aspartic protease inhibitors such as pepstatin, ovomacroglobulin, haloperidol, transit state mimetics, U-81749, H-261, MV7-101, A-75925, A-76928 and A-7OO3. U-81749, H-261, MV7-101, A75925, A-76928 and A-7OO3 are experimental drugs recently described in the literature. According to yet another preferred embodiment of the test device according to the invention, the presence of pepstatin as an aspartic acid protease inhibitor is detected.

According to this assay device of the invention, the target hydrolase includes, but is not limited to: proteases, proteinases, peptidases, lipases, nucleases, homo-oligosaccharidases, hetero-oligosaccharidases, homo-polysaccharidases, hetero-polysaccharidases, phosphatases, sulfatases, neuraminidases and esterases. The particular target hydrolase used in the process will depend on which inhibitor is detected and the choice in the present case will be apparent to those skilled in the art. For example, if the inhibitor of interest is pepstatin, the target hydrolase used in the above assay system is aspartic acid protease. In addition, appropriate concentrations of the target hydrolase, geometric location of the target hydrolase in the assay device, and appropriate time and controlled release technologies and matrix are known to those skilled in the art.

As with other assay devices of the invention, the reporting enzyme or signaling enzyme used in this particular assay device may be a signal-inducing enzyme, i.e. an enzyme whose activity causes a detectable change not subject to inactivation by any agent in the sample, including inactivating hydrolysis by the target hydrolase. incorporated into the test system. Such enzymes include, but are not limited to: peroxidase, phosphatase, oxidoreductase, dehydrogenase, transferase, isomerase, kinase, reductase, deaminase, catalase, urease, and glucuronidase. The selection of the appropriate reporter or signaling enzyme will be readily apparent to those skilled in the art. In this test device according to the invention, suitable reporter enzymes are peroxidases and especially horseradish peroxidase.

The reporter enzyme is immobilized on a solid support, i.e., on an insoluble matrix, gel or resin, such that the reporter enzyme is released by the action of the target hydrolase, unless the target hydrolase is inhibited by the presence of an inhibitor in the sample. Suitable solid carriers according to the invention include, but are not limited to, cellulose, agarose, dextran, polyacrylate, polyacrylamide or derivatives thereof, chitin, sepharose, oxirane, acrylic beads, polymeric dialdehyde, starch, collagen, keratin, elastin, cowhide powder, peptidoglycan bacterial cell walls or fragments thereof, nylon, polyethylene terephthalates, polycarbonates and controlled pore glass.

As explained above, the reporter enzyme can be immobilized on a solid support using a linker molecule, which is a hydrolyzable substrate for an enzymatically active target hydrolase. Such linker molecules of the invention include, but are not limited to: proteins carbohydrates, lipids, peptides, esters, and nucleic acids. The particular linker molecule used to attach the reporter enzyme to the first solid support depends on which target hydrolase is detected and the choice in each particular case is readily apparent to those skilled in the art.

The indicator may be any chemical that undergoes a detectable change due to the reaction or as a result of the culmination of ongoing reactions, unless the target hydrolase is inactivated by the presence of an inhibitor in the sample. Preferred indicators are visual indicators, and in particular chromogenic indicators, i.e. those in which the change in color is a visual change, including the coloring of otherwise colorless material by the reporter or signaling enzyme when released from the solid support by an enzymatically active target hydrolase. inhibitor in the sample.

A variety of chromogenic indicators (i.e., chromogens) and other substances having a similar effect can be used as visual indicators. Preferred chromogenic indicators of the invention, when the releasable reporter enzyme used is peroxidase, are hydrogen peroxide and a chromogen including, but not limited to, one of the following: guaiac, 2-2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid, tetramethylbenzidine) , phenol, 4-aminoantipyrine and 4,5-dihydroxynaphthalene-2,7-disulfonic acid A particularly preferred chromogenic indicator consists of hydrogen peroxide and guaiac, which is a chromogen colorless in a reduced state and dark blue in an oxidized state. As described above, if the visual indicator is a chromogenic indicator, either a liquid or solid chromogenic system may be used.

It follows from the discussion of the structure of a hydrolase assay device that its construction and preferred embodiment are fully applicable to a candidiasis assay device for testing for the presence of an enzymatically active aspartic protease and as an assay device for the presence of an inhibitor. target proteases.

Although it is not intended to limit the invention to any particular design of a test device, the following figures illustrate, not to scale, how such a test device may be constructed.

FIG. 1 is a perspective view of the support structure of the test device prior to application of the indicator and reagents and prior to closure of the chamber. The carrier structure consists of one sheet 11 of a relatively rigid, transparent chemically inert plastic with a fold line 12 that defines a fold dividing sheet in two halves, a lower half 13 and an upper half 12 having the same length and width. The lower half 13 has a composite shaped indent having a central circle 15 and rectangular recesses in the middle, a first rectangular recess 16 and a second rectangular recess 17. In the upper half 14 there are three openings, including the central opening 18. which serves as the sample entry port and the two side openings, the first vent 19 and the second vent 20. The first vent 19 and the second vent 20 are circular and the central vent 18 is circular one sharp edge for easy scraping of the sample from the swab used as a sampling device. The apertures are positioned such that when the plastic is folded according to the folding groove 12 and the upper half 14 contacts the lower half 13, the central opening 18 is above the center of the indentation circle 15 and the first vent 19 is above the first rectangular recess 16 and the second vent 20 is above the second square recess 17 at their outer edge. The first rectangular recess 16 and the second rectangular recess 17 represent the areas of positive and negative control of the test device.

There may be many variations of the test apparatus of Figure 1. The lower half 13 and the upper half 14 may have different lengths or widths for various reasons. The only critical feature is that the indentations in the lower half 13 and the holes in the upper half 14 are positioned relative to the fold line 12, that the holes and indentations are in the correct position if both halves fold according to the fold line 12. In another example, the first rectangular recess 16 and the second rectangular recess 17 in the lower half 13 of the structure may terminate in circular (or semicircular) regions flush with the first vent 19 and the second vent 20 in the upper half 14. The first vent 19 and the second vent 20 may have any shape. It is a fact that if the vents have a different shape than the central sample entry opening 18, it is advantageous for the user not to mistake where to introduce the sample.

Fig. 2 is a cross-sectional view of the test apparatus of Fig. 1 showing the chamber 31 in cross-section after coating and after folding and gluing of the two halves. The inner surface of the transparent polymer lower half 13 is coated with a first adhesive 32 and the inner surface of the transparent polymer upper half 14 is coated with a second adhesive 33. Directly beneath the second adhesive layer 33 is a layer comprising a visual indicator 34 and below it is a reagent layer 35 It is noted that the visual indicator layer 34 and the reagent layer 35 may occupy the entire length and width of the chamber and surround the central opening 18 for sample introduction. and interfere with all areas of the chamber.

The test area and the control area of the chamber are delimited by the horizontal placement of the coatings on the lower half 13 of the chamber. The negative control reagent is contained in a first coating 36 that occupies the lower surface of the first rectangular recess 16 of the chamber (see Figure 1) and the positive control reagent is contained in a second coating 37 similarly situated in the second rectangular recess 17. Alternatively, the control reagents may be located on the upper surface of the chamber rather than on the lower surface. This is indeed beneficial for some tests. A portion of the lower surface below the central aperture 18 of the chamber (see FIG. 1) is coated with a layer 3.8 which may contain the additional reagent used in the test reaction or may not contain the reagent at all. Viewed from the top of the closed test device, the circular test area 41 has a square negative control area 42 at the sides and a positive square control area 43 at the sides. The three segments of the first coating 36, the second coating 37, and the layer 38 may be separated by gaps, or a first break 44 and a second break 45 to slow down or minimize diffusion between them, or to prevent contact between these segments. Similar breaks may also be between the visual indicator layer 34 and the reagent layer 35, directly above the gaps of the first break 44 and the second break 45. The breaks in the visual indicator layer 34 and reagent layer 35 further prevent diffusion of control components or other reagents from the control areas into the test area. In addition to any reagents present, all inward surfaces of reagent 35, first coating 36, second coating 37, and layer 38 may additionally comprise a wetting agent or detergent to promote rapid and complete spillage of the sample along the top and bottom surfaces to fill the chamber. In some cases, the same effect is achieved by a layer of protein.

In some embodiments of the invention, reagents tend to degrade due to prolonged exposure to air or air humidity. In the device shown in FIG. 2, this is prevented by a thin sheet 46 of material that is impermeable to both moisture and air. The thin sheet 46 covers the central sample insertion hole 18 and both vents and seals the interior of the chamber against the environment until the test device is ready for use, whereupon the sheet is easily peeled off. For materials particularly sensitive to water and air, it may also be appropriate to place a waterproof and air impermeable sheet at the bottom of the test device where the thin sheet 46 may be permably attached or peeled off. Additional protection against moisture and air can be achieved by placing the device in a bag that completely encloses the device.

As mentioned above, all the dimensions of the apparatus of Figures 1 and 2 may vary as well as their configuration and shape. However, a typical example is a device where the carrier is Mylar 0.127 mm thick and the adhesive layer is a thin 0.051 mm thick copolymer, a 47 gap width is 0.19 mm and the test area is a circle of 7.938 mm diameter and 0.494 cm ² and a square the areas have an area of 3.175 x 1.588 mm (0.0504 cm ² ). The vents and the circular sample insertion hole in this example are circular with a diameter of 3.2 mm. The chamber content is approximately 12 μΐ.

The test apparatus of the invention is suitable for testing samples for the presence of hydrolase from a wide range of sources, including biological and other sources. Examples are body fluids such as blood, serum, plasma, urine, urethral secretions, tears, vaginal fluid, cerebral effusion, spleen fluid and saliva, as well as non-body fluids such as foodstuffs, pond and swimming water, and solid waste.

DETAILED DESCRIPTION OF THE INVENTION

I. Preparations

A. Preparation of cyanogen bromide activated solid supports

1. Materials

Solid, insoluble carriers (Sepharose 4B, chitin and Sigmacell ^R 20) [from Sigma Chemical CO.]

b. Distilled water and ice from distilled water

c. 4.0 M sodium hydroxide

d. Solid brorakyan

e. Coupling buffer (0.1 M sodium bicarbonate containing 0.6 M sodium chloride)

f. Magnetic stirrer motor and stirring bar; pH meter; exhaust hood

3. Procedure

10 ml of cold distilled water is added to 5 g of wet solid support and the mixture is cooled to 10-15 ° C. The pH of the suspension is adjusted to 10.8 with 4.0 M sodium hydroxide and 100 mg of crushed cyanogen bromide per g wet solid carrier is added. The pH was maintained at 10.8 by adding 4.0 M sodium hydroxide as needed and the temperature of the slurry increased to 18-20 ° C during the activation process. Activation is considered complete when no additional 4.0 M sodium hydroxide additives are required to maintain a pH of 10.8.

At this point, crushed ice was added to cool the reaction mixture and the suspension was filtered on a pre-cooled filter funnel. The filtrate was collected in a suction flask containing solid ferrous sulfate which reactivated the remaining cyanogen bromide and cyanide remaining in the reaction mixture. The solid support is washed with 1 L of cold distilled water and 1 L of coupling buffer for aspiration and stored as a wet paste at 4 ° C.

