CS274496B2 - Method of affinitive-enzymatic compounds preparation for cholesterol's visul indication - Google Patents

Description

The invention relates to the field of bioorganic chemistry and to a process for the production of affinity enzymatic compounds for the visual indication of cholesterol on the skin surface of a patient based on an indicating agent that is affinity 9 and a visual indicating agent.

Most importantly, the compounds of the invention may be used in diagnostics, including early detection of atherosclerosis, refining diagnosis under clinical, outpatient and home conditions, as well as controlling the course of treatment. The compounds may also be used in physiological, histological and histochemical investigations to determine elevated cholesterol content and localization. In addition, these compounds also allow the determination of the reduced cholesterol content in patients 9e detected by the disease, in particular of an oncological nature.

Atherosclerosis and its complications such as heart attacks, insults and gangrene are the main cause of population mortality in all countries of the world.

Many years of research have led to the finding that atherosclerosis is one of the diseases that need to be identified and combated, the main risk factor for atherosclerosis and the accumulation of cholesterol in the body. Prophylaxis of atherosclerosis in the population consists of being identified among the patients who are the most at risk group, who are individually treated, and affected patients are given their way of life. Determining groups of patients at increased risk of atherosclerosis based on the amount of cholesterol accumulated in the body is the most difficult problem in the prophylaxis of atherosclerosis. Known methods for diagnosing atherosclerosis are based on quantitative determination of total cholesterol in venous blood plasma (see Consensus Conference on Lowering Blood Cholesterol's Prevent Heart Desease,

JAMA, 1985, 253, pp. 2080-2086; The Lipid Research Clinics Publication 80-152, Bethesda, Ma., National Institutes of Health, 1980, Vol. 1;

Lipid Research Clinics Programm, JAMA, 1984, 251, pp. 351-364). Cholesterol levels above 260 mg are considered sufficient for patients to be counted at risk. A more detailed diagnosis is made on the basis of blood plasma lipoprotein analysis and the atherogenicity index, which is formulated as the ratio of the difference between total cholesterol ( ^c H _ce ) and high density lipoprotein (Ch 2) cholesterol to high density lipoprotein cholesterol:

^Ch cel ^Ch lvh

I ather.

^Ch lvh

With an atherogenicity index above 3, the patient is considered a member of the risk group and at an index above 5.6 to atheroslarotic patients (see Klimov AN, Phenotipirovanie lipoproteinov, Metodičeskie rekomendacii MZ USSR, M., Medicina, 1975; Goldfourt V., Holtzmann E. , Neufeld HN, Total and High Density Lipoprotein Cholesterol in the Serum and Risk of Mortality, British Medical Journal, 1985, 290, pp. 1239-1243).

The use of these methods requires blood collection, which is traumatic for patients and, moreover, is not entirely safe due to the possibility of transmission of various viral diseases. Plasma lipoprotein fractionation and cholesterol analysis are an expensive and complicated procedure. In addition, in one of the three cases, the total cholesterol content detected and even the results of total phenotyping are not correlated with the atherosclerosis difficulties (see Mjaenikov A.L., Giperton, Bohemia Bolez, 1965, M., Medicina, p. 300).

In addition, experiments conducted in recent years have shown that blood plasma in its entirety cannot fully depict the processes of cholesterol accumulation characteristic of arterial walls and other bradytrophic tissues.

Further improvement of diagnostic methods resulted from the solution of individual issues of pathogenesis of atherosclerosis. It should be emphasized that tissue cholesterol plays a crucial role in the development of atherosclerosis. Tissues that accumulate cholesterol could be detected,

CS 274 496 B2

Like the walls of artsria.

Research in recent years has shown that there is a close relationship between the cholesterol content of the artery walls and the skin. This finding allows the discovery of fundamentally new diagnostic methods, particularly for atherosclerosis.

This relationship between cholesterol content in arterial walls and skin was established by direct determination of cholesterol in skin biopsies. Skin samples were frozen in liquid nitrogen and lyophilized, then cholesterol was extracted with Folch's reagent and determined by conventional chemical or biochemical methods (see Nikitin Ou.P., Gordienko IA, Dolgov AV, Filimonova TI, Sodium Choleeterin in the skin and ego correljacia with lipidny by a syvorotic shrub in both human and painful health care serias, Cardiology, 1S87, XXVII, No. 10, pp. 48-51; Bouisson H., De Graeve, Solera ML et al Ann.Biol.Clin., 1982, 40, pp. 361-407).

