Method detection hydrolases and device for its implementation

 

(57) Abstract:

The invention relates to biotechnology, medical Microbiology, concerns the detection hydrolytically active enzyme, in particular aspartic protease in a sample or preparation. The sample or the drug is in contact with the solid medium in which the immobilized enzyme-reporter (i.e., signalgenerating enzyme) in such a way that under the action of the enzyme active hydrolases, if present in the sample, the enzyme-reporter is separated from the solid media. After contact with the solid carrier, the sample interacts with the indicator. The indicator is any chemical substance that can be detectivemisa change, usually a colour change under the action of the enzyme reporter. Detective change indicator indicates the presence in the sample of enzyme active hydrolases that may indicate the presence of a specific pathogen or pathological conditions, such as candidiasis. The device for implementing the method includes a receiver formed partially in the first and second opposite walls, facing each other, the inner sides with a gap. Wall izge enzyme-reporter and indicator, able to detectivemisa change under the action of the enzyme reporter. In the receiver made the hole for the input sample. The invention provides ease of analysis, is fast and economical to use. 2 S. and 3 C.p. f-crystals, 2 ill., 30 table.

The invention relates to methods for testing for the presence hydrolases activity (i.e., hydrolytic enzymes) in a sample or preparation. In particular, the present invention relates to a method for detection of candidiasis by analysing the presence in the sample enzymatically active aspartic protease.

Prior art

Candida albicans and other Candida species cause a number of widely used and medically serious infections. So, for example, candidiasis of the digestive system often occurs in immunocompromised patients. In addition, vulvovaginal candidiasis is one of the most common diseases in obstetrics and gynecology. According to experts, it was found that approximately three quarters of adult women suffer from at least one of these diseases. (De. Bernardis et al., J. Clin Environ 27(11):2598-2603 (1989)). As a result of this wide rasprastrtimi candidiasis.

These studies showed that the causative factor in the occurrence of candidiasis in humans are mushrooms of the species Candida albicans and other Candida species; and currently, it is believed that the occurrence of candidiasis mainly due to the presence of Candida albicans. In addition, evidence has been obtained showing the role of aspartic protease or (same thing) acid proteases as virulence factor of Candida albicans. It is known that a pure culture of Candida albicans produce aspartic protease under cultivation only in particular conditions. It is also known that pure strains of Candida albicans obtained from women with symptomatic vulvovaginitis, secrete the specified enzyme after culturing these strains specially selected culture medium.

Antigen acid proteinase of Candida albicans, i.e., the antigen aspartic protease was detected immunologically in the vaginal secretions of women, which was dedicated vulvovaginal Candida albicans (De Bernardis et al., Abstracts N F-91, in "Abstracts of Annual Meeting of the American Society for Microbiology Micro, (Anaheim, CA 1990)). However, in patients with symptomatic vulvovaginal candidiasis concentration of antigen acid proteinase of Candida albicans is significantly higher than in asymptomatic nakateleeli 176 to 15.2 ng/ml, whereas its concentration in women who have not been allocated Candida albicans, i.e. women not suffering from clinical candidiasis, is less than 2 ng/ml Asymptomatic carriers of Candida albicans have intermediate levels of antigen 94 of 18.5 ng/ml, These data clearly indicate that acidic proteinase (i.e. aspartic proteases) involved in the pathogenesis of vulvovaginal candidiasis. However, it is known that the aspartic proteases of Candida albicans unstable at the temperature of the body. In addition, immunological detection of antigen aspartic proteases is not possible to determine whether this enzyme in the enzymatic active form.

Acid proteinase of Candida albicans is an extracellular aspartic protease. Aspartic proteases are one of the broad classes of proteases. They contain one or more key aspartic acid residues responsible for the enzymatic activity. Aspartic proteases of Candida albicans has a broad specificity to the protein substrate, such as albumin, hemoglobin, casein, immunoglobulin A and many other proteins. This enzyme operates optimally in acidic conditions (i.e. at pH of 2.5 to 5.5) and is rapidly inactivated at higher pH (i.e. the agents, the chelators or inhibitors of serine proteases.

Many pharmaceutical companies and leading scientists conduct research to study inhibitors of aspartic proteases and their potential therapeutic use. However, these studies are difficult due to the lack of a suitable enzyme analysis for aspartic proteases. For some serine, thiol, metal, acid and alkaline proteases and peptidases are simple colorimetric tests, but these tests may not be used for aspartic proteases. The substrate specificity of this special class of enzymes requires the presence of several hydrophobic amino acids. This property of these enzymes creates significant difficulties in finding a simple synthetic chromogenic substrates, because of hydrophobic amino acids, which serve as substrate for aspartic proteases, known to be hard to dissolve in water. So chromogenic substrates for aspartic proteases are not made by the industry, they are difficult to synthesize and oharakterizovat, and they do not dissolve in water. As a result of these limitations enzymatic activity, opredeleny absolutely insensitive to aspartic protease. Similarly, were described fluorogenic substrates for aspartic proteases, but they also have limited use. First, as mentioned above, the substrate specificity of this class of enzymes requires the presence of several hydrophobic amino acids, resulting in these substrates are practically insoluble. Secondly, while achieving low concentrations, these florigene substrates hydrolyzed very slowly, and therefore fluorogenic analyses require time-consuming. In addition, many biological preparations contain fluorescent substances that can interfere with fluorophenol analysis for the presence of aspartic proteases.

Due to the lack of suitable colorimetric or fluorogenic assays for the detection of aspartic proteases, typically use ultraviolet (UV) spectrophotometric method of analysis. In a typical UV-spectrophotometric method of analysis, aspartic protease is added to the protein solution (such as hemoglobin or albumin), and the mixture was incubated at 30 - 37oC for 0.5-4 hours. After incubation, cooled to the incubation mixture Holodnaya ultraviolet light, remain in the solution. And finally precipitated non-hydrolyzed protein is centrifuged for about 1 hour at temperatures of cooling, after which the supernatant is sucked off and to evaluate the degree of protein hydrolysis determine the optical density (OD) at 280 nm. Although this analysis and can be used to determine the aspartic proteases, however, it is labour intensive and requires a large amount of time.

Therefore, we can say that today there is no easy, simple and rapid method of detecting the presence of enzymatically active aspartic protease. In accordance with this present invention relates to a method of detecting the presence of enzymatically active aspartic protease, which allows to solve the problems associated with the deficiencies of previous methods of analysis. In addition, the methods of the present invention can also be used to detect the presence of other hydrolytic enzymes, i.e., hydrolases.

Brief description of the invention

It was found that enzymatically active aspartic protease of Candida albicans is present in vaginal secretions of women with vulvovaginal candidiasis. Chrome is ATA may serve as an indicator for detection and diagnosis of candidiasis. In line with this, developed a method of detecting candidiasis by testing for the presence of enzymatically active aspartic protease in the sample.

In this method, the sample, such as vaginal discharge, is subjected to contact with a solid carrier. On solid medium, i.e. a medium that is in contact with the sample, immobilized enzyme-reporter (i.e., signalgenerating enzyme). Moreover, the specified enzyme-reporter, immobilized on a solid medium is separated from the specified solid media under the action of the enzyme active aspartic protease, if this enzymatically active aspartic protease present in the sample. After the sample was subjected to interaction with solid media, it is combined with the indicator. An indicator is any chemical substance capable of visible or detectable change (e.g., such as a change of color) under the action of the enzyme reporter. If after contact with the sample, the indicator undergoes a detectable change, then enzymatically active aspartic protease present in the sample, and therefore, can be diagnosed with candidiasis.

Technology release of the enzyme reporter, which was the basis for analysis for the presence of aspartic proteases, can also be used for detection of the presence of lli proteases (these terms are interchangeable), peptidases, lipases, nucleases, homopolysaccharides, heteropolysaccharides, phosphatase, sulfatase, neuraminidase and excerise. In accordance with this, in the present work, we have also developed methods of analysis for the presence in the sample of enzyme active hydrolases.

In these methods, the sample or the product is subjected to contact with a solid carrier. On a solid medium that is in contact with the sample, the immobilized enzyme-reporter (i.e., signalgenerating enzyme). The specified enzyme-reporter immobilized on a solid carrier so that it is separated from that of solid media under the action of hydrolases, if such enzymatic active hydrolase is present in the sample. After contact with the solid carrier, the sample interacts with the indicator. This indicator is any chemical substance having the ability to appear or detectivemisa change (usually a colour change) under the action of the enzyme reporter. Detective change indicator indicates that the sample is enzymatically active hydrolase. Conversely, the absence of detected changes in the indicator indicates that in this sample the enzymatic act is, respectively, candidiasis, the claimed methods of analysis for the presence in the sample of enzyme active hydrolases are fast, accurate, cost-effective and easy to use.

Additionally, the release of the enzyme reporter, which was the basis of the above-described methods can also be used to analyze the presence of any known inhibitor of hydrolytic enzyme, including, without limitation, inhibitors of proteases or proteinases (these terms are interchangeable), peptidases, lipases, nucleases, homopolysaccharides, heteropolysaccharides, homopolysaccharides, heteropolysaccharides, phosphatases, sulfates, neuraminidase esters. In accordance with this, in this work, methods were developed for the analysis of the presence in the sample of inhibitor hydrolases.

In these methods, the sample or the product is subjected to interaction with solid media. On a solid medium that is in contact with the sample, the immobilized enzyme-reporter (i.e., signalgenerating enzyme). The specified enzyme-reporter immobilized on a solid carrier such that it is separated from the solid media under the action of the target hydrolases, provided that the specified celebutante with the target hydrolases and enzyme-reporter, it reacts with the indicator. An indicator is any chemical substance which is capable of detectivemisa change, usually a colour change under the action of the enzyme reporter, if the specified enzyme reporter was separated from the solid media through targeted hydrolases. If the inhibitor target hydrolases not present in the sample, the target hydrolase will facilitate the separation of the enzyme from the carrier, thereby detective change indicator. Conversely, if the inhibitor target hydrolases present in the sample, it will be inhibitorof target hydrolase, and enzyme-reporter will not be separated from the solid media, and therefore, the detected change or response indicator will not be observed. As in the case of the above methods, the claimed methods of detecting the presence of an inhibitor of hydrolases in the sample are fast, accurate, cost-effective and easy to use.

In addition to the methods of analysis for the presence of enzymatically active aspartic proteases and other hydrolytic enzymes, the present invention relates to a stand-alone device intended for analysis of the sample for the presence of candidiasis detected by the Autonomous device, designed for analysis for the presence in the sample of enzyme active hydrolases. These test devices combine the enzyme-reporter, immobilized on a solid medium, the indicator and one or more other reagents in dry form, which are on the plate panel with internal camera, and this camera is empty up until inside is placed the sample. For convenience, the side of the panel, and position of the functional chemical materials on the panel are described on the basis of the horizontal position of the panel, because it is a position usually has a panel during use. In this position of the panel, in particular, for the preferred panels of the present invention, which are thin and flat, the test sample is placed in the specified camera through the holes in the top of the panel. All of the layers that make up the panel, the top layer in this position, i.e., the layer through which the injected sample will be designated as the top layer, the bottom surface of this layer forms the upper surface of the camera. Similarly, the bottom layer of the panel will hereinafter be called the bottom layer of the panel, with the upper surface of the lower layer of the lateral edges of the panel, and thin lateral ends of the camera along the edges of the top and bottom surfaces will hereinafter be called the side walls of the chamber. The segments of any given surface, which are adjacent to any other area in the same horizontal plane, will hereinafter be called horizontally adjacent, and the layers are applied directly on top of other layers, and forming a parallel horizontal plane, will hereinafter be called vertically adjacent.

The top, bottom and both layers panel made of a light transmissive, preferably transparent material. Enzyme-reporter immobilized on solid media; indicator and other components and reagents necessary for the test feature in one or more layers inside the chamber in the form of coatings or on the upper surface of the camera or on the bottom surface of the camera or on both surfaces. An indicator is any chemical compound capable of detectivemisa change, usually a colour change under the action of the enzyme reporter in that case, when it is separated from the solid media using an enzymatic active hydrolases, the presence of which is detected in this analysis. The layer containing indie the La analysis, can be included in the same layer that contains the indicator, or they may be incorporated in separate layers, which are located on the same surface or opposite surface. In some preferred embodiments, the implementation of the present invention, the indicator is in the layer that is applied directly under the surface of the translucent wall, and enzyme-reporter, immobilized on a solid medium is a layer deposited on the opposite wall.

Reagents filling these layers can be selected so that the tests will only need to add the sample and the minimum number of additional reagents, such as the developer. However, in a particularly preferred embodiment of the present invention, these layers contain all the necessary reagents except the sample, so that for analysis it is only necessary to add the sample and nothing more.

All layers prior to their contact with the sample are solid, and the layer containing the indicator preferably is a composition which is insoluble in the liquid sample for analysis which it is intended, therefore, indica is the ima environment, the preferred indicator layer is either an indicator that is not soluble in water, or indicator, which is contained in the matrix, not soluble in water. Thus, when using the indicator contained concentrated in a thin layer that is applied directly to the inner side of the translucent wall, the change in the color of the indicator, which is observed through the translucent wall, occurs in a short period of time, which allows to obtain highly sensitive and rapid results.

The present invention can be adapted and used for analysis for the presence of various hydrolases wide range in samples taken from various sources. In addition, the present invention can be adapted and used for analysis for the presence of inhibitors hydrolases wide range. The test may be conducted using either a reaction or sequence of reactions, the culmination of which leads to detectivemisa change in the indicator; and the number and types of reagents and reactions may vary from one test to another, depending on the detected hydrolases. In some cases, Naya and bottom surfaces, however, they are separated from each other by a gap up until this gap is filled with the test sample. In other cases, the reagents may be in a common layer or in two or more different, but vertically adjacent layers on the top or bottom surface of the chamber that is in no way affecting the reliability of the test. However, in all cases, these layers are constructed and arranged so that reaction, the culmination of which leads to detectivemisa change indicator, occurred only when the chamber is filled with the test sample; and if such detective the indicator's change takes place, it at least is concentrated in the layer adjacent to the transparent wall, and preferably limited to this layer.

In preferred embodiments of the present invention, the testing device includes positive control, negative control, or both, which are activated only by adding the sample. Activation of these control reagents occurs during testing, and detected evidence (for example, staining or its absence), representing both the control and test, and the device, control zones are horizontally adjacent and the test area; and to identify zones of control and differentiating them from the test zone on the bottom or on the top (preferably on top) surface of the device have corresponding labels. The control zone will consist of additional layers containing reagents or other suitable substance, which either directly induce detective change in the indicator, or prevent the occurrence of such change, and which is valid only in the presence of the test sample, regardless of whether there is or there is no "suspected" hydrolase in the sample to be tested. And similarly, the choice of the positive and negative control, and chemical mechanisms by which they operate, as well as the choice of their location (i.e., on the surface, on which there is an indicator, or on the opposite surface of the camera) can vary from one test to another.

In an even more preferred embodiment, the present invention provides for improving the efficiency of testing. For water samples, the introduction of surface-active substances (surfactants) in the layers directly in the same tank. Surfactants may be the only functional ingredient in this layer, or it can be combined with the reaction reagents in this layer. Preferably, both sides of the gap bordered with layers, carrier surfactants. For direct entry of the sample into the chamber, the device is provided with a hole for the input sample, and in the preferred embodiment, the camera has one or more vent holes separated from each other by the hole for the input of the sample and intended to facilitate the filling of the chamber.

Other distinctive features, objectives and advantages of the present invention and its preferred options will be apparent from the following description.

Brief description of drawings

Fig. 1 is a perspective view of the characteristic test device of the present invention.

Fig. 2 is a side view in section of the test device shown in Fig. 1.

Detailed description of the invention and its preferred implementation

In one aspect, the present invention relates to a method of analysis for the presence of enzymatically active hydrolases in the sample, including:

(a) contacting the sample with a solid noticedate hydrolases;

(b) after contacting with the specified solid carrier, the connection of the sample with the indicator that is exposed detectivemisa change under the action of the enzyme reporter; and

(c) monitoring detektivami change indicator; detective change indicator indicates the presence of enzymatically active hydrolases in the sample.

The term "hydrolase" used in the present description, refers to an enzyme that catalyzes the hydrolytic reaction. The method of the present invention can be used to analyze the presence of any known hydrolytic enzyme. Such hydrolytic enzymes, i.e. hydrolases, are, without limitation, the following enzymes: protease or proteases (these terms are interchangeable), peptidases, lipases, nucleases, Homo - or heteropolysaccharides, Homo - or heteropolysaccharides, phosphatase, sulfatase, neuraminidase and esterase. In a preferred embodiment, the method of the present invention can be used to analyze for the presence of proteases, including, but not limited to, aspartic proteases, serine proteases, thiol proteases, metalloproteases, acid protease and alkaline protease. In juvie Homo - or heteropolysaccharides, or Homo - or heteropolysaccharides, including, but not limited to, chitinase, amylase, cellulase and Lisitsin.

The terms "enzyme-reporter" or "enzyme-marker" (these terms are interchangeable) used in the present description, refer to signalgenerator the enzyme, i.e. an enzyme activity which causes detective change. Such enzymes-reporters are not limited to, peroxidase, phosphatase, oxidoreductase, dehydrogenase, transferases, isomerases, kinases, inhibitors, deaminase, catalase, urease and glucuronidase. When choosing enzyme-reporter for use in the method of the present invention, it is necessary to bear in mind that this enzyme reporter shall not be subjected to inactivation under the action of any agent that is present in the sample, including any active hydrolase present in the sample and is involved in inactivating hydrolysis. The selection of the appropriate enzyme-reporter or marker can be easily carried out by a qualified technician. Preferred enzymes-reporters are peroxidase, for example, such as peroxidase horseradish. Enzyme-reporter immobilized on a solid medium, such as insoluble polimerase the specified enzyme-reporter separated from the carrier under the action of hydrolases, the presence of which is necessary to detect in this analysis. In accordance with the present invention is preferable solid carriers are not limited to, cellulose, agarose, dextran, polyacrylate, polyacrylamide or derivatives thereof, chitin, sepharose, exernally balls and dialdehydecellulose polymer, starch, collagen, keratin, elastin, powder from bovine skin, peptidoglycan cell wall of bacteria or its fragments, nylon, polyethylenterephtalate, polycarbonates, and the glass having pores of a certain size. Immobilization of the enzyme reporter on solid media carried out using standard methods and operations, known to every expert.

Enzyme-reporter can be associated directly with a solid carrier. In this case, the insoluble carrier is used directly as substrates for hydrolases. For example, if the analyzed hydrolases is chitinase, reporter or marker enzyme (such as horseradish peroxidase) can be attached directly to insoluble chitin. In the presence of chitinases, horseradish peroxidase will be separated from the solid media. Similarly, if the study of hydrolases is but to the cellulose, and in the presence of cellulose, horseradish peroxidase will be separated from the solid media. And finally, if detectable by hydrolases is lysozyme, the enzyme-reporter, for example horseradish peroxidase, can be attached directly to the peptidoglycan cell wall of bacteria, and in the presence of lysozyme, horseradish peroxidase will be separated from the solid media.

Alternatively, the enzyme-reporter can be immobilized on a solid medium through the use of linker molecules, which represents a hydrolyzable substrate to detectable hydrolases. Such linker molecules are not limited to, proteins, carbohydrates, lipids, peptides, esters, and nucleic acids. The choice of a specific linker molecules used for the attachment of the enzyme reporter to solid media depends on the type of detectable hydrolases, and in any case can easily be carried out by a qualified technician.