B. Preparation of [kappa-kase in-HRPO] conjugates (hydrazide method)

1. Materials

a. Capa-casein and horseradish peroxidase hydrazide (HRPO) [from Sigma Chemical Co.]

b. Distilled water

c. Buffers

i. 50 mM 2- [morpholine] ethanesulfonic acid (MES), pH 6.0 ii. 100 mM sodium acetate buffer, pH 4.0 containing 0.5 M sodium chloride iii. 100 mM sodium borate, pH 9, containing 0.5 M sodium chloride

d. Solid sodium periodate

e. 100 mM formaldehyde

Method

a. Periodic activation of capa-casein

20 mg of kappa casein is dissolved in 2 ml of MES buffer and 8.6 mg of solid sodium periodate is added to the solution. The mixture is incubated on the rotor for 30 minutes in the dark at room temperature. The reaction mixture is then dialyzed against about 300 ml of mM MES, pH 6.0 for about 45 minutes at room temperature, whereupon the dialysis fluid is replaced and continued for a further 45 minutes.

b. Covalent binding of HRPO to kappa casein

To the activated capacasein, 5 mg of HRPO hydrazide (approximately 175 units per 1 mg protein) was added and the mixture was incubated with stirring at room temperature for 4 hours. 200 ml of 100 mM formaldehyde are added to the mixture and incubation is continued at room temperature for an additional 30 minutes. 2 ml of 1M cold acetate buffer, pH 4.0 is added and the conjugate is allowed to precipitate for 30 minutes at 4 ° C. The pellets were removed by centrifugation, redissolved in 2 ml of 100 mM borate buffer, pH 9.0 and stored at 4 ° C before use.

C. Attaching [kappa-casein-HRPO] conjugates (hydrazide method) to cyanogen bromide activated supports

1. Materials

a. Conjugates [kappa-casein HRPO] (preparation B)

b. Distilled water c. P uf r y

i. coupling buffer (0.1M NaHCO 3 containing 0.5 M NaCl) ii. 1.0 OM Tris buffer, pH 8.0 iii. 100 mM sodium acetate buffer, pH 4.0 containing 0.5 M sodium chloride iv. 100 mM sodium borate, pH 9, containing 0.5 M sodium chloride

d. Bromkyan i d activated Sepharose 4B and Sigma 1 ^R 20 May (in case AND) e. Bromkyan i d activated Sepharose 6MB (from Sigma Chemical Co.) 2, Method a. Br o mkya n i d act Sepharose 4B and Sigmace11 ^R 20 May

Dilute 2 ml of [kappa-casein-HRPO] conjugate prepared from HRPO hydrazide as described in Preparation B in 3 ml of coupling buffer and add to 1 g of wet cyanogen bromide activated by Sepharose 4B and Sigmacell ^R. Stir the suspension continuously at room temperature for 2 hours. The solid support is washed with coupling buffer and water and 3 ml of 1.0 M Tris buffer pH 8 is added. The suspension is incubated for 2 hours at room temperature to inactivate the remaining active sites on the solid support. To remove unbound material from the solid support, wash cycles consisting of an acid buffer of pH 4.0 followed by a coupling buffer are used. The final rinse is distilled water and the wet suspension is stored at 4 ° C until use.

b. Commercially Activated Sepharose 6 MB (Sigma Chemical Co.)

The wet gel (1 g wet weight) was washed with 200 ml of 1 mM hydrochloric acid before use as above.

c. Activated chitin

The above procedure A was followed except that 500 mg of wet activated chitin was used instead of 1 g.

D. Preparation of horseradish peroxidase (HRPO) aldehyde

1. Materials

HRPO Type II 200 J / mg (from Sigma Chemical Co.)

b. 20 mM 2- (N-morpholino) ethanesulfonic acid buffer, pH 5.0

c. Solid sodium periodate

d. Biogel ^R P-30 O spherical polyacrylamide gel from Bio-Rad

2. Procedure

25.7 mg solid sodium periodate (40 mmol) is added to 30 mg HRPO in 3 mg MES buffer. The solution is incubated in the dark for 30 minutes at room temperature with continuous rotation. The solution was passed through a column of P-30 in MES buffer to remove excess sodium perorate and the stained HRPO was collected to a total volume of 3 ml. HRPO aldehyde is immediately coupled to the desired protein (hemoglobin, myoglobin or casein).

E. Binding of hemoglobin and myoglobin to cyanogen bromide-activated solid supports

1. Materials

Solid, insoluble carriers

i. Bromocyanate activated by Sepharose SMB, (from Sigma Chemical CO.) i. Sigmacell ^R 20 (as described in Preparation A)

b. Distilled water c, P uf r y:

Coupling Buffer (0.1 M sodium bicarbonate containing 0.5 M sodium chloride) i. 1.0 M Tris buffer, pH 8.0 iii. 100 mM sodium acetate buffer, pH 4.0 containing 0.5 M sodium chloride iv. 100 mM sodium borate, pH 8.0, containing 0.5 M sodium chloride

v. a 0.1% v / v solution of glutaraldehyde in water

2. Procedure

To 1 g of wet solid carrier, 100 mg of either hemoglobin or myoglobin dissolved in 5 ml of coupling buffer is added. The suspension was stirred continuously at room temperature for 2 hours. The solid support is washed with coupling buffer and water and 3 ml of 1.0 M Tris buffer pH 8 is added and the suspension is incubated for 2 hours at room temperature to inactivate the remaining active sites on the solid support. Three wash cycles consisting of an acid buffer pH 4.0 followed by a borate buffer, both containing sodium chloride, are used to remove unbound material from the solid support. For myoglobin-derivatized carriers, a final rinse with distilled water is used and the wet suspension is stored at 4 ° C until use. For carriers derived from hemoglobin, 5 ml of a 1% glutaraldehyde solution are added to the wet suspension of the above procedure and stored at 4 ° C overnight. Three wash cycles consisting of an acid buffer pH 4.0 followed by a borate buffer pH 8.0 were used to remove unbound material from the support. The final rinse is performed with distilled water and the wet suspension is stored at 4 ° C until use.

F. Binding of horseradish peroxidase (HRPO) aldehyde to carriers derivatized with hemoglobin or myoglobin. Ma y er 1 y

a. HRPO aldehyde prepared according to Preparation D

b. Hemoglobin or myoglobin derivatized solid supports prepared according to Preparation E

c. 100 mM sodium cyanogen bromide

d. 1.0 M sodium chloride

e. Distilled water. Procedure ml of HRPO aldehyde (30 mg) obtained from column P-30 in Preparation D is added to 1 g of solid carriers derivatized with hemoglo54in or myoglobin described in Preparation E. The suspension is incubated for 16 hours at 4 ° C and subsequently 4 hours at room temperature. Three wash cycles consisting of distilled water and 1.0 M sodium chloride are used to remove unbound material from the support. The solid support is then incubated with 5 ml of 100 mM cyanogen bromide overnight at 4 ° C. Final rinsing is performed with distilled water and the wet suspension is stored at 4 ° C until use.

6. Preparation of aldehyde-activated Sigmacell ^R20

1.Mat er i á1y

a. Sigmacell ^R 20 (from Sigma Chemical Co.)

b. Distilled water

c. Solid sodium periodate

d. 20 mM sodium bicarbonate pH 9.5. Method

To 1 g of Sigmacell ^R suspended in 10 ml of distilled water add solid sodium periodate (428 mg). The mixture was allowed to rotate continuously for two hours at room temperature. The granulate is removed by centrifugation, washed five times with 10 ml of distilled water and once with 10 ml of 20 mM sodium bicarbonate, pH 9.5. The resin was used immediately after preparation.

H. Covalent binding of hemoglobin and myoglobin to aldehyde-activated Sigmacell ^R20

I. Materials

a. Sigmacell ^R 20 (from Sigma Chemical Co.)

b. Distilled water

c. 100 mM sodium cyanoborohydride

d. Human hemoglobin and equine cardiac 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 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

To 1 ml of aldehyde-activated Sigmacell ^R 20 add 50 mg of either myoglobin or hemoglobin dissolved in 9 ml of sodium bicarbonate, pH 9,5. (Preparation 6). The suspension is allowed to rotate overnight at room temperature and the granules are centrifuged. The granulate is washed with 10 ml of water. 9 ml of 50 mM ethanolamine, pH 9.5, were added and the mixture was allowed to stand at room temperature for one hour. The granulate is isolated, washed successively with 10 ml of acetate buffer, Tris buffer and MES buffer respectively. 20 ml of an aqueous solution of 100 mM sodium cyanoborohydride are added and the mixture is incubated for one hour at constant temperature at room temperature, then incubated overnight at 4 ° C. After overnight incubation, the derivatized support is washed five times with 10 ml aliquots of distilled water and twice with 10 ml aliquots of MES buffer.

is separated from- 9 ml 50 mM ět - rotate 1 him - centrifugation m a

I. Covalent binding of aldehyde HR.PO to hemoglobin or myoglobin immobilized on Sigmacell ^R 20 by activated aldehyde

I. Materials

a. Hemoglobin or myogloibine derivatized Sigmacell ^R 20 (from Sigma Chemical Co.)

b. Distilled water

c. 100 mM sodium cyanoborohydride

d

i. 100 mM 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 HR.PO (Preparation D).

. Procedure mg of desalted HRPO aldehyde (Preparation D) dissolved in 2 ml MES buffer pH 5.0 is added to 1 g of Sigmacell ^R 20 hemoglobin or myoglobin derivatized (Preparation H) and the suspension is rotated for 1 hour at room temperature. followed by overnight incubation at 4 ° C. The support is isolated by centrifugation and washed twice with 10 ml aliquots of distilled water. 20 ml of 100 mM sodium cyanoborohydride are added and the suspension is incubated for 60 hours at 4 ° C. The granulate is washed successively twice with distilled water, acetate buffer, Tris buffer and MES buffer.

J. Covalent Binding of Hemoglobin to Commercial Acrylic Beads

Eupergit ^R C

1.Materials

a. Oxiracrylic beads (Eupergit ^R C, from Sigma Chemical Co.)

b. Human Hemoglobin (Type IV from Sigma Chemical Co.)

c. Buffers and Solutions i-1.0 M potassium phosphate buffer, pH 7.5 containing 0.1% sodium azide (w / v) ii. . 1.0 M sodium chloride iii, 5% (v / v) mercaptoethane no 1, adjusted to pH 8.0 containing 0.5 M sodium hydroxide 100 mM potassium phosphate buffer, pH 7.4 500 mM potassium phosphate buffer, pH 7.4 i v.

in.

vi. knows i

d.

3.5 M sodium thiocyanate Phosphate buffered saline (PBS) (0.01 M sodium phosphate, pH 7.2 containing 0.15 M NaCl) Distilled water

2. Procedure

Dissolve hemoglobin (125 mg) in a buffer, pH 7.4, containing sodium azide and incubate without stirring for 72 hours with 1.0 M phosphate 1 g of Eupergit ^R C. The mixture at room temperature.

The support was washed three times with 1.0 M sodium chloride and five times with distilled water. The carrier is mixed with 2.5 ml of mercaptoethane no preset at pH 8.0 and the suspension is allowed to stand overnight at room temperature. The beads are washed ten times with distilled water, placed in a small sintered glass funnel and washed successively with 50 ml of 0.5 M potassium phosphate buffer, pH 7.4, 0.1 M potassium phosphate buffer, pH 7.4 successively. 3.5 M sodium thiocyanate and finally a large amount of phosphate buffered saline (PBS) (0.01 M sodium phosphate, pH 7.2 containing 0.15 M NaCl). The derivatized beads were treated with 0.1% glycol aldehyde by rotation for 2 hours at room temperature, overnight at 4 ° C, and then washed with distilled water.

