RU2271539C2 - Spectrophotometric measurements in coloration-based biochemical and immunological investigations - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to method for biological material sample analysis in biological or immunological tests, wherein in sample treatment process coloring is observed, and color darkening is correlated with amount of tested substance. At least one color characteristic, such as coloring angle, chromaticity, darkening, and obtained color brightness is measured. Method of present invention is useful in analysis of lung, throat, cervix uteri or spermatic liquid mucilage for diagnosis of cancerous and precancerous conditions.

EFFECT: enhanced assortment of agents for diagnosis of cancerous and precancerous conditions.

14 cl, 4 dwg

Description

This invention relates to biochemical and immunological tests and tests based on the presence of color, in which the sample being the subject of determination or testing is subjected to spectrophotometric measurements to determine its color characteristics, in particular color angle and / or degree of color saturation. It is proved that tests that use such measurements are useful for obtaining quantitative or semi-quantitative results in a wide range of medical tests and procedures for the detection of diseases, as well as diagnostic methods.

As described in more detail below, many diagnostic tests depend on the visual observation and evaluation of the color that appears in the sample of biological material when the sample is treated with reagents that cause color formation in a positive correlation with the amount of analyte, i.e., a specific compound, the content of which should be evaluated (e.g., cholesterol), or specific molecular markers present in the sample that are indicative of a pathological condition, such as cancer.

Tests that require visual determination and evaluation of color changes are convenient and often adequate as a preliminary, subjective assessment of the presence of a detectable compound or disease marker, but they are not essentially quantitative.

The inventor of the present invention unexpectedly found that measuring the angle of the color tone and / or color, as well as the associated color characteristics using reflective spectrophotometry, allows at least semi-quantitative measurements of the test results. From such measurements of one or more specific color characteristics of the test sample, a lot of valuable information is obtained related to the presence or absence of progressive diseases, such as cancer, and also to the stage of their development.

Color Based Cancer Test

From US Pat. No. 5,162,202, it is known that in order to determine colorectal cancer and colon cancer in human patients, it is necessary to examine the rectal mucosa. This mucous membrane is selected on a membrane filter. A cellulose membrane filter is preliminarily prepared by impregnating it with a solution of the galactose oxidase enzyme in phosphate buffer, followed by lyophilization. In use, the cellulose membrane filter is wetted and then brought into contact with the membrane filter on which the mucous sample is applied for 1-2 hours. Then the mucosal filter is washed and treated with basic fuchsin for 15 minutes, washed and dried. Fuchsin bleaching indicates the presence in the mucous membrane of carbohydrate markers of cancerous and precancerous conditions. Such a test is long and difficult to perform and does not provide a high degree of sensitivity, so it can give false negative results.

An advanced test of the rectal mucosa is described in US Pat. No. 5,348,860 (Shamsuddin), published September 20, 1994. In this procedure, a mucosal sample is taken and immobilized on a membrane filter and treated with galactose oxidase in order to oxidize all the vicinal galactose groups in the sample to vicinal aldehydes . They are visualized with Schiff's reagent. This is a faster procedure. Samples that were negative with this method can be further oxidized with iodic acid and then visualized with Schiff's reagent to reduce the likelihood of false positives.

A constant problem with known mucosal tests is the need to visually identify and evaluate staining results. While such studies are adequate as preliminary, subjective assessments of the presence or absence of cancer markers, they are only qualitative. They do not provide reliable quantitative information on the number and concentration of markers that were detected and which could indicate the degree of development of the cancer state, if present. In addition, the medium on which the samples are applied, usually a cellulose membrane, such as a paper filter, may itself contain compounds capable of participating in reactions associated with the appearance of color. This can provide a “background” that complicates the interpretation of the test results and reduces its sensitivity. This requires qualified personnel trained to interpret the test results.

Color Based Cholesterol Test

The association of high serum cholesterol in patients with a tendency to develop atherosclerosis and, consequently, the increased risk of coronary artery disease, heart attack and PVD, has been firmly established, so it is often desirable to monitor cholesterol levels in patients. Typically, cholesterol levels are determined in blood samples taken. Most often, many other diagnostic tests are also carried out on selected blood samples, but most of them must be performed at longer intervals than the determination of cholesterol. The invasive nature of the blood sampling procedure for cholesterol analysis deprives many patients of the desire to check for cholesterol as often as necessary. Accordingly, there is a need to test for cholesterol without invasion.

It is estimated that the skin contains about 11% of total body cholesterol, which is largely a result of epidermal steroid formation and diffusion of cholesterol from blood vessels. It has been postulated that the level of cholesterol in the skin can more accurately reflect the degree of development of atherosclerosis than the amount of cholesterol in the serum.

Nikitin Yu.P., Gordenko I.A., Dolgov A.V. and Filimonova T.A. ("The cholesterol content in the skin and its correlation with the assessment of serum lipids is normal and in patients with coronary heart disease", Cardiology, 1987, No. 10, p. 48-51) and others showed that there is a close correlation between the cholesterol content in arterial walls and cholesterol in the patient’s skin. This suggests the possibility of developing skin tests to determine the patient's cholesterol level.

However, the method described by Nikitin et al. Involves the removal and analysis of skin samples in vitro, which is impractical in clinical use.