The term "indicator" as used in the present description, refers to any chemical compound that undergoes detectivemisa change in the reaction or in the climax of the reactions occurring in the presence of eenie indicates, this product or sample is enzymatically active hydrolase.

Preferred indicators are visual indicators, and, in particular, chromogenic indicators, i.e. indicators such in which the visible change is the change in colour (including education coloring in another colorless material) under the action of the enzyme reporter or enzyme marker, if it is separated from the solid media using an enzymatic active hydrolases, the presence of which is detected in this analysis. Alternatively, the enzyme-reporter may be able to catalyze the formation of a fluorescent signal, a fluorescence signal, a bioluminescent signal, a chemiluminescent signal, or electrochemical signal after its separation from the solid media under the action of hydrolases. In addition, the enzyme-reporter may be able to produce other visible or detected signals, such as clots, agglutination, precipitation, or the formation of zones of clarification. In these cases, the indicator must be a chemical compound or the substrate required for this enzyme reporter or pesthouse using enzyme-reporter, such as horseradish peroxidase, can be used in a variety of chromogenic indicators (i.e., Chromogens) and other compounds having similar effects. Preferred chromogenic indicators in accordance with the present invention contain a hydroperoxide and a Chromogen, including, but not limited to, guaiac resin, 2,2'-Azino-bis(3-ethyl-benazolin-6-sulfonic acid), tetramethylbenzidine, phenol, 4-aminoantipyrine and 4,5-dihydroxynaphthalene-2,7-disulfonate acid. Especially preferred chromogenic indicator is the hydroperoxide and guaiac resin, i.e., a Chromogen, which is colorless in its restored form and dark blue in the oxidized form. Guaiac resin may be (but not necessarily) is purified prior to its use, for example, by solvent extraction. The choice of the most suitable chromogenic indicator for this enzyme-reporter depends on the reaction or reactions that this enzyme is able to catalyze or initiate, and in any case, this choice can easily be carried out by each specialist.

If the visual indicator is applied chromogenic indicator, in this case, can be used with either liquid chromogenic A, such a system should consist of a solvent, cumene hydroperoxide, and the Chromogen, is able to oxidize hydroperoxides in the presence of peroxidase, such as horseradish peroxidase. Alternatively, if you are using solid chromogenic system, such a system should consist of hydroxyperoxide; Chromogen capable oxidized by hydroperoxides in the presence of peroxidase; and solid media, which is impregnated with a Chromogen or immobilized Chromogen, and then dried. In this system, the Chromogen may be impregnated blotting paper or substrate, as was done in the case of drugs Hemoccultor alternatively, the Chromogen may be deposited a thin layer of plastic or other material made in the form of a plate. In the latter case, the Chromogen may be laid up in the form of a solution containing a polymeric material (such as hydroxypropylcellulose, ethylcellulose and so on). If the Chromogen itself is water soluble, it can be introduced into the matrix material, insoluble in water. Alternatively, if the Chromogen is not soluble in water, it may be caused by the layer in the form of a solution in an organic solvent either alone or in continuousy peroxidases, the hydroperoxide may be used in the solid state (such as hyperoxic titanium), or it can be generated in situ. So, for example, hydrogen peroxide can be generated in situ using glucose, atmospheric oxygen and glucosidase. Alternatively, hydrogen peroxide can be obtained in situ by using a dry layer formed after deposition of a suspension of sodium perborate in alcohol. At low pH, perborate sodium spontaneously emit hydrogen peroxide. In the presence of hydrogen peroxide and peroxidase, after its separation from the solid media using hydrolases, the Chromogen is oxidized, resulting in possible to visually observe the colour change. This change in color indicates the presence of enzymatically active hydrolases in the sample or preparation.

This method and other methods of the present invention can be used for simultaneous analysis on the presence of two or more active hydrolases in the sample or preparation. In the said method can be used in mixture of two or more enzymes reporters, immobilized on a solid medium (media) through the substrate crosslinks susceptible to specific hydrolases; and D. the porters. For example, the methods of the present invention can be used for simultaneous detection of a mixture of proteases, lipases, and polysacharides obtaining hydrolytic profile of a given pathogen or pathological process.

In addition, each specialist is clear that to improve hydrolases specificity for differentiation of various hydrolases present in the sample, the reaction conditions in the methods of the present invention (such as the choice of the linker molecules or bridge ties, solid carriers, pH, buffer capacity, buffer identity, salts, etc. can be modified and adjusted. For example, it is known that some hydrolases function at low pH and inhibited at high pH. In contrast, other hydrolases operate at high pH and inhibited at low pH. Thus, by regulating the pH of the analytical mixture can be selective detection for the presence of specific hydrolases. In addition, each specialist is clear that to increase hydrolases activity and specificity, as well as for differentiation of various hydrolases, in the methods of the present invention can be used specific enzyme inhibitors.

This method of the present invention can also be used to analyze the presence of any known hydrolytic enzyme, including but not limited to, hydrolases such as proteases or Proteus the OMO - or heteropolysaccharides, phosphatase, sulfatase, neuraminidase, and esterase. In a preferred embodiment of the present invention, this method is used to analyze the presence of proteases, including (but not limited to) such proteases, as aspartic proteases, serine proteases, thiol proteases, metalloproteases, acid protease and alkaline protease.

Enzyme reporter or the enzyme is a marker used in this method can be any signalgeneration enzyme that does not undergo inactivation of any agent present in the sample, including inactivation by hydrolysis under the action of the active hydrolases present in the sample. Such enzymes reporters include, but are not limited to, peroxidase, phosphatase, oxireductases, dehydrogenases, transferases, isomerases, kinases, inhibitors, deaminase, catalase, urease and glucuronidase. In accordance with the present invention, the preferred enzymes-reporters or enzyme markers are peroxidase, such as horseradish peroxidase.

Enzyme-reporter immobilized on the first solid medium, i.e. insoluble matrix, gel or resin, so that this enzyme reporter was separated from the is raised in this analysis. This enzyme reporter can be directly linked to the first solid carrier and, if a solid carrier is a substrate for hydrolases; or, alternatively, the enzyme-reporter can be immobilized on the first solid carrier via a linker molecule, which is the substrate for the enzyme active hydrolases, the presence of which is detected in this analysis. Such linker molecules are (but are not limited to); proteins, carbohydrates, lipids, peptides, esters, and nucleic acids. The choice of a specific linker molecules used for binding of the enzyme reporter with the first solid medium, depends on the analyzed hydrolase, but in any case, this choice can easily be carried out by each specialist. Immobilization of the enzyme reporter on the first solid medium carried out using standard methods and operations, well known to every expert.

In this method of the present invention, the indicator associated with the second solid carrier which is not in contact with the first solid carrier. In accordance with the present invention the preferred first and second carriers are (but are not limited to: cellulose is, polymeric dialdehyde starch, collagen, keratin, elastin, powder from bovine skin, peptidoglycan cell wall of bacteria or enzymes, polyethylenterephtalate, polycarbonates, and the glass having pores of a certain size. Immobilization of the visual indicator on the second solid medium is performed with the use of standard methods and procedures known to every expert.

The indicator may be any chemical compound that is detectivemisa change in the reaction or in the climax of reactions occurring under the action of the enzyme active hydrolases, if he is present in the sample or preparation. Induced as a result of this detective change in the indicator indicates that the enzymatic active hydrolase is present in the sample. Preferred indicators are visual indicators, and in particular, chromogenic indicators, there are indicators in which the visible change is the change of color, including the formation of color in another colorless material, under the action of the enzyme reporter or enzyme marker, if it is separated from the solid media with POM is este visual indicators can be used in a variety of chromogenic indicators (i.e., Chromogens) a wide range, and other compounds having similar effects. In accordance with the present invention the preferred chromogenic indicators for peroxidation enzymes reporters consist of a hydroperoxide and a Chromogen, which represent the following compounds (but not limited to): guaiac resin, 2,2'-Azino-bis(3-ethyl-benazolin-6-sulfonic acid), tetramethylbenzidine, phenol, 4-aminoantipyrine and 4,5-dihydroxynaphthalene-2,7-disulfonate acid. Especially preferred chromogenic indicator consists of a hydroxide and guaiac resin, i.e., Chromogen, which is colorless in the reduced state and the dark blue in the oxidized state. The choice of the most suitable chromogenic indicator for this enzyme-reporter depends on the reaction or reactions that this enzyme is able to catalyze or initiate, and in any case, this choice can easily be carried out by each specialist.

If the visual indicator for the peroxidase-catalyzed reactions is used chromogenic indicator, and in particular, solid chromogenic system, this system consists of a hydroperoxide; Chromogen, oxidized by hydroperoxides in prisutstvie dried. In this system, the Chromogen may be impregnated blotting paper or media, as was done in the case of drug Hemoccultor alternatively, the Chromogen may be deposited a thin layer of plastic or other material made in the form of a plate. In the latter case, the Chromogen may be laid up in the form of a solution containing a polymeric material (such as hydroxypropylcellulose, ethylcellulose, and so on). If the Chromogen is a water-soluble, it can be introduced into the matrix material is insoluble in water. Alternatively, if the Chromogen is not soluble in water, it may be caused by the layer in the form of a solution in an organic solvent either alone or in combination with soluble or water-insoluble polymer.

In solid chromogenic system used by the hydroperoxide may be in the solid state (such as a hydroperoxide titanium), or it can be generated in situ in the analytical device. In the presence of a hydroperoxide and peroxide, after its separation from the first solid media using hydrolases, the Chromogen is oxidized, resulting in the observed visually detectable colour change. This change is Arata.

In the preferred embodiment, this method of the present invention, detectable by hydrolases is enzymatically active aspartic protease, the method itself

is that a sample is placed in a device containing first and second solid media, while the first solid medium, which is a polyacrylate, immobilized horseradish peroxidase by a molecule of myoglobin, which is a substrate for aspartic protease, and the second solid medium contaktirutm with the first solid carrier and a derivative of cellulose, Immobiliser hydroperoxide and guaiac resin, which is a chromogenic substrate undergoing a colour change under the action of horseradish peroxidase in the presence of hydroperoxide; and the specified pattern is placed in the device so that he was in contact with the first and second solid media as a result, any horseradish peroxidase, released under the action of any enzymatically active aspartic protease present in the sample can diffuse through the sample to the second solid medium; and after that see whether guaiac resin izmeneniia protease present in the sample.

As mentioned above, any specialist it is clear that to increase the specificity of aspartic proteases can be used in certain reaction conditions (such as specifically selected linker molecule, solid support, pH, buffer capacity, buffer identity, salt, etc), and specific inhibitors. For example, by conducting analysis on aspartic protease at a pH of from about 2.5 to 5.0, aspartic proteases can be detected selectively with respect to thiol, serine, metallo -, and alkaline proteases. In addition, selective detection aspartic proteases can be done by adding inhibitors of metalloprotease, thiol proteases, serine proteases and acid or alkaline proteases.

In another aspect of the present invention deals with a method for detection of candidiasis by testing for the presence of enzymatically active aspartic protease in a sample, including:

(a) contacting the sample with a solid medium in which the immobilized enzyme reporter so that this enzyme reporter was released under the action of aspartic protease;

(b) after contact with a specified solid nosystem enzyme reporter; and

(c) monitoring detektivami change indicator; and if detective change occurs, it indicates the presence of enzymatically active aspartic protease in the sample, and therefore the presence of candidiasis.

Enzyme reporter or the enzyme is a marker used in this method can be any signalgeneration enzyme, i.e. an enzyme activity which causes a visible or detectable change and which is not subject to inactivation by any agent present in the sample, including inactivation by hydrolysis under the action of any aspartic protease or any other active hydrolases present in the sample. Such enzymes-reporters are (but are not limited to): peroxidase, phosphatase, oxidoreductase, dehydrogenase, transferases, isomerases, kinases, inhibitors, deaminase, catalase, urease and glucuronidase. The selection of the appropriate enzyme-reporter or enzyme marker can be easily implemented by any specialist. In this method of the present invention, the preferred enzymes-reporters are peroxidase. It is particularly preferred enzyme-reporter is horseradish peroxidase, as estnykh proteases, which may be present in the sample.

Enzyme-reporter immobilized on a solid medium, i.e. insoluble matrix, gel or resin, so that this enzyme reporter was released under the action of aspartic proteases. In accordance with the present invention the solid carriers include, but are not limited to: cellulose, agarose, dextran, polyacrylate, polyacrylamide or their derivatives, chitin, sepharose, exernally granules, polymeric dialdehyde starch, collagen, keratin, elastin, powder from bovine skin, peptidoglycan cell wall of bacteria or its fragments, nylon, polyethylenterephtalate, polycarbonates, and the glass having pores of a certain size. Particularly preferred solid media in this method of the present invention are chitin, polyacrylate, cellulose, and their derivatives, and sepharose. Immobilization reporter enzyme on a solid medium is performed with the use of standard methods of operations, known and understood by each specialist.

Enzyme-reporter can be immobilized on a solid carrier via a linker molecule, which is a hydrolyzable substrate for the enzymatic active and peptides, in this preferred linker molecules are proteins. If the linker molecule is a protein, it is preferred proteins include, but are not limited to): azocasein, casein, Kappa-casein, immunoglobulins, hemoglobin, myoglobin, albumin, elastin, keratin and collagen. In the preferred embodiment, this method of the present invention, the linker molecule is a Kappa-casein, casein, hemoglobin, or myoglobin.

The indicator may be any chemical compound that is detectivemisa change in the reaction or the climax of the reactions occurring under the action of the enzyme active aspartic protease, if it is present in the sample or preparation. Displayed in detective change indicates that the enzymatically active aspartic protease present in the sample, and the presence of enzymatically active aspartic protease in the sample, in turn, indicates the presence of candidiasis.

Preferred indicators are visual indicators, and in particular, chromogenic indicators, i.e. those indicators, which detektivami change is an Ira or enzyme marker, in that case, if it is separated from the solid media by using enzymatically active aspartic protease. Alternatively, the enzyme-reporter may have the ability to catalyze the formation of a fluorescent signal, a fluorescence signal, a bioluminescent signal, a chemiluminescent signal or electrochemical signal after its separation from the solid media under the action of aspartic proteases. In addition, the enzyme-reporter may have the ability to cause other visible or detected signals, such as clots, agglutination, precipitation or the formation of zones of clarification. In these cases, the indicator must be a chemical compound or the substrate required for this enzyme reporter or enzyme marker caused the desired detective change.

If enzymes-reporters or enzyme markers are peroxidase, the visual indicators can be used in a variety of chromogenic indicators (i.e., Chromogens) a wide range, as well as other compounds having similar effects. In accordance with the present invention the preferred chromogenic indicators consist of GIDA, 2-2'-Azino-bis(3-ethyl-benazolin-6-sulfonic acid), tetramethylbenzidine, phenol, 4-aminoantipyrine and 4,5-dihydroxynaphthalene-2,7-disulfonate acid. Especially preferred chromogenic indicator consists of a hydroxide and guaiac resin, i.e., Chromogen, which is colorless in the reduced state and the dark blue in the oxidized state. Guaiac resin may be, but is not necessary, cleaned before use, for example, by solvent extraction. The choice of the most suitable chromogenic indicator for this enzyme-reporter depends on the reaction or reactions that this enzyme is able to catalyze or initiate, and in any case, this choice can easily be carried out by any specialist. As described earlier, if the visual indicator is a chromogenic indicator, in this case can be used with either liquid chromogenic system, or solid chromogenic system.

In another aspect of the present invention deals with a method for detection of Trichomonas vaginalis by testing for the presence of enzymatically active thiol protease in a sample, including:

(a) contacting the sample with a solid carrier, which is effective active model protease;

(b) after contact with a specified solid media, the interaction of the sample with the indicator, which can cause detective change under the action of the enzyme reporter; and

(c) monitoring detektivami change indicator, and, if such detective change occurs, it indicates the presence of enzymatically active thiol protease in the sample, and hence the presence of Trichomonas vaginalis.

As in the methods described above, the enzyme reporter or the enzyme is a marker used in this method can be any signalgeneration enzyme, i.e. an enzyme activity which causes a visible or detectable change and which is not subject to inactivation by any agent present in the sample, including inactivation by hydrolysis under the action of any active hydrolases present in the sample. Such enzymes-reporters are (but are not limited to): peroxidase, phosphatase, oxireductases, dehydrogenases, transferases, isomerases, kinases, inhibitors, deaminase, catalase, urease and glucuronidase. The selection of the appropriate enzyme-reporter or enzyme marker can be easily implemented by any specialist. In this method, the present and the

Enzyme-reporter immobilized on a solid medium, i.e. insoluble matrix, gel or resin, so that this enzyme reporter was released under the action of the enzyme active thiol protease. In accordance with the present invention the solid carriers include, but are not limited to: cellulose, agarose, dextran, polyacrylate, polyacrylamide or derivatives thereof, chitin, sepharose, exernally granules, polymeric dialdehyde starch, collagen, keratin, elastin, powder from bovine skin, peptidoglycan cell wall of bacteria or its fragments, nylon, polyethylenterephtalate, polycarbonates and glass with pores of a certain size. Particularly preferred solid media in this method of the present invention are chitin, polyacrylate, cellulose, their derivatives and sepharose. Immobilization reporter enzyme on a solid medium is performed with the use of standard methods and operations, known and understood by each specialist.

Enzyme-reporter can be immobilized on a solid carrier via a linker molecule, which is a hydrolyzable substrate for the enzyme active thiol protease. In this method, the present and the molecules are proteins. If the linker molecule is a protein, it is preferred proteins include, but are not limited to): azocasein, casein, Kappa-casein, globulin, hemoglobin, myoglobin, albumin, elastin, keratin and collagen. In the preferred embodiment, this method of the present invention, the linker molecule is a Kappa-casein, hemoglobin, or myoglobin.

The indicator may be any chemical compound that is detectivemisa change in the reaction or the climax of the reactions occurring under the action of the enzyme active thiol protease, if it is present in the sample or preparation. Induced in detective change indicates that the enzymatic active thiol protease present in the sample, and the presence of enzymatically active thiol protease in the sample, in turn, indicates the presence of Trichomonas vaginalis.

Preferred are visual indicators, and in particular, chromogenic indicators, i.e. those indicators, which detektivami change is the change of color, including the formation of color in another colorless material under the action of fermentive active thiol protease. Alternatively, the enzyme-reporter may have the ability to catalyze the formation of a fluorescent signal, a fluorescence signal, a bioluminescent signal, a chemiluminescent signal or electrochemical signal after its separation from the solid media under the action of the enzyme active thiol protease. In addition, the enzyme-reporter may have the ability to cause other visible or detected signals, such as clots, agglutination, precipitation or the formation of zones of clarification. In these cases, the indicator must be a chemical compound or the substrate required for this enzyme reporter or enzyme marker caused the desired detective change.

If enzymes-reporters or enzyme markers are peroxidase, the visual indicators can be used in a variety of chromogenic indicators (i.e., Chromogens) a wide range, as well as other compounds having similar effects. In accordance with the present invention the preferred chromogenic indicators consist of a hydroperoxide and a Chromogen, which represent the following compounds (but not limited to) the Pirin and 4,5-dihydroxynaphthalene-2,7-disulfonate acid. The most preferred chromogenic indicator consists of a hydroperoxide and guaiac resin, i.e., Chromogen, which is colorless in the reduced state and the dark blue in the oxidized state. The choice of the most suitable chromogenic indicator for this enzyme-reporter depends on the reaction or reactions that this enzyme is able to catalyze or initiate, and in any case, this choice can easily be carried out by any specialist. As described earlier, if the visual indicator is a chromogenic indicator, in this case can be used with either liquid chromogenic system, or solid chromogenic system.