K. HRPO Aldehyde Covalent Binding (Preparation D) to Eupergit ^R C Hemoglobin Derivatives (Preparation J)

1. Materials:

a. HRPO Aldehyde (Preparation D)

b. Hemoglobin derivatized Eupergit ^R C (Preparation J)

c. 100 mM sodium cyanoborohydride

d. 1.0 M sodium chloride

e. Distilled water

2. Procedure mg of HRPO aldehyde (Preparation D) in 3 ml MES buffer is added to 1 g of hemoglobin derivatized Eupergit ^R C (Preparation J). The suspension is incubated at 4 ° C for 16 hours followed by 4 hours at room temperature. Three wash cycles consisting of distilled water and 1.0 M sodium chloride are used to remove unbound material from the support. The solid support is then incubated with 5 ml of 100 mM cyanoborohydride for 60 hours at 4 ° C. The final rinse is performed with distilled water and the wet suspension is stored at 4 ° C until use.

L.

Covalent binding of casein HRPO to polymeric dialdehyde

1. Material

a. 25 mg of the kappaase-n-HRPO conjugate prepared by hydrazole (preparation B) dissolved in 2 ml of 100 mM borate buffer, pH 9.0

b. Polymeric dialdehyde (from Sigma Chemical Co.) c. P uf r y:

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. 100mM formaldehyde

e. 100 mM sodium cyanoborohydride is discharged by treatment with 3 ml of MES buffer and the suspension is allowed to rotate overnight at formaldehyde temperature and the mixture is removed.

2. Procedure

Using the method described in Preparation B, 2 ml of both kappaase and n-HR.PO conjugate were prepared with one modification: aldehyde. This solution was added to 1 g of polymeric dialdehyde at room temperature for hours and incubated at 4 ° C. 200 ml of 100 mM are added to the suspension, incubated for 1 hour at room temperature, centrifuged, washed with water and resuspended in 3 ml of 100 mM ethanolamine, pH 8.5. After incubating for two hours at room temperature, the resin is washed sequentially with acetate buffer, borate buffer and water. The solid support is then incubated with 5 ml of 100 mM cyanoborohydride overnight at 4 ° C. The final wash is performed with distilled water and the wet suspension is stored at 4 ° C for use.

Binding of total casein or capa-casein labeled with HR.PO (aldehyde method) to acrylic beads Eupergit ^R C

1. Materials

a. Oxiracrylic beads (Eupergit ^R C from Sigma Chemical Co.)

b. Bovine complete casein or capa-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 containing 0.5 M NaCl iii. 100 mM Tris buffer containing 0.5 M NaCl iv. 20 mM MES buffer, pH 5.0

v. 5% (m / v) mercaptoethanol in water vi. 200 mM sodium borohydride in water

d. Distilled water

2. Procedure

c. Covalent binding of casein or casein to Eupergit® C beads

2 ml of a solution containing either complete casein or capa-casein (10 mg / ml) in 100 mM sodium bicarbonate buffer, pH 8.5 containing 0.5 M NaCl are added to 1 ml of Eupergit ^R C beads suspended in water. The mixture was incubated with rotation for 48 hours at room temperature. The granulate is collected by centrifugation and washed successively with a 9 ml aliquot of acetate and Tris buffer followed by two washes of ethanol and ml aliquot and MES buffer. 10 ml of 5% mercapto stirring is added overnight while the carrier is isolated and the mixture is incubated at room temperature. Dilute with casein by dilution and wash four times with 9 ml of MES buffer.

b. Labeling of casein immobilized on Eupergite ^R C by HR.PO

Three ml of aldehyde-activated HRP0 (Preparation D) is added to the casein-derivatized Eupergit ^R C from a) above and the mixture is incubated with rotation for 1 hour at room temperature and 80 hours at 4 ° C. The granulate is isolated by centrifugation and washed with 10 ml portions of water. An aqueous solution of sodium borohydride (5 ml) was added to the granulate and the suspension was incubated for 6 hours at room temperature. The granulate is collected by centrifugation and washed sequentially with 9 ml portions of acetate, Tris, acetate, Tris and MES buffer.

N. Covalent binding of myoginobin-HR.PO to polymeric dialdehyde

- Materials

30 mg of equine cardiac myoglobin dissolved in 2 ml of 20 mM bi carbonate buffer, pH 9.5

b. Polymeric dialdehyde (cellulose dialdehyde from Sigma Chemical Co.)

c. Buffers:

1. 50 mM ethanolamine, pH 9.5 i. 100 mM sodium borate, pH 9.0 containing 0.5 M NaCl iii. 100 mM sodium acetate buffer 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

Two ml of myoglobin solution is mixed with 1 ml of polymer dialdehyde suspension and the suspension is rotated at room temperature overnight. The resin was separated by centrifugation and washed twice with 10 ml aliquots of water. 9 ml of ethanolamine solution are added and the suspension is incubated under rotation for 1 hour at room temperature. The resin was separated by centrifugation and washed successively with 10 ml portions of acetate, Tris and MES buffers. 20 ml of sodium cyanoborohydride are added. The suspension is incubated overnight at 4 ° C. The myoglobin-derivatized polymeric dialdehyde is washed five times with 10 ml aliquots of water and twice with MES buffer.

b. Labeling of myoglobin-derived beads with aldehyde HR.PO (Preparation D)

2 ml of desalted aldehyde HR.PO (Preparation D) is added to the resin and the suspension is incubated for 1 hour with rotation at room temperature and overnight at 4 ° C. The resin is then isolated by centrifugation, washed twice with 10 ml portions of water, suspended in 20 ml of sodium cyanoborohydride and incubated for 60 hours at 4 ° C. The resin was isolated by centrifugation, washed twice with 10 ml portions of water, then successively twice with 10 ml portions of acetate / NaCl and Tris / NaCl buffers. Finally, the resin is washed with 10 ml of MES buffer and stored at 4 ° C until use.

Preparation of HR.PO labeled chitin and its use for chitinase assay

1.Materials

a. Cyanogen-activated chitin (Preparation A)

b. HRPO (type II from Sigma Chemical Co., 78 J / mg)

c. Chitinase (EC 3.2,1.14, isolated from Streptomyces grisius, commercial product of Sigma Chemical Co.)

d. P u f r y;

i. 500 mM Tris / MES buffer, pH 5.4 i. 100 mM Sodium bicarbonate coupling buffer

0.5 M NaCl 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. 1.0 M Tris buffer, pH 8.0

ABTS Assay Mix: 40 mL of 25 mg / mL ABTS (2,2'-azino-di [3-ethylbenzthiazolinesulfonic acid] diammonium salt).

a. Preparation of HRPO derivatized chitin

500 mg of cyanogen bromide activated chitin (Preparation A) is suspended in 5 ml of coupling buffer, 5 mg of HRPO is added and the suspension is stirred at room temperature for 2 hours without interruption. Solid material. Wash with distilled water and coupling buffer. 3 ml of 1.0 M Tris buffer, pH 8.0 is added to the carrier and the suspension is stirred for 2 hours at room temperature, then washed three times successively with acetate and borate buffer and finally with water.

b. The HRPO-labeled chitinase assay of 50 mq of choline is suspended

MES / Tris. Add 50 μΐ of chitinase solution (50 incubate for 4 hours to sediment chitin with 100 μΐ of the test mixture at 37 C. C. Reaction and mix 10 μl aliquot of ABTS. Release of chitinase chitin bound HRPO is detected by green flux formation.

in 200 μΐ of buffer J / ml) and centrifuge the mixture by centrifugation of the supernatant catalysed in color. Preparation of cyanogen bromide activated sepharose casein or kappa-HRPO aldehyde. Materials

a. Bromocyanate-activated Sepharose (Preparation A)

b. 10 mg / ml casein or capa-casein in coupling buffer (0.1 M sodium bicarbonate containing 0.5 M NaCl)

c. Buffers and solutions:

1. 50 mM. et. hano laminate, pH 9.5. 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 20 mM MES buffer, pH 5.0

v. 10 mM Tris buffer, pH 8.5 containing 0.5 M NaCl knows. 100 mM sodium borohydride in water

d. HRPO aldehyde (Preparation D)

2. Procedure ml of the casein or kappa casein solution is added to 1 ml of cyanogen-activated sepharose and the suspension is incubated for 2 hours with rotation at room temperature. 7 ml of ethanolamine solution are added and the mixture is incubated for 1 hour at room temperature. The resin was isolated by centrifugation and washed successively with a 9 ml aliquot of acetate and Tris buffer containing sodium chloride, followed by washing twice with 9 ml MES buffer.

3 ml of desalted HRPO aldehyde (Preparation D) was added and the mixture was rotated for 1 hour at room temperature, followed by in63 cubic overnight at 4 ° C. The resin was isolated by centrifugation and washed twice with 10 ml of water. The resin was suspended in 10 ml of sodium borohydride and incubated at room temperature for 2 hours. Finally, the resin is isolated by centrifugation and washed successively with 9 ml buffers containing 0.5 M NaCl: acetate, Tris, acetate and Tris buffer. The resin is finally washed with 9 ml of HES buffer and stored in MES buffer at 4 ° C.

Q. Covalent binding of myoglobin and bovine serum albumin to finely powdered Eupergit ^R C acrylic beads (Eupergit ^R C) and subsequent covalent labeling with horseradish peroxidase (aldehyde method).

Surgery 1: Covalent binding of myoglobin and bovine serum albumin to Eupergi acrylic beads. herein ^{R is} C ^(R Eupergit C)

M a ter e 1 y

Oxiracrylic beads (Eupergit ^R C, 150 pm beads from Sigma Chemi ca 1 Co.)

Equine cardiac myoglobin (type IV) or bovine serum albumin (from Sigma Chemical Co.)

Buffers and solutions:

1.0 M potassium phosphate buffer, Sodium (weight to volume) pH 7,5 containing 0,1 % and z i du 1.0 M NaCl 5% (by volume) mercaptoe t hano 1 set to pH 8.0 0, 5 N sodium hydroxide 1 00 M potassium phosphate puf r, PH 7.5 500 M potassium phosphate puf r, pH 7.5 3, 5 Sodium thiocyanate

i i.

i i i i v.

in.

v i ..

v i i

d.

. Phosphate buffered saline (PBS) (0.01 M sodium phosphate, pH 7.2 containing 0.15 M NaCl)

Distilled water

2. Procedure

One gram of oxiracrylic beads is broken by hand in a mortar to a fine powder. Dissolve bovine serum albumin (125 mg) in 5 ml of 1.0 M phosphate buffer, pH 7.5 containing sodium azide and add 1 g of finely ground Eupergit ^R C. The mixture is incubated without stirring for 72 hours at room temperature. rooms. By centrifugation, the support is washed three times with 1.0 M NaCl and five times with 20 ml of distilled water. The carrier is mixed with 2.5 ml of mercaptoethanol preset to pH 8.0 and the suspension is allowed to stand overnight at room temperature. The beads are washed ten times by centrifugation with distilled water and sequentially with 50 ml of the following buffers: 0.5 M Drase phosphate buffer, pH 7.5; 0.1 M dra with 1 nophosphate buffer, pH 7.5; 3.5 M sodium thiocyanate and finally large volumes of phosphate buffered saline (PBS) (0.01 M sodium phosphate, pH 7.2 containing 0.15 M NaCl).

Operation 2: Covalent labeling with horseradish peroxidase (aldehyde method)

Horseradish peroxidase aldehyde (Preparation D) is used to label the above-described myoglobin or albumin derivative-derived acrylic beads from operation 1 using procedure F.