US Pat. Nos. 5,489,510 and 5,587,295 (Lopukhin et al.) Describe a non-invasive diagnostic test that is performed on a patient’s skin surface and that shows cholesterol levels in the skin. In the test described in these patents, reagents are used in the form of compounds having an affinity for enzymes that are bifunctional in nature. Such a bifunctional compound AB comprises a binding agent A (eg, digitonin), which is capable of selectively forming stable complexes with skin cholesterol in order to give the bifunctional compound an overall affinity for cholesterol; and imaging agent B, for example, an enzyme, such as peroxidase, to detect the presence of bifunctional compounds associated with skin cholesterol. In the practical use of this test, a complex of a binding agent A and a visualizing agent B, possibly in combination with a bridging agent C, to improve the sensitivity of the test, for example the bifunctional compound A-C-B, can be applied to the skin of the palm of the hand of the patient. The bridging agent C may be a high molecular weight polyfunctional compound, such as a polysaccharide or protein; it serves to separate the imaging agent and the binding agent in order to minimize the steric hindrance of the reaction of the cholesterol binding agent. After an appropriate incubation period, to ensure that the complex bonds with the skin cholesterol, the surface is completely washed with clean water to remove unbound reagents. Then, the reacted surface is treated with indicator agent D in order to react with visualizing agent B to form a color. The higher the level of cholesterol, the higher the degree of formation of bonds of a bifunctional compound with the skin and the higher the degree of appearance of color.

A cholesterol test based on the aforementioned patents of Lopukhin et al. Was developed at a commercial level and put into commercial practice. This includes providing a kit including reagents and a color card or reader. Most reagents are contained in a vial that the user places on the test surface in the palm of his hand after removing the protective cap. After exposure, the user imposes an indicator agent and visually assesses the degree of occurring color change using a color map or uses a reader.

One of the drawbacks of this test is the need for a visual assessment of color changes. Since it is assumed that the test can be performed by unskilled personnel, such as a patient, the visual assessment of the resulting color change is subjective and substantially non-quantitative. This can provide a meaningful indication of cholesterol levels and therefore potential problems, but not quantitative measurements of the type that the attending physician usually prefers. This assessment can easily be influenced by the nature and color of the background, namely the skin.

The objective of the invention is the creation of a new method for determining and measuring the result of a biochemical or immunological test, based on the formation of color, not associated with a subjective visual assessment. More specifically, the object is to provide a method of measuring, quantifiable, the amount of a detectable compound in a sample of biological material that has been subjected to a biochemical or immunological test, causing color formation in a positive correlation with the amount of said detectable compound.

The objective of the invention is also to provide a new test for fluid and semi-fluid secretions of the body, as well as a kit for use in this test, suitable for the diagnosis of cancer.

In addition, a more specific objective of the present invention is the creation of a new test of rectal mucous and other secretions, liquid and semi-liquid, including stool, and mixtures thereof, and a kit for use in this test, in which one or more of the above disadvantages. In the following description, the term “semi-solid contacting the colon” is used to refer to mucus, stool, and other liquid and semi-solid substances obtained from the patient’s rectum or colon, and mixtures thereof, which provide suitable analysis material for use in the method of this invention .

The objective of the invention is also the provision of a new diagnostic, conducted without invasion test for cholesterol.

In addition, a more specific task is to create such a test for cholesterol, which can give at least semi-quantitative results.

From the standpoint of overcoming the shortcomings of previous diagnostic tests based on the need for visual assessment of color changes, this invention in its broadest aspect uses some specific parameters that can be determined using a spectrophotometer, but which were not previously used for these applications. These colorimetric parameters are examined and analyzed to provide studies and diagnoses with increased sensitivity and specificity. The color resulting from the test or test is measured at various wavelengths; measure the color tone angle and / or color in order to provide relevant information related to the presence or absence of detectable compounds, disease states or other conditions that are the purpose of the test or test based on the formation of color. In some cases, measuring additional color characteristics, such as brightness or saturation, can further improve the sensitivity of the test or test.

In accordance with another aspect of the present invention, there is provided a method for diagnosing liquid or semi-solid secretions of a patient’s body to prove abnormalities in the tissues or organs from which these secretions are secreted, which includes taking a sample of liquid or semi-solid secretions of the patient’s body, placing at least a portion of this sample on the substrate, usually white, staining the sample on the substrate and the manifestation of the color of the painted sample, determining the specific color characteristics of the developed color of the sample by spectrophotometry and classifying the sample as a normal or abnormal based on the value thus obtained specific coloring characteristics.

In accordance with yet another aspect of the present invention, there is provided a system for analyzing liquid and semi-solid body secretion samples obtained from human patients in order to diagnose the presence or absence of abnormalities in patients by using the determination of a specific color characteristic obtained in a sample selected from a color angle, color or saturation and brightness, and the specified system includes:

a white, cellulose-free substrate with a porous “granular” surface, for acceptance and retention of the sample during processing;

a galactose oxidase source adapted to deposit galactose oxidase on the surface of a substrate for selectively enzymatically oxidizing a sample deposited on this surface;

a Schiff reagent source adapted to deposit Schiff reagent on an oxidized sample on a substrate to exhibit characteristics suitable for analysis;

and means for presenting the sample with the developed color to a portable reflective spectrophotometer capable of detecting and outputting a specific color characteristic selected from the angle of the color tone, color or color saturation and brightness, in stained samples on the indicated substrate.

In accordance with yet another more specific aspect of the invention, there is provided a method for diagnosing rectal semi-solid samples in contact with the colon to prove abnormalities in a patient, which includes taking a semi-solid sample in contact with the colon in a patient, placing at least a portion of this sample on a substrate, usually white, staining the sample on the substrate with galactose oxidase, color manifestation of the stained sample using Schiff's reagent, determination of a specific color characteristic the color of the sample using spectrophotometry and the classification of the sample as normal or abnormal in accordance with the value of the thus obtained specific color characteristics.