However, due to its traumatic nature and complexity, this method is unsuitable for serial examinations.

US-PS 4458686 discloses a method for determining glucose and glucose. of ethanol in the blood directly under the skin and on its surface, the possibility of determining cholesterol using cholesterol oxidase is also reported. The methods for determining the amount of these compounds are based on stoichiometric changes in oxygen concentration using a specific redoxferment for the substrate to be analyzed, preferably oxidoreductase. Quantitative measurements of changes in oxygen concentration are carried out electrochemically, for example polarographically, by means of special devices and electrodes which have been specially developed for these processes. These complicated instruments require a highly qualified personnel for diagnosis. This leads to a limitation of the applicability of these methods in serial examinations.

PCT / US 84/00888 discloses a complex of a detection means and a visual-indicating means in which these components are combined directly or by means of an enhancement means. This complex serves to determine small amounts of specific molecules such as lipids in biological tissues. This lipid assay procedure, however, can only be used under laboratory conditions and requires biological fluid or tissue to be removed from patients first, which means that the method is traumatic, multi-stage and complicated in its performance.

Data for the correlation between the cholesterol content of the skin and the degree of atherosclerotic vascular damage are obtained on skin biopsies. This method is not only traumatic, but also has a number of other disadvantages, since 1 mm thick biopsies contain different layers of skin, namely a horn layer (average thickness of 0.1 mm) and connective tissue, i.e. the skin itself (darma), which again contains two layers, namely Stratum papillare and Stratum reticulare. Both of these layers are well supplied with blood, therefore the respective biopsies also contain blood vessels and blood, as well as sweat and sebaceous glands as well as their secretions. Directly beneath the skin is tissue of subcutaneous fat, which may sometimes belong to a biopsy. Thus, the inhomogeneity of the biopsy may distort the results of the skin cholesterol accumulation assay. In this regard, the method that allows the determination of the cholesterol content of the skin surface in the skin horn layer is most accurate.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for the production of affinity enzymatic compounds for the visual indication of cholesterol immediately on the skin of a patient, particularly in the corneal layer of the skin, without the need for blood and skin biopsy removal. Any skin area can be used for diagnosis. The most suitable is the surface of the hand, which does not contain any sebaceous glands, whose secretions as well as the corneal layer of the skin contain cholesterol, which may affect the results of diagnosis.

According to the invention, this object has been achieved by a process for the production of affinity enzyme compounds which are bifunctional compounds. These selectively bind to the free cholesterol of the patient's skin and can then be monitored visually. For the preparation of such a compound

CS 274 496 B2 requires at least two components, namely diagnostic agent A, selected from the group of bifunctional substances which they produce when applied to the skin! A free complex cholesterol of the skin, a solid complex, and a composition allowing visual indication of B that allows visual detection of cholesterol-bound bifunctional compounds.

The compounds obtainable according to the invention lead to a maximum simplification of the diagnosis of atherosclerosis, are not traumatic and also do not require any special equipment. The use of these compounds for the diagnosis of atherosclerosis allows the subjects to be divided into three groups: atherosclerosis patients, at-risk groups, and healthy subjects.

Affine enzymatic compounds for the visual indication of cholesterol and their use possible diagnostic methods (three-point methods) They are also suitable for serial diagnostics. The method is very simple and does not require any special qualification by medical personnel; it can also be carried out at home.

The compounds of the invention can also be prepared from diagnostic composition A and visualization B by chemical activation of the functional groups of one of the two compositions. In this case, upon addition of one reagent to another containing activated chemical groups at defined relationships between the amounts of reagents A and B, a solid chemical bond is formed, thereby forming high molecular weight bifunctional compounds for visual indication on the skin of the patient.