R. Binding of myoglobin Sigmace11 ^R -20 to HRPO gutara ldehyde

1. Materials

a. Myoglobin covalently bound to cyanogen bromide-activated Sigmace 1 1u ^R -2 O (Preparation E)

b. 0.5 M MES buffer, pH 5.0

c. 1% (v / v) glautara ldehyde in water

d. HRPO Hydrazide (from Sigma Chemical Co. 200 J / mg)

e. 100 mM formaldehyde

f. 100 mM sodium cyanoborohydride

g. 0.5 M sodium chloride

2. Procedures

To 1 g of myoglobin-conjugated Sigmace11 ^R 20 is added 1 ml of glacial dehydrogenate solution and the suspension is rotated for 30 min at room temperature and washed with water. 5 mg of HRPO hydrazide in 2 ml of 100 mM Mes buffer was added to the washed resin and the suspension was rotated for 4 hours at room temperature. Add 200 μΐ of 100 mM formaldehyde solution and incubate for 30 minutes at room temperature. The resin was washed with water, suspended in 5 ml of 100 mM sodium cyanoborohydride and incubated overnight at 4 ° C. The resin was then washed with distilled water and 0.5 M NaCl and stored as a wet paste until use.

S. Preparation of guaiac solution and guaiac leaves

1. Guaiac, a hydroxypropylcellulose solution (that is guaiac ink)

Dissolve 150 g of powdered guaiac in 660 ml of warm stirred ethanol. To the resulting solution was added 1330 mL of distilled water and the resulting suspension was allowed to cool to room temperature. After 2 hours, the supernatant was decanted and the remaining suspension was collected.

250 ml of a 10% by weight solution of hydroxypropylcellulose in ethanol are mixed with an additional 250 ml of ethanol and added to the guaiac suspension. The mixture was stirred until the guaiac residue was completely dissolved.

2. Gua i akové sheets

Pipette 1 ml of the above guaiac solution onto the polyethylene side of the 254 x 254 mm MyL / polyethylene laminate (0.178 mm Mylar / 3 mL of polyethylene). The guaiac solution is spread over the leaf surface using a standard knit wire test rod and allow to dry at room temperature.

T. Preparation of Sodium Perborate Suspension (i.e., Sodium Perborate I nc) The finely divided solid sodium perborate is mixed with a sufficient volume of a 10% (by weight) solution of hydroxypropyl cellulose in anhydrous alcohol to form 1 liter of suspension.

II. Attempts

Experiment I

This experiment relates to the release of HRPO from [Sepharose-casein HRPO] and (Sepharose-kappa-casein HRPO) by an aspartic protease released into a culture of Candida albicans.

activated with cyanogen bromide) (Method

A. Materials: (Sepharose HRPO aldehyde coupling)

1. Bromocyanate-activated derivative (Pr pr and P) and metal

Sepharose 4B (Preparation A) pre-bound with casein or capa-casein subsequently labeled with HRPO aldehyde (Preparation D)

2. Asparagus protease producing a Candida albicans culture (ATCC 28366) propagated and grown in liquid culture as described in Jorn 1 of General Microbiology, 129, pp. 431-388 (1983).

Suaiak Covered Leaves (Preparation S)

Buffers and solutions of hydrogen peroxide

500 mM MES buffer, pH 6.0 P os tup y mg (wet weight) of [Sepharose-casein HRPO] conjugate (or Kapa-casein of the new derivative) is suspended in 300 μΐ either in a Candida albicans culture containing an active secreted aspartic protease or 300 μΐ of the same Candida albicans culture cooked for 20 minutes to inactivate the aspartic protease. The mixture was allowed to rotate for 15 minutes at room temperature and the suspension was centrifuged to sediment the solid conjugate and Candida albicans cells.

μΐ of the clear supernatant is mixed with 10 μΐ of hydrogen peroxide solution and 10 μΐ of MES buffer. Add 20 μΐ of each solution to the surface of a guaiac coated sheet and examine the sheet for blue coloration.

Color

I nterpretace

O No noticeable blue tint ± Possible blue color

0,25 Finest blue color visually detected

0.50 Clear blue color

1.00 Dark blue color

1.50 Blue color with an intensity of 1.0 to 2.0

2,00 Darkest blue color possible in trial. system

C. Results

A dark blue color is formed on the guaiac coated sheet at the site of addition of the Sepharosa-protein-HRPO supernatant treated with Candida albicans. No color is seen at the site to which the boiled culture-treated Sepharosapro tei n-HRPO supernatant was added.

Enzyme source

Color

Candida albicans culture 1.5

W e r e c tio n 1 t u r a 0

D. Interpretation

The active aspartic protease secreted into the growth medium by Candida albicans cells hydrolyzes the Sepharose-bound casein or kappa-casein while releasing soluble active HRPO. Soluble HRPO catalyzes the oxidation of guaiac with hydrogen peroxide, producing a blue color. Thus, blue staining on guaiac coated leaves is a convenient way to detect an aspartic protease.

Boiling the culture inactivates the aspartic protease secreted into the growth medium by Candida and 1bicans cells. Therefore, the casein bound to Sepharose or kappa-casein is not hydrolyzed and soluble active HRPO is not released from the carrier. Centrifugation of the segregated HR.PO bound to Sepharose leaves no aspartic protease isolated by HRPO in solution. The oxidation of guaiac with hydrogen peroxide is not catalysed and a barely noticeable blue color develops. The release of HRPO from the carrier is not simply the result of non-specific release of HRPO by salts or other compounds present in the growth medium.

Experiment II

This experiment relates to the release of HRPO from [Eupergit ^R C acrylic bead-casein HRPO] conjugates and [Eupergit ^R C acrylic bead-kappa-casein HRPO] by aspartic protease released into Candida culture and 1 bica ns.

A. Materials: (Eupergit ^R C activated acrylic beads) (aldehyde coupling method HRPO)

1. Oxiracrylic beads (Eupergit ^R C are treated with casein or capa-casein (Preparation M) and subsequently labeled with HRPO aldehyde (Preparation D)

2. Asparagus protease producing a Candida albicans culture (ATCC)

28366) propagated and grown in liquid culture as described in Uornal of General Microbiology, 129, pp. 431-438 (1983).

3. Guaiak-covered leaves (Preparation S)

4. Buffers and solutions

and 20 mM hydrogen peroxide

b. 500 mM MES buffer, pH 6.0

B. Procedures The mg (wet weight) conjugate of [Eupergit ^R C-casein-HRPO] (or the new equivalent kappa-casein) is suspended in 300 μΐ of either a Candida albicans culture containing secreted aspartic protease or 300 μ1 of the same Candida albicans culture that was cooked for 20 minutes to inactivate the aspartic protease. The mixture was allowed to rotate for 15 minutes at room temperature and the suspension was centrifuged to sediment the solid conjugate and Candida albicans cells.

Mix 80 μΐ of the clear supernatant with 10 μΐ of hydrogen peroxide solution and 10 μΐ of MES buffer. Place 20 μΐ of each solution on the surface of the guaiac coating sheet and investigate the formation of a blue color on the sheet.

Color

Interpretation

No appreciable blue tint ± Possible blue color

0,25 The finest blue color visually detected

0.50 Clear blue color

1.00 Dark blue color

1.50 Blue color with an intensity of 1.0 to 2.0

2,00 Darkest blue color possible in trial. system

C .. Resources

A dark blue color is formed on the guaiac coated sheet at the Eupergit supernatant addition point. ^R C-protein-HRPO treated Candida albicans. No coloration is seen at the site to which the cooked culture-treated Eupergit supernatant was added. ^R C-for thi n-HRPO.

Enzyme source Coloring

Candida albicans culture 1.5

Vařenákultura 0

D. Interpretation

The active aspartic protease secreted into the culture medium by Candida albicans cells hydrolyzes capacasein or casein bound to Eupergit ^R C to release soluble active HRPO. By centrifugation of the sediment, HRPO is bound to Eupergit ^R C and leaves only soluble aspartic protease solubilized by HRPO in solution. Soluble HRPO catalyzes the oxidation of guaiac with hydrogen peroxide, producing a blue color. Thus, the formation of a blue color on guaiac-coated leaves is a convenient way to detect and cure the protease.

Cooking the culture inactivates the aspartic protease secreted into the growth medium by Candida and 1bicans. Therefore, casein bound to Eupergit ^R C or kappa-casein is not hydrolyzed and does not release soluble active HRPO from the carrier. Centrifugation of the sedated HR.PO bound to Eupergit ^R C leaves no aspartic protease soiled by HRPO in solution. The oxidation of guaiac with hydrogen peroxide is not catalysed and a barely noticeable blue color develops. The release of HRPO from the carrier is not simply the result of non-specific release of HRPO by salts or other compounds present in the growth medium.

Experiment III

This experiment relates to the release of HRPO from [Sepharose-hemog1 ob inHR.POJ or [Sepharose-myog1 ob in-HRPO] by pepsin and an aspartic protease from Aspergillus saitoi.

A. Materials: (cyanogen bromide activated Sepharose) (HRPO aldehyde bond method)

1 · Bromocyanate-activated Sepharose 4B (Preparation A) first derivatized with covalently bound hemoglobin or myoglobin (Preparation E) and subsequently labeled with HRPO aldehyde (Preparation D and F). The conjugate was treated overnight with 100 mM sodium cyanoborohydride at room temperature and washed with 0.5 M NaCl before use.

2. Assay solutions: a. Commercially available aspartic protease (type XIII) from Aspergillus saitoi (30 mg / ml, 0.6 J / mg); b. pepsin (1 mg / ml, 2900 J / mg) and c. bovine serum albumin (BSA) (2 mg / ml in water) (all from Sigma Chemical Co.)

3. Guaiac impregnated paper in the form of commercially available Hemoccult ^R strips (from SmithKline Diagnostics)

4. Solutions and solutions

a. 0.02% (v / v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0

b. 100 mM acetate buffer, pH 4.0

Β. Procedures mg (wet weight) of [Sepharose-hemog1 ob in-HRPO] or (myog1 obine equivalent) conjugate is suspended in 75 μΐ acetate buffer, pH 4.0 and 25 μΐ of the test solution is added. The suspension is incubated for 15 minutes at room temperature and centrifuged to complete the solid phase conjugate. Add 5 μΐ of supernatant to the guaiac strip followed by 5 μΐ of hydrogen peroxide solution.

Color

O ±

0.25, 0.5, 2.0

Interpretation

No appreciable blue tint Fine blue tint possible

The finest blue color visually detected Clear blue color

Dark blue color

The darkest blue color in trial. system

C. Results

A dark blue color is formed on the guaiac coated sheet at the location where the supernatant samples containing aspartic protease from Aspergillus saitoi have been added. Only a very fine color is seen at the site to which supernatant fractions containing only BSA alone (2 mg / ml) or buffer were added.

Enzyme source

Asparagus protease Pepsin BSA

Buffer

Color

2,0

2,0

D. Interpretation

The active aspartic protease Aspergillus saitoi and pepsin and the aspartic protease from porcine stomach hydrolyzes Sepharose-bound hemoglobin or myoglobin, releasing soluble active HRPO. Centrifugation sediments the Sepharose-bound HRPO and leaves only the enzyme purified by HRPO in solution. Soluble HRPO catalyzes the oxidation of guaiac with hydrogen peroxide, producing a blue color. Thus, blue staining on guaiac-coated leaves is a convenient way of detecting active Aspergillus aspart protease or porcine pepsin.

Neither BSA nor buffer has aspartic protease activity. Thus, Seharose-bound hemoglobin or myoglobin is not hydrolyzed and soluble active HRPO is not released from the carrier. Centrifugation of the sedimented HRPO bound to Sepharose does not leave any aspartic protease with HRB stabilized in solution. The oxidation of guaiac with hydrogen peroxide is not catalysed and a barely noticeable blue color develops.