In accordance with another aspect of the present invention, there is provided a kit for analyzing semi-solid samples in contact with the colon and obtained from human patients for the purpose of diagnosing the presence or absence of rectal abnormalities in a patient, including:

usually a white, cellulose-free substrate for sample collection;

galactose oxidase source;

Schiff reagent source;

and a portable reflective spectrophotometer capable of detecting and delivering a specific color characteristic selected from a color tone angle, chroma or color saturation, and brightness of colored samples on said substrate.

In accordance with another aspect of the present invention, there is provided a test in which liquid or semi-solid reagents are applied to a patient's skin to bind skin cholesterol with subsequent color development in the reagents, wherein the degree of color development is directly related to the amount of cholesterol in the skin. However, instead of a visual assessment, liquid or semi-solid reagents in which the color manifests itself are analyzed colorimetrically to determine the degree of manifestation of color, from where you can at least semi-quantitatively obtain the level of cholesterol. Selected colorimetric parameters, such as hue angle or hue, are independent of color density, intensity or brightness (L) and simply measure the hue of the color. This largely eliminates the uncertainties introduced by the background color of the skin, so this test can be performed on the surface of the patient’s skin. Instrumental colorimetric (spectrophotometric) analysis gives objective figures that are at least semi-quantitative and show the patient's cholesterol level.

In accordance with this invention, in yet another aspect, a method for determining cholesterol levels in a patient’s skin is provided, which comprises:

applying to the surface of human skin a reagent that selectively binds to skin cholesterol;

the implementation of the chemical reaction of the manifestation of color with the cholesterol of the skin thus formed is a binding reagent with the formation of a colored complex;

and spectrophotometric analysis of the thus obtained colored complex in order to obtain a given color characteristic of the colored complex.

Another aspect of this invention is a kit for determining cholesterol levels in the skin of a human patient, including:

a source of a detection agent capable of binding to the patient’s skin cholesterol to form compounds on the skin surface;

a source of a visualizing agent capable of reaction with an agent-detector compound — a binding agent to form an optically altered complex;

a source of the developing agent and means for applying the developing agent to an optically altered complex for developing color therein;

and means for holding and presenting said optically altered complex to a portable reflective spectrophotometer to determine a specific characteristic of its color, selected from a color tone angle, chroma, or saturation.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following description of a preferred embodiment of the present invention with regard to the aforementioned aspect of the invention relating to a method and kit for determining patient cholesterol levels, reference is made to the figures of the drawings, wherein:

figure 1 is a schematic illustration of a strip for the test used according to this invention in determining cholesterol levels in the skin of a patient;

figure 2 is an image of a spectrophotometric reader when used to measure cholesterol in the skin according to this invention and in its open position;

figure 3 depicts the same as figure 2, but the spectrophotometer is in the closed position;

figure 4 is a detailed image of the lower part or pads, spectrophotometer shown in figure 2 and figure 3.

As is evident from the discussion above, the main invention of the applicant — the use of colorimetric measurements, in particular color saturation and / or color tone angle to determine test results and tests based on color formation — finds application in a number of aspects and embodiments. Thus, two aspects of the present invention and preferred embodiments thereof will be described below, separately and under separate headings.

Spectrophotometers suitable for use in all aspects of the present invention are portable, reflection-based instruments that provide accurate measurement of color characteristics, such as hue angle, luminance and chroma, or color saturation, in which the incident beam of the spectrophotometer is reflected from a dyed sample and incident on device receiving device. They are commercially available. A specific example of a suitable instrument is a Model CA22 Spectrophotometer distributed by X-Rite, Grand Falls, Michigan, U.S.A. It is equipped with appropriate software so that it can be connected to a computer to ensure accurate reading of the color tone angle of the painted sample during the test. This spectrophotometer senses reflected light with a wavelength of about 400-700 nm, that is, in most of the visible spectrum, usually at intervals of about 20 nm.

It is known that color can be defined and expressed in terms of the angle of the color tone. The concept of “color tone angle” is defined and discussed in standard textbooks such as “Principles of Color Technology”, Fred W. Billmeyer and Max Saltzman; Ed. John Wiley and Sons) (see especially chapters 1 and 2), incorporated herein by reference. The “color tone” is the color or tone of the sample, independent of its brightness or intensity, and the “color tone angle” for the color or tone is the determination of the wavelength of its reflected color using its angular position relative to the standard three-dimensional ellipsoidal coordinate system of the continuum of the general spectrum of visible light. The continuum of visible light (color) is presented on an angle scale from 0 to 360 °, and the angular values read by a spectrophotometer operating in reflected light are converted into a linearized form to obtain the transformed "color tone angle" used in the method of this invention.

(i) Cancer test

It was unexpectedly discovered, in accordance with this invention, that the presence of the entire range of pathologies, including pathologies of the intestines, lungs, cervix and others, can be determined by determining the angle of the color tone or other specific color characteristics mentioned above obtained on liquid or semi-solid secretion of the body from appropriate tissue or body organ. The reaction and spectrophotometric analysis of rectal mucus will serve to diagnose colon cancer. Thus, in individuals with colorectal cancer, samples of mucus from the rectum after staining and color development, as described, have higher color tone angles than those with normal intestines. Thus, a color tone angle or other specific color characteristic of a stained sample can be used to distinguish individuals with cancer of the intestine from those that do not. More specifically, it was found that samples taken from cancerous lesions had color tone angles typically in the range of 375-425 °, the upper quartile of measurements for clinical samples. In addition, since the test result is interpreted using a portable spectrophotometer, there is no requirement that the test results be obtained by qualified, trained personnel.