As in the case of the analysis of the aspartic protease, in this method for increasing the activity and specificity of thiol proteases can be used in certain reaction conditions and specific inhibitors. For example, in order in this analytical system could function, and hence could be detected only thiol protease, this system can be added inhibitors (such as pepstatin for the inhibition of aspartic proteases, soybean trypsin inhibitor for the inhibition of trypsin, EDTA or DRs, not related to the group of thiol proteases). In addition, many thiol protease active at a pH of about 7.4, while aspartic protease at this pH value will be inactivated.

It is important to note that the technology of the release of the enzyme reporter in accordance with the present invention can also be used for detection of the presence or absence of inhibitor hydrolases in the sample. Many biological processes, including regulation of blood pressure, blood clotting, replication of bacteria, etc. require very specific carefully modelirovaniya hydrolases. Moreover, it is known that many drugs, pesticides and herbicides act by inhibiting specific hydrolases. Under certain circumstances, it is necessary to determine the concentration in the blood of a therapeutic agent that inhibits hydrolase, or to determine the level of product contamination potential pesticide inhibitor hydrolases, etc., So in these cases it is necessary to carry out the detection is not very hydrolases, and its inhibitor.

Accordingly, in another aspect, the present invention deals with a method of analysis for the presence of inhibi the m carrier, on which the immobilized enzyme reporter so that this enzyme reporter was released under the action of the target hydrolases in that case, if the target hydrolase not inactivated by the presence of the inhibitor;

(b) after contact with the target hydrolases and solid media, the interaction of the sample with the indicator, which can be detectivemisa change under the action of the enzyme reporter; and

(c) monitoring detektivami change indicator, and, if such change occurs, it indicates that the inhibitor target hydrolases in this sample is missing.

To detect the presence of the inhibitor hydrolases in the sample to the analytical system enter a specific number of target hydrolases, and subsequent testing is aimed at detecting the ability of the sample to inhibit the target hydrolase. If the inhibitor target hydrolases is absent in the sample, the target hydrolase will facilitate the separation of the enzyme reporter from solid media, resulting in the indicator will be detective change. Conversely, if the inhibitor target hydrolases present in the sample, the target hydrolase will ingibirovannym be detective change or the detected response. While it is not necessary that the inhibitor present in the sample, completely inhibited target hydrolase added to the analytical system. You only need to target hydrolase inhibited in a quantity sufficient to produce noticeable changes in the expected detektiruya the answer.

This method of the present invention can be used to analyze the presence or absence of any known inhibitor of hydrolytic enzyme, including but not limited to, inhibitors of proteases or proteinases (these terms are interchangeable), peptidases, lipases, nucleases, Homo - or heteropolysaccharides, Homo - or heteropolysaccharides, phosphatases, sulfates, neuraminidase and esterases. In the preferred embodiment, this method is used to analyze the presence of protease inhibitors, including but not limited to, inhibitors of aspartic proteases, inhibitors of serine protease inhibitors of thiol protease inhibitors metalloprotease, inhibitors of acid protease and inhibitors of alkaline protease. In a more preferred embodiment, this method is used to analyze the presence of inhibitors of aspartic proteases, such as the H-261, MV7-101, A-75925, A-76928 and A-7003 are experimental drugs that were previously described in the literature. In an even more preferred embodiment, the implementation of this method of the present invention provides for the detection of pepstatin, which is an inhibitor of aspartic proteases.

In accordance with the method of the present invention, the target hydrolases include, but are not limited to): proteases, proteinases, peptidases, lipases, nucleases, Homo - or heteropolysaccharides, Homo - or heteropolysaccharides, phosphatase, sulfatase, neuraminidase and esterase. The choice of a specific target hydrolases used in the above method depends on the inhibitor, the presence of which is necessary to determine in this analysis, and in any case, this choice can be made easier by each specialist. For example, detektivami inhibitor is pepstatin, as the target hydrolases, in the above described analytical system should be used aspartic protease.

Enzyme reporter or the enzyme is a marker used in this method of the present invention can be any signalgeneration enzyme, i.e. an enzyme activity which is outstudy in the sample, including inactivation by hydrolysis under the action of the target hydrolases present in the sample. Such enzymes-reporters are (but are not limited to): peroxidase, phosphatase, oxireductases, dehydrogenases, transferases, isomerases, kinases, inhibitors, deaminase, catalase, urease and glucuronidase. The selection of the appropriate enzyme-reporter or enzyme marker can be easily implemented by any specialist. In this method of the present invention, the preferred enzymes-reporters are peroxidase, and in particular horseradish peroxidase.

Enzyme-reporter immobilized on a solid medium, i.e. insoluble matrix, gel or resin, so that this enzyme reporter was separated from the solid media under the action of the target hydrolases, if this target hydrolase not inactivated by the presence of the inhibitor. In accordance with the present invention the solid carriers include, but are not limited to: cellulose, agarose, dextran, polyacrylate, polyacrylamide or derivatives thereof, chitin, sepharose, exernally granules, polymeric dialdehyde starch, collagen, keratin, elastin, powder from bovine skin, peptidoglycan cell wall of the EPA.

Enzyme-reporter can be immobilized on a solid carrier via a linker molecule, which is a hydrolyzable substrate for the enzyme active target hydrolases. Such linker molecules are (but are not limited to proteins, carbohydrates, lipids, peptides, esters and nucleic acids. The choice of a specific linker molecules used for binding of the enzyme reporter with solid media, depends on the specific target hydrolases added in an analytical system that can be easily implemented by each specialist.

The indicator may be any chemical compound that causes detective change in the reaction or the culmination of reactions occurring under the action of the enzyme active target hydrolases in if this hydrolase not inactivated by the presence of the inhibitor in the sample or preparation. Preferred are visual indicators, and in particular, chromogenic indicators, i.e. those indicators, which detektivami change is the change of color, including the formation of color in another colorless material under the action of the enzyme reporter or enzyme marker, if the TA target hydrolase not inactivated by the presence of the inhibitor in the sample or preparation.

As visual indicators can be used in a variety of chromogenic indicators (i.e., Chromogens) a wide range, as well as other compounds having similar effects. In accordance with the present invention the preferred chromogenic indicators for peroxidation enzymes reporters consist of a hydroperoxide and a Chromogen, which represent the following compounds (but not limited to): guaiac resin, 2,2'-Azino-bis(3-ethyl-benazolin-6-sulfonic acid), tetramethylbenzidine, phenol, 4-amino-antipyrine and 4,5-dihydroxynaphthalene-2,7-disulfonate acid. Especially preferred chromogenic indicator consists of a hydroperoxide and guaiac resin, i.e., Chromogen, which is colorless in the reduced state and the dark blue in the oxidized state. As described earlier, if the visual indicator is a chromogenic indicator, in this case can be used with either liquid chromogenic system, or solid chromogenic system.

In another aspect of the present invention is considered a testing device for testing for the presence of enzymatically active hydrolases in the sample, where the specified devices, the other internal surfaces with a gap between them, the first, second or both walls made of translucent material; enzyme-reporter, immobilized on a solid medium on the inner surface of the first or second wall, and the enzyme-immobilized reporter so that it is released under the action of hydrolases; indicator contained on the inner surface of the first or second walls and having the ability to be detectivemisa change under the action of the enzyme reporter; and the hole in the specified receiver for inputting sample.

In accordance with this aspect of the present invention the specified receiver device is flat and thin and has such a size that makes it easy to hold in hand. In accordance with this camera is preferably flat and shallow, and its length and width much greater than the depth, and the depth remains largely constant. This camera is preferably shallow, i.e., having a depth sufficient to ensure spontaneous wetting of the walls of the chamber with the drug in order to achieve maximum contact between the drug and the coating dried reagents on the top and bottom surfaces. Of particular interest p is a constant distance between these surfaces will also minimize the distance through which the reagents are located on the surface opposite to the surface coated with her visual indicator, will have to diffuse before reaching the indicator.

Based on these considerations it should be noted that the depth of the chamber is not critical to this invention and may vary. Basically the depth camera, which is in the range from about 3 to 50 mils (0.003 to 0,050 inch; 0,0076-0.127 centimeters), and preferably from about 5 to 15 (0,005-0,015 inch; 0,0127-0,0381 cm), allows you to get the best results. For any given depth, the transverse dimensions of the camera (i.e. the distance between its side walls) will be determined by the sample size, which should be adapted to this device, but otherwise these dimensions are not critical, except when they determine the size and shape of the test area on the outer surface of the device. For example, the transverse dimensions should be such that the test area was large enough to make observations and at the same time small enough so that the cell was completely filled with the preparation of the appropriate size. The size of the drug can various surface is assumed to be approximately 0.1 cm2up to 10 cm2and preferably from 0.3 cm2up to 3 cm2. In addition, the internal volume of the chamber in a typical device may vary, but for most types of samples the most appropriate and suitable amount is from about 3 μl to 300 μl.

The testing device of the present invention can be used to analyze the presence of any known hydrolytic enzyme, including but not limited to): protease or proteases (these terms are interchangeable), peptidases, lipases, nucleases, Homo - or heteropolysaccharides, Homo and heteropolysaccharides, phosphatase, sulfatase, neuraminidase and esterase. In a preferred embodiment of the present invention, the testing device can be used to analyze for the presence of proteases, including (but not limited to, serine proteases, aspartic proteases, thiol proteases, metalloproteases, as well as acidic and alkaline protease. In another preferred embodiment, the testing device of the present invention can be used to analyze for the presence of Homo - or heteropolysaccharides or Homo - or heteropolysaccharides, including (but not limited the range of this test device can be any signalgeneration enzyme i.e. enzyme activity which causes a detectable change and which is not affected by inactivation of any agent present in the sample, including inactivation by hydrolysis under the action of the active hydrolases present in the sample. Such enzymes-reporters are (but are not limited to): peroxidase, phosphatase, oxidoreductase, dehydrogenase, transferases, isomerases, kinases, inhibitors, deaminase, catalase, urease and glucuronidase. The selection of the appropriate enzyme reporter or enzyme marker can be easily implemented by any specialist. Preferred enzymes reporters in this embodiment of the present invention are peroxidase, such as horseradish peroxidase.

Enzyme-reporter immobilized on the first solid medium, i.e. insoluble matrix, gel or resin, so that this enzyme reporter was separated from the solid media under the action of hydrolases, the presence of which is detected in this analysis. In accordance with the present invention is preferable solid carriers include, but are not limited to: cellulose, agarose, dextran, polyacrylate, polyacrylamide or derivatives thereof, chitin, sepharose, oxirane the douping the cell wall of the bacteria or its fragments, nylon, polyethylenterephtalate, polycarbonates and glass with pores of a certain size. Immobilization of the enzyme reporter on the first solid medium carried out using standard methods and operations are well known and understood by each specialist.

This enzyme reporter can be directly linked to the first solid carrier, or alternatively, it can be immobilized on the first solid carrier via a linker molecule having a hydrolyzable bond, which is the substrate for the enzyme active hydrolase, detected in this analysis. Such linker molecules are (but are not limited to: proteins, carbohydrates, lipids, peptides, esters and nucleic acids. The choice of a specific linker molecules used for binding of the enzyme reporter with the first solid medium, depends on the creation of hydrolases, which can be easily carried out by each specialist.

In a test device of the present invention, the indicator immobilized on the second solid medium, which is not in contact with the first solid carrier. In accordance with the present invention is preferable solid carriers include, but are not the sludge granules, polymeric dialdehyde starch, collagen, keratin, elastin, powder from bovine skin, peptidoglycan cell wall of bacteria or its fragments, nylon, polyethylenterephtalate, polycarbonates and glass with pores of a certain size. Immobilization of the indicator on the second carrier is carried out using standard methods known to every expert.

The indicator may be any chemical compound that causes detective change in the reaction or in the climax of the reactions occurring in the case, if the sample or the drug is present enzymatic active hydrolase. Induced as a result of this detective change in the indicator indicates that the enzymatic active hydrolase is present in the sample or preparation. Preferred indicators are visual indicators, and in particular chromogenic indicators, i.e. indicators such in which the visible change is the change of color, including the formation of color in another colorless material under the action of the enzyme reporter or enzyme marker in that case, if it is separated from the solid media using hydrolases, the presence of which detec the Finance fluorescent signal, fluorescence signal bioluminescent signal, a chemiluminescent signal or electrochemical signal after its separation from the solid media under the action of hydrolases. In addition, the enzyme-reporter may cause other visible or detected signals, such as clots, agglutination, precipitation or the formation of zones of clarification. In these cases, the indicator must be a chemical compound or the substrate required for this enzyme reporter or enzyme marker has produced the desired detective change.

As visual indicators can be used in a variety of chromogenic indicators a wide range (i.e., Chromogens) and other compounds having similar effects. If the enzyme-reporters or enzyme markers are used peroxidase, the preferred chromogenic indicators consist of a hydroperoxide and a Chromogen, which represent the following compounds (but not limited to): guaiac resin, 2-2'-Azino-bis(3-ethyl-benazolin-6-sulfonic acid), tetramethylbenzidine, phenol, 4-aminoantipyrine and 4,5-dihydroxynaphthalene-2,7-disulfonate acid. The most preferred homogeneousand condition and dark blue in the oxidized state. Before using guaiac resin may be, but not necessarily purified, for example, by solvent extraction. The choice of the most suitable chromogenic indicator for this enzyme-reporter depends on the reaction or reactions that this enzyme is able to catalyze or initiate and can easily be carried out by any specialist.

For visual chromogenic indicators use solid chromogenic system. This solid chromogenic system consists of a hydroperoxide; Chromogen that can be oxidized by hydroperoxides in the presence of peroxidase; and solid media, which is impregnated with a Chromogen or immobilized Chromogen, and then dried. In this system, the Chromogen may be impregnated blotting paper or media, as was done in the case of specimens Hemoccultor alternatively, the Chromogen may be deposited a thin layer of plastic or other material made in the form of a sheet. In this latter embodiment, the Chromogen may be laid up in the form of a solution containing a polymeric material (such as hydroxypropylcellulose, ethylcellulose and so on). If the Chromogen is a water-soluble, totsa in water, it can be applied in the form of a solution in an organic solvent, both separately and in combination with soluble or water-insoluble polymer.

In the specified chromogenic system can be used hydroperoxide in solid form (for example, the hydroperoxide titanium), or it can be generated in situ in the test device. So, for example, hydrogen peroxide can be generated in situ by applying dried glucose and glucose oxidase on the inner surface of the test device or by applying a suspension of sodium perborate in alcohol on the inner surface of the test device. At low pH there is a spontaneous release of hydrogen peroxide of sodium perborate. In the presence of hydrogen peroxide and peroxidase (for example, percoidei horseradish), after its separation from the first solid media using hydrolases Chromogen is oxidized, resulting in the observed visually detectable colour change. As mentioned above, this change in color indicates that the enzymatic active hydrolase is present in the sample or preparation.

Any specialist it is clear that to improve the specificity and activity GIII (such as a solid carrier, linker molecules, pH, buffer capacity, buffer identity, salts, etc.,) in the inventive test device can be modified and adjusted. In addition, each specialist is clear that to improve the activity and specificity of hydrolases and differentiation of various hydrolases present in the sample, stated in the test device can be added specific enzyme inhibitors.

The test device is equipped with an opening for entry of the sample into the chamber. This hole is preferably on the same wall, on which there are changes in visual indicator, i.e., light transmissive wall. The specified hole must be of the form that would be suitable for a portable device used to transfer the sample from its source, and therefore, this hole may vary in accordance with different types of portable devices that can be used for this purpose. Examples of portable devices include syringes, pipettes, a rod with a swab for taking a swab and the medical mirror. Can be used and other tools that are well known in the art. Basically, the hole has a round shape, is who can do the scraping for best separation of samples.

Preferred variants of the test devices have additional features that facilitate the passage of the liquid required to fill the chamber and thereby to bring all reagents in contact with the drug. One of these distinguishing features is that the camera provide one or more vent holes for air passage. To increase the surface area wetted by the sample, these vents must be located at an adequate distance from the hole to enter the sample. In order to avoid obtaining incorrect or uncertain data, in devices where the activated samples positive and negative control zones are located inside the chamber and are horizontally adjacent to the test area, the ventilation openings must be located so that the sample was achieved by both the control area and fill them in. As discussed below, in one of the preferred designs of this device the test area is between control areas, and positive and negative control areas do not have a common border, although each of them has a common border with the test area. directly above the test area, one vent is located above each of the two control zones, or near the outer edges of these zones, resulting in the sample fills the first test area, and then both the control area.

Another feature in the preferred embodiment of the present invention, facilitate the movement of fluid in the device, is that along the inner surface of the chamber is placed surfactant. This substance may be placed along one or the other of the upper and lower surfaces, and preferably along both surfaces and may be included in the form of dry solute in the matrix of the medium constituting the uppermost layer or coating on the surface. In some cases, this layer may also contain one or more reagents involved in the test reactions. In other cases, the surfactant may be the only functional component of this layer.

Surface-active substances (surfactants) can be used for preparations containing water, representing the majority of biological drugs. Suitable surfactants are those substances, to the E. of a wide range of substances, with surface-active action. Such substances may be detergents, wetting agents or emulsifiers, however, they can vary widely in its chemical structure and electronic properties and represent anionic, cationogenic, switcheroonie and nonionic substances. As examples can serve alkyl-alkoxysilane, alkyl-arylsulfonate, esters of glycerol formed with the fatty acids derived on the basis of lanolin, polyoxyethyleneglycol, polyoxyethylenes, polyoxyethylene fatty acids and esters, polyoxyethylene fatty alcohols and ethers, poly(etilenglikolevye) fatty acids and esters, polyoxyethylene fatty esters and oils, condensation products of polyoxypropylene/and polyoxyethylene block polymers, complex arbitrarily fatty acids, sulfo derivatives succinate, Alkylglucoside and derivatives holeva acid. Some of these substances are in the form of finished products under the following trademarks; Lubrol, Brij, Tween, Tergitol, Igepal, Triton, Teepol and many others.

The formation of solid layers of the indicator reagent can be carried out by applying a material in liquid form with the e l e C for example, a solution, suspension or unhardened liquid state of matter, the next stage of rejection which can be carried out, for example, by solvent evaporation or solidification of the substance. This substance can be combined with other materials, for example, in order:

(1) facilitate the application liquid on the surface by modifying the viscosity of the liquid;

(2) formation of a continuous smooth solid layer, which would remain homogeneous and are not subject to disintegration and granulation over time or after the application of other layers;

(3) modification of solubility of the layer of the solvents used in the layers applied on top of this layer; or give this layer the ability to dissolve in solvents that do not dissolve layers deposited below this layer; or to achieve all these goals simultaneously. To achieve one or all of these purposes, the preferred additives are polymeric materials. As examples can serve as cellulose and its derivatives with substituents, selected in such a way as to achieve the desired solubility characteristics. If the test device prednaznachennyi in a matrix of solid material, which is not soluble in water. This prevents the indicator from the migration of the layer and transmits light from the surface. And if this indicator is insoluble in water, it will form a coherent layer which will remain intact.