Experiment IV

This experiment relates to the release of HRPO from [Sepharose-caseinHRPO] or [Sepharose-kappa-casein-HRPO] by pepsin and an aspartic protease from Aspergillus saitoi.

A. Materials: (cyanogen bromide-activated Sepharose) (HRPO hydrazide bond method)

1. The cyanogen-activated Sepharose 4B (Preparation A) is reacted with kappa-casein-HRPO or casein-HRPO (hydrazide process) (Preparation B and C).

Commercial (mg / ml, paper)

2. Test solutions: a. Aspergillus saitoi (30 2900 J / mg) and c. Albumin (all from Sigma Chemical Co.).

3. Guaiac-impregnated strips of Hemoccult ^R available asparagus protease (6 J / mg); b. pepsin (1 mg / ml, serum (BSA) (2 mg / ml in water) as commercially available

4. Buffers and solutions

a. 0.02% (v / v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0

b. 100 mM acetate buffer, pH 4.0

B. Procedures mg (wet weight) of conjugate [Sepharose-kase i n-HR.PO] or (kappa-casein of the new equivalent) is suspended in 75 μ. 1 acetate buffer, pH 4.0 and add 25 μl! The suspension is incubated for 15 minutes at room temperature and centrifuged to remove the solid phase conjugate. Add 5 μΐ of supernatant to the guaiac strip followed by 5 μΐ of hydrogen peroxide solution.

Coloring Interpretation

O No appreciable blue tint ± Fine blue tint possible

0,25 'The finest blue color visually detected

0.5 Clear blue color

1.0 Dark blue color

2,0 Darkest blue color possible in trial. system

C. Results

A dark blue color is formed on the guaiac coated sheet at the site of addition of the supernatant samples containing aspartic protease from Aspergillus saitoi or pepsin. Only a finer color is seen at the site to which aliquots of supernatant containing only BSA alone (2 mg / ml) or buffer were added.

Enzyme source

Asparagus protease Pepsin BSA

Color

2,0

2,0 ±

Buffer ±

D, Interpretation

The active aspartic protease Aspergiilus saitoi and porcine pepsin hydrolyzes Sepharose-bound casein or capa-casein, releasing soluble active HRPO. Centrifugation sedimentes Sepharose-bound HRPO and leaves only the enzyme purified by HRPO in solution. Soluble HRPO catalyzes the oxidation of guaiac with hydrogen peroxide, producing a blue color. Thus, blue staining on guaiac-coated leaves is a convenient way to detect asparagus protease or pepsin.

Neither BSA nor buffer has aspartic protease activity. Thus, Seharose-bound casein or capa-casein is not hydrolyzed and soluble active HRPO is not released from the carrier. Centrifugation of the sedimented HRPO bound to Sepharose leaves no aspartic HRPO-solubilized protease in solution. Oxidation of the guaiac with hydrogen peroxide is not catalyzed and hardly noticeable blue stains are formed.

Attempt V

This experiment relates to the release of HRPO from [chitin-kappa-caseinHRPO] by pepsin and an aspartic protease from Aspergiilus saitoi.

A. Materials: (Bromocyanate-activated chitin) (HRPO hydrazide bond method)

1. The cyanogen-activated chitin (Preparation A) is reacted with kappa-casein-HRPO (hydrazide method) (Preparation B and O).

Assay solutions: a. Commercially available aspartic Aspergiilus saitoi (30 mg / ml, 0.6 J / mg);

protease

b. pepsin (1 mg / ml, (2 mg / ml in water)

Bovine serum albumin (BSA) (all from Sigma Chemical Co.)

3, Guemoco Impregnated Hemoccult ^R Strip Paper (by SmithKline Diagnostics)

4. Buffers and solutions

a. 0.02% (v / v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0 commercially available

b. 100 mM acetate buffer, pH 4.0

8. Procedures

Suspend 40 mg (wet weight) of the conjugate [chi-thi-n-kappa-casein-HRPO] in 75 μΐ acetate buffer, pH 4.0, and add 25 µl of the test solution. The suspension is incubated for 15 minutes at room temperature and centrifuged to remove the solid phase conjugate. Add 5 μΐ of supernatant to the guaiac strip followed by 5 μΐ of hydrogen peroxide solution.

Coloring ±

0.2 5 0.5 1.0 2.0

Interpretation

No appreciable blue tint Fine blue tint possible

The finest blue color visually detected Clear blue color

Dark blue color

The darkest blue color in trial. system

C. Results

A thorn-blue color is formed on the guaiac coated sheet in the addition of samples of the supernatant containing aspartic protease from Aspergillus saitoi or pepsin. Only barely perceptible staining is seen at the site to which aliquots of the supernatant containing only BSA alone (2 mg / ml) or buffer were added.

Enzyme source

Color

Asparagus Protease 2.0

Pepsin 2.0

BSA ±

Buffer ±

D. Interpretation

The active aspartic protease Aspergillus saitoi and porcine pepsin hydrolyze Sigmacell ^R 20 bound hemoglobin or myoglobin, releasing soluble active HRPO. Centrifugation sediments Sigmacell ^R 20 bound HRPO and leaves only the enzyme solubilized by HRPO in solution. Soluble HRPO catalyzes the oxidation of guaiac with hydrogen peroxide, producing a blue color. Thus, blue staining on guaiac-coated leaves is a convenient way to detect asparagus protease or pepsin.

Neither BSA nor buffer has aspartic protease activity. Thus, Sigmacell ^R 20 bound hemoglobin or myoglobin is not hydrolyzed and soluble active HRPO is not released from the carrier. Centrifugation of the granulated HRPO bound to Sigmacell ^R 20 leaves no aspartic protease with HRB isolated in solution. The oxidation of guaiac with hydrogen peroxide is not catalysed and a barely noticeable blue color develops.

Experiment VI

This experiment involves the release of HRPO from ^[R 2 -O-Sigmace11 the hemoglobin I. obi N-HRPO] or [S ce GMA and ^R 1 1 2 0 1 g of myo-obi-HRP n O] pepsinern and aspartic protease from Aspergillus saitoi.

A. Materials: (Bromocyanate-activated Sigmace11 ^R 2O) (HRPO aldehyde bonding method)

1. Bromocyanate-activated Sigmacell ^R 20 (Preparation A) first derivatized with covalently bound hemoglobin or myoglobin (Preparation E) and subsequently labeled with HRPO aldehyde (Preparation D-F). The conjugate was treated overnight with 100 mM sodium cyanoborohydride at room temperature and washed with 0.5 M NaCl before use.

2. Assay solutions: a. A commercially available aspartic protease from Aspergillus saitoi (30 mg / ml, 0.6 J / mg); b. pepsin (1 mg / ml, 2900 J / mg) and c. bovine serum albumin (BSA) (2 mg / ml in water) (all from Sigma Chemical Co.)

3. Suaiak impregnated paper in the form of commercially available Hemoccult ^R 4 strips. Buffers and solutions

a. 0.02% (v / v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0

b. 100 mM ace dad pu.fr, pH 4.0

B, Procedures

Suspend 40 mg (wet weight) of the [Sigmacell ^R 20 -hemogl obi n-HRPO] conjugate in 75 μΐ acetate buffer, pH 4,0 and add 25 μl of the test solution. The suspension is incubated for 15 minutes at room temperature and centrifuged to complete the solid phase conjugate. Add 5 μl. 1 supernatant per guaiac strip followed by 5 μl of hydrogen peroxide solution.

Color

Interpretation

0 No visible blue color ± Fine blue possible color 0,2 5 The finest blue color visually found 0, 5 Clear blue color 1.0 Dark blue color 2,0 The darkest all blue color possible ve Zkus. s ys t

C. Results

A dark blue color is formed on the guaiac coated sheet at the site of addition of the supernatant samples containing aspartic protease from Aspergillus saitoi or pepsin. Only a very fine color is seen at the site to which aliquots of BSA supernatant (2 mg / ml) or buffer were added.

Enzyme source

Asparagus protease

Pepsin

BSA

Buffer

Color

2,0

2,0 ±

D. Interpretation

The active aspartic protease Aspergillus saitoi and porcine pepsin hydrolyze Sigrnacell ^R 20 bound hemoglobin or myoglobin, releasing soluble active HRPO. By centrifugation of the sediment, 5 µgmacell ^R 20 is bound HRPO and leaves only the enzyme solubilized by HRPO in solution. Soluble HRPO catalyzes the oxidation of hydrogen peroxide to form a blue color. Thus, blue staining on guaiac-coated leaves is a convenient way to detect asparagus protease or pepsin.

Neither BSA nor buffer has aspartic protease activity. Thus, Sigmacell ^R 20 bound hemoglobin or myoglobin is not hydrolyzed and soluble active HRPO is not released from the carrier. Centrifugation of the sedated HRPO bound to Sigmacell ^R 20 leaves no aspartic protease solubilized by HRPO in solution. The oxidation of guaiac with hydrogen peroxide is not catalysed and hardly noticeable blue coloration is produced.

Experiment VII

This experiment relates to the release of HRPO from [Sigmace 11 ^R 20 O-kappa casein-HRPO] by pepsin and aspartic protease from Aspergillus saitoi

A. Materials: (Bromocyanate-activated Sigmacel1 ^R 20) (hydrocide bond HRPO)

1. Cyanogen-activated 3gmacell ^R 20 (Preparation A) reacted with both kapapaase and n-HRPO (hydrazide process) (Preparation B and C)

2. Assay solutions: a. Commercially available aspartic protease from Aspergillus saitoi (30 mg / ml, 0.6 J / mg); b. pepsin 2900 U / mg) and c. albumin (all from Sigma Chemical Co.)

3. Guaiac-impregnated Hemoccult ^R strip paper. P ufrya solutions

a. 0.02% (v / v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0

b. 100 mM acetate buffer, pH 4.0 (1 mg / ml, bovine serum (BSA) (2 mg / ml in water) as commercially available

Β. Progresses

It suspends ss 40 mg (wet weight) of the [Sigmacell ^R 20-1 kappa-kase and n-HRPO] conjugate in 75 μΐ acetate buffer, pH 4.0 and add ss 25 μΐ of the test solution. The suspension is incubated for 15 minutes at room temperature and centrifuged to wound the solid phase conjugate. Add 5 μΐ of supernatant per guaiac strip followed by 5 μΐ of hydrogen peroxide solution.

Coloring Interpretation 0 No visible color ± Fine blue color possible 0.25 No finer blue color visually detected 0, 5 Clear blue color 1.0 Dark blue color 2,0 The darkest blue color in trial. system

C. Results

A dark blue color is formed on the guaiac coated sheet at the site of addition of a sample of supernatant containing aspartic protease from Aspergillus saitoi or pepsin. Only a very fine color is seen at the site to which aliquots of BSA supernatant (2 mg / ml) or buffer were added.

2Source of enzyme

Asparagus protease Pepsi n

Color

BSA

Buffer

D, Interpretation

The active aspartic protease Aspergillus saitoi and porcine pepsin hydrolyze Sigmacell ^R 20 bound capa-casein to release soluble active HR.PO. The centrifugation sedimented Sagmacell ^R 20 bound to HRPO and leaves only the enzyme salted with HRPO in solution. The soluble HRPO catalysis is the oxidation of guaia to hydrogen peroxide to produce a blue color. Thus, blue staining on guaiac coated leaves is a convenient way to detect asparagus protease or pepsin.