□ the following compounds are suitable for detecting A-affinity cholesterol:

steroid glycosides containing, as an aglycone, a cyclopentanoperhydrophenethrenic fragment of the furostanol or spirostanol type and an oligosaccharide residue containing from 3 to 10 monosaccharides of linear or branched structure (see PKKintja, Strojenie i biologic9kaja activity of steroid glicosides rjada spirostana i furostana, 1987; 142 / or triterpenglycosides containing aglycone σς- or (5-amyryl-, lupan-, hopan-, dammaran-, linostane- or holostane series) and an oligosaccharide of 2 to 8 branched or unbranched residues (see GEDekanosidze, V.Oa. Church, TV Sargienko,

N.I. Uvarova, Issledovanie triterpenových glikosidov (Tbilisi, Mecniereba, 1982) or hydrophobic proteins that can form a cholesterol-selective complex compound (see lANKlimov, GVTitova, KAKoževnikova, Biochimija, 1982, ev. 47, no. 2, p. 226) - 232,

2. A.N.Klimov, K.A.Kozevnikova, N.N.Klujeva et al. Voprosy med.chimii, 1984, Vol. 30, No. 3, pp. 86-90;

3. G. V. Titova, N. N. Klujeva, K. A. Kozevnikova et al., Biochimija, 1980, vol. 45, No 1, p.

- 55) or protein toxins capable of forming complex compounds 9 by cholesterol, derived from bacteria, marine micro-organisms, insects or reptiles (see V V. Dálin, NGFiš, Belkovye toksiny mikrobov, Moscow, ed. Medicine, 1980) or polyene antibiotics that are capable of forming a selectively complex compound with cholesterol (see 1. PP.Katzenstein AMSpiegelvogel, AWNorman, O. Antibiot. 27, 12, 1974, pp. 943-951; Gong Shang-Shyng, Wang) Hsi-Hua, Clin., Microbiol., 1976,9, (1-2), pp. 19-30; 3. Readingphonephine O. et al., Biochim.Biophis.Acta, 1982, 685 ( 2), 9 (219-224) or high affinity enzymes whose substrate is cholesterol and which show high affinity).

The following enzymes are suitable as visualizing agent B: acstylcholinesterase, tyrosinase, glucose-6-f9fate dehydrogenase, glucooxidase, glucoamylase, galactosidase, psroxidase, alkaline or acid phosphatases, α-chymotrypsin or pyrophosphatase.

The object of the invention is to provide a cholesterol A-affinity detecting agent selected from the group of stsroid glycosides containing, as an aglycone, a cyclopsntanopsrhydrophenanthronine residue of the furostanol or spirostanol series and an oligosaccharide residue containing from 3 to 10 monosaccharides of straight or branched containing aglycone - nsbo / 3-amyryl, nsbo lupannsbo hopan- or dammaran nsbo linostan- nbo holostane series and oligosaccharide in 2 to 8

CS 274 496 B2 branched or unbranched residues, or from a group of hydrophobic proteins which can form a complex compound with cholesterol, or from a group of protein toxins derived from bacteria, marine microorganisms, insects or reptiles that can form a complex compound with cholesterol, or groups of polyene antibiotics, which can form a selectively complex compound with cholesterol, or from a group of enzymes that show high affinity for cholesterol, activate by detection by means of chemical methods in aqueous medium in molar ratio detection agent A: activator = 1: 1 - 1:10 at detection concentration reagent A 1 to 20 mg / ml at 0 to 25 ° C, pH 4 to 11 for 0.1 to 24 hours, and visualization agent B, selected from the group of acetylch ^r ' ¹ nesterase, is added to the prepared solution, tyrosinase, glucose-6-phosphate dehydrogenase, glucooxidase, glucoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, -chymotrypsin or pyrophosphatase in an A: B molar ratio of 20: 1 to 1: 1 and the solution is maintained at a temperature of 0 to 25 ° C for 1 to 24 hours until the final product is obtained.

Oak is mentioned earlier, the functional groups of the visualizing agent B may also be subjected to the pre-chemical activation. In this case, the chemical activation of the visualizing agent B is carried out in an aqueous medium at a molar ratio of visualizing agent B: activator = »1: 1 - 1:10 at of visualization reagent B 1 to 20 mg / ml at 0 to 25 ° C, at pH 4 to 11, for 0.1 to 24 hours and then the detection solution A, which is one of the former substances, is added to the solution obtained, at a molar ratio of A: B = 20: 1 - 1: 1 and the solution is incubated at 0 to 25 ° C for 1 to 24 hours until the final product is obtained.

