DD289604A5 - METHOD FOR THE PRODUCTION OF AFFIN-ENZYMATIC COMPOUNDS FOR THE VISUAL DETECTION OF CHOLESTERINE ON THE SKIN SURFACE OF A PATIENT BASED ON A CHLOROESININAFFINATE-PROOF AND A VISUALIZING AGENT AND ITS USE - Google Patents

Abstract

Used as detecting agent A are steroid glycosides, triterpene glycosides, hydrophobic proteins, proteinaceous toxins or polyene antibiotics which have an affinity for cholesterol, and as visualisation agent B are enzymes. To increase the sensitivity of the prepared compounds, the detecting agent A and the visualisation agent B are immobilised on a crosslinking agent C, for which low or high molecular weight compounds are used. <IMAGE>

Description

For this 1 page drawings

Field of application of the invention

The invention relates to the field of bioorganic chemistry, and to a method of making affine enzymatic compounds for the visual detection of cholesterol on the skin surface of a patient based on a cholesterol-affinity-based detection agent and a visualization agent.

Most effectively, the compounds of the present invention can be used in diagnostics, including for the early detection of atherosclerosis, for clarification of the diagnosis under clinical, ambulatory and domestic conditions as well as for the control of the course of treatment. The compounds listed can also be used in physiological, histological and histochemical studies to detect elevated levels of cholesterol and to locate them. In addition, the compounds also allow the determination of reduced cholesterol content in patients with certain pathologies, especially oncological character.

Characteristic of the known state of the art

Atherosclerosis and its complications, such as infarcts, insults and gangrene, are one of the main causes of population mortality in all WoIt countries. Several years of research have shown that atherosclerosis is one of the diseases that must first be detected and prevented, with the main factor for the risk of atherosclerosis being cholesterol accumulation in the organism. The prevention of atherosclerosis in the population requires, first and foremost, that the most vulnerable risk group is identified among the patients, according to which it is to be treated in a differentiated manner and the patients concerned change their way of life. The identification of patient groups with an increased risk of atherosclerosis due to the accumulated in the organism cholesterol amount is the most difficult to the problem of atherosclerosis prophylaxis. The known methods of atherosclerosis diagnosis are based on the quantitative determination of total cholesterol in venous blood plasma, (see Consensus Conference on Lowering Blood Cholesterol to Prevent Heart Disease, JAMA, 1985, 253, pp. 2080-2086; The Lipid Research Clinics Population Studies Data Book;

Publication 80-152; Bethesda, Ma., National Institutes of Health, 1980, Vol. 1; Lipid Research Clinics Program, JAMA, 1984, 251, pp. 351-364.) Cholesterol levels in excess of 260 mg are considered to be sufficient in some cases to account for the patient at risk. A detailed diagnosis can be made on the basis of the analysis of blood plasma lipoproteins and the atherogenicity index, which represents the ratio of the difference between total cholesterol (Ch ₀ ,,) and cholesterol of the high density lipoproteins (Ch, _hd ) to the high density lipoproteins cholesterol:

Chg "-

l "th" r. = XT

ch

With an atherogeneity index of over 3, the patient is included in the risk group and with an index of more than 5.6 to the atherosclerotic patients (see Klimov AN, "Fenotipirovanie lipoproteinov", Metodiæeskie rekomendacii MZ SSSR, M., Medicine, 1975, Goldfourt V. , Holtzman E., Neufeld HN, Total and High Density Lipoprotein Cholesterol in the Serum and Risk of Mortality, British Medical Journal, 1985, 292, pp. 1239-1243).

The use of these methods requires a blood sample, which is traumatic and external to the patient. It is not dangerous to do so, taking into account the possibility of transmitting various viral infections. Fractionation of plasma lipoproteins and analysis of cholesterol remains a complicated and tense process. In addition, in one out of three cases, total cholesterol and even full phenotyping results are not correlated with the severity of atherosclerosis (see Mjasnikov AL, "Gipertoniieskaja bolezn '", 1965, M., Medicine, p. 300).

Moreover, studies conducted in recent years have revealed that blood plasma can not fully reflect the processes of cholesterol accumulation characteristic of the arterial wall and other bradytrophic tissues.

A further improvement of the diagnostic procedures resulted from the solution of some fundamental questions of the pathogenesis of atherosclerosis. It has been shown that tissue cholesterol plays the crucial role in the development of atherosclerosis. It could be found Gowebe, which accumulate the cholesterol analogous to the Arterienwandung. Studies in recent years have shown that there is a close correlation between the cholesterol content in the arterial wall and in the skin. This has allowed the development of fundamentally new diagnostic methods, in particular for atherosclerosis,

The aforementioned correlation between the cholesterol content in the arterial wall and in the skin was determined by direct determination of cholesterol in skin biopsies. The skin samples were frozen in liquid nitrogen and lyophilized, after which the cholesterol was extracted with Folch's reagent and determined by conventional chemical or biochemical methods (see Nikitin Ju. P "Gordienko IA, Dolgov A.V" FiIi onova TI, "Soderianie cholesterina ν koJe i ego korreljacija s lipidnym pokazatelem syvorotki krovi u zdorovych ljudej i bol'nych iSemlcOskoj bolezn'ju serdca ", Kardiologija, 1987, XXVII, No. 10, pp. 48-51; Bouisson hl. De Graeve, Solera ML et al., "Ann. Biol. CHn." 1982, Vol. 40, pp. 3-1-407). However, due to its traumatic nature and the complexity of its implementation, this method is unsuitable for screening.

US Pat. No. 4,458,686 describes a method for the determination of blood-localized glucose or ethanol immediately under the skin and on the skin surface. Also indicated is the possibility of determining cholesterol using cholesterol oxidase. The method for determining the amount of these substances is based on the stoichiometric change in the oxygen concentration when used for the substrate to be determined specific Redoxfermente, preferably oxidoreductase. In this case, the quantitative measurement of the change in the oxygen concentration takes place electrochemically, for example polarographically with special devices and a specially developed electrode. The complicated equipment required for this requires the diagnosis of a highly qualified personnel. All of this inevitably limits the applicability of this method to screening.

From the published PCT / US application 84/00888 a complex of a detection and visualization Miti is known in which they are connected to each other directly or via a crosslinking agent. This complex is used to determine small amounts of specific molecules, such as lipids in biological tissues. However, this method of detecting lipids is only practicable under laboratory conditions and requires that a biological fluid or tissue be previously removed from the patient, which means that the procedure is intrinsically traumatic, multi-stage and complicated to perform.