Neither BSA nor buffer has aspartic protease activity. Thus, Sigmace 11 ^R 20 O-bound capa-casein is not hydrolyzed and soluble active HRPO is not released from the carrier. Centrifugation of the sedimented HRPO bound to Sigmacell ^R 20 leaves no aspartic HRPO-solubilized protease in solution. The oxidation of guaiac with hydrogen peroxide is not catalysed and a barely noticeable blue color develops.

Experiment VIII

This experiment concerns the release of HRPO from [Sigmacell ^R 20 kapakasein-HRPQ] (hydrazide method) or [Sigmace11 ^R2O ~ mnyogi ob in / hemog1 ob i-HRPO] (aldehyde method) by aspartic protease from Asp ergi 1 1 ussaitoi or pepsin m.

(Cyanogen bromide-activated Sigmace11 ^R2O) activated Sigmacell ^R2O (Preparation A) Let (hydrazide method) (PREPARATION B).

Alternatively, the cyanogen bromide activated Sigmacell ^R 20 (Preparation E) is derivatized and then bound to the HRPO aldehyde (Preparation D).

a. Commercially available asparagus

A. Materials:

1. cyanogen bromide react with kappa-casein-HRPO protease (1 mg / ml,

Test solutions:

Aspergillus saitoi (30 mg / ml, 0.6 J / mg); b. pepsin

2900 J / mg) and c. Bovine serum albumin (BSA) (2 mg / ml in water) (all from Sigma Chemical Co.)

3. Guaiac-impregnated paper in the form of commercially available Hemoccult ^R strips

4, Buffers and solutions

0.02% (v / v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0 b, 100 mM acetate buffer, pH 4.0

B. Procedures

Suspend 40 mg (wet weight) of the [Sigmacell ^R 20-1kapa-casein-HRPO] conjugate in 75 μΐ acetate buffer, pH 4,0 and add 25 μΐ of the test solution. The suspension is incubated for 15 minutes at room temperature and centrifuged to remove the solid phase conjugate. Add 5 μl of supernatant per guaiac strip followed by 5 μl of hydrogen peroxide solution.

Color Interpretation 0 No visible blue color ± Fine blue color possible 0, 25 The less blue color is visually detected 0.5 Clear blue bar va 1.0 Dark blue color 2,0 The darkest blue color in trial. system

C. Results

A dark blue color is formed on the guaiac by the sheet coating at the point of adding η. Aspergillus saxifluorobacteria containing psoriasis or aspirin. Only very slight discoloration is noticeable at the site to which portions of BSA supernatant (2 mg / ml) or buffer were added.

Source enzyme u

Asparagus protease

P e p 3 i n

BSA

P u f r

Color

2,0

2,0 ±

+ ·

D. Interpretation

The active aspartic protease Aspergillus saitoi and porcine pepsin hydrolyze Sigmacell ^R 20 bound capa-casein to release soluble active HRPO. Centrifugation sediments Sigmacell ^R 20 bound HRPO and leaves only the enzyme soluble HRPO in solution.

Soluble HRPO catalyzes the oxidation of guaiac with hydrogen peroxide, producing a blue color. Thus, blue staining on guaiac-coated leaves is a convenient way to detect asparagus protease or pepsin.

Neither B5 A nor the buffer has the activity of a parasite protease. Thus, Sigmacell ^R 20 bound capacasein is not hydrolyzed and soluble active HRPO is not released from the carrier. Centrifugation of the sedimented HRPO bound to Sigmacell ^R 20 leaves no aspartic HRPO-isolated aspartic protease in solution. The oxidation of guaiac with hydrogen peroxide is not catalysed and a barely noticeable blue color develops.

IX

This experiment involves the release of HRPO from [Sigmacell ^R2O the hemoglobin-1-obi ηn HR.PO] or [S i GMA ce 1 1 ^R O ~ 2 g of 1 mnyo obi N-HR AFTER] by aspartic protease released into Candida albicans culture.

A. Materials: (aldehyde-activated Sigmace 11 ^R 2 O) (aldehyde bond method HR PO). Aldehyde-activated Sigma 1 ^R 2 O (Preparation 6) is first dehydrated with covalently bound hemoglobin or myoglobin (Preparation H) followed by HRPO aldehyde (Preparation A and P)

2. Candida albicans (ATCC 28356) culture producing aspartic protease propagated and grown in liquid culture as described in Journal of General Microbiology 129, pp. 431-38 (1983).

3. Guaiak-impregnated paper (Addition S)

4. Buffers and solutions

and 20 mM hydrogen peroxide

b. 500 mM MES buffer, pH 6.0

B. Procedures

Suspend 10 mg (wet weight) of the [Sigmacell ^R -20-protein-HRPO] conjugate in 300 μΐ of either a Candida albicans culture containing secreted aspartic protease or 300 μΐ of the same Candida albicans culture that has been boiled for 20 minutes to inactivate the aspartic protease. The mixture was allowed to rotate for 15 minutes at room temperature and the suspension was centrifuged to sediment the solid conjugate and Candida albicans cells.

Mix 1 μl of the clear supernatant with 10 μl of hydrogen peroxide solution and 10 μl of MES buffer. Place 20 μl of each solution on the surface with a guaiac liner and examine for blue staining.

Coloring ±

0.25 0.5 1.0 1, 5 2.0

Interprstace

No noticeable blue tint Fine blue tint possible

No finer blue color visually detected Clear blue color

Thorn blue color

Blue color with an intensity of 1.0 to 2.0 Darkest blue color possible in the trial. system

C. S ubject

A dark blue color is formed on the guaiac coated sheet at the point where Sepida-HRPO-treated Candida albicans has been added. There is no discoloration at the site where the cooked culture-treated Sepharose-HRPO protein has been introduced.

Source enzyme u

Color

Candida albicans culture 1.5

Cooked culture 0

D, Interpretation

Asparagus protease secreted into the culture medium by Candida albicans cells hydrolyzes the protein bound to Sigmacell ^R 20 and releases soluble, active HRPO. Centrifugation sediments the HRPO bound to Sepharose and leaves only the soluble aspartic protein in the solution. Soluble HRPO catalyzes the oxidation of guaiac with hydrogen peroxide, producing a blue color. Thus, the formation of blue staining on guaiac-coated leaves is a convenient way to detect an aspartic protease.

Cooking culture inactivates the aspartic protease secreted into the culture medium by Candida albicans cells. Therefore, the protein bound to Sigmacell ^R 20 is not hydrolyzed and soluble active HRPO is not released from the carrier. Centrifugation of the granulated HRPO bound to Sigmecell ^R 20 leaves no aspartic protease isolated by HRPO in solution. The oxidation of guaiac with hydrogen peroxide is not catalysed and a barely noticeable blue color develops. The release of HR.PO from the carrier is not simply the result of non-specific release of HRPO from salts or other compounds present in the culture medium.

Attempt X

This is an attempt to test a kappa-kasei η-H R PO] and a p arag o v o u o t A. Mat e r i á 1 v *

1. A commercially available polymer di a 'is reacted with kappa-casein-HRPO B and L).

HRPO for [polymeric dialdehyde from Aspergillus s.

H R P0 is a hydrazide process from Sigma Cemical Co. (hydrazide process) (Preparation of

Assay solutions: a. Commercially available aspartic protease from Aspergillus saitoi (30 mg / ml, 0.6 J / mg); b. pepsin (1 mg / ml, 2900 J / mg) and c. bovine serum albumin (BSA) (2 mg / ml in water) (all from Sigma Chemical Co.)

3. Guaiac-impregnated paper in the form of commercially available Hemoccult ^R strips

4. Buffers and solutions

a. 0.02% (v / v) hydrogen peroxide in 200 mM phosphate buffer, pH 7.0

b. 100 mM acetate buffer, pH 4.0

B. Procedures

Suspend 40 mg (wet weight) of the conjugate [polymeric and 1-aldehyde-kappa-casein-HRPO] in 75 μl! acetate buffer, pH 4.0 and add 25 μΐ of the test solution. The suspension is incubated for 15 minutes at room temperature and centrifuged to remove the solid phase conjugate. Add 5 μΐ of supernatant per guaiac strip followed by 5 μl of hydrogen peroxide solution.

Color Interpretation 0 No visible blue color ± Fine blue color possible 0,2 5 The finest blue color visually detected 0, 5 Clear blue color 1.0 Dark blue color 1, 5 Blue color with intensity of 1.0 to 2.0 2,0 Darkest blue color maybe in and try. system

C .. Results

A dark blue color is formed on the guaiac coating sheet at the point of addition of the pernatant containing the p and r and g from the Aspergillus s or i or p p s i n. Only a very fine color is seen at the site to which aliquots of BSA supernatant (2 mg / ml) or buffer were added.

Source e n z ymu

Asparagus protease Peps i n

Color

BSA

P u f r

D. I n t e r e r a t c

The active aspartic protease Aspergillus saitium and porcine pissin hydrolyzes polymer dialdehyde-bound capa-casein to release soluble active HRPO. Centrifugation sediments the polymer dialdehyde-bound HRPO and leaves only the enzyme HR.PO solubilized in solution. Soluble HR.PO catalyzes the oxidation of guaiac with hydrogen peroxide, producing a blue color. The formation of a blue color on guaiac-coated leaves is therefore a convenient way to detect an asparagus protease or pepsin.

Neither BSA nor buffer has aspartic protease activity. Thus, polymeric dialdehyde-bound capacasein is not hydrolyzed and soluble active HRPO4 is not released from the carrier. Centrifugation of the sedimented HR.PO bound to the polymeric dialdehyde leaves no aspartic protease in the solution with HRB.PO. The oxidation of guaiac with hydrogen peroxide is not catalysed and a barely noticeable blue color develops.

Experiment XI

This experiment relates to the release of HR.PO from [polymeric dialdehyde n-HRPO] by an aspartic protease from Candida albicans.

A. Materials: (polymeric dialdehyde) (HP PO aldehyde binding method).

1. Commercially available polymeric dialdehyde from Sigma Chemical Co. is reacted with myoglobin (Preparation N) followed by HR.PO aldehyde (Preparation N).

2. Candida albicans culture (ATCC 28366) producing an aspartic protease propagated and grown in liquid culture as described in Journal of General Microbiology 129, pp. 431-438 (1983)

3. Guaiac-impregnated paper (Preparation S). P u fr y a solution y a. 20 mM hydrogen peroxide

b. 500 mM MES buffer, pH 6.0

B .. · Procedures

It suspends with 10 mg (wet weight) conjugate [polymer di and 1-aldehyde-myogline-HRPO] in 300 μΐ of either a Candida albicans culture containing secreted aspartic protease or 300 μ 300 of the same Candida albicans culture that has been boiled for 20 minutes to inactivate the aspartic protease. The mixture was allowed to rotate for 20 minutes at room temperature and the suspension was centrifuged to sediment the solid conjugate and C cells and d and d and a and b and c.

μΐ of the clear supernatant is mixed with 10 μΐ of hydrogen peroxide solution and 10 μΐ of MES buffer. Place 20 μΐ of each solution on the surface with a guaiac liner and examine for blue staining

2, a b and rve ηί In terpretace

0 Be z blue color + Fine blue color possible 0, 25 No finer blue color visually detected 0, 5 Clear blue color 1, o Dark blue color 1.5 M o d rá color of intensity 1.0 to 2.0 2,0 No j t m and higher blue color possible in try. system

C. Results

A dark blue color is formed on the guaiac coating sheet at the point where the polymeric diahydroxy-myoglobin-HRPO from p r a c a c d a d d a d b a c a n s has been added. At the point where the cooked culture-treated di and 1-hydroxydeogin-HRPO were introduced, none of the aforesaid is evident.