According to the invention, the chemical activation of detection means A or visualization means B is carried out by methods known per se by azide, carbodiimide succinimide or by periodate oxidation.

In those cases where a high molecular weight enzyme is used as the visualization agent B and low molecular weight compounds, such as glycosides, as detection reagent A, there may be no more than one visualization agent B molecule in the final product molecule. binding with the skin cholesterol in the final product, may exceed the content of visualizing agent B only a few times, consistent with the number of functional groups in the ferment molecule capable of coupling with detection agent A without any loss of enzymatic activity. In such compounds, the content of visualizing agent B such as an enzyme is limited. As a result, the sensitivity of the compounds obtained is relatively low and the need to increase the exposure time at diagnosis.

The steroid glycosides used as detection agent A and having in their composition as aglycone a cyclopentane perhydrophenanthrene residue of the furostanol or spirostanol series and an oligosaccharide fragment containing 3 to 10 monosaccharides of direct or branched structure include in particular: agavosld A, agavoside B, agavoside C, agavoside F, agavoside H, agavoside G, trillin, funcoside C, funcoside D, funcoside F, funcoside G, funcoside G, funcoside I, dioscin, gracillin, protodioscin, cicubasaponin, Juncoside E, juncoside H, lanatigonin, desglucodongin, , rocoside O, rocoside E, funcoside E, alliumoside C, polygonatonin, tigogenintetraoside, tigogeninhexaoside, capsicoside, ammumoside B, ammumoside C, ammumoside D, desglucodesramnoparillin, desparlucidide as, parillide, asparillide, paraffinide, asparagoside H, protoyuccoside H, yuccoside B, lanatigoside, monoside, bioside, trioside, purpureagitoside, gecogenin, rocogen in, diogenin, tigogenin, protopolygametoside, tomatonin, laxogeninlycotetraoside, alliogeninlycotetraoeid, caroteoside A, cyclosievorsioside C, alliofusoside A, allio3piroside A, cyclosieversloside F, thiososonosinide F, theasososonoside F, theasosonosinide F, theasosonosinide F

CS 274 496 B2

Of these steroid glycosides, funcosides C, D, E, F, G or I, dioscin, roco9id, lanatigonin, digitonin and tomatonin are particularly preferred. Most preferred are digitonin and tomatonin.

Preferred triterpenglycosides used as Detection Agent A, which contain an oC- or amyryl, lupan, hopan, dammaran, lanostane or holostane series aglycone and an oligoeacharide having 2 to 8 branched or unbranched residues. They are aescin, avenacin, theasaponin, - hederin, cauloside C, stichinoside A, cyclamin, quinine eaponins of sugar beet, hypsoside and triterpenglycosides obtained from Fatsia japonica.

Preferred hydrophobic proteins that are used as Detection Agent A and are capable of selectively forming a complex compound with cholesterol are Folch meilin proteins, lysosome and mitochondria proteins, fibrinogen, immunoglobulins, cerebron, myoglobin, cytochrome C, cytochrome lipoprotein, or apobiloteins .

Of the protein toxins useful as Detection Agent A, capable of selectively forming a complex compound with cholesterol and derived from bacteria, marine microorganisms, insects and reptiles, streptolysin, pneumolysin, listeriolines, Θ-toxin CI, derived from Perfrigens type A, are preferred. C, α-toxin CI new types A and C, haemolysin C.I. histolyticum, haemolysin C.I, botulinum type C and D, tetanolysin, cerebrolysin, alveolysin, turingeolysin or cytotoxin from actinia Metridium senile.

Preferred polyenantibiotics used as detection means A which can form a complex compound with choleterol are amphotericin B, philipin or nistatin.

Preferred high affinity enzymes having high affinity for cholesterol and used as detection means A are cholesterol oxidases, cholesterol hydrogenases or cholesterol esterases.