Similarly, lung cancer and precancerous conditions of the lung can be diagnosed by exposing mucus and sputum from the lungs to similar staining and spectrophotometric analysis. Cancer and precancerous condition of the cervix can be diagnosed using the same procedures applied to mucus from the cervix. Seed secretions, such as sperm, can be analyzed in the same way for cancer of the reproductive organs, such as testicular cancer. Throat mucus can be similarly analyzed to detect and diagnose throat cancer. Mucus from the throat and lungs can be obtained using known procedures, such as bronchoscopy or broncho-alveolar swabs. A fluid taken from the nipples of the breast is body fluid, which can likewise be analyzed by the method of the invention in a breast cancer test.

An essential point of the preferred embodiments of the present invention is the use of a porous fiberglass membrane on which the sample is oxidized and its color appears. Such fiberglass material is largely devoid of color-forming particles, so there are no particles that will undergo enzymatic oxidation and take part in the subsequent color development reaction. In accordance with this, the manifestation of a background color that can distort or hinder diagnostic tests is effectively eliminated. In addition, this membrane is essentially pure white in color, which further reduces the background “noise” with respect to which the results are read.

An additional characteristic of the fiberglass membrane in this aspect of the present invention is the surface porosity, which allows the additional spread of the mucus sample on it so as to expose additional carbohydrate markers in the sample to participate in oxidation and color reactions and, therefore, to improve the sensitivity of the method tests.

A specific, preferred example of a porous fiberglass membrane for use in this invention is a material sold by the Laboratory Department of Whatman Inc. titled “Whatman 934-AH Glass Microfiber Filter”, a borosilicate glass filter medium having high loading capacity and high retention efficiency at high flow rates. It is recommended for use in collecting cells grown in culture, and in methods for counting scintillation in a liquid. This, however, is only an example, and other, mainly purely white, carbohydrate-free fiberglass substrates with surface porosity, capable of efficiently distributing a semi-solid sample in contact with the colon can also be used.

In a specific method using a preferred aspect of the present invention, for determining colon cancer using an enzymatic oxidation reaction to detect color, a test sample is first taken from a patient. Lubricate the operator’s finger with a glove. The finger is inserted into the patient's anus and rotated 360 ° to obtain a representative sample of a semi-solid in contact with the colon, such as mucus from the rectum. The finger is removed from the rectum, and the sample is smeared on the surface of the white membrane filter described above, superimposed on the rectal mucus test card with the appropriate coating, protective layer and identification, and this card is analyzed.

For analysis, the substrate of the mucus test card from the rectum is removed and 50 μl of a standard galactose oxidase solution is applied to the test card. In a standard process, incubation lasts 10 minutes. Then the card is immersed in double-distilled water for 30 seconds, and then 1 ml of Schiff's reagent is applied for 3 minutes. Then the color is shown by passing the card through four washes with water, 10 minutes each wash. Then the card is allowed to dry and a determination is made by reading the angle of the color tone using a portable spectrophotometer of the type previously described. The color tone angle below a certain set value (350 ° in the case of mucus from the rectum) indicates the normal, healthy nature of the tissue. Values above a certain set value (370 ° in the case of mucus from the rectum) indicate the cancerous nature of the tissue.

Intermediate samples can be subjected to universal oxidation to facilitate a definitive diagnosis. Based on the knowledge that in any sample only a fraction of the vicinal hydroxyl groups on the carbohydrate marker is oxidized enzymatically with the formation of a color, it is possible to subsequently oxidize all remaining such groups with a powerful oxidizing agent such as periodate, and then show color again and determine it. If this results in a large difference compared to the original result, this indicates that this sample should be classified as if it gave a higher result first. If only a small difference is obtained or a noticeable difference is not obtained, then it is safer to group this sample with samples that give lower results.

From values of the color tone angle that are simply read on the spectrophotometer in this way, the operator can determine without any subjective interpretation whether the sample was taken from a patient who has a healthy, normal large intestine, or if the colon is infected with cancer, with a significantly reduced probability of false positive or erroneous negative definitions compared to previous diagnostic methods. The same standard stages of selection, staining, incubation and color development are applied and the same standard reagents are used, so that the new method according to this invention can be adopted by existing diagnostic laboratories with minimal interruption and economic costs.

Very similar methods are applied to other mucus samples from other organs and tissues of the body. Enzymatic galactose oxidase oxidation followed by a Schiff reagent is preferred as a method for conducting a color development reaction for subsequent color analysis of the present invention. However, the present invention is not limited thereto. You can use any method that allows for a selective reaction leading to the manifestation of a color characteristic in the development of cancer. For example, for color development, a direct reaction of a sample with a Schiff reagent without an enzymatic reaction step can be used. In all cases of spectrophotometric analysis of the developed color in order to determine the objective parameter of the hue or color tone, for example, the color tone angle used in this invention, such color parameters were found to correlate with the presence and degree of development of various types of cancer.

(ii) Non-invasive cholesterol test

In a preferred method of this aspect of the present invention, liquid or semi-solid biochemical reagents are used, exhibit a color indicative of cholesterol in the skin of a patient, and the color thus developed is subjected to spectrophotometric analysis. In accordance with this invention, the exact nature and essence or shade of the color thus obtained, which are characterized, for example, by its color tone angle, correlates with the amount of complex formed and, therefore, with the cholesterol content in the skin. This measurement of color characteristic is objective and at least semi-quantitative. Accordingly, it is carried out regardless of the background color of the skin, which does not affect this measurement to any significant extent.