2dr o j e n y y

Color

CultureCandidaalbicans 1,5

Vařenákultura O

D. I n t e r e r a t c

The active aspartic protease secreted into the growth medium by Candida albicans cells hydrolyzes the polymeric dialdehyde myoglobin and releases soluble, active HRPO. By centrifugation, HRPO bound to polymeric dialdehyde sits and leaves only the soluble aspartic protease HRPO in solution. The soluble HRPO kata88 lyses the oxidation of guaiac with hydrogen peroxide, thereby forming a blue weld in it.

Cooking culture inactivates the aspartic protease secreted into the culture medium by Candida albicans cells. Thus, the polymer-bound dialdehyde-bound protein is not hydrolyzed and soluble active HRPO is not released from the carrier. Centrifugation of the sediment in the HRPO bound to the polymeric dialdehyde leaves no aspartic protease with 1 µbis of HRPO in solution. The oxidation of guaiac by hydrogen peroxide is not catalyzed and hardly noticeable blue coloration is produced. The release of HRPO from the carrier is not simply the result of non-specific release of HRPO by salts or other compounds present in the growth medium.

Experiment XII

This experiment concerns the release of HRPO from both [Sepharose SMB-Kapaka and n-HRPO] vaginal fluid crystals:

. Normal vaginal fluid

2. Normal vaginal fluid to which a culture of Candida albicans has been added

3. Vaginal fluid from women with clinically diagnosed vulvovaginal candidiasis

A. Materials: (Commercially available cyanogen bromide activated Sepharose 6 MB) (HRPO hydrazine bond method)

1. Sepharose 6 MB-kappa-casein-HRPO conjugate (Preparation C). Vaginal fluid samples obtained on standard dacron swabs. The samples are frozen until ready for use. The samples are thawed and centrifuged in specially prepared tubes to allow the undiluted vaginal fluid to be extracted from the swab. The entire sample from each swab is tested. If necessary, make up to 75 μΐ final volume.

Where indicated, 25 μΐ of Candida albicans culture (see experiment 1) is added to vaginal fluid samples of clinical vaginal candidiasis.

3. Guaiak-impregnated filter paper adds rain and μΐ to the sample.

Candida albicans (vi: obtained from women without (commercially available Hermoccult ^R strips). Buffers and solutions

a. 6 m M hydrogen peroxide

b. 25 mM mM glycyl buffer, pH 3.0

Suspension of kappa-casein-HRPO1 in the liquid. The suspension suspension is centrifuged at mg (wet weight) of conjugate [Sepharose75 μΐ of treated or unprocessed vaginal is incubated for 15 minutes at room temperature and sedimented with solid conjugate and other particles of μ 1 clear with uper natan tuss and placed on the surface with a guaiac. Then add 5 μl of hydrogen peroxide solution and examine the leaf for blue staining.

Interpretation

O ±

0.25, 0.5, 2.0

No visible blue Possible fine blue

The finest blue tint is visually apparent

Clear blue color

Dark blue color

The darkest pale blue color possible in the trial. system

C. Results

A dark blue color is formed on the guaiac coating sheet at the site where the supernatant of vaginal fluid from women infected with vaccinia candidiasis has been added. No staining occurs at the site where the supernatant of the vaginal fluid from control women has been applied. A strong blue color is also formed on the guaiac-impregnated paper at the location where the supernatant of vaginal fluid from normal women has been found, supplemented with medium from cultivated Candida albicans.

Sample number

Color

7 7 5 6 candidiasis 2,0 7701 candidiasis 2,0 7749 candidiasis 2,0 7760 candidiasis 2,0 77 5 3 candidiasis 2,0 7 7 8 5 candidiasis 2,0 7779 candidiasis 2,0 77 99 candidiasis 2,0 7 768 no r has 1 η í 1.0 7770 normal 0, 0 7744 normal 00 7745 n o r r i n i 0, 0 77 5 0 no r has 1 η í 0, 0 7 7 6 1 norm 1 ηí 0, 0 0001 normal 0, 0 0001 normal + Candida culture 2,0 77 4 7 normal + Candida culture 2,0 7 7 4 7 normal + Candida culture 1.0 7 7 4 0 normal + Candida culture 2,0 7 7 5 7 normal + Candida culture 2,0

D. Interpretation

Vaginal fluid from women with clinically proven vulvovaginal candidiasis contains an aspartic protease that hydrolyzes at pH 3.0 in 15 mi release of soluble, active

Sepharose 6 MB bound kappa-casein at room temperature at HRPO. The centrifuged sedimented on Sepharose 6 MB bound HRPO radiates only the released salt of 1 HRB in solution. Hermoccult ^R commercial strip oxidisers catalyzed by soluble HRPO produce a blue color. Thus, blue staining on the Hercocult® ^R strips is a convenient way to detect Candida-derived aspartic protease in the vaginal fluid and therefore the vagagaginal candidiasis.

Vaginal fluid from normal women without vulvovaginal candi91 diasis does not contain an aspartic protease and does not hydrolyze on Sepharose 6 MB bound capa-casein at pH 3.0 for 15 minutes at room temperature and does not release soluble, active HRPO. By centrifugation dispersed on Sepharose 6 MB bound HRPO leaves undiluted solubilized HRPO in solution. In the absence of the HRPO catalyst, there is no oxidation on the commercial Hermoccult® strips and no blue color is produced. The lack of blue staining on the Hermoccult ^R strips therefore provides a convenient way to detect in aspirin-deficient aspartic protease-induced vaginal fluid and, therefore, women who do not have vulva gin α candidiasis in normal women.

Finally, when the medium from Candida culture and culture is added to the vaginal fluid of Norwegian women without a vaccinating candidiasis, hydrolysis to Sepharose S MB bound capacasein occurs at pH 3.0 for 15 minutes at room temperature and released soluble, active HRPO. Centrifugation sedimented on Sepharose 6 MB bound HRPO leaves the released solubilized HRPO in solution. The oxidation of commercial Hermoccult® strips catalyzed by soluble HRPO produces a blue color. The formation of blue staining on Hermoccult® strips demonstrates that release of aspartic protease into cultured Candida albicans cell media can be easily and conveniently detected, although it is added to vaginal fluid from normal women.

Experiment XIII

This experiment concerns the release of HRPO from [Eupergit ^R C-myog1 obin -HRPO] and [SigmacellR 20-myog1 obin.HRPO] vaginal fluid samples:

1. Normal vaginal fluid

2. Vaginal fluid from women with clinically agglutinated vulvovagine α1 η í candidiasis

A, Materials: (Bromocyanate-activated Sigmace11 ^R20 -myog1 ob in-HRPOpreparation A and I) and (Eupergit ^R C-myog1 obin-HRP0-Preparation Q) (HRPO aldehyde bond method)

1. Finely ground oxiranacrylic beads bound to myogin-aluminium-HRPO aldehyde (Preparation Q)

2. Sigmacell ^R 20 (cyanogen bromide activated, Preparation A) bound to myog1-obine-HRPO aldehyde (Preparation I)

3. Vaginal fluid samples obtained on standard dacron swabs. The samples are frozen until ready for use. The samples are thawed and centrifuged in specially prepared tubes to allow the undiluted vaginal fluid to be extracted from the swab. The entire sample from each swab is tested.

4. Guaiac impregnated filter paper (commercially available Hemoccult ^R strips). P ufryaroztoky

and 6 mM hydrogen peroxide

b. 250 mM glycylglycine buffer, p H 3, O

c. 1.0 M Acetate Buffer, p H 4.0

B. Procedures

Suspend 20 mg (wet weight) of the conjugate to Eupergit ^R bound myog1 obn-HRPO in undiluted vaginal fluid and 10 μΐ of 1M acetate buffer. The suspension is centrifuged and the state particles are centrifuged.

Incubate 10 µl of the clear supernatant at 10 ° C to sedimentate the solid conjugate, place on the surface with guaiac po in the leaf, then add 5 µ 5 of hydrogen peroxide solution and examine the leaf for blue staining.

mg (wet weight) conjugate per Sigmace1 1 ^R2 2 O bound myog1 ob in-HRPO is suspended in undiluted vaginal fluid and 10 μl of 1 250 mM acetate buffer. The suspensions are incubated for 15 minutes at room temperature and the suspension is centrifuged to sediment the solid conjugate and other particles.

Place the ΐΐ of the clear supernatant on the surface with a guaiac of the leaf, followed by the addition of 5 μΐ of hydrogen peroxide solution and examine the leaf for blue staining.

Color

Interpretation

No visible blue color

0.25, 0.5, 2.0

Results

Possible fine blue color No finer blue color visually detected

Clear blue color Dark blue color

The darkest all blue color possible in try. system

A dark blue color is formed on the guaiac coated sheet at the site where the supernatant of vaginal fluid from women infected with vaccinia candidiasis has been added. In the place where it was applied

vaginal supernatant no discoloration. lni fluid from control of women nevyt vo ř Sample number Diagnosa S u b s three For b a r v e η í 7823 candidiasis S i gma c e1 1 ^R 2,0 7824 candidiasis S i gmace11 ^R 2,0 7844 c a n d i d i and s i s S i gma c e 1 ^R 2,0 78 4 8 candidiasis S igmation 1 1 ^R 2,0 78 20 kontra 1 a S igmation 1 1 ^R 0.0 78 2 1 controls and S i gmace1 1 ^R 0, 0 7826 control S i gma c e 1 ^R 0, 0 7 8 3 1 k o n t r o 1 a S i gma cel 1 ^R 0.0 789 1 candidiasis Eupergi t ^R C 2,0 78 2 1 candidiasis Eupergi t ^R C 2,0 79 14 control Eupergit ^R C + / - 7 9 15 control Eupergit t ^R C 0, 0

D. Artist c e

Active aspartic protease, secreted into vaginal fluid cells of women with clinical Candida albicans, hydrolyzes both Sigmacell ^R 20 bound protein and Eupergit ^R bound protein, releasing soluble, active HRPO. Centrifuged sedimented polymer-bound HRPO leaves only released solubilized HRPO in solution. The soluble HRPO catalyzed oxidation of guaiac by hydrogen peroxide produces a blue coloration. Thus, blue staining on the Hermoccult ^R bands is a convenient way to detect active aspartic protease in the vaginal fluid and, therefore, the vaginal candidiasis.

The active aspartic protease is not found in the vaginal fluid of control women, i.e., women without clinical candidiasis. Thus, vaginal fluid samples from female women do not hydrolyze the polymer-bound protein and do not release soluble, active HRPO from any carrier. Centrifugation of the sedented HRPO-bound polymer does not leave the HRPO solubilized HRPO in solution. In the absence of HRPO, the supernatant from the control samples does not catalyze the oxidation of guaiac with hydrogen peroxide and does not produce a blue color. The lack of blue staining on guaiac-impregnated paper thus provides a convenient way of identifying women who have not been infected with Candida and Ib and c.

Experiment XIV

This experiment concerns the different hydrolytic activity of several microbial proteases on [Sigmace1 and ^R- Myoglobin-HRPO]

A. Materials. [5 igmace 1 1 ^R - Mog 1 obi η - HRPO] (Preparations E and F)

Asparagus protease producing Candida albicans (ATCC culture)

28366) a

Bc

δ

(B)

Trichomonas cell suspension culture Cell suspension of Mobiluncus curtisii vagina 1 s (ATCCF 3001) (ATCC 35241)

Buffers and solutions

0.2% hydrogen peroxide solution Potassium phosphate buffer pH 7.5, 300 mM sodium acetate buffer pH 4.0, 100 mM guaiac impregnated paper (Hermoccult ^R business strips) Procedure

Suspend 20 mg of substrate ^[R Sigmace11 -Myog1 Obin-HRPO] 75 μΐ appropriate buffer (see table) and treated with 25 μ 1 of cell culture suspension. The reaction mixture is incubated for 10 to 30 minutes, after which the sample is centrifuged to remove the solid phase conjugate and debris. Apply 5 μΐ of supernatant to the guaiac strip and develop 5 μΐ of hydrogen peroxide solution. The reaction conditions and color development are shown in the following table.