Data on the correlation between the cholesterol content in the skin and the degree of atherosclerotic damage to the vessels are obtained on skin biopsies. This procedure is not only traumatic, but also has a number of other disadvantages, since 1 mm thick biopsy specimens include various skin layers, namely the horny layer (average thickness 0.1 mm) and connective tissue, that is the actual skin (derma) again comprises two layers, namely the stratum papillare and the stratum reticulare. These two layers are well supplied with blood, which is why biopsies contain blood vessels and blood, as well as sweat and sebaceous glands and their secretions. Immediately below the dermis is the subcutaneous fatty tissue, which may also belong to the bioptate. The inhomogeneity of the bioptates can thus distort the results of the determination of cholesterol accumulated in the skin. In this regard, one most accurate method is to enable the determination of the cholesterol level on the skin surface in the horny layer of the derma.

Object of the invention

The present invention provides a process for the production of affine-enzymatic compounds for the visual detection of cholesterol directly on the skin.

The compounds according to the invention lead to a maximum simplification of Atherosklerosediagnostik, are not traumatic and do not require special equipment.

-6- 289 604 Presentation of the Essence of the Invention

The invention has for its object to develop a process for the preparation of affine-enzymatic compounds for the visual detection of cholesterol directly on the skin of the patient, in particular in the horny layer of the epidermis without having to remove the patient's blood and skin biopsies , For diagnosis, any skin area can be used. Most suitable, however, is the palm, as it contains no sebaceous glands, the excretions of which, like the horny layer of the skin, contain cholesterol, which influence the results of the diagnosis

This object has been achieved according to the invention by a process for the preparation of affin-enzymatic compounds, clio bifunctional, represent elle compounds. These selectively combine with the free cholesterol of the patient's skin and can then be visualized visually. For the preparation of such a compound at least two components are required, namely a detection agent A, selected from the group of substances that with the free skin cholesterol to address the entire bifunctional compound on the free cholesterol of the skin with this selectively a solid Complex and a visualization agent B which visually visualizes the bifunctional compound linked to the cholesterol.

The inventively produced compounds lead to a maximum simplification of Atherosklerosediagnostik, are not traumatic and also require no special equipment. The use of these compounds for the diagnosis of atherosclerosis makes it possible to assign the persons examined to one of the following three groups: persons suffering from atherosclerosis, risk group and healthy subjects.

The affine enzymatic compounds which can be prepared according to the invention for the visual detection of cholesterol and the diagnostic methods based on their use ("three-point method") are also suitable for screening The method is very simple to use and requires no special qualification on the part of medical personnel; be done at home.

The compounds according to the invention can also be prepared from the detection agent A and the visualization agent B by chemical activation of the functional groups of one of the two agents. In this case, after one has added to each of the two agents containing the activated chemical groups, each having the other agent, at certain proportions between the agents A and B to form a solid chemical bond, which higher molecular weight bifunctional compounds for the obtains visual evidence of cholesterol on the skin of the patient.

As Nachweiemittel A, which is a Cholesterinaffinans, the following compounds are suitable:

Sterol glycosides containing as aglycone the furan and spirostanol cyclopentanone hydrophenantrene fragment and an oligosaccharide fragment containing from 3 to 10 straight or branched monosaccharides (see PK Kintja, "Stroenia i biologiäeskaja aktivnost 'steroidny glikozidov rjada spirostana i furostana", Kiäinev, VI. Etinca , 1987, p. 142) or

Asteroidal glycosides containing an aglycone of the α or β-amyryl, lupane, hopane, deaminan, linostan or holostane series and an oligosaccharide of 2 to 8 branched or unbranched radicals (see GE Dekanosidze, V.Ja. Cirva , TV Sergienko, NI Uvarova, Issledovanie triterpenovych glikozidov ", Tbilisi, Cf. Mecnierebf., 1982) or

Hydrophobic proteins capable of selectively forming a complex compound with cholesterol (see I. AN Klimov, GVTitova, KA Koievnikova, Biochimija, 1982, Vol. 47, No. 2, pp. 226-232, 2. KA Koievnikova, NN Kljueva et al Voprosy med chimii, 1984, Vol. 30, No.3, pp. 86-90, 3rd GV Titova, NN Kljueva, KA Koievnikova et al., Biochimija, 1900, Vol.45 , Ni.1, p.51-55) or

proteinaceous toxins derived from bacteria, marine microorganisms, insects or snakes, which can selectively form a complex with cholesterol (see MV Dalin, NG Fiä, "Belkovye toksiny mikrobov", Moscow, VIg. "Medicina", 1980) or

Polyene antibiotics which are able to selectively form a complex compound with cholesterol (see 1. J. P. Katzenstein,

A.M. Spielvogel, A.W. Norman "J. Antibiot." 27, 12, 1974, pp. 943-951; 2. Jong Shang-Shyng, Wang Hsi-Hua, "Clin. J. Microbiol.", 1976, 9 / 1-2 /, p 19-30;

3. Reading Josephine D. et al., "Biochem. Biophis. Acta ", 1982, 685, 121, p.219-224) or

high-affinity enzymes, which have cholesterol as a substrate and show a high affinity for this.

As a visualization agent B, the following enzymes are: acetylcholinesterase, ^T vrosinase, Glukoso-6-phosphate dehydrogenase, Glukooxidase, glucoamylase, galactosidase, peroxidase, alkaline or acidic phosphates, a-chymotrypsin or Pyrophosphatast ·.

The object is further achieved in that the detecting agent A, which is a Cholesterinaffinans and is selected from Steroidglykosiden containing as Aglykon the Cyclopentanperhydrophenantrenfragment furostanol and Spirostanolreihe and an oligosaccharide fragment with 3 to 10 unbranched or branched monosaccharides, or under Triterpenglykosiden, the an aglycone of the α- or β-amyryl, or Lupan or Hopan or Dammaran or Linostan or holostane series and an oligosaccharide from 2 to 8 branched or unbranched radicals, or from hydrophobic proteins, with the cholesterol selectively a complex compound or from proteinaceous toxins derived from bacteria, marine microorganisms, insects or snakes, and selectively complexed with cholesterol. Or from polyene antibiotics which are capable of selectively forming a complex with cholesterol, or of enzymes having high affinity for cholesterol by chemical methods in an aqueous medium at a molar ratio of reagent A to activator of 1: 1 to 1:10, a detection agent concentration of 1 to