For those variants of the present invention, where the device is injected indicator a positive and negative control, or both for each control can be used one or more reagents. Such additional reagents can be introduced into one of the layers in the horizontal part of this layer, or they can be deposited as a separate layer that is vertically adjacent to the horizontal part of the existing layer. So thanks to the position of these additional reagents to determine the control area, which are horizontally separated from each other and from the test area.

The choice of reagents suitable for positive or negative controls, dependent hydrolases, the presence of which is detected in this analysis, the type of visual indicator used to detect the presence of this hydrolases and purpose of reage use of known chemical compounds in most cases, the choice of a suitable reagent can be easily carried out by each specialist. For example, the reagent positive control may be a sample of the hydrolases,

similar hydrolases or any other substance with similar mechanism of action that initiates or induces reaction or cascade of reactions leading together to detectivemisa change indicator. The layer containing the reagent can be either on the top or on the bottom surface of the chamber, provided that the reagent will not initiate or to induce detective change until, until you have entered the sample, but it should act regardless of present or absent enzymatic active hydrolase in the sample. Reagent negative control may be inhibiting substance, such as denaturing, inhibiting or any other inactivating agent that prevents or blocks the reaction or cascade reactions, thus preventing detective change occurring regardless of present or absent enzymatic active hydrolase in a sample or preparation.

Both control act is more efficient, if the control reagents will be in the layers located on the same surface as the layer (or layers) containing a different reagent (or reagents). In other cases, the best results can be achieved if the control reagents will be in layers on the surface of the chamber which is opposite the surface carrying the other reagent (or reagents) so that the control reagent is separated from the other reagent (or reagents) by an air gap. In a preferred embodiment of the present invention, the control zone devices contain all components and reagents used in the test zone and control reagents, which are either injected into the horizontally-defined areas of the same layer one or more layers used in the test zone, or applied as separate layers in such horizontally delineated areas. To obtain clear boundaries of control areas and for protection of the test zone from the activation or deactivation of the control reagents, it is often preferred that the layers on the boundaries of the control area from the test area, there were breaks in order to minimize the zones. These discontinuity in the layers may be present along the top surface, bottom surface, or along both surfaces.

As mentioned above, the control zone is activated preferably by the same drug used for the test zone. This success is due to the fact that the control area is like a continuation of the test zone, and they are all in the same camera test device with unhindered movement of fluid between the various zones. In the preferred embodiment, which uses positive and negative control zone, these control zones are separated from each test area, which is located between them. Filling in all of these areas with one making the sample can be carried out with the device holes for the input of the sample and vent holes described above. Since the detected changes or lack of them are detected by a light transmissive wall of the unit, the identification of both positive and negative control areas by using appropriate labels to be put on the external, the which is inert and rigid enough to maintain the indicator layer, and transparent enough to monitor the change of the indicator, if applicable. Can be used translucent or transparent material, preferably non-absorbing materials, preferred are transparent materials. Examples of the transparent polymer materials suitable for use in the above-mentioned purposes are polyethylenterephtalate (such as Mylar) and polycarbonates (such as Lexan). The opposite (i.e., lower) wall of the device may also be made of transparent or translucent material and an opaque material, because the observation of the results of the analysis, as well as for positive and negative control is carried out only from one side of the device. If the bottom wall is transparent, the detection of changes in the test zone and control zone or in one and the other through the top wall may be reinforced by applying strains or coating on any surface of the bottom wall with the use of a colored or reflective material for mi. For example, the sheet of polymeric material with the corresponding grooves defining the shape of the camera, with holes for the input of the sample and vents can be layered on each other. The depth camera, as well as its shape and transverse dimensions are determined by the thickness of the Central sheet, whereas the position of the holes will be governed by the top sheet. Coverage indicator and reagent can be applied on the top sheet, the bottom sheet or both sheets, if necessary, after which the sheets are combined, forming a layered structure. For reliability, these sheets can then be bonded together by any standard method, for example, by welding or bonding using adhesives.

Especially preferred method of receiving device of the present invention is that, for this purpose use just one sheet of transparent or any other translucent polymer material, which is subjected to embossing or any other mechanical or chemical treatment in certain areas so that the inner surface of the chamber contained a recess or groove of the same depth. This deepening is on one half lestremiinae areas of the sheet put coverage indicator and reagent after which the sheet is folded so that its half containing the specified holes superimposed on the other half, thus forming a closed chamber with an exact match of the areas representing the top and bottom surface of the camera. The opposite surface of the sheet (i.e., the surface of the spread) are fastened together by the means indicated in the previous paragraph to obtain the laminate.

In the preferred method of fastening the two halves of the sheet provides for the use of the adhesive activated by heat bonding under pressure, water-based or solvent-based. Avoid contact with reagents involved in the test, the adhesive may be applied on the peripheral areas, remote from the camera, or it may cover the entire surface of the sheet, but in this case, the adhesive must be applied before the coating of the indicator and reagents. In the latter case, it is necessary to use glue that is transparent, inert wetted and compatible for any other parameters with layers, which are applied to the adhesive. There are many types of adhesives that meet the specified requirements, and selecting the most appropriate is the SPECTA present invention relates to a testing device for analysis of a sample for the presence of candidiasis by detection of the presence of enzymatically active aspartic protease, where the specified test device includes: a receiver formed partially in the first and second opposite walls facing each other of the inner surfaces and with a gap between them, and the first, second or both walls made of translucent material; enzyme-reporter, immobilized on a solid medium on the inner surface of the first or second wall so that this enzyme reporter is released under the action of aspartic protease; indicator immobilized on the inner surface of the first or second wall, and having the ability to be detectivemisa change under the action of the enzyme reporter; the hole in the specified receiver for introduction of the sample.

Enzyme reporter or the enzyme is a marker used in this test device can be any signalgeneration enzyme that does not undergo inactivation of any agent present in the sample, including inactivation by hydrolysis under the action of any active aspartic protease or any other active hydrolases present in the sample. Such enzymes-reporters are (but are not limited to): peroxidase, fosa and glucuronidase. Particularly preferred enzymes-reporters are peroxidase, such as horseradish peroxidase.

Enzyme-reporter immobilized on the first solid medium, i.e. insoluble matrix, gel or resin, so that this enzyme reporter was released under the action of aspartic proteases. Enzyme-reporter can be immobilized on a solid carrier via a linker molecule having a hydrolyzable bond, which is a substrate for aspartic protease. In this test device such linker molecules are proteins and peptides, while the preferred linker molecules are proteins. If the linker molecule is a protein, it is preferred proteins include, but are not limited to): azo-casein, casein, Kappa-casein, globulin, hemoglobin, myoglobin, albumin, elastin, keratin and collagen. In a preferred embodiment, the test device of the present invention the linker molecule is a Kappa-casein, casein, hemoglobin, or myoglobin.

In a test device of the present invention, the indicator immobilized on the second solid medium which does not contact the first solid carrier. In soo): cellulose, agarose, dextran, polyacrylate, or their derivatives, chitin, sepharose, exernally granules, polymeric dialdehyde starch, collagen, keratin, elastin, powder from bovine skin, peptidoglycan cell wall of bacteria or its fragments, nylon, polyethylenterephtalate, polycarbonates and glass with pores of a certain size. Immobilization of the indicator on the second carrier is carried out using standard methods known to every expert.

The indicator may be any chemical compound that is detectivemisa change in the reaction or the climax of the reactions occurring in the case, if the sample or the drug is present enzymatic active hydrolase. Displayed in the result detective change indicates that the enzymatically active aspartic protease present in the sample, and the presence in the sample enzymatically active aspartic protease in turn indicates the presence of candidiasis. Preferred indicators are visual indicators, and in particular chromogenic indicators, i.e. indicators such in which the visible change is the change of color, including education, what if he is separated from the solid media by using enzymatically active aspartic protease, the presence of which is detected in this analysis.

As visual indicators can be used in a variety of chromogenic indicators (i.e., Chromogens) a wide range, as well as other compounds having similar effects. If the released enzyme-reporter used the peroxidase, in accordance with the present invention the preferred chromogenic indicators consist of a hydroperoxide and a Chromogen, representing one of the following compounds (but not limited to): guaiac resin, 2-2'-Azino-bis(3-ethyl-benazolin-6-sulfonic acid), tetramethylbenzidine, phenol 4-aminoantipyrine and 4,5-dihydroxynaphthalene-2,7-disulfonate acid. Especially preferred chromogenic indicator consists of a hydroperoxide and guaiac resin, which is colorless in the reduced state and a dark blue in the oxidized state.

If the visual indicator is a chromogenic indicator for peroxidases or pseudoperoxidase, this case is solid chromogenic system, which consists of a hydroperoxide, a Chromogen, speedyway a Chromogen or where immobilized Chromogen, and then dried. In this system, the Chromogen may be impregnated blotting paper or media, as was done in the case of specimens Hemoccultor alternative Chromogen may be deposited a thin layer of plastic or other material made in the form of a sheet. In this latter embodiment, the Chromogen may be applied in the form of a solution containing a polymeric material (such as hydroxypropylcellulose, ethylcellulose and so on). If the Chromogen itself is water soluble, it can be included in the matrix of material which is not soluble in water. Alternatively, if the Chromogen is not soluble in water, it can be applied in the form of a solution in an organic solvent, both separately and in combination with soluble or water-insoluble polymer.

In the specified solid chromogenic system can be used hydroperoxide in solid form (for example, the hydroperoxide titanium), or it can be generated in situ in a given test device. In the presence of a hydroperoxide and peroxide after its separation from the first solid medium under the action of the enzyme active aspartic protease, a Chromogen is oxidized, resulting in n is Alstom about what enzyme active aspartic protease present in the sample or the drug, which in turn indicates the presence of candidiasis. In the latter aspect the present invention relates to a testing device for analysis of a sample for the presence of the inhibitor target hydrolases, where the aforementioned testing device includes: a receiver formed, in part, first and second opposite walls facing each other of the inner surfaces and with a gap between them, and the first, second or both walls made of translucent material; a target hydrolase located on the inner surface of the first or second wall and is able to undergo inactivation in the presence of inhibitor; enzyme-reporter, immobilized on a solid medium on the inner surface of the first or second wall so that this enzyme reporter is released under the action of the target hydrolases in if this hydrolase not inactivated by the presence of the inhibitor; the indicator is immobilized on the inner surface of the first or second wall and can be detectivemisa change under the action of an enzyme-report is hydrolases in the sample in the test device enter a specific number of target hydrolases or immediately before testing, or, preferably, in the manufacturing process of this testing device. Implementation testing involves determining the ability of the sample to inhibit the target hydrolase. If the inhibitor target hydrolases is absent in the sample, the target hydrolase will facilitate the separation of the enzyme reporter from solid media, resulting in the indicator will be detected the induction response. Conversely, if the inhibitor target hydrolases present in the sample, the target hydrolase will be inhibited, resulting in an enzyme-reporter cannot be detected from the solid media, and the indicator will not be detective change or the detected response. It's not necessary that the inhibitor present in the sample, completely inhibited target hydrolase added to the test device. You only need to target hydrolase inhibited in a quantity sufficient to produce noticeable changes in the expected detektiruya the answer. Elements of positive and negative control, available on the test device, allow for comparison of responses to illustrate the appearance of the article in stateliest described technology release of the enzyme can be adjusted by imposing certain quantities of target hydrolases in the target device. Similarly, if necessary, the time during which the target hydrolase exposed any inhibitor present in the sample, can be regulated by physical or chemical separation of the target hydrolases from the immobilized enzyme-reporter. For example, using a coating of immobilized enzyme-reporter in the matrix slow release, pH-degradiruemosti coating or other material with controlled release and relatively slow dissolution, can be adjusted temporary access target hydrolases to the immobilized enzyme-reporter. Alternatively, the target hydrolase may be present in a particular place or in a specific chemical form that is directly accessible to any inhibitor present in the sample. Therefore, before the target hydrolase will be available immobilized enzyme-reporter, it may be first exposed to the inhibitor for a period of time sufficient to effect the inhibition. Because access to the immobilized enzyme, the reporter may be subjected to the provisional regulation, then this test can be detected presence is retene can be used to analyze the presence or absence of any known inhibitor of hydrolytic enzyme, including (but not limited to, inhibitors of proteases or proteinases (these terms are interchangeable), peptidases, lipases, nucleases, Homo - or heteropolysaccharides, Homo - or heteropolysaccharides, phosphatases, sulfates, neuraminidase and esterases. In the preferred embodiment, this testing device is used to analyze the presence of protease inhibitors, including but not limited to): inhibitors of aspartic proteases, inhibitors of serine protease inhibitors of thiol protease inhibitors metalloprotease, inhibitors of acid protease and inhibitors of alkaline protease. In a more preferred variant of this test device is used to analyze the presence of inhibitors of aspartic proteases, such as pepstatin, ovomacroglobulin, haloperidol, replicas of the transition state, U-81749, H-261, MV7-101, A-75925, A-76928 and A-7003. U-81749, H-261, MV7-101, A-75925, A-76928 and A-7003 are experimental drugs that were previously described in the literature. In an even more preferred variant of this test device of the present invention detektivami inhibitor of aspartic proteases is pepstatin.

In accordance with izaberete teensy, peptidases, lipases, nucleases, Homo - or heteropolysaccharides, Homo - or heteropolysaccharides, phosphatase, sulfatase, neuraminidase and excerise. The choice of a specific target hydrolases used in this test device, depends on the inhibitor, the presence of which is determined in this analysis, and can be easily carried out by a specialist. For example, if detektivami inhibitor is pepstatin, as the target hydrolases in the above-described test device should be used aspartic protease. In addition, appropriate concentration of the target hydrolases, geometric localization of the target hydrolases in the test device, as well as appropriate techniques and matrix temporary and controlled release.

As in other test devices of the present invention, the enzyme-reporter or the enzyme is a marker used in this particular test device can be any signalgeneration enzyme, i.e. an enzyme activity which causes a visible or detectable change and which is not subject to inactivation by any agent present in the sample, including the inactivation through the-reporters are (but are not limited to): peroxidase, phosphatase, oxireductases, dehydrogenases, transferases, isomerases, kinases, inhibitors, deaminase, catalase, urease and glucuronidase. The selection of the appropriate enzyme-reporter or enzyme marker can be easily implemented by any specialist. In this test device of the present invention, the preferred enzymes-reporters are peroxidase, and in particular horseradish peroxidase.

Enzyme-reporter immobilized on a solid medium, i.e. insoluble matrix, gel or resin, so that this enzyme was released under the action of the target hydrolases in that case, if the task hydrolase not inactivated by the presence of the inhibitor. In accordance with the present invention the solid carriers include, but are not limited to: cellulose, agarose, dextran, polyacrylate, polyacrylamide, or their derivatives, chitin, sepharose, exernally granules, polymeric dialdehyde starch, collagen, keratin, elastin, powder from bovine skin, peptidoglycan cell wall of bacteria or its fragments, nylon, polyethylene terephthalate, polycarbonates and glass with pores of a certain size.

As discussed above, the enzyme-reporter can be immobilized on tvarchive target hydrolases. Such locarnini molecules are (but are not limited to: proteins, carbohydrates, lipids, peptides, esters and nucleic acids. The choice of a specific linker molecules used for binding of the enzyme reporter with solid media, depends on the specific target hydrolases entered in this test device, and can be easily carried out by each specialist.

The indicator may be any chemical compound that is detectivemisa change in the reaction or the climax of the reactions occurring under the action of the enzyme active target hydrolases in that case, if the task hydrolase not inactivated by the presence of the inhibitor in the sample or preparation. Preferred are visual indicators, and in particular chromogenic indicators, i.e. those indicators, which detektivami change is the change of color, including the formation of color in another colorless material under the action of the enzyme reporter or enzyme marker in that case, if it is separated from the solid carrier by means of enzymatic active target hydrolases, provided that this target hydrolase not inactivated due at the turn supported by various chromogenic indicators (i.e., Chromogens) a wide range, as well as other compounds having similar effects. In accordance with the present invention the preferred chromogenic indicators for peroxidation enzymes reporters consist of a hydroperoxide and a Chromogen, which represent the following compounds (but not limited to): guaiac resin, 2-2'-Azino-bis(3-ethyl-benazolin-6-sulfonic acid), tetramethylbenzidine, phenol, 4-aminoantipyrine and 4,5-dihydroxynaphthalene-2,7-disulfonate acid. Especially preferred chromogenic indicator consists of a hydroperoxide and guaiac resin, i.e., Chromogen, which is colorless in the reduced state and the dark blue in the oxidized state. As described earlier, if the visual indicator is a chromogenic indicator, in this case can be used with either liquid chromogenic system, or solid chromogenic system.

It should be noted that the previous discussion concerning the structure of the test device for testing for the presence of hydrolases, its construction and the preferred variants of this device are fully applicable to the testing device for testing a sample for the nalitch is to test the device for analysis of a sample for the presence of the inhibitor target hydrolases.

Although the present invention is not limited to any specific construction of the test device, however, in the drawings accompanying this description and are drawn not to scale, illustrates one of the possible methods of making such devices.

In Fig. 1 shows the supporting structure of this device is shown in perspective before applying the indicator and reagents and to close the camera. This supporting structure consists of a single sheet 11 made of the relative rigid, transparent, chemically inert plastic, which marked the bend line 12, dividing the sheet into two halves 13, 14 having the same length and width. The lower half 13 includes the deepening of complex shapes consisting of a circle 15 in the center with two rectangular extensions 16, 17 arranged in different directions. The upper half 14 has three openings including a Central opening 18, which serves as a hole for the input sample and two lateral holes 19, 20, which serve as vent holes. These two ventilation holes 19, 20 are round, and the hole for the input of the sample 18 is a circle with a straight edge, sdelannoe are thus when plastic folding on the fold line 12 and the contact of the upper half 14 with the lower half 13, the insertion opening for the sample 18 is located above the center of the circular part 15 of the recess, and the ventilation holes 19, 20 are located on two rectangular extensions 16, 17 at the edges farthest from the center. These two rectangular extension 16, 17 represent positive and negative control area of this unit.

The device shown in Fig. 1, can be produced in numerous variants. For example, two halves 13, 14 may have different length, width or both. This design has only one significant distinction, which is that of deepening in the lower half and the holes in the upper half must be located relative to the fold line so that when the sum of these two halves of the bend line, the said recesses and holes are combined as needed to work with this device. As another example, you can specify the design, where the rectangular extension 16, 17 in the lower half of this device may result in a circle (or semicircle) for full compliance with the ventilation holes 19,tertia, the shape of which differs from the shape of the openings for entry of the sample 18, have the advantage that the user will not be able to confuse these holes with a hole for the input sample.

In Fig. 2 shows a side view in section of the device illustrated in Fig. 1, which shows the camera 31 (in section) after coating and after the addition of the two halves to each other with their subsequent hermetic bond. The inner surface of each of the two halves 13, 14 transparent polymer cover the adhesive 32, 33, respectively. Directly below the upper layer 33 is a layer containing a visual indicator 34, and below this layer of the indicator is the reagent layer 35. It should be noted that the layer of the visual indicator 34 and the reagent layer 35 may extend along the entire length and width of the camera, surrounding the hole to enter the sample 18 and extending to all areas of the camera.