It is usually convenient to apply all reagents in the appropriate sequence to the surface of the patient’s skin, color on the surface of the skin, and then spectrophotometrically examine the color-causing complex while it remains on the skin. The entire test can be done in less than five minutes. The skin surface chosen for the test should be substantially free of sebaceous glands, since such glands introduce a cholesterol-containing secretion, which can distort the results. The sole of the foot and palm of the hand are such suitable areas of the skin, with the palm of the hand being the most convenient for use in this test.

The kit includes means for holding and presenting the color-forming complex for analysis using a portable spectrophotometer. These agents may be a container in the form of a strip adhering to the skin with one or more windows passing through it so that the reagents contained in the windows can come into contact with the patient's skin. The design of the container will be largely determined by the physical characteristics of the spectrophotometer. Instead of using a reagent container, an inert thixotropic agent can be added to the reagents to limit their spread over the skin surface and to prevent mixing of test reagents with control reagents applied to adjacent skin areas.

As with other color-based tests to which this invention is applicable, a hallmark of a non-invasive cholesterol test in accordance with this aspect of the invention is that the color tone angle of the stained complex associated with skin cholesterol correlates with the content cholesterol in the skin.

Suitable chemicals for use in accordance with this aspect of the invention are typically those described in the aforementioned patents of Lopukhin and others, the descriptions of which are incorporated herein by reference. Their precise selection is not an essential or restrictive feature of the present invention; their use in combination with each other should lead to the chemical manifestation of color as a result of the formation of a bond with skin cholesterol. The term “bond formation” is used here in its broad sense of a chemical reaction with the attachment of one chemical particle to another, as well as a specific interaction of the “capture” type (based on affinity), which often occurs in biochemical systems.

Thus, binding agent A is selected from the group of substances capable of selectively forming stable complexes with free skin cholesterol in order to give the entire bifunctional compound in which it is included an affinity for cholesterol. It can form a stable complex by direct reaction with cholesterol before or after it chemically attaches to imaging agent B, either directly or via bridging agent C.

Representative classes of compounds useful as cholesterol binding agent A include:

steroid glycosides containing, as aglycon, a cyclopentane perhydrophenanthrene fragment of the furostanol or spirostanol series, and an oligosaccharide fragment comprising from 3 to 10 monosaccharide residues with linear or branched structures (Hinta PH "Structure and biological activity of steroidal glycosides, Chisinau, Chisinauides, and Chisinau series 1987, p.142), specific preferred examples of which are funcosides C, D, E, F, G and I, dioscin, rocosides C, D and E, lanotigonin, digitonin and tomatoine;

triterpene glycosides containing aglycone of the alpha or beta-amiryl, lupan, hopan, dommaran, linostan or holostan series, and oligosaccharides containing branched or linear saccharide residues (G. Dekanosidze, V. Yu. Chirva, T. V. Sergienko ., Uvarova NL "Study of triterpene glycosides", Tbilisi, Metznireba, 1982);

hydrophobic proteins capable of selectively forming a complex compound with cholesterol (Khimov A.N., Titova G.V., Kozhevnikov H.A., Biochemistry, 1982, V. 47, No. 2, pp. 226-232; Khimov A.N. ., Kozhevnikov H.A., Klyueva N.N. et al. Questions of Medical Chemistry, 1984, T. 30, No. 3, pp. 86-90; Titova G.V., Khilyueva N.N., Kozhevnikov X .A. Et al., Biochemistry, 1980, Vol. 45, No. 1, pp. 51-55);

protein toxins capable of selectively forming complex compounds with cholesterol. They are obtained from bacteria, marine microorganisms, insects or snakes (Dalin MV, Fish NG "Protein toxins of microorganisms", Moscow, Medicine, 1980); or

polyene antibiotics capable of selectively forming complex compounds with cholesterol (IJKatzenstein, AMSpielvogel, AWNorman, J. Antibiot, 27, 12, 1874, pp.943-951; Jong Shan Shyng, Wang Hsi-Hua, Clin. J. Microbiol ., 1976, 9, (1-2), pp. 19-30; Readio Josphine D. et al. Biochim. Biophys. Acta, 1982, 685 (2), pp. 219-24); or

enzymes with high affinity, the substrate of which is cholesterol, and which have a high affinity for it. All of the above publications are incorporated herein by reference.

The most preferred choice for cholesterol binding agent A is digitonin.

The visualizing agent B is usually an enzyme, since enzyme / substrate reactions leading to a color change are particularly useful. Specific examples of suitable enzymes include acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, beta-D-galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin and pyrophosphatase. A preferred choice is peroxidase, for example horseradish peroxidase (HRP).

The use of a bridging agent C improves the technical implementation of the method and facilitates the preparation of the final desired complex A-C-B, from which coloring can occur, while the functional activity of agents A and B is maintained. The most preferred AC-B complexes are those that use steroid glycosides containing, as aglycon, a cyclopentane perhydrophenanthrene fragment from the rows of furostanol or spirostanol and oligosaccharide fragments comprising from 2 to 10 monosaccharide residues with linear or branched structures, such as as an affinity for cholesterol, a bridging agent A. It is especially desirable to use a bridging agent C if agent A is selected as the cholesterol binding agent digitonin, and HRP was chosen as the visualizing agent, since HRP is a relatively large molecule, which, if it was directly connected to digitonin, could sterically hinder the reaction of digitonin with skin cholesterol. It is preferable to use high molecular weight polyfunctional compounds as bridging agent C for such purposes. Their use allows a wide range to regulate the ratio of agents A and C in the final complex. Such high molecular weight polyfunctional bridging agents C may be various polysaccharides, proteins or synthetic polymers, that is, any suitable high molecular weight compounds containing functional groups of a primary amine, carboxyl, hydroxyl, aldehyde, acid halide, mixed anhydrides, iminoether, azide, hydroxide, azimide or hydroxide epoxy group. The most preferred high molecular weight polyfunctional bridging agents C are copolymers of acrylic acid or maleic acid or maleic anhydride and N-vinylpyrrolidone. Asymmetric, low molecular weight bifunctional compounds such as bromine cyanide, trichlorotriazine or 2-amino-4,6-dichloro-3-triazine can also be used.