20 mg / ml, a temperature of 0 to 25T: a pH of 4 to 11 within 1 to 24 hours is activated, after which the visualization agent B is added to the resulting solution, selected from the enzymes acetylcholinesteraae, tyrosinase, glucoso-6 Phosphorus dehydrogenase, glucosooxidase, glucosoamylase, galactosidase, peroxidase, alkaline or acid phosphates, α-chymotrypsin or pyrophosphatase at a molar ratio of A: B of 20: 1 to 1: 1 and the solution at a temperature of 0 to 2b ^J C 1 to Kept for 24 hours until the final product is received. As already stated above, the functional groups of the visualization agent B can also be chemically activated beforehand. In this hall dio chemical activation of the visualization agent B takes place in an aqueous medium at a molar ratio of visualizing agent B to activating agent of 1: 1 to 1:10, a visualizing agent concentration of 1 to 20 mg / ml, a temperature of 0 to 25 ° C and a pH Value of 4 to 111 m over a period of 1 to 24 hours, after which the resulting solution is added to the detecting agent A, which is one of the above-mentioned substances, at a molar ratio of AB of 20: T to 1: 1 and the solution at a temperature from 0 to 25 ° C for 1 to 24 hours until the final product is obtained.

According to the invention, the chemical activation of the detection agent A or of the visualization agent B takes place according to the known azide, carbodiimide or succinimide method or according to the method of periodate oxidation. In cases where a higher molecular weight compound, namely an enzyme, is used as visualization agent B and low molecular weight compounds such as glycosides are used as detection agent A, the molecule of the end product can not have more than 1 molecule of visualization agent B. In this case, the molar content of detection agent A, which allows the binding to the cholesterol of the skin in the final product, the visualization agent content may exceed only a few times, and that according to dom content of functional groups in the enzyme molecule, for the linkage with the detection agent A without significant loss of enzymatic activity are suitable. In such compounds, the content of enzyme used as the visualization agent B is limited. This results in the relatively low sensitivity of the compounds obtained, which is why the skin must be exposed for a longer time in the diagnosis.

For the extension of the technical possibilities of the use of affine enzymatic compounds for the visual detection on the skin of the patient and to increase their sensitivity, it is proposed to use the detection agent A with the visualization agent B with the aid of a crosslinking agent C selected from low molecular weight asymmetric bifunctional compounds such as cyanogen bromide , Trichlorotriazine or 2-amino-4,6-dichloro-S-triazine. Thereby, the compounds can be prepared in two ways, either first the Nachwoismittel A can be connected to the crosslinking agent C, after which the visualization agent B is added to the intermediate product (A + C) to obtain the final product or it becomes first the intermediate product (B + C) is formed, then the detection agent A is added. In the former case, the affinate-enzymatic compounds are obtained by ordinary dissolution in an aqueous salt buffer solution (pH 5 to 9) of the detecting agent A selected from the listed substances at a concentration of 1 to 20 mg / ml, followed by dissolving the solution low molecular weight asymmetric bifunctional crosslinking agent C selected from the group of cyanogen bromide, trichlorotriazine or 2-amino-4,6-dichloro-8-triazine in a molar ratio of A: C of 1: 0.5 to 1:10, the resulting solution at 0 to 20 ° C for 1 to 20 hours, then the visualization agent B, selected from the above substances, at a molar ratio of A: B from 20: 1 to 1: 1 added and the solution at 0 to 20 ° C 1 for up to 48 hours until the final product is received.

In the preparation of the affin-enzymatic compounds via the intermediary product B + C, the visualization agent B selected from the above-mentioned substances is dissolved in a conventional manner in an aqueous salt buffer solution (pH 5 to 9) at a visualization agent concentration of 1 to 20 mg / ml and adding to the solution the low molecular weight asymmetric bifunctional crosslinking agent C selected from cyanogen bromide, trichlorotriazine or 2-amino-4,6-dichloro-S-triazine at a molar ratio of B: C of 1: 0.5 to 1:10 , holding the resulting solution at 0 to 20 ° C for 1 to 20 hours, then adding the detecting agent A selected from the above-mentioned substances at a molar ratio of A: B of 20: 1 to 1: 1 and the solution at 0 to 20 ° C for 1 to 48 hours until the final product is obtained.

The maximum sensitivity of the affin-enzymatic compounds for the visual Nachwels of cholesterol on the skin of the patient can be achieved, however, only if one uses as a crosslinking agent high molecular weight polyfunctional compounds such as polysaccharides, proteins or synthetic polymers, that is, any higher molecular weight compounds, any functional group selected from the group of primary amine, carboxyl, hydroxyl, aldehyde, hydrohalide, mixed anhydride, imino ether, azide, hydrazide, maleimide, isocyanate or epoxide. In this case, the detection agent A, which is one of the listed substances or mixtures thereof, and the visualization agent B immobilized, which is also one of the listed substances independently of one another, are immobilized on one of these compounds. Such affine enzymatic compound for the visual detection of cholesterol on the skin surface of the patient allows the ratio of detection agent A to visualization agent B depending on the degree of affinity of the selected detection agent A for the cholesterol of the skin or the activity of the enzyme B used within to vary over a very wide range. For example, using a Detecting Agent A having a weaker affinity for the cholesterol of the skin, its relative molar content in the final product can be substantially increased.

Such affine-enzymatic compounds are prepared by using the detection agent A, which is one of the substances enumerated above, or mixtures thereof, and the visualization agent B, which is also one of the substances enumerated above, in a molar ratio AB of 20: 1 1 to 1, it dissolves in an aqueous salt buffer solution having a pH of 5 to 9 at a Konzentrati Dn of A and B of 0.1 to 20mg / ml, the resulting solution with a high molecular weight polyfunctional Verf C means polysaccharides, proteins or synthetic polymers, ie any higher molecular weight compound which represents any functional group selected from the group of primary amine, carboxyl, hydroxyl, aldehyde, hydrohalide, mixed anhydride, azide, hydrazide, maleimide, isocyanate or epoxide, in a molar ratio of A + B: C of 1: 0.3 to 1:10 and the resulting solution at a temperature of 0 to 2O ⁰ C1 to 20 For hours until receipt of the final product.