The test and control zones of the chamber are defined by the horizontal position of the coatings on the bottom wall 13 of the camera. Reagent for the negative control contained in one of the coatings 36, which is the bottom surface of one of the two rectangular extensions 16 of the camera (see Fig. 1), and a reagent for positive contri 17. Alternatively, control reagents can be placed not on the lower surface of the chamber, and on its upper surface. For some analyses, such arrangement of the reagents is preferred. Part of the bottom surface under the Central circular part 15 of the camera (see Fig. 1), covered with a layer 38, which may contain additional reagents used in the analytical reaction, or it may not contain any reagents. For example, if you look at the enclosed chamber from the top, with two diametrically opposite sides of the circular test area 41 are rectangular areas of the negative control 42 and the positive control 43. Three segments 36, 37, 38 may be separated by gaps or gaps 44, 45 to slow or minimize diffusion between these segments or contact between the contents of these segments. Directly above the gaps 44, 45 in the lower layer can be similar breaks in any of the layers of the visual indicator and reagent, or in both of these layers 34, 35. Gaps in the layers of the visual indicator and reagent serve as an additional barrier for diffusion control components or other reagents of the control zones in ispytatelei wetting agent or detergent to promote rapid and complete distribution of the sample along the top and bottom surfaces with filling the whole chamber. In some cases, a similar effect can be achieved using a layer of protein.

In some embodiments, implementation of the present invention as reagents may deteriorate with prolonged exposure to air or airborne moisture. In the apparatus shown in Fig. 2, this phenomenon can be prevented by the use of thin sheet material 46, which is breathable and moisture. This sheet covers the opening for the input of the sample and both the vent holes, thereby protecting the camera from the effects of the environment prior to its use, and when this device is ready to use, this sheet is easily removed. If the materials used are especially sensitive to the effects of water or air, it is desirable that the bottom surface of this device was covered with a moisture - impermeable sheet covering that may be either permanent or removable. Additional protection from moisture and air can be accomplished by placing the device in the package, which completely covers the device.

As mentioned above, the size of the device, pakatan logit design, in which the supporting sheet is a Mylarhaving a thickness of 5 mils (0.005 inches, 0,0127 cm); the adhesive layer is a low density polyethylene with a thickness of 2 mils (0.002 inch, 0,0051 cm); width of the gap 47 is 7.5 mils (0,0075 inch 0,019 cm), the area of the test zone is a circle of a diameter of 5/16 inch (area: 0,0766 square inch, 0,494 cm2), and the sizes of negative and positive control zones are each 1/8 and 1/16 inch (area: 0,0078 square inch, 0,0504 cm2). In this example, the ventilation holes are round and the dimensions of these holes and the holes for inserting the sample are (each) 1/8 inch (0,32 cm) in diameter. The chamber volume is approximately 12 μl.

The test device of the present invention can be used to analyze samples for the presence of hydrolases, originating from different sources a wide range, including biological sources and other Examples of such sources can serve as a physiological fluid, such as blood, serum, plasma, urine, discharge from the urethra, tears, vaginal secretions, cervical exudate, cerebrospinal fluid, and saliva, as well as nafisi the tx2">

Examples

1. Getting

A. preparation of solid media, activated with bromine cyan

1. Materials

a. Insoluble solid media (Sepharose 4B, chitin, and Sigmacell20 (bought by the company Sigma Chemical Co));

b. Distilled water and ice from distilled water;

c. 4.0 M NaOH;

d. Solid CNBr;

e. Binding buffer (0.1 M NaHCO3containing 0.5 M NaCl);

f. The engine has a magnetic stir bar and rod for mixing, pH meter and chemical fume hood.

2. The order of execution

10 ml of cold distilled water is added to 5 g of the wet, washed solid media and the resulting mixture is cooled to a temperature of 10-15oC. Then the pH of this suspension is adjusted to a value 10,8 and support, if necessary with the help of 4.0 NaOH, then add 100 mg of solid powdered CNBr per 1 g of wet solid media. Then the temperature of the suspension is increased to 18-20oC during the activation process. Activation is considered complete if for maintaining a pH of about 10.8, no longer requires the addition of 4.0 M NaOH. At this stage for cooling the reaction mixture, add the crushed ice and the suspension is filtered through a pre-cooled funnel of sintered stellately CNBr and cyanide, remaining in the reaction mixture. Solid support was washed with cold distilled water (1 l) and binding buffer (1 l), in a vacuum, and then incubated at 4oC in the form of a wet paste.

B. Obtaining conjugates [Kappa-casein-HRPO] (gersuny method)

1. Materials

a) Kappa-casein and hydrazide of horseradish peroxidase (HRPO) (purchased by the company Sigma Chemical Co.).

b) Distilled water;

c) Buffers:

i) 50 mm 2-[Morpholino(econsultancy acid)] (MES) buffer, pH 6,0

ii) 100 mm sodium buffer (pH of 4.0) containing 0.5 M NaCl

iii) 100 mm sodium borate (pH 9,0) containing 0.5 M sodium chloride;

d) Solid periodate sodium;

e) 100 mm Formaldehyde.

2. The order of execution

a) Activation of periodate Kappa-casein

20 mg Kappa-casein dissolved in 2 ml of MES buffer and to the solution was added 8.6 mg of solid periodate sodium. Then the resulting mixture was incubated on a rotor for 30 minutes in the dark at room temperature. The reaction mixture deleteroute approximately 300 ml of 50 mm MES, pH 6.0 for approximately 45 minutes at room temperature, after which the liquid to replace dialysis and dialysis hold for another 45 minutes.

b. Covalent binding of HRPO from kapporet in activated cialisovernight Kappa-casein and the mixture was incubated for 4 hours at room temperature, stirring the while. To this mixture is added 200 microliters 100 mm formaldehyde and incubated at room temperature for another 30 minutes. Then to the mixture is poured 2 ml of 1 M cold acetate buffer (pH 4,0) and conjugate precipitated for 30 minutes at a temperature of 4oC. the Resulting precipitate is removed by centrifugation, re-dissolved in 2 ml of 100 mm borate buffer (pH 9,0) and stored at a temperature of 4oC, then use as directed.

C. Binding of conjugates /Kappa-casein-HRPO/ (hydrazide method) with the media, activated bromine cyan

1. Materials

(a) Conjugates /Kappa-casein-HRPO/ (example B);

b) Distilled water;

c) Buffers:

i) Binding buffer (0.1 M NaHCO3containing 0.5 M sodium chloride);

ii) 1.0 M Tris buffer, pH 8.0;

iii) 100 mm acetate-sodium buffer (pH 4,0), sumeragi 0.5 M NaCl;

iv) 100 mm bharatmatrimony buffer (pH 9,0) containing 0.5 M NaCl;

(d) Sepharose 4B and Sigmacell20, activated with bromine cyan (example A);

(e) Sepharose 6 MB (from the company Sigma Chemical Co.), activated bromine cyan.

2. The order of execution

a) Sepharose 4B and Sigmacell20, activated with bromine cyan

2 ml of conjugate Kappa-casein-HRPO obtained from the guide who authorized Sepharose 4B or SigmacellThe resulting suspension is continuously stirred for two hours at room temperature. Solid support was washed with binding buffer and water, and then beat 3 ml of 1.0 M Tris buffer, pH 8. Then, the suspension is incubated for 2 hours at room temperature to inactivate the remaining active sites on the solid media. To remove unbound materials from the media using three cycles of leaching, each of which carry acetate buffer with pH 4.0, and then binding buffer. The final washing is carried out using distilled water and a wet slurry before using stored at a temperature of 4oC.

b) Industrial activated Sepharose 6 MB (Sigma Chemical Co.).

The wet gel (1 g of wet weight) was washed with 200 ml of 1 mm hydrochloric acid before use in accordance with the above description.

C) Activated chitin

Repeat the progress described in example A except that instead of 1 g using 500 mg of wet activated chitin.

D. Obtaining aldehyde of horseradish peroxidase (HRPO)

1. Materials

a. HRPO, Type II, 200 units/mg (from the company Sigma Chemical Co);

b. 20 MMD> P-30 (polyacrylamide gel in the form of spheres, supplied by the firm Rio-Rad).

2. The order of execution

of 25.7 mg (40 millimoles) of solid periodate sodium added to 30 mg of horseradish peroxidase in 3 ml of MES buffer. The resulting solution was incubated in the dark for 30 minutes at room temperature with continuous rotation. This solution is passed through a column of P-30 in MES buffer to remove excess periodate sodium and dyed horseradish peroxidase ordinary collect in full 3 ml of the resulting aldehyde of horseradish peroxidase (HRPO) directly attached to the desired protein (hemoglobin, myoglobin, or casein).

E. Binding of hemoglobin and myoglobin with brachionichthyidae solid media

1. Materials

a. Insoluble solid media;

i. Brachionichthyidae sepharose 6MB (supplied by the company Sigma Chemical Co.

ii. Sigmacell20 (described in example A);

b. Distilled water

c. Buffers:

i. Binding buffer (0.1 M NaHCO3containing 0.5 M NaCl)

ii. 1.0 M Tris buffer, pH 8.0.

iii. 100 mm acetotartrate buffer (pH of 4.0) containing 0.5 M NaCl

iv. 100 mm sodium buffer (pH 8.0) containing 0.5 M NaCl

v. 0.1% (on the a, dissolved in 5 ml of binding buffer, added to 1 g of wet solid media. The resulting suspension is continuously stirred for 2 hours at room temperature. Then, the solid support is washed with binding buffer and water, then add 3 ml of 1.0 M Tris-buffer (pH 8) and the suspension incubated for 2 hours at room temperature to inactivate the remaining active sites on the solid media. To remove unbound materials from the media, using three cycles of washing, each of which carry acetate buffer (pH 4,0), and then borate buffer containing sodium chloride. For media derivatizing by myoglobin, using the final rinse with distilled water, and the moist suspension stored at a temperature of 4oC until its use. For media derivatizing hemoglobin, to damp the suspension obtained in accordance with the above procedure, add 5 ml of 0.1% glutaraldehyde solution and the resulting suspension is incubated over night at a temperature of 4oC. To remove unbound materials from media use three rinse cycle, each of which hold acetate (pH of 4.0), and then borate buffer (pH 8.0). Pospolitaya.

F. Linking aldehyde of horseradish peroxidase (HRPO) with media derivationally hemoglobin or myoglobin

1. Materials

a. HRPO aldehyde obtained by the method described in example D.

b. Solid carriers, derivateservlet hemoglobin or myoglobin and obtained by the method described in example E

c. 100 mm cyanoborohydride sodium

d. 1.0 M NaCl

e. The distilled water.

2. The order of execution

3 ml of HRPO aldehyde (30 mg) obtained from column P-30 (example D), added to 1 g of the solid media, derivatizing hemoglobin or myoglobin and described in example E. the Resulting suspension is incubated for 16 hours at a temperature of 4oC and then for 4 hours at room temperature. To remove unbound materials from the media using three cycles of washing, each of which is carried out using distilled water and 1.0 M NaCl. Then, the solid support is incubated with 5 ml of 100 mm cyanoborohydride over night at a temperature of 4oC. After final washing with distilled water the moist suspension stored at 4oC until its use.

C. Receiving allegedclaiminga Sigmacellthe profiled water

c. Solid periodate sodium

d. 20 mm sodium Bicarbonate, pH of 9.5.

2. The order of execution

Solid periodate sodium (428 ml) are added to 1 g Sigmacellsuspended in 10 ml of distilled water. The resulting mixture was continuously stirred by rotation for 2 hours at room

temperature. The formed precipitate is removed by centrifugation, and then washed five times with 10 ml of distilled water and once with 10 ml of 20 mm sodium bicarbonate pH of 9.5. The product resin used immediately upon receipt.

H. Covalent binding of hemoglobin and myoglobin with allegiantairlines Sigmacell20

1. Materials

a. Allegiantairlines Sigmacell20 (get G)

b. Distilled water

c. 100 mm cyanoborohydride sodium

d. Human hemoglobin and myoglobin the hearts of horses (supplied by the company Sigma Chemical Co.

e. Buffers:

i. 20 mm sodium bicarbonate, pH 9.5 to

ii. 50 mm ethanolamine, pH 9.5 to

iii. 100 mm M Tris buffer (pH 8.0) containing 0.5 M sodium chloride

iv. 100 mm acetotartrate buffer (pH of 4.0) containing 0.5 M NaCl

v. 20 mm MES buffer, pH 5.0.

2. The order of execution

50 mg of myoglobin or hemoglobin, races the er (G). The resulting suspension was stirred by rotation overnight at room temperature, and the formed precipitate is removed by centrifugation. The precipitate after centrifugation washed with 10 ml of water. After adding 9 ml of 50 mm ethanolamine (pH 9,5) the mixture is continuously stirred by rotation at room temperature for one hour. The formed precipitate was separated by centrifugation and washed subsequently with 10 ml of acetate buffer, 10 ml of Tris-buffer and 10 ml of MES buffer. After adding 20 mg of an aqueous solution of 100 mm cyanoborohydride sodium, the mixture is incubated for one hour at room temperature and with continuous rotation, and then incubated overnight at 4oC. then derivationally media five times washed with 10 ml aliquot of distilled water and twice washed with 10 ml aliquot of MES buffer.

I. Covalent binding of HRPO aldehyde with hemoglobin or myoglobin immobilized on allegiantairlines Sigmacell20

1. Materials:

a. Sigmacell20, derivationally hemoglobin or myoglobin (get (H)

b. Distilled water

c. 100 mm cyanoborohydride sodium

d. Buffers:

i. 100 mm Tris-BS pH 5.0.

e. Allegedclaiminga HRPO (example D).

2. The order of execution

20 mg of desalted HRPO aldehyde (example), dissolved in 2 ml of MES buffer (pH 5.0), add to 1 g Sigmacell20, derivatizing hemoglobin or myoglobin (example H) and the resulting suspension is stirred with rotation for 1 hour at room temperature, and then incubated overnight at a temperature of 4oC. the Medium is separated by centrifugation and washed twice 10 ml aliquot of distilled water. After adding 20 ml of 100 mm cyanoborohydride sodium suspension incubated for 60 hours at a temperature of 4oC. the Resulting precipitate consistently (twice), washed with distilled water, acetate buffer, Tris buffer (MES buffer.

J. Covalent binding of hemoglobin made with industry EupergitC (granules acrylic acid)

1. Materials

a. Exernally granules ( EupergitC from the company Sigma Chemical Co.)

b. Human hemoglobin (type IV from the company Sigma Chemical Co.)

c. Buffers and solutions:

i. 1.0 M califofnia buffer (pH 7.5), containing 0.1% sodium azide (wt./about.)

ii. 1.0 M NaCl

iii. 5% (vol./about.) mercaptoethanol, brought to pH 8

vi. 3.5 M sodium thiocyanate

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

d. The distilled water.

2. The order of execution

Hemoglobin (125 mg) is dissolved in 5 ml of 1.0 M phosphate buffer (pH 7.5) containing sodium azide and added to 1 g of EupergitC. the resulting mixture was incubated for 72 hours at room temperature, while stirring. The media three times washed with 1.0 M NaCl and five times with distilled water. Then the carrier is mixed with 2.5 ml of mercaptoethanol, previously brought to pH 8.8 and the resulting suspension was incubated overnight at room temperature. The product in the form of pellets washed 10 times with distilled water, and then placed in a small funnel fused glass and sequentially washed with 0.5 M califorina buffer (50 ml), pH 7.5; 0.1 M califorina buffer with pH 7.5 (50 ml), 3,5 M sodium thiocyanate (50 ml) and finally with 50 ml of a large volume of phosphate-buffered saline (PBS) (0.01 M sodium phosphate, pH 7.2 and containing 0.15 M sodium chloride). Derivateservlet granules treated with 0.1% (vol./about.) the glutaraldehyde by rotation for 2 hours at room temperature, and then during the night when tempea HRPO (example D) with EupergitC, derivational hemoglobin (get (J)

1. Materials:

a. HRPO aldehyde (example D)

b. EupergitC, (derivationally hemoglobin (example J)

c. 100 mm cyanoborohydride sodium

d. 1.0 M NaCl

e. The distilled water.

2. The order of execution

30 mg of HRPO aldehyde (example D) in 3 ml of ME-buffer mixed with 1 g of EupergitC (example J), derivatizing hemoglobin. The resulting suspension is incubated for 16 hours at a temperature of 4oC and then for 4 hours at room temperature. To remove unbound materials from the media used three cycles of leaching, each of which is carried out with distilled water and 1.0 M NaCl. Then the solid media were incubated with 5 ml of 100 mm cyanoborohydride for 60 hours at a temperature of 4oC. then finally washed with distilled water, and the moist suspension stored at a temperature of 4oC until its use.

Covalent binding of casein-HRPO with a polymeric dialdehyde

1. Materials:

a. 25 mg conjugate [Kappa-casein-HRPO] obtained hydrazide (example B) and dissolved in 2 ml of 100 mm borate buffer (pH 9,0)

b. Polymeric dialdehyde (pasta, containing 0.5 M NaCl

iii. 100 mm nitroacetate buffer with a pH of 4.0, containing 0.5 M NaCl

iv. 50 mm MES buffer, pH 6,0

d. 100 mm formaldehyde

e. 100 mm cyanoborohydride sodium.

2. The order of execution

2 ml conjugate [Kappa-casein-HRPO] obtained by the method described in example B except that the treatment with formaldehyde does not hold. The resulting solution is mixed with 3 ml of MES buffer and this mixture was added to 1 g of polymeric dialdehyde. Then the suspension is stirred by rotation at room temperature for 6 hours and incubated overnight at a temperature of 4oC. thereafter, to the suspension add 200 ál of 100 mm formaldehyde and the mixture is incubated for one hour at room temperature. The resulting resin is removed by centrifugation, washed with water and re-suspended in 3 ml of 100 mm ethanolamine pH of 8.5. After 2-hour incubation with rotation at room temperature the resin is successively washed with acetate buffer, borate buffer and water. Then, the solid support is incubated with 5 ml of 100 mm cyanoborohydride over night at a temperature of 4oC. then finally washed with distilled water, and the moist suspension stored at a temperature of 4oC wppa-casein with EupergitC (acrylic beads)

1. Materials:

a. Exernally beads ( EupergitC supplied by the company Sigma Chemical Co.)

b. Whole bovine casein or Kappa-casein (supplied by the company Sigma Chemical Co.

c. Buffers and solutions:

i. 100 mm sodium bicarbonate (pH 8.5) containing 0.5 M NaCl

ii. 100 mm nitroacetate buffer (pH of 4.0) containing 0.5 M NaCl

iii. 100 mm Tris buffer (pH 8.0) containing 0.5 M NaCl

iv. 20 mm MES buffer, pH 5.0.

v. 5% (vol./about.) mercaptoethanol in water

vi. 200 mm borohydride sodium in water.

d. The distilled water.