Indicator Agent D typically contains an enzyme substrate used as imaging agent B, and additional compounds necessary to make the reaction between the enzyme and the substrate visible. A specific example of such an indicator agent D when using the peroxidase enzyme as visualizing agent B is an agent containing hydrogen peroxide, N, N-diethyl-p-phenylidene sulfate together with appropriate stabilizers. The indicator agent D is selected in combination with imaging agent B from a number of compounds known in the art that will produce color formation reactions in combination with the selected enzyme.

A kit for carrying out the test according to this invention is proposed. This kit includes the necessary reagents in appropriately sealed packages, such as bottles or bottles equipped with a dropper, a container or other holding means, in which it is possible to carry out color-forming reactions on the patient's skin in such a way as to prevent the reagents from spreading over too large a surface, and from of which the obtained color can be presented for research and measurement in a means for determining and obtaining data on a specific characteristic of the color, such as the angle Vetovo tones, such as a portable reflective spectrometer. This container is usually an adhesive tape provided with one or more cut-out windows, originally provided with a protective layer on the back side to protect the adhesive layer. Preferably, this container has at least two or three windows, so that control experiments can be carried out during the test. In order to facilitate the correct conduct of the experiments during the test and control experiments, these windows are usually made different in appearance from each other, for example, they have different shapes.

Figure 1 shows such a container for use in this invention in the form of strips 10 for dough having a rectangular shape. This strip includes a porous base 10 with a layer 12 of skin-compatible adhesive material temporarily protected by a peelable film 14. The first central window 16 for testing purposes, having a circular cross section, passes through the porous substrate of the strip and through the adhesive layer 12. Second window 18 for positive a control having a rhombic cross-section and a third negative control window 20 having a square cross-section, similarly made into a foam base are strips 10 on the sides of the central window 16. Different-looking shapes con assist the operator in carrying out tests, facilitating the correct choice of window for the respective purposes.

The test is preferably carried out on the skin of the palm of the hand of the patient. The reagent container, in the form of adhesive tape, is applied to the skin for some time so that the bottom of the window contacts the skin. Reagents drip into the windows, coloring occurs in the windows, and then the color is determined by a spectrophotometer, without removing the strip from the skin. For this purpose, a specially designed spectrophotometer is used, which is another aspect of the present invention. The spectrophotometer transmits the received data to a computer for analysis. The spectrophotometer is designed to ensure proper placement on top of the test cell.

Accordingly, in this aspect of the present invention, there is provided a spectrophotometer adapted to transmit signals obtained by measuring color in reflected light from a test sample to a computer, this spectrophotometer having a housing, light emitting means located in the housing, a lower part of the housing with an opening through which you can direct the light emitted by this tool, and the recess on the lower surface of the specified lower part, adapted in such a way as to cover the strip for the test with windows, superimposed on the surface of the skin of the patient, to ensure exact correspondence of the position of the spectrophotometer and the detected sample in the window of the specified strip with windows for the test. Preferably, the bottom of the spectrophotometer is hinged to the housing so that it can be properly set to register correctly on the test strip in the open position and then attached to the spectrophotometer housing for measurement. It is also preferred that the hinged lower part and the spectrophotometer body are provided with electrical contacts acting as a switch to turn on the light source of the spectrophotometer when the hinged lower part is attached to the spectrophotometer body.

Figure 2, 3 and 4 schematically shows a spectrophotometer. It has a housing 22 with an electrical connection (not shown) to a computer programmed accordingly to analyze the results obtained by the reader. There is a lower part 24, at position 26, hinged to the housing 22. The lower part 24 has an opening in position 28. A groove 30 extends along the width of the lower surface of the lower part 24, which protrudes upward from the lower surface. The groove is closed on one side of the bottom. The width of the groove 30 is made in such a way that it exactly and strictly corresponds to the strip 10 for conducting the test shown in figure 4. When the measurement is to be taken, the end of the test strip 10 corresponds to the closed end of the groove, and this, in combination with the placement of the test strip 10 within the width of the groove 30, ensures that the window 16 where the test is carried out matches the light beam that must be emitted from the housing 22 of the reader through the hole 28. The spectrophotometer contains appropriate detector devices for sampling the reflected signals from the window in the window 16 and transmitting them for analysis and reading by the computer. The housing 22 and the lower part 24 are provided with corresponding to each other electrical contacts 32, 34, which are closed as a switch when the lower part 24 is connected to the housing 22, including the light for measurement.

Next, a specific test procedure will be described in the form of a specific, but non-limiting example of the practice of the diagnostic test of this invention.