Preferred steroid glycosides which are used as detection agent A and contain as Aglykon a Cyclopentanperhydrofenantrenment the furostanol or Spirostanolreihe and an oligosaccharide fragment with 3 to 10 unbranched or branched monosaccharides are Agavosid A, Agavoside B, Agavoside C, Agavosid D, Agavosid F, agavoside H , Agavoside G, trillin, funcoside C, funcoside D, funcoside F, funcoside G, funcoside I, dioscine, gracillin, protodioscin, cicubasaponin, juncoside E, juncoside H, lanatigonin, desglucodigitonin, digitonin, gitonin, rocoside C, rocoside D,

Rocoside E _1, Funcoside E ₁ Alliumoside C, Polygonatcnin, Tigogenin Tetraoside, Tigogeninhexaoside, Capsicoside, Ammumoside B, Ammumoside C, Ammumoside D, Ammumoside E, Desglukodosramnoparillin, Desglukoparillin, Parillin, Sarsaparilloside, Asparagoside C, asparagoside D, asparagoside G, asparagoside H, protoyuccoside H, yukkoside B, lanatigoside, monoside, bioside, Trioside, purpureagitoside, Gekogenin, Rocogenin, Oiogeiiin, Tigogenin, Protopolygametoside, Tomatonin, Laxogenin lycotetraoside, alliogenin lycotetraoside, carotenoside A, cyclosiovorsioside H, acantofiloside C, alliofusoside A, Alliospiroside A, cyclosieversioside F, T'leasaponin, acantofiloside B, tigonin (total), glycoside of the plant Calha polypetala or Cyclosieversioside G. Among the above-mentioned steroid glycosides, the funcosides C, D, E, F, G, I, Dioscln, Rocoside, Lanatigonin, Digitonin and Tomatonin particularly preferred. Most preferred are digitonin and tomatonin. Preferred triterpene glycosides which are used as detection agent A and an aglycone of the α- or β-amyryl, lupan, Hopan, Dammaran, Lanostan or holostane series and a Oligosaccharld from 2 to 8 branched or unbranched radicals

Aescin, Avenacin, Theasaponin, A-Hederin, Cauloside C, Stichinoside A, Cyclamine, Chinovin, Saponins

Sugar beet, hypsoside and triterpene glycosides derived from Fatsia japonica. Preferred hydrophobic proteins, which are used as Ndchweismittol A and those with the cholesterol Are selectively the Meilinprotein according to Folch, proteins of lysosomes and Mitochondrion, fibrinogen, immunoglobulins, cerebron, myoglobin, trypsin, cytochrome C, cytochrome P-450 or apoei whites

of lipoproteins.

Preferred proteinaceous toxins used as detection agent A selectively cholesterol one Complex compound can be formed and recovered from bacteria, marine microorganisms, insects or snakes

are Streptolysin o, Pneumolysin, Listerio! / sin, o-toxin C.I "derived from Perfrigeno type A and C, δ-toxin Cl. novyj

Type A and C, hemolysin Cl. histolyticum, hemolysin Cl. botulinum type C and D, tetanolysin, cerebral / sin, alveolysin, Door enolysin or cytotoxin from the actinia Metridium senile. Preferred polyene antibiotics used as detection agent A and complexed with cholesterol Widen are Amphotericin B, Philipin or Nistatin. Preferred high affinity enzymes that have high affinity for cholesterol and are used as detection A are Cholesterol oxidases), cholesterol dehydrogenases or cholesterol esterases. Among the listed cholesterol affinity agents A such as glycosides, hydrophobic proteins, proteinaceous toxins, Polyene antibiotics and high-affinity enzymes, the glycosides are preferred because they are chemically most stable and both

without endangering their ability to react with organic solvents and high temperatures in the synthesis

Cholesterol of the skin to form a complex, forfeit. The low molecular weight of the glycosides allows the Preparation of a High Molar Content Means of Detecting Agent, Which Means a Variety of Interactions

ensured between the middle and the cholesterol of the skin.

Particularly preferred among the glycosides are the steroid glycosides, which in comparison to the triterpene glycosides with the Cholesterol most effective to be able to form a complex. The hydrophobic proteins, proteinaceous toxins, polydntibiotics and high affinity enzymes can also be successfully used for

However, the production of a diagnostic agent can be used, but cause due to their low chemical

Resistance and its inactivating ability have certain limitations in the process of making the

affine enzymatic compounds. In addition, the high molecular weight of these detecting agents somewhat reduces the

Sensitivity of the final compound, resulting in an increased concentration of the agent and for the detection of cholesterol on the skin

longer exposure time required.

It is understandably desirable to carry out the reactions under mild conditions, that is, at neutral pH,

low ionic strength, low temperatures and shorter reaction time.

As visualization agent B enzymes are used, which due to the reaction with the detection agent A the

affinenzymatic product for the visual detection of cholesterol on the patient's skin. They are selected from the group acetylcholinesterase, tyrosinase, glucoso-6-phosphate dehydrogenase, glucosooxidase, glucosoamylase,

Galactosidase, peroxidase, alkaline or acid phosphatase, α-chymotrypsin or pyrophosphatase. All of these enzymes can be used together with any of the above-listed Detecting Agent A, preferably

however, are steroid glycosides.

The use of a crosslinking agent (C) extends the technological possibilities of the process for producing a A compound for the visual detection of cholesterol on the skin surface of a patient, wherein the functional activity of the Means A and B is maximally preserved. Particular preference is given to compounds for the preparation of which as cholesterol-sensitive detection agent A steroid glycosides

which contain as aglycone the cyclopentane perhydrophenantraene fragment of the furostanol and spirostanol series and an oligosaccharide fragment with 3 to 10 unbranched or branched monosaccharides.

Such steroid glycosides have high chemical resistance and can under severe conditions, that is at

high temperatures and, if appropriate, by dissolving in organic solvents immobilized on a cross-linking polymer, which ensures their high content in the final product. The compound for the visual detection of

Cholesterol on the skin surface of the patient is obtained by immobilizing the detection agent A on the Crosslinking agent C under severe conditions and subsequent immobilization of the visualization enzyme B on the Crosslinking agent C and the reagent A containing under mild conditions, so that its

enzymatic activity is maintained.

As crosslinking agent C asymmetric low molecular weight bifunctional compounds, such as cyanogen bromide, Trichlorotriazine or 2-amino-4,6-dichloro-S-triazine. The use of higher molecular weight polyfunctional compounds as crosslinking agent C allows the ratio of Detecting agent A to visualizing agent B in the compound for visual detection of cholesterol within a wide range Range to vary, for example, to an optimal ratio between the number of link centers of Connection with the skin surface (detection means A) and the amount of visualization agent B to get.

As higher molecular weight polyfunctional crosslinking agent C are used polysaccharides, proteins or synthetic) polymers, that is, any higher molecular weight compounds containing any functional group selected from the group primary amine, carboxyl, hydroxyl, aldehyde, hydrohalide, mixed anhydride, imino ether, azide, hydrazide, Maleimide, isocyanate or epoxide.