Of the aforementioned cholesterolfinic detection agents A such as glycosides, hydrophobic proteins, protein toxins, polyene antibiotics and high affinity enzymes, the most preferred glycosides are chemically stable and resistant to both organic solvents and high synthesis temperatures without losing their ability forming a complex of 9 cholesterol skin. The low molecular weight of the glycosides makes it possible to obtain a composition with a high molar content of the detection agent, which provides a high number of points for the reaction of the compositions with the skin cholesterol.

The most preferred of the glycosides are steroid glycosides, which most efficiently produce triterpenic glycosides! complex with cholesterol.

Hydrophobic proteins, bikinic toxins, polyene antibiotics, and high affinity enzymes are also successfully used for the preparation of a diagnostic reagent, but the method for preparing the affine enzymatic compound is somewhat limited due to their low chemical stability and the possibility of their inactivation. In addition, the high molecular weight of these detecting agents slightly decreases the sensitivity of the final compound, which increases the concentration of the composition and the exposure time to indicate skin cholesterol.

Of course, attempts are made to perform reactions under mild conditions, i.e., at neutral pH, low ionic strength, low temperatures, and shortened reaction time.

As visualizing agent B, enzymes are used which react with Detection A to provide an affine enzymatic product for visual indication of cholesterol on the skin of a patient selected from the group consisting of: acetylcholinesterase, tyrosinases, glucose-6-phosphate dehydrogenases, glucooxidase, glucosoamylase, galactosidase , peroxidase, alkaline or acidic phosphatases, X-chymotrypsin or pyrophosphate.

All of these enzymes can be used with any of the above detection reagent A, but steroid glycosides are preferred,

Most preferred are compounds in which atheroid glycosides having the cyclopentanoperhydrophenatrenic residue of furostanol nobo splrostanol series and an oligosaccharide fragment ranging from 3 to 10 unbranched or branched monosacids are used as the affinity-detecting agent A after CS 274 496 B2.

Such steroid glycosides exhibit high chemical stability and can be mobilized to the crosslinking polymer under harsh conditions, i.e. at high temperatures and, if necessary, by dissolution in organic solvents, ensuring their high content in the final product.

The molar ratios of the components involved in the reaction are selected according to the molecular weights of the selected reagents A or 8, the degree of affinity for skin cholesterol and the activity of the selected enzyme.

The invention is further elucidated by means of exemplary embodiments and figures which show: Fig. 1 - the marks used Fig. 2 - obtained compounds and their interactions with cholesterol 9 on the skin surface of a patient.

Example 1

100 mg of tomatonin (A) are mixed with 9 10 ml of water, 40 mg of sodium periodate are added and kept at 20 ° C for 4 hours. To 1 ml of the obtained solution of ss, add 1 ml of a 10 mg / ml solution of γΛ-chymotrypsin (8) in 0.2 M phosphate buffer Q pH 7.5 and maintain the mixture at 4 ° C for 12 hours. An aqueous solution of the final product is thus obtained.

Example 2 mg of horseradish peroxidase (B) is dissolved. in 1 ml of water, 0.5 mg of sodium periodate is added and the mixture is kept at 4 ° C for 8 hours. 3 mg of cholinesteroxidase (A) in 1 ml of 0.2 M phosphate buffer pH 7.5 was added to the obtained solution and the mixture was kept at 4 ° C for 12 hours. A solution of the final product is obtained.

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

The diagnosis using the compounds obtained according to the invention is not based on an accurate qualitative determination of cholesterol, but on the possibility of classifying the patient in one of three groups: atherosclerosis patients, at risk groups and practically healthy people. In order to classify a patient into one of these three groups, it is necessary to evaluate the cholesterol content of the horn layer of the epidermis of the skin that is characteristic of each of these groups.

Therefore, three concentrations should be selected for each compound prepared according to the invention, which are further used. These three different concentrations of the affinity-enzymatic composition are applied in the form of three points to the skin of the patient, preferably the palm. The maximum concentration of compound used ensures staining of the spot or spot in all humans, including healthy ones, with a minimum cholesterol content in the skin. This stain is used as a control stain for the reaction with cholesterol. A solution with a minimum concentration of the compound prepared according to the invention provides a coloration only in patients who are atherosclerosis at a clinical stage and who have the highest cholesterol content in the skin. A solution containing a moderate concentration of a compound of the invention provides for color development in both sick people and those belonging to the risk group (increased cholesterol content), but does not provide coloration in healthy people.