2. The order of execution

a. Covalent linking of casein or Kappa-casein with EupergitC

2 ml of a solution containing whole casein or whole Kappa-casein (10 mg/ml) in 100 mm buffer solution of sodium bicarbonate (pH 8.5) containing 0.5 M NaCl, added to 1 ml of EupergitC in the form of granules, suspended in water. The resulting mixture is incubated, rotating within 48 hours at room temperature. The formed precipitate was separated by centrifugation and washed subsequently with 9 ml aliquot of acetate buffer and Tris buffer and then twice washed with 9 ml aliquot ME-buffer. After adding 10 ml of a 5% mercaptoethanesulfonate casein, separated by centrifugation and washed 4 times 9 ml of MES buffer.

b. Tagging casein immobilized on EupergitC using HRPO

3 ml of HRPO, activated aldehyde (example D), added to the casein derivateservlet EupergitC from the above stage (A) and the resulting mixture was incubated, with mixing by rotation for 1 hour at room temperature, and then for 60 hours at a temperature of 4oC. the Resulting precipitate was separated by centrifugation and washed with two 10 ml aliquot of water. After adding to the precipitate aqueous solution of sodium borohydride (5 ml), the suspension is incubated for 6 hours at room temperature. The formed precipitate was separated by centrifugation and washed subsequently with 9 ml aliquot acetate, Tris, Acetate, Tris, and MES buffer.

N. Covalent binding of horseradish peroxidase, activated myoglobin with a polymeric dialdehyde

1. Materials:

a. 30 mg of myoglobin in the heart of the horse, dissolved in 2 ml of 20 mm sodium bicarbonate buffer with a pH of 9.5

b. Polymeric dialdehyde (dialdehyde cellulose supplied by the company Sigma Chemical Co.

c. Buffers:

i. 50 mm ethanolamine, pH 9.5 to

ii. 100 mm sodium borate (
v. 100 mm Tris buffer (pH 8.0) containing 0.5 M NaCl

vi. 100 mm formaldehyde

vii. 100 mm cyanoborohydride sodium in water.

2. The order of execution

a. Covalent binding of myoglobin with a polymeric dialdehyde

2 ml of myoglobin mixed with 1 ml suspension of polymeric dialdehyde and the suspension is stirred by rotating overnight at room temperature. The resulting resin was separated by centrifugation and washed twice 10 ml aliquot of water. After adding 9 ml ethanolamine solution, the suspension is incubated rotating for one hour at room temperature. Then the resin is isolated by centrifugation and washed subsequently with 10 ml aliquot acetate, Tris and MES buffer. Then add 20 ml of cyanoborohydride sodium. The resulting suspension is incubated over night at a temperature of 4oC. Polymeric dialdehyde, derivationally by myoglobin, five times washed with 10 ml aliquot of water and twice washed with MES buffer.

b. Tagging myoglobin-derivatizing acrylic beads using HRPO aldehyde (application D)

To the resin is poured 2 ml of desalted HRPO aldehyde (example D), and the resulting suspension is incubated with rotation in the course is t by centrifugation, twice washed with 10 ml of the aliquot of water, suspended in 20 ml of cyanoborohydride sodium and incubated for 60 hours at a temperature of 4oC. the resulting mixture was centrifuged and the obtained resin was twice washed with 10 ml of the aliquot of water, and then twice washed with four 10-ml aliquot of the acetate /NaCl and Tris/NaCl buffer. The resulting resin was washed with 10 ml of MES buffer and stored at a temperature of 4oC until its use.

O. Receiving HRPO-labeled chitin and its use for analysis for the presence of chitinases

1. Materials:

a. Chitin, activated with bromine cyan (example A)

b. HRPO (Type II from the company Sigma Chemical Co., 78 units/mg)

c. The chitinase (EC 32114, isolated from Streptomyces grisius and purchased by the company Sigma Chemical Co.

d. Buffers:

i. 500 mm Tris/MES-buffer, pH 5,4

ii. Binding buffer, 100 mm sodium bicarbonate containing 0.5 M NaCl

iii. 100 mm nitroacetate buffer (pH of 4.0) containing 0.5 M NaCl

iv. 100 mm natriuretic buffer (pH 8.0) containing 0.5 M NaCl

v. 1.0 M Tris buffer, pH 8.0

e. Analytical ABTS-a mixture of 40 μl of 25 mg/ml ABTS (2,2'-Azino-di[3-ethylbenzthiazolinesulfonic acid, demoniaca salt]: 10 µc a 1% (vol. /about.) peroxide vtoroy of horseradish

500 mg brachionichthyidae chitin (example A) are suspended in 5 ml of binding buffer, and then add 5 mg HRPO, and the resulting suspension is continuously stirred for 2 hours at room temperature. The solid material is washed with distilled water and a binding buffer. After adding to the media 3 ml of 1.0 M Tris-buffer (pH 8.0), the suspension is stirred at room temperature for 2 hours, and then three times successively washed with acetate and borate buffers, and finally with water.

b. Analysis for the presence of chitinases

50 mg of chitin labeled with HRPO, suspended in 200 μl of MES/Tris buffer. After adding 50 μl of solution chitinases (50 units/ml), the mixture is incubated for 4 hours at a temperature of 37oC. the Reaction mixture was centrifuged for deposition of chitin, and 10-µl aliquot of the supernatant is mixed with 100 ál analytical ABTS-mixture. Catalisano the chitinase release of HRPO associated with chitin, detects the appearance of green color of the solution.

P. Getting brachionichthyidae [sepharose-casein] or aldehyde Kappa-casein-HRPO

1. Materials:

a. Brazian enabled sepharose (example A)

b. 10 mg/ml casein or Kappa-casein in binding buffer (0.1 M Bika is rija (pH 9,0), containing 0.5 M NaCl

iii. 100 ml acetotartrate buffer (pH of 4.0) containing 0.5 M NaCl

iv. 20 mm MES buffer, pH 5.0

v. 100 mm Tris buffer, pH 8.5, containing 0.5 M NaCl

vi. 100 mm borohydride sodium in water

d. HRPO aldehyde (example D)

2. The order of execution

2 ml of a solution of casein or Kappa-casein is added to 1 ml sepharose, activated bromine cyan, and the resulting suspension is incubated with rotation for 2 hours at room temperature. After adding 7 ml ethanolamine mixture incubated for 1 hour at room temperature, then centrifuged and washed subsequently with 9 ml aliquot of acetate buffer and Tris buffer containing sodium chloride, and then twice washed with 9 ml of MES buffer.

Then add 3 ml of desalted HRPO aldehyde (example D) and the mixture is stirred by rotating for 1 hour at room temperature, then incubated overnight at a temperature of 4oC, centrifuged and the resulting resin was washed twice with 10 ml water. Then, the resin is suspended in 10 ml of the sodium borohydride and the mixture was incubated for 2 hours at room temperature and then centrifuged the resin is successively washed with acetate buffer containing 0.5 EOM, containing 0.5 M NaCl (9 ml). The resulting resin was washed with 9 ml of MES buffer and stored in MES buffer at a temperature of 4oC prior to use.

Q. Covalent binding of myoglobin and bovine serum albumin with beads coated with finely sprayed acrylic coating ( EupergitC) and subsequent covalent tagging using horseradish peroxidase (aldehyde method)

Stage 1: Covalent binding of myoglobin and bovine serum albumin with beads coated with acrylic coating ( EupergitC)

1. Materials:

a. Exernally beads ( EupergitC, 150 n supplied by the company Sigma Chemical Co.)

b. Myoglobin heart of a horse (type IV) or bovine serum albumin (from the company Sigma Chemical Co.)

c. Buffers and solutions:

i. 1.0 M califofnia buffer having pH 7.5, containing 0.1% (wt./about.) sodium azide

ii. 1.0 M NaCl

iii. 5% (vol./about.) mercaptoethanol, brought to pH 8 with 0.5 n sodium hydroxide

iv. 100 mm califofnia buffer, pH 7.5

v. 500 mm califofnia buffer, pH 7.5

vi. 3.5 M thiocyanate sodium

vii. Phosphate-buffered saline (PBS) (0.01 M sodium phosphate pH 7.2 and 0.15 M NaCl)

d. The distilled water.

2. The order of execution

1 g oxyradical serum (125 mg) is dissolved in 5 ml of 1.0 M phosphate buffer (pH 7.5), containing sodium azide and added to 1 g of finely ground product ( EupergitC). The resulting mixture was incubated without stirring for 72 hours at room temperature and centrifuger media three times washed with 1.0 M NaCl and five times with 20 ml of distilled water. Then the carrier is mixed with 2.5 ml of mercaptoethanol, previously brought to pH 8.0 and the suspension is kept over night at room temperature. Then the beads are washed 10 times with distilled water using centrifugation, and then by centrifugation washed with 0.5 M califorina buffer with pH 7.5 (50 ml), 0.1 M califorina buffer with pH 7.5 (50 ml), 3,5 M sodium thiocyanate (50 ml) and finally washed with a large amount of phosphate buffer saline (PBS) (0.1 M sodium phosphate (pH of 7.2) containing 0.15 M sodium chloride).

Stage 2: Covalent tagging using horseradish peroxidase (aldehyde method)

For marking balls with acrylic coating of the above stage 1, derivatizing the myoglobin or albumin in accordance with the method described in example F, using the aldehyde of horseradish peroxidase (example D).

R. Linking The R>
1. Materials:

a. Myoglobin, covalently associated with brachionichthyidae Sigmacell20 (example E).

b. 0.5 M MES buffer, pH 5.0

c. 1% (vol./about.) glutaradehyde in water

d. HRPO-hydrazide (from the company Sigma Chemical Co., 200 units/mg)

e. 100 mm formaldehyde

f. 100 mm cyanoborohydride sodium

g. 0.5 M NaCl

2. The order of execution

1 ml solution of glutaraldehyde is added to 1 g Sigmacell20 paired with myoglobin, and the resulting suspension stirred by rotating at room temperature for 30 minutes and then washed with water. To the washed resin add 5 mg HRPO-hydrazide in 2 ml 100 ml mm MES-buffer, and the resulting suspension is stirred by rotating for 4 hours at room temperature. After adding 200 μl of 100 mm formaldehyde solution, the mixture is incubated for 30 minutes at room temperature. The resulting resin was washed with water, suspended in 5 ml of 100 mm cyanoborohydride sodium and incubated over night at a temperature of 4oC. Then the resin is washed with distilled water and 0.5 M NaCl, then store in the form of a wet paste until use.

Obtaining a solution of guaiac resin and sheets of guaiac resin

1. The solution containing guaiac resin and is heated and mixed in ethanol. To the resulting solution was added 1330 ml of distilled water, and the resulting suspension is cooled to room temperature. After 2 hours, the supernatant decanted, and the residual suspension defend.

250 ml of 10% (wt./wt.), solution hydroxypropylcellulose in ethanol is mixed with an additional quantity of ethanol (250 ml) and the resulting solution was added to guaiac suspension. Then the mixture is stirred until complete dissolution of the residue guaiac resin.

2. The leaves of guaiac resin

1 ml of the above solution of guaiac resin is applied with a pipette on the polyethylene side of the plate (10 inch x 10 inch = 254 cm x 254 cm) plate of Mylar/polyethylene (7 mil Mylar/3 ml polyethylene). A solution of guaiac resin is sprayed over the surface of the sheets by means of standard laboratory rod of twisted wire, and then allow to dry at room temperature.

T. obtaining a suspension of sodium perborate (i.e. stripperbraces reagent)

20 g of finely ground solid sodium perborate mixed with a sufficient volume of 10% (by weight) solution hydroxypropylcellulose in anhydrous alcohol to obtain one liter of suspension.

II. Experiments:

The experimental the n-HRPO] and [sepharose-Kappa-casein-HRPO] using aspartic protease, allocated by the culture of Candida albicans.

A. Materials: (sepharose activated with bromine cyan) (method binding with HRPO aldehyde)

1. Brachionichthyidae sepharose 4B (example A) first derivativesa covalently linked casein or Kappa-casein (example P), and then labeled using HRPO aldehyde (example D)

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

3. The sheets coated with guaiac resin (example S)

4. Buffers and solutions:

a. 20 mm hydrogen peroxide

b. 500 mm MES buffer with a pH of 6.0

B. the Order of execution

10 mg (wet weight) of the conjugate sepharose-casein-HRPO (or equivalent Kappa-casein) are suspended in 300 μl of culture Candida albicans containing the active secreted aspartic protease, or 300 μl of the same culture Candida albicans, heated to boiling for 20 minutes to inactivate aspartic protease. The resulting mixture is subjected to rotation for 15 minutes at room temperature, and then centrifuged suspended for the deposition of solid conjugate and cells of Candida albicans.

80 ál of the clear supernatant is mixed with the itih guaiac resin and observe the formation of blue color (see table. 1).

C. Results:

Dark blue color seen on that side of the sheet coated with guaiac resin, which caused the supernatant conjugate [sepharose-protein-HRPO] treated culture of Candida albicans. On the square, which was added to the supernatant conjugate [sepharose-protein-HRPO] processed boiled culture, education staining was not observed (see tab. 2).

Interpretation:

Active aspartic protease, secreted in the environment of the cultivation of cell cultures of Candida albicans, hydrolyzed in separate associated with casein or Kappa-casein, thereby releasing soluble active horseradish peroxidase. Centrifugation was besieging sepharose associated with HRPO, leaving only the aspartic protease solubilizing HRPO in solution. Soluble HRPO was katalizowania oxidation of guaiac resin with hydrogen peroxide, thereby producing the appearance of a blue coloration. Therefore, the formation of blue color on plates coated with guaiac resin, allows a convenient way of detecting aspartic protease.

Boiling culture inactivates aspartic protease, secreterial on Wednesday culturing cells of Candida albican is sidasa of horseradish was not separated from the carrier. Centrifugation was besieging sepharose associated with HRPO, leaving in solution aspartic protease solubilizing HRPO. Catalyzing oxidation of guaiac resin under the action of hydrogen peroxide did not occur, and the resulting blue color was barely detectable. Department of HRPO from the media was nothing more than just the result of nonspecific release of HRPO under the action of salts or other constituents present in the environment of cultivation.

Experiment II

This experiment involves the separation of horseradish peroxidase from ordinary conjugates [ EupergitC (acrylic granules)-casein-HRPO] and conjugates [ EupergitC (acrylic granules)-Kappa-casein-HRPO] by aspartic protease secreted by the culture of Candida albicans:

A. Materials: EupergitC (activated acrylic granules (HRPO aldehyde method binding).

1. Exernally granules EupergitC-treated casein or Kappa-casein (example M), and then labeled with HRPO aldehyde (example D)

2. Grown and propagated in liquid culture medium culture of Candida albicans (ATCC 28366), producing aspartic protease as described in the Journal of General Micro is hydrogen peroxide

b. 500 mm MES buffer with a pH of 6.0

B. the Order of execution

10 mg (wet weight) conjugate [ EupergitC-Casein-HRPO] (equivalent to Kappa-casein suspended in 300 μl of culture Candida albicans containing the active secreted aspartic protease or 300 μl of the same culture Candida albicans, boiling for 20 minutes to inactivate thus aspartic protease. Then the mixture is stirred by rotation for 15 minutes at room temperature and the resulting suspension is centrifuged to precipitate solid conjugate and cells of Candida albicans.

80 ál of the clear supernatant is mixed with 10 μl of hydrogen peroxide solution and 10 μl of MES buffer. 20 μl each of these solutions are applied to the surface of the sheet coated with guaiac resin, and this sheet is evaluated on the formation of blue color (see tab. 3).

C. Results:

Dark blue color was formed on the leaf area covered with guaiac resin, to which was added the supernatant conjugate [ EupergitC-protein-HRPO] treated culture of Candida albicans. On the square, which was added to the supernatant conjugate [ EupergitC-protein-HRPO] processed is heated to the boiling point of culture, education oraclemobile environment cells of Candida albicans hydrolyzed EupergitC associated with casein or Kappa-casein, thereby releasing soluble active horseradish peroxidase. Centrifugation was besieged EupergitC associated with HRPO, leaving only the aspartic protease solubilizing HRPO in solution. Soluble HRPO was katalizowania oxidation of guaiac resin under the action of hydrogen peroxide, producing thereby the formation of a blue color. Therefore, the formation of blue color on plates coated with guaiac resin, gives the possibility of detecting aspartic protease.

The heated culture inactivates aspartic protease, secreterial in the culture medium by cells of Candida albicans. So EupergitC associated with casein or Kappa-casein, not hydrolizable and soluble HRPO was not separated from the carrier. Centrifugation was besieging C associated with HRPO, leaving in solution only aspartic protease solubilizing HRPO. Catalyzing oxidation of guaiac resin under the action of hydrogen peroxide did not occur, and the resulting blue color was barely detectable. Department of horseradish peroxidase from the media was not just the result of nonspecific Department HRPO under the action of the Sol is speriment provides for the separation of horseradish peroxidase from ordinary [sepharose-hemoglobin-HRPO] or [sepharose-myoglobin-HRPO] under the action of pepsin and aspartic protease, secreted by Aspergillus saitoi

A. Materials: (Sepharose activated with bromine cyan) (aldehyde method binding HRPO)

1. Brachionichthyidae sepharose 4B (example A), first derivativesa covalently linked hemoglobin or myoglobin (example E), and then labeled with HRPO aldehyde (example D and F). The conjugate was treated overnight with 100 mm cyanoborohydride sodium at room temperature and washed before use 0.5 M sodium chloride.

2. Solutions for analysis:

a) manufactured by the industry aspartic protease (type XIII) from A-pergillus saitoi (30 mg/ml, 0.6 units/mg);

b) pepsin (1 mg/ml, 2900 units/mg) and

c) bovine serum albumin (BSA) (2 mg/ml in water) (all of these components were supplied by the company Sigma Chemical Co.)

3. Paper impregnated with guaiac resin, in the form of microscopic preparations (Hemoccult)., produced by the industry (firm Smittkline Diagnostics).

4. Buffers and solutions:

a. 0,2% (about. /about.) hydrogen peroxide in 200 mm phosphate buffer, pH 7.0

b. 100 mm acetate buffer, pH 4,0

B. the Order of execution

40 mg (wet weight) conjugate [sepharose-hemoglobin-HRPO] (or sepharose-myoglobin-HRPO) is suspended in 75 μl of acetate buffer with a pH of 4.0, and then relax the ut, then centrifuged to remove the solid phase conjugate. On a microscopic preparation coated with guaiac resin, put 5 μl of the supernatant, and then 5 μl of peroxide solution (see tab. 5).

C. Results:

Dark blue color was observed on the part of the leaf, guaiac impregnated with resin, to which was added the supernatant samples containing aspartic protease from Aspergillus saitoi or pepsin. Part of the sheet, to which was added an aliquot of supernatant, containing only one BSA (2 mg/ml) or buffer was visible very faint blue tint (see tab. 6).

D. Interpretation:

Active aspartic protease from Aspergillus saitoi and pepsin, aspartic protease, isolated from the stomach of the pig, hydrolyzed in separate associated with hemoglobin or myoglobin, thereby releasing soluble, active HRPO. Centrifugation was besieging sepharose associated with HRPO, leaving in solution the only enzyme solubilizing HRPO. Soluble HRPO was katalizowania oxidation of guaiac resin under the action of hydrogen peroxide, producing thereby the formation of a blue color. Therefore, the formation of blue color on plates coated with guaiac resin, gives the possibility of detecting active aspartic paraginomai protease. So sepharose associated with hemoglobin or myoglobin, not hydrolethalus, and soluble, active HRPO was not separated from the carrier. Centrifugation was besieging sepharose associated with HRPO, leaving aspartic protease solubilizing HRPO in solution. Catalyzing oxidation of guaiac resin under the action of hydrogen peroxide did not occur, and the resulting blue color was barely detectable.

Experiment IV

This experiment involves the separation of HRPO from [sepharose-casein-HRPO] or [sepharose-Kappa-casein-HRPO] under the action of pepsin and aspartic protease derived from Aspergillus saitoi.