As higher molecular weight polyfunctional crosslinking agent C comonomer preferred acrylic acid or maleic anhydride copolymers in question.

Particularly preferred as higher molecular weight polyfunctional agent C is the N-vinylpyrrolidone-maleic anhydride copolymer. The cited crosslinking agents C are used with any detection agent A and any visualization agent B. This always gives you a final product A-C-B.

Particular preference is given to compounds in which Storoid glycosides are used as detection agent A.

As regards the agent 8, alkaline phosphatase and peroxidase are preferred here. Particularly preferred is the peroxidase of Kraren (horseradish).

Activating agents for the detection agent A and the visualization agent B are compounds which convert the functional groups of these agents into their reactive form. Groups such as primary amine, carboxyl and α-glycol can be activated by methods known per se, such as the azide, carbodiimide, succinimide or periodate method.

Particular preference is given to the periodate and the carbodimide methods, since these methods ensure the maximum gentle synthesis conditions and thereby allow maximum retention of the activity of the immobilized enzyme,

As aqueous salt buffer solutions, solutions obtained from compounds which ensure a buffering action in a pH range of 5 to 9 and which do not cause inactivation or inhibition of the enzymes are used. Such solutions may be, for example, borate, citrate, phosphate and other buffer solutions. Usually, however, to use solutions having a salt concentration which ensures a constant pH of the solution during the immobilization, but does not exceed values at which it comes to denature the enzyme.

Some of the detection agents A, such as steroid glycosides, triterpene glycosides and polyene antibiotics, dissolve poorly in water or do not over-scour, which is why organic solvents are used in their solution. As such, polar aprotic solvents, for example dimethyl sulfoxide, dimethylformamide, hexamethanol, a mixture of dimethylformamide with toluene (2: 1) or a mixture of dimethylformamide with hexane (2: 1) are suitable.

The conditions for the preparation of the cholesterol detection compounds (temperature and hold time in the thermostat) depend on the choice of components A, B and C. Be used as a visualization agent B enzymes, the reaction temperature must not exceed 2O ⁰ C. Preferably, the reaction is carried out at 4 ⁰ C in buffer solutions having a pH of 5 to 9. If synthetic polymers are used as crosslinking agent C and glycosides as the detection agent A, the reaction can be carried out in organic solvents and at high temperatures (up to 12O ⁰ C) and a reaction time is sufficient, which is sufficient for the achievement of a maximum yield of end product. The molar ratios of the reaction components are chosen according to the molecular weight of the agents A and B chosen, the degree of affinity for cholesterol of the skin and the activity of the chosen enzyme.

Exemplary embodiments

The invention will be explained in more detail with reference to embodiments and drawings in which it is shown. Showing:

1: the reference numerals,

Fig. 2: the compounds obtained and their interaction with the cholesterol on the skin surface of the patient according to

Claims 1 and 2, Figure 3: the compounds obtained and their interaction with the cholesterol on the skin surface of the patient according to

Claims 3 and 4 and 4: the compounds obtained and their interaction with the cholesterol on the skin surface of the patient according to claims 5 and 18.

example 1

100mg Tomatonin (A) are introduced into 10 ml of water, followed by adding 40 mg of sodium periodate and holds at 20 ⁰ C for 4 hours. Thereafter, 1 ml of the resulting solution is added with 1 ml of a solution of α-chymotrypsin (B) (10 mg / ml) in a 0.2M phosphate buffer of pH 7.5 and the mixture is kept at 4 ° C. for 12 hours to obtain an aqueous solution of the final product.

Example 2

3 mg of peroxidase (B) of the Krens are dissolved in 1 ml of water and treated with 0.5 mg of sodium periodate, after which the mixture is kept at 4 ° C. for 8 hours. The resulting solution is added with 3 mg of cholesterol oxidase (A) and 1 ml of 0.2M phosphate buffer of pH 7.5 and kept at 4 ° C for 12 hours to give a final solution of the product.

Example 3

10 mg of polyacrylic acid (C) are dissolved in 10 ml of acetate buffer with a pH of 4.8 and admixed with 15 mg of n-cyclohexyl-N '- (2-morpholinyl- (4-ethyl)) - carbodiimide-mp-toluenesulfonate. The mixture is kept at ⁰ ° C. for 1.5 hours. The resulting solution is admixed with 5 ml of buffer containing 5 mg of O-streptolysin (A) and 5 mg of peroxidase (B) of the krene. The solution is kept at 20 ° C for 2 hours to give the final product.

Example 4

100ng Agavoside G (A) are placed in 5 ml of water and with 100 mg of cyanogen bromide (C), keeping the pH at 11 by addition of 1M NaOH solution. The mixture is kept for 30 minutes at 4 ⁰ C, after which the pH word is lowered by the addition of phosphoric acid to 8.5. To 1 ml of the solution thus obtained is added 3 ml of a 0.5M phosphate buffer solution having a pH of 8.5 and containing 6 mg of alkaline phosphatase (B). The mixture is kept at 4 ° C for 12 hours, yielding a solution of the final product.

Example 5

500mg Nistalin (A) and 250 mg of ethylene-maleic anhydride copolymer (C) are dissolved in 5 ml of dimethylformamide and kept for 3 hours at 5O ⁰ C in an argon stream. The product obtained is precipitated with 20 ml of ether and dried in vacuo at 20 ° C. 1 ml of a solution of β-galactosidase (B) (2 mg / ml) in a 0.2M phosphate buffer with a pH of 7.5 is added 6 ml of the product obtained and kept boi 4 ° C for 12 hours. A solution of the final product is obtained.

Example β

50mg aescin (A) and 100 mg of N-vinylpyrrolidone Maloinsäureanhydrid copolymer (C) are dissolved in 1 ml DMSO and kept for 2 hours at 100 ⁰ C. The product obtained is precipitated with 3 ml of acetone and dried over P ₂ Oj for 4 hours at 100 ⁰ C in vacuo.

Thereafter, 1 ml of a solution of 1 mg / ml of a-chymotrypsin <Λ) is placed in 0.2 M phosphate buffer at a pH of 7.5 with 8 mg of the product obtained and kept at 6 ⁰ C for 15 hours. This gives a solution of the final product.

In the present example, the product obtained is characterized by a high molar content of detecting agent A. The step of preparing the detecting agent A and higher molecular weight crosslinking agent C in advance allows maximum preservation of the enzymatic activity of the visualizing agent B due to the gentle conditions of immobilization of the enzyme.