Thus, three different concentrations of the compound obtained according to the invention are applied to the patient's skin. In the presence of a single stain, the patient belongs to a group of healthy people, in the presence of two stained stains to the risk group and in the presence of three stained stains to patients with atherosclerosis in the clinical stage.

A few square centimeters of skin selected on the skin is sufficient to perform the diagnosis

CS 274 496 B2 any part of the body. Oako's most advantageous appearance is the inner side of the hand, which is easily accessible and, moreover, does not contain fat glands.

The compound obtained according to the invention is applied to the skin surface in three different concentrations in an amount of 10 to 20 (µl) and incubated for 1 minute. Excess compound that did not react with skin cholesterol was washed away. To visualize the skin cholesterol-reacted compound of the present invention, a substrate of the corresponding enzyme used as visualizing agent B is deposited on these parts of the surface in the preparation of said compound which reacts to produce a colored product resulting from the enzymatic reaction with the compound prepared according to the invention. (T.T.Ngo and H.M.Lenhoff, Enzyme-mediated Immunoassay, 1985, Plenum Press, New York, in the Russian translation of this book p. 11) and A.Leninger "Biochemistry, publ. Mir, Moscow 1976, p 198.

Depending on the substrate selected for the enzymatic reaction, the colorless substrate solution is colored red, yellow, blue, green, violet or other color solution, or the color solution is discolored.

For example, if horseradish peroxidase is used as visualizing agent B, then any of the following solutions can be used as substrate of this enzyme immediately after preparation:

solution 1: ABTS (2,2'-azinobis-) 3-ethyl-benzthiazoline-6-sulfonic acid)) 1 mM, 0.002% hydrogen peroxide in phosphate citrate buffer pH pH 4.3, solution 2: o-phenylenediamine 4 mM, 0.004% hydrogen peroxide in phosphate citrate buffer pH b 5, solution 3: 3.3 ', 5.5 ^from tetramethylenebenzidine 0.4 mM, 0.004% hydrogen peroxide in acetate buffer pH 6.0.

However, other peroxidase substrates used in a wide range of immunoenzymatic assays that are useful in the present invention are also known.

If alkaline phosphatose is used as the visualizing agent B in the production of the affinity-enzymatic compound for the visual detection of cholesterol, a solution containing p-nitrophenol phosphate (2.5 mM) and magnesium chloride (0.5 mM) in sodium chloride may be used as the substrate for this enzyme. diethylamine buffer (pH 9.5), or any other alkaline phosphatase substrate used in immunoenzymatic analysis.

Research results show a high correlation of vascular damage.

200 people with verified diagnosis, which are listed in the table, into the environment.women diagnostic methods, with atherosclerotic grade

familyly iechemic- ischemia coronary heart disease healthy conditional ischemic ké 9rdeč- lower ba + hypertonio + persons hyperlipi- cardiac hyper- ni choro- limbs ischemic cho · demie disease tonie ba + hyper- roba bottom tonie limbs number patients 9 7 44 30 8 66 30 M 5 2 13 12 - - 15 Dec 2 4 5 31 18 8 66 15 Dec average age 2-45 M-40 2-55 M-47 2-50 M-52 2-54 M-52 48 54 2-24 M-26 plasma choleste- rol 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 + 34.1 index atheroge- 10.0 + 3.3 5.4 + 1.9 5.4 + 1.7 7.6 + 2.9 5.1 + 1 ., 8 5.7 + 2.6 3.6 + 1.3

CS 274 496 B2

Table - continued family-related hyperlipidemia ischemic heart disease hypertonia ischemic heart disease + hypertonia ischemia of lower extremities ischemic healthy heart disease ba + hypertonia + ischemic disease of lower extremities

exam 3 spots 3 spots 3 spots 3 spots 3 spots 3 spots 1 stain on the skin according to u all u 3 at 10 at 24 at 6 u all u all invention persons 2 spots 2 spots 2 spots 2 spots oeob persons

u3 u3 u6 u2

W - women, M - men

Claims (2)