A. Materials: (sepharose activated with bromine cyan) (method binding HRPO and hydrazide)

1. Brachionichthyidae sepharose 4B (example A), left for the reaction with [Kappa-casein-HRPO] or casein-HRPO (hydrazide method) (example B and C)

2. Solutions for analysis:

a) manufactured by the industry aspartic protease, derived from Aspergillus saitoi (30 mg/ml, 0.6 units/mg);

b) pepsin (1 mg/ml, 2900 units/mg); and

c) bovine serum albumin (BSA) (2 mg/ml in water) (all these enzymes were supplied by the company Sigma Chemical Co.)

3. Paper impregnated with guaiac resin, in the form of microscopic is (about./about.) hydrogen peroxide in 200 mm phosphate buffer, pH 7.0.

b. 100 mm acetate buffer, pH 4,0

B. Procedure:

40 mg (wet weight) conjugate [sepharose-casein-HRPO] or conjugate [sepharose-Kappa-casein-HRPO] suspended in 75 μl of acetate buffer with pH 4.0, and then add 25 ál of the solution for analysis. The resulting suspension is incubated at room temperature for 15 minutes and then centrifuged to remove the solid phase conjugate. To microscopic preparation coated with guaiac resin, add 5 ál of the supernatant, and then 5 μl of hydrogen peroxide solution (see tab. 7).

C. Results:

Dark blue color was observed on the part of the sheet coated with guaiac resin, to which was added the supernatant samples containing aspartic protease, derived from Aspergillus saitoi or pepsin. On the square, to which was added an aliquot of supernatant, containing only BSA (2.0 mg/ml) or buffer were found only very weak staining (table. 8).

D. Interpretation:

Active aspartic protease from Aspergillus saitoi and porcine pepsin hydrolyzed in separate associated with casein or Kappa-casein, thereby releasing soluble, active HRPO. Centrifugation was besieging sepharose associated with HRPO, leaving in solution leash is xida hydrogen, producing thereby the formation of a blue color. Therefore, the formation of blue color on plates coated with guaiac resin, allows a convenient way of detecting aspartic protease or pepsin.

Neither BSA nor the buffer does not have the activity of aspartic proteases. So sepharose associated with casein or Kappa-casein, was not hydrolyzed, and soluble, active HRPO was not separated from the carrier. Centrifugation was besieging sepharose associated with HRPO, leaving aspartic protease solubilizing HRPO in solution. Catalyzing oxidation of guaiac resin under the action of hydrogen peroxide did not occur, and the resulting blue color was barely detectable.

Experiment V

This experiment involves the separation of HRPO from conjugate [chitin-Kappa-casein-HRPO] under the action of pepsin and aspartic protease derived from Aspergillus saitoi.

A. Materials: (chitin, activated with bromine cyan) (method binding HRPO and hydrazide)

1. Chitin, activated with bromine cyan (example A) and left to react with [Kappa-casein-HRPO] (hydrazide method) (example B and C)

2. Analytical solutions:

a) manufactured by the industry asparagine the>c) bovine serum albumin (BSA) (2 mg/ml in water) (all these drugs were supplied by the company Sigma Chemical Co.)

3. Paper impregnated with guaiac resin, in the form of microscopic preparations Hemoccultproduced by the industry (firm Smitkline Diagnostics).

4. Buffers and solutions:

a. 0,02% (vol./about.) hydrogen peroxide in 200 mm phosphate buffer, pH 7.0

b. 100 mm acetate buffer, pH 4,0.

B. the Order of execution

40 mg (wet weight) conjugate [chitin-Kappa-casein-HRPO] suspended in 75 μl of acetate buffer, pH 4.0, and then add 25 ál of the analytical solution. The resulting suspension is incubated for 15 minutes at room temperature, and then centrifuged to remove the solid phase conjugate. On a microscopic preparation with guaiac resin was added 5 μl of the supernatant, and then 5 μl of hydrogen peroxide solution (see tab. 9).

C. Results:

Dark blue color was formed on the leaf area, guaiac impregnated with resin, to which was added the supernatant samples containing aspartic protease, derived from Aspergillus saitoi or pepsin. On the square, to which was added an aliquot of supernatant, containing only BSA (2.0 mg/ml) or buffer was discovered, rashomama from Aspergillus saitoi and porcine pepsin, hydrolyzed Sigmacell20 associated with hemoglobin or myoglobin, thereby releasing soluble, active HRPO. Centrifugation was besieged Sigmacell20 associated with HRPO, leaving in solution the only enzyme solubilizing HRPO. Soluble HRPO was katalizowania oxidation of guaiac resin under the action of hydrogen peroxide, producing thereby the formation of a blue color. Therefore, the formation of blue color on plates coated with guaiac resin, allows a convenient way of detecting aspartic protease or pepsin.

Neither BSA nor the buffer is not possessed by the activity of aspartic proteases. So Sigmacell20 associated with hemoglobin or myoglobin, was not hydrolyzed, and soluble, active HRPO was not separated from the carrier. Centrifugation was besieged Sigmacell20 associated with HRPO, leaving in solution aspartic protease solubilizing HRPO. Catalyzing oxidation of guaiac resin under the action of hydrogen peroxide did not occur, and the resulting blue color was barely detectable.

Experiment VI

This experiment involves the separation of HRPO from convergenoe protease, derived from Aspergillus saitoi

A. Materials: (brazian activated Sigmacell20 ) (allegedy method binding HRPO)

1. Brazian activated Sigmacell20 (example A), first derivationally covalently linked hemoglobin or myoglobin (example E), and then labeled with HRPO aldehyde (example D-F). The conjugate was treated overnight with 100 mm cyanoborohydride sodium at room temperature and washed before use 0.5 M sodium chloride.

2. Analytical solutions:

a) manufactured by the industry aspartic protease, derived from Aspergillus saitoi (30 mg/ml, 0.6 units/mg);

b) pepsin (1 mg/ml, 2900 units/mg); and

c) bovine serum albumin (BSA) (2 mg/ml in water) (all these drugs were supplied by the company Sigma Chemical Co.)

3. Paper impregnated with guaiac resin, in the form of microscopic preparations Hemoccultproduced by the industry.

4. Buffers and solutions:

a. 0,02% (vol./about.) hydrogen peroxide in 200 mm phosphate buffer, pH 7.0

b. 100 mm acetate buffer, pH 4,0

B. execution Order 40 mg (wet weight) conjugate [ Sigmacell20 - hemoglobin-HRPO] or conjugate [ Sigmacell20-myoglobin/HRPO] suspended in 75 μl acetatee 15 minutes at room temperature, and then centrifuged to remove the solid phase conjugate. To microscopic drug with guaiac add 5 ál of the supernatant, and then 5 μl of hydrogen peroxide solution (see tab. 11).

C. Results:

Dark blue color formed on the leaf area covered with guaiac resin, to which was added the supernatant samples containing aspartic protease, derived from Aspergillus saitoi or pepsin. On the square, which after BSA (2.0 mg/ml) or buffer was added to an aliquot of supernatant was noticeable only very weak staining (see tab. 12).

D. Interpretation:

Active aspartic protease from Aspergillus saitoi and porcine pepsin hydrolyzed Sigmacell20 associated with hemoglobin or myoglobin, thereby releasing soluble, active HRPO. Centrifugation was besieged Sigmacell20 associated with HRPO, leaving in solution the only enzyme solubilizing HRPO. Soluble HRPO was katalizowania oxidation of guaiac resin under the action of hydrogen peroxide, producing thereby the formation of a blue color. Therefore, the formation of blue color on plates coated with guaiac resin, allows a convenient way of detecting aspartic nature>20 associated with hemoglobin or myoglobin, was not hydrolyzed, and soluble, active HRPO was not separated from the carrier. Centrifugation was besieged Sigmacell20, linked to horseradish peroxidase, leaving aspartic protease solubilizing HRPO in solution. Catalyzing oxidation of guaiac resin under the action of hydrogen peroxide did not occur, and the resulting blue color was barely detectable.

Experiment VII

This experiment involves the separation of HRPO from conjugate [ Sigmacell20-Kappa-casein-HRPO] by pepsin and aspartic protease derived from Aspergillus saitoi

A. Materials: ( Sigmacell20, activated with bromine cyan) (method binding HRPO and hydrazide)

1. Brachionichthyidae Sigmacell20 (example A), left to react with the Kappa-casein-HRPO (hydrazide method, example B, and C)

2. Analytical solutions:

a) manufactured by the industry aspartic protease, derived from Aspergillus saitoi (30 mg/ml, 0.6 units/mg);

b) pepsin (1 mg/al, 2900 units/ml); and

c) bovine serum albumin (BSA) (2 mg/ml in water) (all these drugs were supplied by the company Sigma Chemical Co.)

3. Paper impregnated with guaiac who very and solutions:

a. 0,02% (vol./about.) hydrogen peroxide in 200 mm phosphate buffer, pH 7.0.

b. 100 mm acetate buffer, pH 4,0

B. the Order of execution

40 mg (wet weight) conjugate Sigmacell20-Kappa-casein-HRPO suspended in 75 μl of acetate buffer, pH 4.0, and then add 25 ál of the analytical solution. The resulting suspension is incubated for 15 minutes at room temperature, and then centrifuged to remove the solid phase conjugate. On the drug with guaiac put 5 μl of the supernatant, and then 5 μl of hydrogen peroxide solution (see tab. 13).

C. Results:

Dark blue color was formed on the leaf area covered with guaiac resin, to which was added the supernatant samples containing aspartic protease, derived from Aspergillus saitoi or pepsin. On the square, which after BSA (2.0 mg/ml) or buffer was added to an aliquot of supernatant was noticeable only very weak staining (see tab. 14).

D. Interpretation

Active aspartic protease from Aspergillus saitoi and porcine pepsin hydrolyzed Sigmacell20 associated with Kappa-casein, thereby releasing soluble active horseradish peroxidase. Centrifugation was besieged Sigmacell20,alitwala oxidation of guaiac resin under the action of hydrogen peroxide, producing thereby the appearance of a blue coloration. Therefore, the formation of blue color on plates coated with guaiac resin, allows a convenient way of detecting aspartic protease or pepsin.

Neither BSA nor the buffer does not have the activity of aspartic proteases. So Sigmacell20 associated with Kappa-casein, was not hydrolyzed, and soluble, active HRPO was not separated from the carrier. Centrifugation was besieged Sigmacell20 associated with HRPO, leaving in solution aspartic protease solubilizing HRPO. Catalyzing oxidation of guaiac resin under the action of hydrogen peroxide did not occur, and the resulting blue color was barely detectable.

Experiment VIII

This experiment involves the release of horseradish peroxidase from ordinary [ Sigmacell20-Kappa-casein-HRPO] (hydrazide method) or [ Sigmacell20-myoglobin]hemoglobin-HRPO (aldehyde method) via aspartic protease derived from Aspergillus saitoi or pepsin.

A. Materials: ( Sigmacell20, activated with bromine cyan)

1. Sigmacell20 activated with bromine cyan (example A), derivatizing Kappa-ka is alternately derivateservlet hemoglobin or myoglobin (example E), and then associated with HRPO aldehyde (example D)

2. Analytical solutions:

a. manufactured by industry aspartic protease, derived from Aspergillus saitoi (30 mg/ml, and 0.6 units/mg);

b. pepsin (1 mg/ml, 2900 units/mg); and

c. bovine serum albumin (BSA) (2 mg/ml in water) (all these drugs were supplied by the company Sigma Chemical Co.).

3. Paper impregnated with guaiac resin, in the form of microscopic preparations Hemoccultproduced by the industry.

4. Buffers and solutions:

a. 0,02% (vol./about.) hydrogen peroxide in 200 mm phosphate buffer, pH 7.0

b. 100 mm acetate buffer, pH 4,0

B. the Order of execution

40 mg (wet weight) conjugate [ Sigmacell20-Kappa-casein-HRPO] suspended in 75 μl of acetate buffer, pH 4.0, and then add 25 ál of the analytical solution. The resulting suspension is incubated at room temperature for 15 minutes, then centrifuged to remove the solid phase conjugate. On the drug with guaiac put 5 μl of the supernatant, and then 5 μl of hydrogen peroxide solution (see tab. 15).

C. Results:

Dark blue color was observed on the leaf area, guaiac impregnated with resin, to which was added the samples with the whether aliquots of the supernatant, containing BSA (2.0 mg/ml) or buffer was visible only a very weak staining (see tab. 16).

D. Interpretation:

Active aspartic protease from Aspergillus saitoi and porcine pepsin hydrolyzed Sigmacell20 associated with Kappa-casein, thereby releasing soluble, active HRPO. Centrifugation was besieged Sigmacell20 associated with HRPO, leaving in solution the only enzyme solubilizing HRPO. Soluble HRPO was katalizowania oxidation of guaiac resin under the action of hydrogen peroxide, thereby producing the appearance of a blue coloration. Therefore, the formation of blue color on plates coated with guaiac resin, allows a convenient way of detecting aspartic protease or pepsin.

Neither BSA nor the buffer does not have the activity of aspartic proteases. So Sigmacell20 associated with Kappa-casein, was not hydrolyzed, and soluble active horseradish peroxidase was not separated from the carrier. Centrifugation was besieged Sigmacell20 associated with HRPO, leaving in solution aspartic protease solubilizing HRPO. Catalyzing oxidation of guaiac resin under the action of hydrogen peroxide did not occur, and the resulting blue OK the Denia HRPO of the conjugate [ 20-hemoglobin-HRPO] or [ Sigmacell20-hemoglobin-HRPO] by aspartic protease secreted by the culture of Candida albicans

A. Materials: ( Sigmacell20, the activated aldehyde) (HRPO aldehyde method binding)

1. Aldehyde-aktivirovaniya Sigmacell20 (example G), which was first derivateservlet covalently linked hemoglobin or myoglobin (example H), and then noted HRPO aldehyde (example D and F)

2. Grown and propagated in liquid culture medium culture of Candida albicans (ATCC 28366), producing aspartic protease with a method described in Journal of General Microbiology (1983) 129:431-438.

3. The sheets coated with guaiac resin (example S)

4. Buffers and solutions:

a. 20 mm hydrogen peroxide

b. 500 mm MES buffer, pH 6,0.

B. Procedure:

10 mg (wet weight) conjugate [ Sigmacell20 protein-HRPO] suspended in 300 μl of culture Candida albicans containing secreted aspartic protease or 300 μl of the same culture Candida albicans, boiling for 20 minutes to inactivate aspartic protease. The resulting mixture is stirred by rotation for 15 minutes at room temperature, after which the resulting suspension is centrifuged to precipitate solid conjugate is 10 μl of MES buffer. 20 μl each of these solutions are applied to the surface of the sheet coated with guaiac resin, and observe formation of blue color (see tab. 17).

C. Results:

Dark blue color was formed on the leaf area covered with guaiac resin, to which was added conjugate [sepharose-protein-HRPO] treated culture of Candida albicans. On the square, which was added by the conjugate [sepharose-protein-HRPO] processed is heated to the boil culture, the appearance of staining was not observed (see tab. 18).

D. Interpretation:

Aspartic protease, secreted into the culture medium with cells of Candida albicans, hydrolyzed Sigmacell20 associated with the protein, thereby releasing soluble, active HRPO. Centrifugation was besieging sepharose associated with HRPO, leaving in solution only aspartic protease solubilizing HRPO. Soluble HRPO was katalizowania oxidation of guaiac resin under the action of hydrogen peroxide, producing thereby the formation of a blue color. Therefore, the formation of blue color on plates coated with guaiac resin, allows a convenient way of detecting aspartic protease.

Heat to a boil culture Sigmacell20 associated with the protein, was not hydrolyzed, and soluble, active HRPO was not released from the carrier. Centrifugation was besieged Sigmacell20 associated with HRPO, leaving in solution aspartic protease solubilizing HRPO. Catalyzing oxidation of guaiac resin under the action of hydrogen peroxide did not occur, and the resulting blue color was barely detectable. Department of HRPO from the specified media was not just the result of separation HRPO under the action of salts or other components present in the culture medium.

Experiment X

This experiment involves the separation of HRPO from conjugate /polymeric dialdehyde-Kappa-casein-HRPO/ by pepsin and aspartic protease derived from Aspergillus saitoi.

A. Materials: (Polymeric dialdehyde) (HRPO-hydrazide method binding)

1. Manufactured by the industry polymeric dialdehyde from the company Sigma Chemical Co. subjected to reaction with Kappa-casein-HRPO (hydrazide method) (example B and L).

2. Analytical solutions:

a) manufactured by the industry aspartic protease, derived from Aspergillus saitoi (30 mg/ml, and 0.6 units/mg)

b) pepsin (1 mg/ml, 2900 units/mg); and

and, impregnated with guaiac resin, in the form of microscopic preparations Hemoccultmanufactured by industry

4. Buffers and solutions:

a. 0,02% (about. /about.) hydrogen peroxide in 200 mm phosphate buffer pH 7.0

b. 100 mm acetate buffer, pH 4,0

B. Procedure:

40 mg (wet weight) conjugate [a polymeric dialdehyde-Kappa-casein-HRPO] suspended in 75 μl of acetate buffer, pH 4.0, and then add 25 ál of the analytical solution. The resulting suspension is incubated at room temperature for 15 minutes, then centrifuged to remove the solid phase conjugate. On the drug with guaiac put 5 μl of the supernatant, and then 5 μl of hydrogen peroxide solution (see tab. 19).

C. Results:

Dark blue color was formed on the leaf area, guaiac impregnated with resin, to which was added the supernatant samples containing aspartic protease from Aspergillus saitoi or pepsin. On the square, which after BSA (2.0 mg/ml) or buffer was added to an aliquot of supernatant was visible only a very weak staining (see tab. 20).

D. Interpretation

Active aspartic protease from Aspergillus saitoi and porcine pepsin hydrolyzed polymeric dialdehyde, Erny dialdehyde, associated with HRPO, leaving in solution the only enzyme solubilizing HRPO. Soluble HRPO was katalizowania oxidation of guaiac resin under the action of hydrogen peroxide, producing thereby the formation of a blue color. Therefore, the formation of blue color on plates coated with guaiac resin, gives the possibility of detecting aspartic protease or pepsin.

Neither BSA nor the buffer does not have the activity of aspartic proteases. Therefore, polymeric dialdehyde associated with Kappa-casein, hydrolysed, and soluble, active HRPO not released from the carrier. Centrifugation was besieged polymeric dialdehyde associated with HRPO, leaving in solution aspartic protease solubilizing HRPO. Catalyzing oxidation of guaiac resin under the action of hydrogen peroxide did not occur, and the resulting blue color was barely detectable.

Experiment XI

This experiment involves the separation of HRPO from conjugate [a polymeric dialdehyde-myoglobin-HRPO] by aspartic protease.

A. Materials: (polymeric dialdehyde) (HRPO aldehyde method binding)

1. Manufactured by the industry polymeric dialdehyde from the company Sigma Chemical Co., proteaseresistant environment culture Sigma Chemical Co. (ATCC 28366), producing aspartic protease with a method described in Journal of General Microbiology, (1983), 129:431-438.