Use of the compounds for visual cholesterol detection on the skin of the patient:

The diagnostics to be carried out with the aid of the compounds according to the invention should not accurately measure the amount of cholesterol, but should make it possible to ascertain whether the patient is suffering from atherosclerosis, belongs to the risk group or is practically healthy. In order to be able to assign the patient to one of these three groups, the cholesterol content characteristic of each group must be determined in the horny layer of the epidermis of the skin.

For this purpose, three different concentrations of the affinity-enzymatic agent are prepared for each compound prepared according to the invention. These are then applied in the form of three points on the skin of the patient, preferably on the palm. The maximum concentration of the respective compound then leads to a discoloration of the applied point or spot in all persons, including healthy ones, who have the lowest cholesterol content in the skin. This is then used, as it were, as a control spot for the reaction to cholesterol. The solution with the minimum concentration of the compound according to the invention only leads to discoloration in atherosclerotic patients in the clinical stage, which have a maximum cholesterol content in the skin. The solution with an intermediate concentration of the compound according to the invention leads to a discoloration in individuals suffering from atherosclerosis as well as those belonging to the risk group (elevated cholesterol content), but not in healthy persons.

Three different concentrations of the compound obtained according to the invention are thus applied to the skin of the patient. If only one discolored spot is identified, the person is considered to be healthy; for two discolored spots, the patient belongs to the at-risk group and for three color spots of the atherosclerotic patient group, it is in the clinical stage.

To carry out the diagnosis, a skin area of a few square centimeters on any part of the body which is particularly preferred, but the inner surface of the hand, which is easily accessible and also has no sebaceous glands.

The compound prepared according to the invention is applied in three different concentrations to the skin surface in an amount of 10 to 20μΙ and thermostated for 1 minute. The excess of the compound which does not combine with cholesterol of the skin is washed away. For the visualization of the compound of the invention which has been linked to the cholesterol of the skin, the substrate of the corresponding enzyme which was used as the visualization agent B in the preparation of the compound, which in turn is used in the enzyme reaction with the preparation according to the invention, is applied to the same skin areas Compound forming the colored product (see TT Ngo and HM Lenhoff, "Enzymemediated Immunoassay" 1985, Plenum Press, New York, in the Russian translation of this book, p

A. Leninger "Biochemistry", Publisher "Mir", Moscow, 1976, p. 198).

Depending on the chosen substrate for the enzyme reaction, the colorless substrate solution turns red, yellow, dark blue, green, violet or otherwise, or the colored solution decolorizes.

If, for example, Krenperoxidase was used as the visualization agent B, as substrate of this Enzynvi any solution of the following solutions can be used immediately after preparation:

Solution 1: ABTS (2,2'-azino-bis (3-ethyl-benzthioazoline-6-sulfonic acid)) 1 mM, hydrogen peroxide 0.002% ^! n Phosphate nitrate buffer with pH = 4.3;

Solution 2: o-phenylenediamine4mM, hydrogen peroxide 0.004% in phosphate nitrate buffer pH = 5; Solution 3: 3,3 ', 5,5'-tetramethylenebenzidine 0.4mM, hydrogen peroxide 0.004% in acetate buffer pH 6.0. However, other peroxidase substrates, such as are widely used in immunoenzyme analysis, may also be used.

For example, when alkaline phosphatase was used as the visualization agent B in the preparation of the affine-enzymatic compound for visual cholesterol detection, as the substrate for this enzyme, a solution containing p-nitrophenol phosphate (2.5 mM) and magnesium chloride (0.5 mM ) in a diethylamine buffer (pH 9.5), or any other alkaline phosphatase substrate used in immunoenzyme analysis.

The results of the examination of 200 persons with verified diagnosis, which are summarized in the table, showed a high correlation between the diagnostic method according to the invention and the atherosclerotic damage of the vessels.

table

Family be Atherogenitäts- 10.0 ± 3.3 ischemia Hyper ischemia ischemia ischemia healthy caused hyper- index 3 spots of the heart tonie of the heart + the lower one of the heart + people lipemia Fiction at all hypertension extremities Hypertension + proper skin per person Ischemia of sample sonen lower extre mitäten number of patients 9 7 44 30 8th 66 30 F 5 2 13 12 - - 15 M 4 5 31 18 8th 66 15 By average age F-45M-40 F-47 B5M F-50M-52 F-54 M-52 48 54 F-24M-26 Plasma- cholesterol 421.4 ± 135.6 252.0 ± 54.6 238.4 ± 51.4 290.5 ± 78.6 218.6 ± 51.5 239.6 + 41.3 188.0 ± 34.0 5.4 ± 1.9 5.4 ± 1.7 7.6 ± 2.9 5.1 ± 1.8 5.7 ± 2.6 3.6 ± 1.3 3 spots 3 spots 3 spots 3 spots 3 spots 1 spot at 3 at 10 at 2 at 6 at all at all 2 spots 2 spots 2 spots 2 spots union union at 4 at 3 at 6 at 2 people people

F - women, M men

Claims (15)