A method for the preparation of affinity enzymatic compounds for the visual indication of cholesterol on the skin surface of a patient based on a detection reagent and a visualizing agent, characterized in that affinity detection reagent A selected from the group consisting of steroid glycoeids having or a spirostanol series and an oligosaccharide fragment containing 3 to 10 monosaccharides of a straight or branched series, including agavoside A, agavoside 8, agavoside D, agavoside F, agavoside H, agavoside G, trillin, funcoaid C, funcoeid , funcoside G, funcoside 1, dioscin, gracillin, protodioscin, cicubasaponin, juncoeid E, juncoside H, lanatigonin, deaglucodigitonin, digitonin, gitonin, rocoside C, rocoside D, rocoside E, funcoside E, alonumoside t, capsicoside, ammunoeid B, ammunoside C, ammunoside D, ammunoside E, dssglukodssramnoparillin, desglucoparillin, parillin, sarsaparilloside, aeparagoside C, asparagoside G, asparogoside H, protoyuoccoside H, yuoccoside B, lanatigoside, monoside, bioside, trioside, purpureagitoside, gecogenin, rocogenin, diogeninetopetide, togen cyclosieversioside H, acantophiloside C, alliofusoside A, alliospiroside A, cyclosieversioeid F, theasaponin, acantophiloside B, tigonin (total), the glycoside of Calha polypetala or cyclosieversioside G, or from the group of trlterpenglycosides containing n-glycone / glycone / , hopane, damaran, lanostane or holostane series and oligosaccharide with 2 to 8 branched or unbranched residues, comprising the following set of aescin, avenacin, theasaponin, - hederin, cauloside C, stichino3id A, cyclamin, quinovin, saponins sugar beet sugar beet sugar obtained from the plant Fgtsia japonica, Detection Reagent A selected from the group of hydrophobic proteins, which are meilin proteins according to Folch, lysosome proteins and mitochondria, fibrinogen, lmmunoglobulins, cerebron, myoglobin, cytochrome C, cytochrome P-450 or lipoprotein apoproteins, from the group of protein toxins capable of forming a cholesterol complex compound obtained from bacteria, marine microorganisms, insects and snakes, which includes streptolysin o-, pneumolysin, listeriolysin, Φ-toxin CI obtained from Perfringons type A and C, toxin C.I. new types A and C, hasmolysin C.I.histolyticum, haemolysin C.I.botulinum type C and O, tetanolysin, cerebrolysin, alveolysin, turingsolysin nobo cytotoxin from Metridium senile actinia, CS 274 496 B2 for groups of polyenantibiotics including amphotaricin B, philipin or nistatin, for groups of enzymes having high affinity for cholesterol, including cholesterol oxidases, cholestarol hydrogenases or cholesterol esterases, activates detection agent A: activator = 1: 1 to 1 in aqueous medium at molar ratio: 10, at a concentration of Detection Reagent A of from 1 to 20 mg / ml at a temperature of from 0 to 25 ° C pH of pH 11 to 1, for a period of 1 to 24 hours, the zigzag solution is reacted with visualization agent B which consists of acetylcholinesterase, tyrosinase, glucoso-6-phosphate dehydrogenase, glucose oxidase, glucosoamylase, galactosidase, peroxidase, alkaline or cylaphine foaphatase, α-chymotrypeine or pyrophosphatase, in a molar ratio of A: B = 20: 1 to 1: 1 and 1: 1 held for 1 to 24 hours at a temperature of from 0 to 25 ° C, and can be replaced with Detection Reagent B in the activation step. 2. A method according to claim 1, wherein the visualizing agent selected from the group set forth in point 1 is activated in an aqueous medium at a visualization agent B: activator ratio of 1: 1 to 1:10 and at a visualization agent B concentration of from 1 to 1. 20 ng / ml at a temperature of 0 to 25 ° C and a pH of 4 to 11 for 1 to 24 hours and the solution obtained is reacted with Detection Reagent A selected from the group referred to in point 1, at a molar ratio of A: B> 20: 1 to 1: 1 and the solution is maintained at 0 to 25 ° C for 1 to 24 hours.