3. Leaves, guaiac impregnated with resin (example).

4. Buffers and solutions:

1. 20 mm hydrogen peroxide

2. 500 mm MES buffer, pH 6,0

B. Procedure:

10 mg (wet weight) conjugate [a polymeric dialdehyde-myoglobin-HRPO] suspended in 300 μl of culture Candida albicans containing secreted aspartic protease, or 300 microlitres the same culture of Candida albicans, boiling for 20 minutes to inactivate aspartic protease. The resulting mixture was stirred by rotation for 20 minutes at room temperature, and then the resulting suspension centrifuged to precipitate solid conjugate and cells of Candida albicans.

80 ál of the clear supernatant is mixed with 10 μl of hydrogen peroxide solution and 10 μl of MES buffer. 20 μl each of these solutions are applied to the surface of the sheet impregnated with guaiac resin, and observe formation of blue color (see tab. 21).

C. Results:

Dark blue color was formed on the leaf area covered with guaiac resin, to which was added conjugate [a polymeric dialdehyde-myoglobin-HRPO] , obrabotan the surveillance culture, visible coloration absent (see tab. 22).

D. Interpretation

Active aspartic protease, secreted into the culture medium by the cells of Candida albicans, hydrolyzed polymeric dialdehyde associated with myoglobin, thereby releasing soluble, active HRPO. Centrifugation was besieged polymeric dialdehyde, linked to horseradish peroxidase, leaving in solution only aspartic protease solubilizing HRPO. Soluble HRPO was katalizowania oxidation of guaiac resin under the action of hydrogen peroxide, producing thereby the formation of a blue color. Therefore, the formation of blue color on plates coated with guaiac resin, allows a convenient way of detecting aspartic protease.

The heated culture iactiveaware aspartic protease, secreted into the culture medium by the cells of Candida albicans. Therefore, polymeric dialdehyde associated with myoglobin, was not hydrolyzed, and soluble, active HRPO was not separated from the carrier. Centrifugation was besieged polymeric dialdehyde associated with HRPO, leaving in solution aspartic protease solubilizing HRPO. Catalyzing oxidation of guaiac resin under the action of peroxy the Rena from the specified media was not just the result of nespetsificheskogo selection HRPO under the action of salts or other components, present in the culture medium.

Experiment XII

This experiment involves the separation of HRPO from conjugate [sepharose 6MB-Kappa-casein-HRPO] using samples of vaginal discharge:

1. Normal vaginal secretion

2. Normal vaginal discharge with the addition of culture Candida albicans

3. Vaginal secretions from women with clinically diagnosed vulvovaginal candidiasis.

A. Materials: (Manufactured by industry sepharose 6MB activated with bromine cyan) (HRPO-hydrazide method binding)

1. Conjugate [sepharose 6MB-Kappa-casein-HRPO] (example C)

2. Samples of vaginal secretions obtained on a standard Dacron swabs.

These samples should be stored frozen until use. Then the samples are thawed and centrifuged in special tubes for extraction undiluted vaginal secretions from the swab. All samples from each swab analyze. If necessary, the sample add distilled water to obtain a final volume of 75 µl.

To samples of vaginal discharge women without a clinical diagnosis of vulvovaginal candidiasis, add 25 ál of the cult(manufactured by industry analytical preparations Hemoccult.

4. Buffers and solutions:

a. 6 mm hydrogen peroxide

b. 250 mm glycylglycine buffer, pH 3.0

B. the Order of execution

30 mg (wet weight) conjugate [sepharose-Kappa-casein-HRPO] suspended in 75 μl of treated or untreated vaginal secretions. The resulting suspension is incubated at room temperature for 15 minutes and then centrifuged to precipitate solid conjugate and other debris.

On paper impregnated with guaiac resin, put 5 ál of the clear supernatant, and then 5 μl of hydrogen peroxide solution and observe formation of blue color (see tab. 23).

C. Results:

Dark blue color was formed on the square of paper impregnated with guaiac resin, to which was added the supernatant vaginal secretions from women, infetsirovanna vulvovaginal candidiasis. Square paper impregnated with guaiac resin, to which was added the supernatant of vaginal discharge in women from the control group painting was missing. The darkest blue colour is also formed on the square of paper impregnated with guaiac resin, to which was added the supernatant of vaginal discharge normal genshinoe women with clinically diagnosed vulvovaginal candidiasis contains aspartic protease, which hydrolyzes conjugate [sepharose 6MB-Kappa-casein] (pH 3.0) for 15 minutes at room temperature, thereby releasing soluble, active HRPO. Centrifugation was besieging sepharose 6MB associated with HRPO, leaving in solution only released solubilizing HRPO. Soluble HRPO was katalizowania oxidation produced by the industry of drugs Hemoccultproducing thereby the appearance of a blue coloration. Therefore, the formation of blue color on the drugs Hemoccultallows a fast and convenient way detection, derived from Candida, aspartic protease in the vaginal discharge, and hence vulvovaginal candidiasis.

Vaginal discharge is a normal woman who is not suffering vulvovaginal candidiasis, do not contain aspartic protease, and therefore the tests at pH 3.0 for 15 minutes at room temperature, the conjugate sepharose 6MB-Kappa-casein is not hydrolizable, and soluble, active HRPO was not released. After deposition of the conjugate sepharose 6MB-HRPO by centrifugation in solution remained associated solubilization HRPO. In the absence of HRPO-oxidation catalyst commercial preparations HemoccultN. the cultallows you to get quick and easy method of examination normal women on the lack of allocated Candida aspartic protease in the vaginal discharge, and consequently, the lack of vulvovaginal candidiasis.

And, finally, adding medium from cultures of Candida albicans to vaginal discharge normal women, not suffering vulvovaginal candidiasis occurred hydrolysis of the conjugate [sepharose 6MB-Kappa-casein] at pH 3.0, for 15 minutes at room temperature, and was released soluble, active HRPO. During the precipitation of the conjugate sepharose 6MB-HRPO by centrifugation the solution was released solubilization HRPO. This soluble HRPO catalyzed oxidation of commercial preparations Hemocculthydrogen peroxide and observed a blue color. Therefore, the formation of blue color on the drugs Hemoccultindicates that the aspartic protease released into the culture medium by the cells of Candida albicans, can be quickly and efficiently located, even if this environment is added to vaginal discharge normal women.

Experiment XIII

This experiment involves the separation of HRPO from conjugate [ Espergit

1. Normal vaginal discharge

2. Vaginal secretions of women with clinically diagnosed vulvovaginal candidiasis.

A. Materials: (brachionichthyidae Sigmacell20-myoglobin-HRPO; example A and I), and ( EspergitC-myoglobin-HRPO; Example Q) (HRPO aldehyde method binding)

1. Finely chopped exernally granules associated with myoglobin-HRPO aldehyde (example Q)

2. Sigmacell20, activated with bromine cyan (example A) and associated with the myoglobin-HRPO aldehyde (example I).

3. Samples of vaginal secretions obtained on a standard Dacron swabs. These samples should be stored frozen until use. Then the samples are thawed and centrifuged in special tubes for extraction undiluted vaginal secretions from the swab. All samples from each swab analyze.

4. Filter paper impregnated with guaiac resin (manufactured by industry drugs Hemoccultfor analysis)

5. Buffers and solutions:

a. 6 mm hydrogen peroxide

b. 250 mm glycylglycine buffer with pH 3.0

c. 1.0 M acetate buffer, pH 4,0.

B. Procedure:

20 mg (wet weight) conjugate the buffer. The resulting suspension is incubated at room temperature for 10 minutes and then centrifuged to precipitate solid conjugate and other debris.

On paper impregnated with guaiac resin, put 5 ál of the clear supernatant, and then 5 μl of hydrogen peroxide solution and observe formation of color.

20 mg (wet weight) conjugate Sigmacell20-myoglobin-HRPO suspended in undiluted vaginal secretion and 10 μl of 250 mm acetate buffer. The resulting suspension is incubated at room temperature for 15 minutes and then centrifuged to precipitate solid conjugate and other debris.

On paper impregnated with guaiac resin, put 5 ál of the clear supernatant, and then 5 μl of hydrogen peroxide solution and observe formation of blue color (see tab. 25).

C. Results:

Dark blue color was formed on the area of the leaf, guaiac impregnated with resin, to which was added the vaginal secretions of women infected with vulvovaginal candidiasis. On the square sheet impregnated with guaiac resin, to which was added vaginal discharge women from the control group, the color is not formed (see ydalenia women with clinically diagnosed candidiasis using cells of Candida albicans hydrolyzed protein, associated with Sigmacell20, and protein associated with Espergit, thereby releasing soluble, active HRPO. Centrifugation was besieged HRPO associated with the polymer, leaving in solution only aspartic protease solubilizing HRPO. Soluble HRPO was katalizowania oxidation of guaiac resin under the action of hydrogen peroxide, thereby leading to the formation of a blue color. Therefore, the formation of blue color on paper impregnated with guaiac resin, allows convenient and efficient method of detecting active aspartic protease in the vaginal discharge and, therefore, vulvovaginal candidiasis.

Active aspartic protease were not found in vaginal secretions of women in the control group, i.e. women who do not have clinically diagnosed candidiasis. Therefore, in the case of samples of vaginal secretions of women in the control group associated with the polymer is hydrolysed protein, and soluble, active HRPO is not separated from the carrier. After deposition of HRPO associated with the polymer by centrifugation, the solution was left aspartic protease solubilizing HRPO. In the absence of HRPO, supernatant from control obrazets lack of education blue coloration on the guaiac impregnated with tar paper is a convenient method to identify women not infected vulvovaginal candidiasis.

Experiment XIV

This experiment involves the differentiation of various hydrolytic activity of some microbial proteases using conjugate Sigmacell-myoglobin-HRPO.

A. Materials:

1. [ Sigmacell-myoglobin-HRPO] (example E and F).

2. Culture of Candida albicans (ATCC 28866), producing aspartic protease.

3. The culture of Trichomonas vaginalis (ATCC 3001).

4. A cell suspension Mobbluncus curtisii ATCC 35241) in physiological solution.

5. Buffers and solutions:

a. 0,02% solution of hydrogen peroxide

b. 300 mm califofnia buffer with a pH of 7.5

c. 100 mm acetotartrate buffer with a pH of 4.0.

6. Paper impregnated with guaiac resin (manufactured by industry drugs Hemoccult).

B. Procedure:

20 mg of substrate [ Sigmacell-myoglobin-HRPO] suspended in 75 μl of the appropriate buffer (see tab. 27), and then add 25 ál of a mixture of cell culture/suspension. The reaction mixture is incubated for 10-30 minutes, after which the sample is centrifuged to remove the solid phase conjugated substrate and cell debris. On the solution of hydrogen peroxide. The reaction conditions and the stage of manifestation of color are given in table. 27.

C. Results:

Adding to drugs with guaiac reaction supernatant from tubes, incubated with buffer at pH 4.0 or pH 7.5 for 30 minutes, manifested by hydrogen peroxide, staining was not observed. A similar result (i.e., no color) was obtained heated to boiling cultures of Candida albicans (pH of 4.0 and 7.5), Trichomonas vaginalis (pH 4.0 and 7.5) or Mobiluncus curtisii (pH of 4.0 and 7.5). Culture of Candida albicans was produced bright blue color after 10 minutes of incubation at a pH of 4.0, but was not produced color at pH 7.5, even after 30-minute incubation. Trichomonas vaginalis was produced bright blue color after a 30 minute incubation at pH 7.5, but after a 30 minute incubation at pH of 4.0 was observed barely noticeable coloration. Culture Mobiluncus curtisii after 30-minute incubation, did not yield something blue coloration nor when 4,0 nor when 7,5 (see tab. 28).

I. Interpretation

After a 30 minute incubation at room temperature using a 300 mm phosphate buffer at pH 7.5 and 100 mm acetate buffer at pH 4,0, HRPO was not separated from the solid media. Similarly heated to the boiling suspension of Candida albicans, Trichomonas vaginalis or Mob using culture Candida albicans HRPO was separated from the solid media at a pH of 4.0 after 10-minute incubation, but when pH 7.5, even after 30-minute incubation, the release of HRPO was not observed. The obtained results correspond to the pH-profile set for aspartic proteases of Candida albicans, which is active at low pH and low or completely inactive at high pH. Conversely, when using a culture of T. vaginalis HRPO easily separated from the solid media at a pH of 7.4 after 30-minute incubation, but at a pH of 4.0 released a barely detectable amount of HRPO. This result is also consistent with known pH profile obtained for thiol proteases of T. vaginalis (i.e., which is active at high pH, but less active or completely inactive at low pH). And finally, Mobiluncus curtisii, for which there is no data concerning section proteases, not released HRPO neither at pH of 4.0, nor at pH 7.5, even after 30-minute incubation.

Thus, the test under different pH conditions allows us to differentiate three microorganism; Candida albicans, which causes the formation of coating by 10-minute incubation at room temperature at pH 4,0; Trichomonas vaginalis, which causes the formation of color after 30 minutes at pH 7.5; and Mobiluncus curtisii, which does not cause the formation of coating by a 30 minute incubation nor p is x proteases and their inhibitors on the substrate [ EspergitC-myoglobin-HRPO]

A. Materials:

1. Sprayed [ EspergitC-myoglobin-HRPO] (example Q).

2. Manufactured by industry aspartic protease from the company Sigma Chemical Co. (Type XIII, secreted by Aspergillus saitoi 0.6 units/mg activity; 40 mg/ml).

3. Trypsin (serine protease) from bovine pancreas (2900 units/mg activity) supplied by the company U. S. Biochemicals 2 mg/ml

4. Papain (thiol protease) from the milky juice of papaya (12 units/mg activity) (2 mg/ml) supplied by the company Sigma Chemicals Co.

5. Culture of Candida albicans (ATCC 28366), producing aspartic protease.

6. Hydrochloride tosylchloramide (TLCK) (50 mm in ethanol) (from the company Sigma Chemicals Co.)

7. Pepstatin A (2 mg/ml) from a microbial source, supplied by the company Sigma Chemicals Co. (ethanol solution).

8. Buffers and solutions:

a. Califofnia buffer pH 7.0, 200 mm.

b. Califofnia buffer pH of 7.4, 100 mm.

c. Acetotartrate buffer pH of 4.0, 100 mm.

d. 0,02% solution of hydrogen peroxide.

e. Absolute ethanol.

9. Paper impregnated with guaiac resin (manufactured by industry drugs Hemoccult).

B. the Order of execution
C-myoglobin-HRPO]. The enzymes and control (enzymes, subjected to boiling for 15 minutes) and incubated with 20 mg of substrate and buffers (the number indicated in the table. 29) for 15 minutes before analysis.

In the second part, the enzyme is pre-incubated with their respective inhibitors and suitable buffers for 15 minutes. Then they are added to the substrate and incubated for another 15 minutes at room temperature.

In both cases, the reaction mixture is centrifuged to remove the solid phase conjugate. The supernatant (5 μl) are added to Hemoccult-drugs and take 5 ál of hydrogen peroxide.

C. Results

1. Part I: adding to the guaiac drugs with the manifestation hydroxide hydrogen, reaction of supernatants of tubes containing only one buffer at pH 4.0 or pH of 7.5, was produced only very weak visual color. Similar results were observed when the reaction of supernatants of tubes containing heated to boiling trypsin at pH of 7.4, culture Candida at pH 4.0 and at pH of 7.4. Bright blue color was formed with the use of trypsin at pH of 7.4, Candida at pH 4.0 and papain at pH 7.0, is not subjected to temperatures up to boiling.

D. Interpretation

Trypsin (serine protease) hydrolyzed solid-phase substrate at a pH of 7.4, releasing soluble HRPO, which catalyses the formation of blue color shown on the guaiac drugs. Similar results were observed for aspartic proteases of Candida albicans at pH 4.0 and papain (thiol protease) at pH 7.0. The heat prevented the release of HRPO under the action of each of these enzymes. Therefore, each of the three enzymes of various types has the ability to the hydrolytic cleavage of soluble HRPO from the media when appropriate reaction conditions. Neither buffers nor thermoinactivation enzymes are not released soluble HRPO, which suggests that the release of HRPO is not simply a nonspecific release induced by salts, etc. in the culture medium or the incubation mixture.

TLCK, a protease inhibitor, can inhibit both serine and thiol proteases, inhibit Tatin, a well-known inhibitor of aspartic proteases, inhibited the formation of color produced aspartic protease of Candida albicans. Therefore, the specificity of detection hydrolases can be achieved by carrying out the incubation in the presence of known inhibitors of specific enzymes or inhibitors of specific classes of enzymes.

The foregoing description is presented for purposes of illustration. Each specialist is clear that the operating conditions, materials, stage procedures, and other parameters described methods and test devices can be modified or replaced, without going beyond being and scope of the invention.

1. Method detection hydrolases, wherein contacting the sample with an immobilized on a solid carrier of the enzyme reporter carried out in such a way that the enzyme reporter is released under the action of hydrolases present in the sample, after the specified contact is exercised by the interaction of the sample with the indicator, and the specified indicator can be detectivemisa change under the action of the enzyme reporter, but not sensitive to the specified detector is. the procedure under item 1, characterized in that hydrolase is a protease selected from the group comprising aspartic proteases, serine proteases, thiol proteases, metalloproteases, acid protease and alkaline protease.

3. The method according to p. 1, characterized in that the enzyme-reporter is peroxidase.

4. The method according to p. 1, wherein the test sample is placed in a device containing first and second solid media, while the first solid medium immobilized enzyme reporter so that it is released under the action of the hydrolases, and the second solid medium, which is not in contact with the first solid medium, immobilized indicator that can be detectivemisa change under the action of the enzyme reporter, and the sample is placed in the device so that he was in contact with the first and second solid media, resulting enzyme-reporter, released under the action of the active hydrolases present in the sample diffuses through the sample to the second solid medium, and observe whether the specified indicator detectivemisa change and, if such detective change impetusto to detect hydrolases, characterized in that includes a receiver formed partially in the first and second opposite walls, facing each other, the inner sides and having between them a gap, and the first, second or both walls made of translucent material on the inner surface of the first or second wall on a solid medium immobilized enzyme reporter so that it is released under the action of hydrolases present in the sample, on the inner surface of the first or second wall is an indicator capable of detectivemisa change only under the action of the enzyme reporter, and the receiver made the hole for the input sample.

 

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The invention relates to the field of Bioorganic chemistry, namely to a new 6-(moselhotel-L-prolyl - L-arginyl)aminonaphthalene-1-isobutylamino formula:

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as a substrate for the determination of thrombin

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FIELD: medicine.

SUBSTANCE: according to the following stages, flow cytometry is used to detect living cells, damaged cells, VNC cells and dead cells of a microorganism in a tested sample: a) the stage of processing the tested sample with enzyme chosen from lipolytic enzymes and protease with activity to destruct the cells differing from those of the microorganism, colloid protein particles or lipids found in the analysed sample; b) the stage of processing the tested sample with topoisomerase inhibitor and/or DNA-gyrase inhibitor; e) the stage of processing the tested sample been processed at the stages a) and b) with a kernel-dyeing agent, and d) the stage of detecting microorganisms in the tested sample processed by the kernel-dyeing agent with using flow cytometry.

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19 dwg, 8 tbl, 8 ex

FIELD: food industry.

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3 tbl

FIELD: biotechnologies.

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28 cl, 11 dwg, 2 tbl, 1 ex

FIELD: medicine.

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FIELD: biotechnology.

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6 cl, 9 dwg, 4 tbl, 6 ex

FIELD: pharmacology.

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22 cl, 5 tbl, 49 ex

FIELD: medicine, oncology, molecular pharmacology.

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9 cl, 6 dwg, 4 tbl, 1 ex

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