Kit components include a dropper bottle containing a detector solution (a combination of digitonin and horseradish peroxidase in an aqueous buffer solution containing bromnitrodioxan and methylisothiazolone with a concentration of less than 0.01% as stabilizers, 1.5 ml), with a brightly colored lid (green); a dropper bottle with a more concentrated detector reagent solution, in the same buffer solution and stabilized with less than 0.01% bromonitrodioxane and methylisothiazolone, as a positive control (1.5 ml), with a brightly colored lid (red); a bottle with a dropper containing an indicator - a reagent solution (4.0 ml of a solution of 3.3 ', 5.5'-tetramethylbenzidine (TMB) and hydrogen peroxide with 5% N, N-dimethylformamide as a stabilizer) for reaction with a detector and RS - reagents that combine with skin cholesterol, with the formation of a bluish-green color, with a brightly colored lid (blue); foamed substrates, as shown in FIG. 1, to which reagents, alcohol swabs and appropriate user manuals can be supplied. These chemicals are stable when refrigerated at 2-8 ° C for a long time. The system also includes a spectrophotometer, as shown in FIGS. 2 and 3, connected to a suitably programmed computer, and calibration plates for use with the spectrophotometer. The kit, as supplied, does not include a spectrophotometer, with the exception, apparently, of the initial sale; the same spectrophotometer is reused with a subsequently refilled kit.

First, the spectrophotometer is calibrated accordingly by inserting the calibration plate into its clamping device and closing the bottom cover to illuminate the calibration plate and obtain calibration data on the spectrophotometer and computer. A signal is issued that the calibration has completed successfully.

The patient's hand is washed thoroughly with soap and water, rinsed and dried well. Then, the outside of the patient’s palm is thoroughly cleaned with an alcohol swab with sufficient force to ensure thorough cleaning. After the hand has dried, remove the protective film 14 from the strip for the test 10, which is then glued to the cleaned surface of the skin of the palm of the patient. The patient turns his hand on a paper towel placed on the table and presses it firmly to ensure that the substrate is firmly attached to the palm of the hand.

The reagents are then added to the appropriate reagent windows. One drop (42 μl) of the detector solution is added to the round window for test 16, one drop of the positive control solution is added to the diamond-shaped window 18, and no liquid is added to the third, square window 20 at this stage. The added solutions were incubated for 1 minute; while the patient holds the hand on which the test is carried out motionless. The patient then turns his hand over and presses the foam backing onto a paper towel to remove fluid from the windows. Visually check that the substrate and the windows for the test were completely dry. Then the patient leaves his hand on a flat surface, palm up.

Then, one drop of the indicator, including the previously unused square window, is added to all three windows, and the reaction is allowed to pass within 2 minutes, while the patient holds his hand motionless. Immediately after this, the reader 22 is placed in a position above the window 16 for the test, close and determine the color signal developed in the window for the test, transmitted to the spectrophotometer and analyzed by the computer, to obtain the value of the angle of the color tone.

A visual inspection is undertaken of the positive control window 18 and the negative control window 20. If the liquid in the negative control window is colorless, and the liquid in the positive control window is colored, then the test is carried out correctly. On the stain formed by the positive control, no quantitative measurements are performed. This is a color obtained at a very high concentration of a reagent solution, which gives a color even with very small amounts of cholesterol on the skin; it is simply an indicator of the performance of the reagents and serves other control purposes.

The liquid remaining on the strip for the test is removed, and the strip for the test is removed from the palm, followed by cleaning the palm with a swab moistened with alcohol.

The currently used spectrophotometer preferably measures the absorbance of the light reflected from the test sample and converts it using a suitable algorithm into a color tone angle. In the specific case of obtaining a color during the horseradish peroxidase - TMB reaction described above, absorption conditions at 450 nm, A ₄₅₀ nm, are suitable measurement conditions. The absorbance at 450 nm was experimentally determined in order to relate it to the color tone angle by the ratio:

h ° (degrees) = 490.45A _{450 nm} +57.124

This ratio was determined by measuring the optical density of a series of successively diluted reaction samples at 450 nm, measuring the color tone angle of the same samples, and plotting from the results of a regression curve to determine the dependence. Similar dependences can be obtained experimentally in the same way for other selected tests, during which dye is formed, performed using different enzyme-substrate pairs giving different colors, which allow converting the optical density readings into values of the color tone angle.

Although specific diagnostic tests and test kits have been given above, one skilled in the art can understand that a large number of color-based tests and analyzes can be improved with the new measurements described here based on hue angle or color saturation, for example:

(i) obtaining quantitative results in solid-phase immunological tests, such as spotting, immunochromatographic tests and penetrating sample tests (the sample is applied to a membrane with an absorbent device behind it; then the analyte is captured and identified by the addition of a labeled detector, such as an antibody, labeled with enzyme or gold);

(ii) obtaining quantitative results in microparticle tests using colored beads;

(iii) as an alternative to densitometry in the analysis of stained gels;

(iv) to obtain quantitative results in the analysis in the spot on Western'y.

The method according to this invention provides a simple approach to quantifying such results, thus complementing the information obtained during testing, the possibility of applying to the test results more advanced methods of statistical analysis.

Accordingly, variations of this invention should not be construed as a departure from its essence and scope of claims, and all such modifications, as obvious to a person skilled in the art, should be included in the scope of the claims.