1. A process for the preparation of affine-enzymatic compounds for the visual detection of cholesterol on the skin surface of a patient on the basis of a detection agent A, which is an affinant of cholesterol, and a visualization agent B, characterized in that A) the detection agent A, which is selected from the group of steroid glycosides which as aglycone the cyclopentanperhydrophenantrenfragment the furostanol or Contain spirostanol series and an oligosaccharide fragment with 3 to 10 unbranched or extended monosaccharides, or from the group of triterpene glycosides containing an aglycone of the α- or β-amyryl, lupane, hops, deaminan, linostane or holostane series and an oligosaccharide having 2 to 8 branched or unbranched radicals, or from the group of the hydrophobic ones Proteins which are able to selectively form a complex compound with the cholesterol, or from the upι uppe the proteinaceous toxins which are able to selectively form a complex compound with the cholesterol and are obtained from bacteria, marine microorganisms, insects or snakes, or from the group of polyene antibiotics which are able to selectively form a complex compound with the cholesterol, or from the group of enzymes which show a high affinity for cholesterol, by chemical methods in an aqueous medium at a molar ratio of reagent A: activator = 1: 1 to 1:10 and a concentration of the reagent A of 1 to 20 mg / ml in a Temperature is chemically activated from 0 to 25 ° C and a pH of 4 to 110.1 to 24 hours and the resulting solution with the visualization agent B selected from the group of the enzymes acetylcholinesterase, tyrosinase, glucoso-6-phosphate dehydrogenase, Glucosooxidase, glucosoamylase, galactosidase, peroxidase, alkaline or acid phosphates, α-chymotrypsin or pyrophosphatase, in M A: B = 20: 1 to 1: 1 and the resulting solution is kept at a temperature of 0 to 25 ⁰ C for 1 to 24 hours, or B) the visualization agent B selected from the group of the enzymes acetylcholinesterase, tyrosinase, glucoso-6-phosphate dehydrogenase, glucosooxidase, glucosoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, chymatrypsin or pyrophosphatase by chemical methods in an aqueous medium at a molar ratio of visualization agent B Activator = 1: 1 to 1:10 and a concentration of the visualizing agent B of 1 to 20 mg / ml at a temperature of 0 to 25 ⁰ C and a pH of 4 to 11 1 to 24 hours chemically activated and the solution obtained in accordance with the detection agent A, selected from the group of seroid glycosides containing as aglycone the cyclopentane perhydrophenantrenostrag furostanol or Spirostanolreihe and an oligosaccharide fragment with 3 to 10 unbranched or branched monosaccharides, or from the group ofTerrepenglykoside, the aglycone of the α- or ß-Amyryl, Lupan, Hopan, Dammaran, Linos tan or holostane series and an oligosaccharide of 2 to 8 branched or unbranched radicals, or from the group of hydrophobic proteins, which are able to selectively form a complex compound with the cholesterol, or from the group of proteinaceous toxins capable of selectively forming a complex compound with the cholesterol and being derived from bacteria, marine microorganisms, insects or snakes, or from the group of polyene antibiotics which are capable of selectively complexing with the cholesterol, or from US Pat Group of enzymes showing a high affinity for cholesterol, in the molar ratio A: B = 20: 1 to 1: 1 is added and the solution is kept at 0 to 25 ⁰ C1 for 24 hours, or C) by means of A, which is selected from the group of steroid glycosides which as Aglykon the Cyclopentanperhydrophenantrenfragment the furostanol or Spirostanolreihe and an oligosaccharide fragment with 3 to 10 unbranched or branched monosaccharides or from the group of triterpene glycosides containing an aglycone of the α- or β-amyryl, lupane, hops, deaminan, linostane or holostane series and an oligosaccharide of 2 to 8 branched or unbranched radicals, or from the group the hydrophobic proteins, which are able to selectively form a complex compound with the cholesterol, or from the group of proteinaceous toxins, which are able to selectively form a complex compound with the cholesterol and are obtained from bacteria, marine microorganisms, insects or snakes, or from the group polyene antibiotics which selectively form a complex with cholesterol, or from the group of high affinity enzymes which show a high affinity for cholesterol in an aqueous saline buffer solution having a pH of from 5 to 9 at a concentration of A equal to 1 to 20 mg / ml is dissolved and the solution with a low molecular weight asymmetric bifunctional crosslinking C medium selected from the group of cyanogen bromide, trichlorotriazm or 2-amino-4,6-dichloro-S-triazine in the molar ratio A: C = 1: 0.5 to 1:10 added and oia solution for 1 to 20 hours at a temperature of 0 to 2O ⁰ C, after which the visualization agent B, selected from the group of the enzymes acetylcholinesterase.Tyrosinase.Glukosa ephosphat dehydrogenase, glucosooxidase, glucosoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, a-chymotrypsin or pyrophosphatase, in the molar ratio A B = 20: 1 to 1: 1 added and the solution is kept for 1 to 48 hours at a temperature of 0 to 2O ⁰ C,
or D) one the visualization agent B selected from the group of the enzymes acetylcholinesterase.Tyrosinase.Glukoso-e-phosphate dehydrogenase, glucosooxidase, glucosoamylase, Ga'aktosidase, peroxidase, alkaline or acid phosphatase, a-chymotrypsin or pyrophosphatase in an aqueous salt buffer solution pH of 5 to 9 and at a visualization agent B concentration of 1 to 20 mg / ml, the low molecular weight asymmetric bifunctional crosslinking agent C selected from the group of cyanogen bromide, trichlorotriazine or 2-amino-4,6-dichloro-S-triazine in the molar ratio B: C = 1: 0.5 to 1:10, and holding the solution for 1 to 20 hours at a temperature of 0 to 20 ⁰ C and then with the corresponding detection agent A, which is selected from the group of Steroid glycosides containing as aglycone the cyclopentane perhydrophenantrenfragment of the furostanol or Spirostanolreihe and a Oligosaccharidragragment with 3 to 10 unbranched or branched monosaccharides e or from the group of titer sterol glycosides containing an aglycone of the α- or β-amyryl, lupane, hops, deaminan, linostane or holostane series and an oligosaccharide having 2 to 8 branched or unbranched radicals, or from the group the hydrophobic proteins, which are able to selectively form a complex compound with the cholesterol, or from the group of proteinaceous toxins, which are able to selectively form a complex compound with the cholesterol and are obtained from bacteria, marine microorganisms, insects or snakes, or from the group the polyene antibiotics, which are able to selectively form a complex compound with the cholesterol, or from the group of enzymes which show a high affinity for cholesterol, in the molar ratio A: B = 20: 1 to 1: 1 and the solution 1 to 48 hours at a temperature of 0 to 2O ⁰ C holds, or E) the detection agent A which is selected from the group of steroid glycosides containing as aglycone the cyclopentane perhydrophenantraene fragment of the furostanol or spirostanol series and an oligosaccharide fragment with 3 to 10 unbranched or branched monosaccharides, or from the group of triterpene glycosides which have an aglycone of the α- or ß-amyryl, lupane, Hopan, Dammaran, Linostan or holostane series and an oligosaccharide from 2 to 8 branched or unbranched radicals, or from the group