Coloring Interpretation +

0.25 0.5 0.5 2.0

No appreciable blue tint Fine blue tint possible

The finest blue color visually detected Clear blue color

Dark blue color

The darkest blue flask maybe in trial. system

:. Results

No coloration occurs when the reaction sapernatant from the tubes incubated with the tray at pH 4.0 or 7.5 is applied to guaiac strips for 30 minutes and developed with conductor peroxide. A similar result (i.e., uncoloured) is observed for cooked ns u i t i r C and n d i d a 1 b i cans (p H 4.0 and 7.5). Candida a 1 b i c a c t i s t o c t o c t e r i n i o n i o n o t incubation at H o H 4.0 but not pH 7.5 even after a 30 minute incubation. Trichononas va gin and 1s produces a strong blue color after 30 min inhalation at pH 7.5, but only a barely perceptible color after 30 min incubation at pH 4.0. The culture of Mobiluncus Hurtisii produces no blue color after 30 minutes at either 3 H 4.0 or p H 7.5.

Enzyme source (common cultures) pH incubation Incubation time 2 a b a r v e η í Candida albicans 4.0 10 min 2,0 Candida albicans 7.5 3 0 min 0, 0 cooked Candida albicans 4.0 3 0 m i n 0.0 cooked Candida albicans 7 5 3 0 min 0, 0 Mo b iuncus curt i s 4.0 3 0 min 0.0

Mobiluncus Curtisia 7.5 Cooked Mobiluncus Curtisia 4.0 Cooked Mobiluncus Curtisia 7.5 Trichomonas vaginalis 4.0 Trichomonas vaginalis 7.5 Cooked Trichomonas vaginalis 4.0 Cooked Trichomonas vaginalis 7.5 Buffer 4.0 Buffer 7.5

D. Interpretation

30 m i n 0, 0 30 m i n 0, 0 3 0 m i n 0, 0 30 m i n 0, 25 30 m i n 2,0 30 rn i n 0, 0 30 m i n 0, 0 30 mi n 0.0 30 m i n 0, 0 mM phosphate p u f r s m

p u f r e m at p H at room temperature,

Trichomonas vagina is at pH 7.5 or 100 mM acetate 4.0 at 30 min. Of the intestinal fluid. Similarly cooked suspensions of Candida albicans or Mobiluncus curtisia will not release HRPO at pH 7.5 or 4.0 at pH 7.5 or 4.0. Incubate at room temperature for 30 minutes. However, Candida albicans culture releases HRPO from the solid support at pH 4.0 at 10 minutes, but does not release it at pH 7.5, even at the 30-minute incubation period. This is consistent with the pH profile of the aspartic protease Candida albicans, which is active at low pH values but is inactive or inactive at high pH values. Unlike. hence, T. vaginalis culture releases HRPO from the solid support at pH 7.4 at a 30 minute incubation interval, but releases barely perceptible amounts of HRPO at pH 4.0. This behavior is also consistent with the known pH profile of T.vaginalis thio protease (this is active at high pH but much less active at low pH). Finally, Mobiluncus curtisia, which is known to release HRPO nor. at. pH 4.0, even at pH 7 incubation interval.

When testing under different pH conditions, a distinction can be made between three microbes: only Candida albicans causes coloring at 10 minutes at room temperature at pH 4.0; only Trichomonas vaginalis causes staining secretes proteases, neu5 at 30 minutes at pH 7.5 at 30 minutes and Mobiluncus curtisii does not stain at 30 minutes at either pH 7.5 or 4.0.

Attempt XV

This experiment concerns different activity of different proteases and their inhibitors on substrate [Eupergit ^R -Myog1 obin-HRPO]

A. Materials

1. Powdered [Eupergit ^R -Myog1 obin-HRPO] (Preparation Q)

2. A commercially available aspartic protease from Sigma Chemical Co (Type XIII, from Aspergillus saitoi, activity 0.6 J / rng) 40 mg / ml

3. Bovine pancreatic trypsin (serine protease) (activity 2900 J / mg) from U.S. Pat. Biochemicals, 2 mg / ml.

4. Papain (thi Protease a) from papaya latex (activity 12 J / mg) from Sigma Chemical Co., 2 mg / ml

5. Asparagus protease producing Candida albicans culture (ATCC 28366)

5. Hydroch ⁵ 1 o r i d it s y l 1 y s i n c h 1 o r me t b y 1 k e t o n u (TLCK) 5 0 m M h e t ha - η o 1 u o d S i gm and Che mi c a i Co. 7. P e p s t a t i n A from a microbial source from Sigma Chemical What? 2 mg / ml v ethanol 8. Buffers a solutions

a phosphate potassium PH 7.0, 200 mM b f o s f o r e n a n u d r a s e1 pH 7.4, 1 00 mM c. Sodium acetate buffer r ρ H 4,0, 1 00 M

d. Hydrogen peroxide solution 0,02¾

e. Absolute ethanol

9. Guaiac-impregnated paper (Hsrmoccutt ^R business strips)

B Procedure

This test is performed in two parts. First, the activity of various proteases on the substrate (Eupergit ^R- Myoglobin-HRPO) is determined, and the enzymes and controls (enzymes cooked for 15 minutes) are incubated with 20 mg of substrate and buffers (see amounts in the table) for 15 minutes prior to assay.

In the second part, the enzymes are preincubated with their respective inhibitors and corresponding buffers for 15 minutes. These are then added to the substrate and incubated at room temperature for a further 15 min at RT.

In both cases, the reaction mixture is centrifuged to remove the solid phase of the conjugate. Apply the supernatant (5 μL) to the Hermocclt ^R strips with 5 μΐ hydrogen peroxide.

Color Interpretation 0 No visible blue color ± Fine blue color possible 0, 25 The finest blue tint visually found 0, 5 Clear blue color 1.0 Dark blue color 2,0 The darkest blue color in trial. with yst é mu

C .. Resources

1. Part I: The supernatants of the reaction mixture from tubes containing buffer alone at pH 4.0 or pH 7.5 produce only the finest detectable coloration, if any. applied to guaiac strips and developed with hydrogen peroxide. The same result is observed with the supernatants of the reaction mixture from tubes containing cooked tripsyn at pH

7, 4, v a í e n o u to 1 turu C a n d i d a at p H 4.0 and boiled papain at pH 7, 4. S i 1 n é blue the color is formed in p ř í fall of uncooked tryp- s i nu at pH 7.4, uncooked k u, 11 u r y Candida ex i pH 4, 0 and uncooked p and p and i ; n u pri PH 7, 0.

Part II: A TLCK protease inhibitor capable of inhibiting both serine and thiol type proteases inhibits staining for both trypsin (serine protease) and papain (thiol protease). Pepstatin, a known aspartic protease inhibitor, prevents the formation of Candida albicans aspartic protease staining.

Table - Part I Z d r oj e n z ymu (o b j .- 25 μ 1) Buffer volume = 75 μΐ) Color Tr yps i n pH 7.4 2.00 Tryps i n ( boiled) pH 7.4 0.25 Ku 1 tura Candida pH 4.0 1.50 Candida cooked p H 4.0 0.25 Pa pa i n pH 7.0 1.00 Papin (cooked) PH 7, 4 0, 25 buffers alone 0, 2 5 T a b u 1 to a - Part II Enzyme Solution E t h a η o 1 Buffer p H, Vol. Ahar v s n i inhibitor (checks). 3) Trypsin TLCK (10 μ. and 7, 4 6 5 µ 1 1.0 Tr yps i n - - 7.4 75 μΐ 2,0 P and p and i n TLCK (25 μ! ) 2 5 μΐ 7.0 50 μΐ 0, 5 P and p and i n - 7.0 5 0 μ 1 1.0 A with p and r and g. P e ps dad (25μ1) 4.0 5 0 μ. and 0, 5 P r o t e 3 z a A with p and r and g. - 25 μΐ 4,0 50 μΐ 1.5 protease

D .. Interpretation

Trypsin, a sarin protease, hydrolyzes the solid phase substrate at pH 7.4 to release soluble HRPO, which catalyses the formation of a blue color on the induced guaiac bands. The same is true for the aspartic protease Candida albicans at pH 4.0 and for pa p a n, t h i o o u u protease at p H 7.0. Cooking induces the release of HRPO for each enzyme. Thus, each of the three different types of enzymes is capable of hydrolytically releasing soluble HRPO from the carrier under suitable reaction conditions. No buffer or heat activated enzymes release soluble HRPO, suggesting that the release of HRPO does not result from simple and non-specific release caused, for example, by salts in the growth medium or incubation mixture.

TLCK, a protease inhibitor capable of inhibiting both serine and thiole proteases, can be stained by both trypsin (serine protease) and papain (thiol protease). Pepstatin, a known aspartic protease inhibitor, inhibits the formation of Candida albicans aspartic protease staining. by incubation in the presence of known specific enzyme inhibitors or inhibitors of specific enzyme classes, the specificity of the hydrolase detection can thus be achieved.

These experiments merely illustrate the invention, but do not limit it in any way. It will be apparent to those skilled in the art that the operating conditions, individual operations and other parameters of the methods and tests and test devices described herein may be further modified or replaced without prejudice to the spirit and scope of the invention.

Method of detecting the presence of enzymatically active hydrolase in a sample, in particular detecting candidiasis by detecting the presence of an enzymatically active protease of aspartic acid.

JUDr. Petr KALENSKÝ

Claims (4)

PATENT CLAIMS A method for detecting the presence of an enzymatically active hydrolase in a sample, characterized in that: (a) contacting the sample with a solid support containing the immobilized reporter enzyme released by the hydrolase; (b) the sample, after contact with the solid support, is combined with an indicator which is sensitive to a detectable change by the reporting enzyme; and (c) any detectable change in the indicator is observed, which is an indication of the presence of enzymatically active hydrolase in the sample. The method of claim 1, wherein the hydrolase is a protease selected from the group consisting of an aspartic protease, a serine protease, a thiol protease, a metal loprotease, an acidic protease, and an alkaline protease. Process according to claim 1, characterized in that peroxidase is used as the hydrolase. 4. A method for detecting the presence of an enzymatically active hydrolase in a sample, the method comprising placing the sample in a test apparatus comprising a first and a second solid support, the first solid support carrying a hydrolase-releasing reporter enzyme immobilized thereon and the second solid support which not in contact with the first solid support, it comprises an immobilized indicator that is sensitive to detectable changes after activation of the reporter enzyme, and the sample is placed in the apparatus in contact with the first and second solid supports and released by the reporter enzyme in the presence of any hydrolase activity diffuses through the sample into the second solid support, observing for any detectable change in the indicator, indicating the presence of enzymatically active hydrolase in the sample. A method for detecting candidiasis by detecting the presence of an enzyme104 wherein the shield hydrolase contained on the inwardly facing surface of either the first or second opposite wall is sensitive to inactivation by the presence of an inhibitor from a reporter enzyme immobilized on a solid support on the inwardly facing surface of the first or second wall the shield hydrolases, if not inactivated by the presence of inhibitor, from an indicator contained on the inwardly facing surface of the first and second walls, the indicator being a substance sensitive to the detected change induced by the reporting system, and from an opening in the sample insertion box. JUDr.Petr