Claims (24)

1. The method of analysis of a sample of biological material in biochemical or immunological tests for any detectable substance, including the following operations: the processing of the sample, in which a color is formed corresponding to the amount of the analyte in this sample; measuring a hue angle or color saturation of the resulting color with a suitable spectrophotometer or other equivalent device, and analysis of measurements of the color tone angle or color saturation to determine the presence or concentration of the specified analyte in the sample. 2. The method according to claim 1, where the specified sample of biological material is mainly liquid or semi-solid body secrets taken from a patient person for diagnosis for abnormalities, and the specified substance is essentially composed of those in this sample markers indicating the presence of cancer, and the measured value of the angle of the color tone or color saturation is used in order to classify the sample as normal or abnormal. 3. The method according to claim 2, where these secrets are mucus from the lungs, mucus from the throat, mucus from the cervix, seminal fluid or fluid taken from the breast nipples. 4. The method according to claim 2 or 3, where the specified sample is applied to a white base as a whole, and the specified processing to form a color involves a reaction with an enzyme. 5. The method of analysis of a sample of biological material according to claim 1, where the specified sample of biological material is a semi-solid sample in contact with the colon, taken from a patient for diagnosis for the presence of anomalies; said detectable substance consists essentially of markers indicating the presence of anomalies; the operation of processing the sample involves applying this sample to a white substrate as a whole and obtaining the color of this sample through an enzymatic reaction and the measured value of the angle of the color tone or color saturation is used to classify the sample as normal or abnormal. 6. The method according to claim 3, where these markers are carbohydrate markers. 7. The method according to claim 5 or 6, where the substrate does not contain cellulose. 8. The method according to claim 7, where the substrate is a fiberglass. 9. The method according to claim 5 or 6, where the substrate is essentially pure white. 10. The method according to any one of paragraphs.5, 6 or 8, where the specified contacting with the colon semi-solid sample is mainly rectal mucus. 11. The method according to claim 7, where the specified contacting with the colon semi-solid sample is mainly rectal mucus. 12. The method according to claim 9, where the specified contacting with the colon semi-solid sample is mainly rectal mucus. 13. The method according to claim 1, where the specified sample of biological material is the surface of the skin of the patient, and the specified determined substance is cholesterol of the skin. 14. A kit for analyzing liquid or semi-solid samples of body secrets obtained from human patients for diagnosing the presence or absence of abnormalities in a patient by determining a color tone angle or color saturation for a color displayed on a sample, said system including a white, cellulose-free substrate with a porous “granular” surface for acceptance and retention of the sample during development; a galactose oxidase source adapted to deposit galactose oxidase on the surface of the substrate to conduct selective enzymatic oxidation of the sample on it; a Schiff reagent source adapted to apply the Schiff reagent to the oxidized sample on the substrate to develop a color suitable for analysis; and a device for presenting a sample with a developed color to a portable reflective spectrophotometer capable of determining the angle of the color tone and color saturation, characterized by the color of the painted samples on the specified substrate. 15. A kit for the analysis of semi-solid samples in contact with the colon obtained from human patients for the diagnosis of the presence or absence of rectal abnormalities in a patient, including a generally white, cellulose-free substrate for sample acceptance; Schiff reagent source and a portable reflective spectrophotometer capable of detecting and providing data on the color tone angle and color saturation characterizing the color of the stained samples on the indicated substrate. 16. The kit according to clause 15, where the specified substrate is fiberglass. 17. A method for determining the level of cholesterol in the skin of a patient, comprising applying to the patient’s skin surface a reagent that selectively binds to skin cholesterol; conducting a chemical reaction of the manifestation of color with the reagent associated with cholesterol of the skin, with the formation of a colored complex and spectrophotometric analysis of the thus obtained colored complex to obtain the values of the angle of the color tone or color saturation, characterizing the level of cholesterol in the skin. 18. The method of claim 17, wherein said reagent that selectively binds to skin cholesterol is selected from the group consisting of (i) steroid glycosides containing, as aglycon, a cyclopentane perhydrophenanthrene fragment of the furostanol or spirostanol series, and oligosaccharide fragments comprising from 3 to 10 monosaccharide residues with linear or branched structures, (ii) triterpene glycosides containing an aglycon of the alpha or beta-amiryl series, lupan, hopan, dommaran, linostan or holostane, and oligosaccharides containing branched or linear saccharide residues, (iii) hydrophobic proteins capable of selectively complexing with cholesterol, (iv) protein toxins capable of selectively forming complex compounds with cholesterol, (v) polyene antibiotics capable of selectively forming complex compounds with cholesterol, and (vi) enzymes whose substrate is cholesterol and which exhibit high affinity for cholesterol, and in which the formation of the specified colored complex is achieved by treating the specified binding agent on the skin surface first with a visualizing agent and then with an indicator agent. 19. The method according to p, where the formation of the specified colored complex is achieved by sequential processing of the specified cholesterol binding agent on the skin surface with a bridging agent, a visualizing agent and an indicator agent. 20. The method of claim 18 or 19, wherein said cholesterol binding agent is digitonin. 21. The method according to p. 18 or 19, where the specified visualizing agent is a peroxidase enzyme, and the specified agent-indicator contains hydrogen peroxide and N, N-diethyl-p-phenylidene sulfate together with appropriate stabilizers. 22. The method according to claim 20, where the specified visualizing agent is a peroxidase enzyme, and the specified indicator agent contains hydrogen peroxide and N, N-diethyl-p-phenylidene sulfate together with appropriate stabilizers. 23. A kit for determining the level of cholesterol in the skin of human patients, including a source of a detection agent capable of binding the patient’s skin cholesterol to form a combination associated with it on the skin; a source of a visualizing agent capable of forming a bond with a coupled combination of a detector agent — a binding agent to form an optically altered complex; a source of the developing agent and means for applying the developing agent to an optically modified complex for developing color in it, and a device for holding said optically altered complex and for presenting it in a portable reflective spectrophotometer for determining a color tone angle or color saturation therein characterizing the manifest color. 24. The kit of claim 23, wherein said device for holding an optically altered complex and presenting it to a spectrophotometer includes a container in the form of a strip adhering to the skin having at least one window passing through it to hold the reagents in contact with the skin the patient.

Images