of hydrophobic proteins, which are able to selectively form a complex compound with the cholesterol or from the group of proteinaceous toxins which are capable of selective complex formation with the cholesterol and are obtained from bacteria, marine microorganisms, insects or snakes, or from the group of polyene antibiotics which are capable of selectively forming a complex compound with the cholesterol, or from the group of high-affinity enzymes, which show a high affinity for cholesterol, or a mixture of said substances with the visualization agent B, selected from the group of enzymes acetylcholinesterase, tyrosinase, glucoso-6-phosphate dehydrogenase, glucose oxidase, glucosoamylase, galactosidase, peroxidase, alkaline or acid phosphites or A-chymotrypsin in the molar ratio A: B = 20: 1 to 1: 1 in an aqueous salt buffer solution having a pH of 5 to 9 at a concentration of A and B of 0.1 to 20 mg / ml dissolves, the resulting solution with the higher molecular weight polyfunctional crosslinking agent C, which contain polysaccharides, proteins or synthetic polymers containing any functional group selected from the group of primary amine, carboxyl, hydroxyl, aldehyde, hydrogen halide, mixed anhydride, azide, hydride, maleimide, isocyanate or epoxide , in the molar ratio (A + B): C = 1: 0.5 to 1:10 and the resulting solution for 1 to 20 hours at a Te temperature of 0 to 2O ⁰ C holds, or F) the detection agent A, which is selected from the group of Steroidglykoside containing as Aglykon the Cyclopentanperhydrophenantrenment of the furostanol or Spirostanolreihe and an oligosaccharide fragment with 3 to 10 unbranched or branched monosaccharides, or triterpene glycosides, the aglycone de; α- or ß-amyryl, lupane, Hopan, Dammaran-, Linostan or Halostanreihe and an oligosaccharide from 2 to 8 branched or unbranched radicals, or polyene antibiotics, which are able to selectively form a complex compound with the cholesterol, or mixtures previously dissolving these substances in an aprotic polar solvent, which comprises the higher molecular weight polyfunctional crosslinking agent C selected from the group consisting of polysaccharides, proteins or synthetic polymers containing any functional group selected from the group of primary amine, carboxyl, hydroxyl, aldehyde, hydrohalide, mixed anhydride , Iminoether, azide, hydrazide, maleimide, isocyanate and epoxide, at a concentration of agents A and C of 1 to 20 mg / ml in the ratio A: C = 1: 1 to 1: 10 added, the solution 0.5 to 6 Hours at a temperature of 20 to 120 ° C, then in a conventional manner, the product obtained (AC) precipitates out of the solvent, the obtained P roduct 4 to 10 hours at a temperature of 80 to 120 ⁰ C in vacuo over P ₂ O ₆ holds, an aqueous salt buffer solution having a pH of 5 to 9, in which the visualization agent B, selected from the group of the enzymes, acetylcholinesterase , Tyrosinase, glucoso-6-phos-glucosoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, -Chyi notrypsin or pyrophosphatase, dissolved in a concentration of 1 to 10 mg / ml .st, with the obtained dry product (AC) in one Concentration of 2 to 20 mg / ml and the solution for 2 to 12 hours at a temperature of 4 to 8 ° C holds. 2. The method according to claim 1 E), characterized in that one uses as a higher molecular weight crosslinking agent C copolymers of acrylic acid or maleic anhydride. 3. The method according to claim 2, characterized in that is used as a relatively high molecular weight crosslinking agent C, the N-vinylpirrolidone-maleic anhydride copolymer ve. 4. The method according to any one of claims 1 to 3, characterized in that are used as steroid glycosides, which are used as the detection means A agavoside A, agavoside B, agavoside C, agavoside D, agavoside F, agavoside G, agavoside H, imillin, funcoside C, Funcoside D, Funcoside F, Funcoside G, Funcoside I, Dioscine, Gracillin, Protodioscin, Cicubasaponin, Juncoside E, Juncoside H, Lanatigonin, Desglukodigitonin, Digiton, Gitonin, Rocoside C, Rocoside D, Rocoside E, Funcoside E, Alliumoside C , Polygonatonin, tigogenin tetraoside, tigogeninhexaoside, capsicoside, ammumoside B, ammumoside C, ammumoside D, ammumoside E, desglucodesramnoparillin, desglucoparillin, parillin, sarsararilloside, asparagoside C, asparagoside D, asparagoside G, asparagoside H, protoyuccoside H, yuccoside B, lanatigoside, monoside , Bioside, Trioside, Purpureagitosid, Gekogenin, Rocogenin, Diogenin, Tigogenin, Protopolygametoside, Tomatonin, Laxogengencetetraoside, Alliogeninlycotetraoside, Karoteoside A, Cyclosieversioside H, Acantofiloside C, Alliofuso sid A, alliospiroside A, cyclosieversioside F, theasaponin, acantofiloside B, tigonin (total), glycoside of the plant Calha polypetala or cyclosieversioside G. 5. The method according to claim 4, characterized in that one uses as the detection means A, the cos D, E, F, G or I, dicosine, rocoside, Lanatigonin, digitonin or Tomatonin. 6. The method according to claim 4, characterized in that one uses as detection means A digitonin or Tomatonin. 7. The method according to any one of claims 1 to 3, characterized in that as a triterpene glycosides for use as detection agents Aescin, avenacin, theasaponin, hederin, Caulosid C, Stichionoside A, cyclamine, quinovine, saponins of sugar beet, hypsoside or Triterpenglykoside used the plant Fatsia japonica. 8. The method according to any one of claims 1 to 3, characterized in that as hydrophobic proteins for use as detection means A the Mellinprotein Floch, the proteins of lysosomes and mitochondrion, fibrinogen, immunoglobulins, cerebron, myoglobin, trypsin, cytochrome C, Cytochrome P-450 and the apoei whites of the lipoproteins used. 9. The method according to any one of claims 1 to 3, characterized in that as proteinaceous toxins for .'ürwendung as the detection means A Streptolysin o, pneumolysin, listeriolysin, O-toxin C. l "won from Perfrigens type A and C, -Toxin CI novyj Type AoderC, hemolvsin CL histolyticum, hemolysin CI botulinum type C and D, tetanolysin, cerebrolysin, alveolysin, t'üringeolysin or cytotoxin from the actinia Metridium senile vwwendet. 10. The method according to any one of claims 1 to 3, characterized in that one uses as polyene antibiotics for use as detection agent A amphotericin B, philipine or nistatin. 11. The method according to any one of claims 1 to 3, characterized in that one uses as high-affinity enzymes for use as detection means A cholesterol oxidases, cholesterol dehydrogenases or cholesterol esterases. 12. The method according to any one of claims 1 to 11, characterized in that one uses for the chemical activation of the detection means A and the visualization agent B, the known carbodiimide, succinimide or azide process or the process of periodate oxidation. 13. The method according to claim 12, characterized in that one uses for the chemical activation of the detection means A and the visualization agent B, the carbodimide process or the process of periodate oxidation. 14. The method according to claim 1 F), characterized in that is used as the aprotic polar solvent dimethylsulfoxide, dimethylformamide, hexamethanol, a mixture of dimethylformamide and toluene in the ratio 2: 1 or a mixture of dimethylformamide and hexanol in the ratio 2: 1. 15. The method according to any one of claims 1 C) to 1 F) and 14, characterized in that one uses as an aqueous salt solution solutions selected from the borate, citrate, or phosphate series, which has a buffer effect in the pH range of 5 to 9 guarantee.