Spectrophotometric measurements in coloration-based biochemical and immunological investigations

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

 

This invention relates to biochemical and immunological tests and tests based on the presence of painting, in which the sample which is the subject of a definition or test, is subjected to spectrophotometric measurements to determine its color characteristics, in particular the angle of color 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 to detect diseases and diagnostic methods.

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

Tests that require visual detection and assessment of change colors that are convenient and often adequate as a preliminary, subjective OC the NCI availability of the designated compound or marker of the disease, but they essentially are not quantitative.

The author of the present invention unexpectedly found that the measurement of the angle of hue and/or chroma, as well as the associated color characteristics using reflective spectrophotometry allow at least semi-quantitative measurement of test results. From such measurements one or more specific color characteristics of the sample receive a lot of valuable information related to the presence or absence of progressive disease, such as cancer, as well as with the stage of their development.

Test for cancer based on the measured color

From U.S. patent 5162202 known that to determine colorectal cancer and cancer of the colon patients-people need to examine the mucosa of the rectum. This take away mucous membrane filter. Membrane filter made of cellulose prepared by impregnation with a solution of the enzyme galactosidase in phosphate buffer followed by lyophilization. When using a membrane filter made of cellulose moistened, and then brought into contact with the membrane filter, which caused a sample of mucous, for 1-2 hours. The filter is then coated with a mucous membrane is washed and treated with basic fuchsin for 15 minutes, washed and dried. Discoloration fuchsin which indicates the presence of mucous in carbohydrate markers of cancer and precancerous conditions. This test is long and complicated in execution, and does not provide a high degree of sensitivity, so it can give false negative results.

Advanced test mucous membrane of the rectum is described in U.S. patent 5348860 (Shamsuddin), published on 20 September 1994 In this procedure, a sample of mucous select and immobilized membrane filter and treat galactosialidosis in order to carry out the oxidation of all vicinal groups of galactose in the sample to vicinal aldehyde groups. Their visualize the Schiff reagent. This is a faster procedure. Samples that this method gave a negative result, it is possible to oxidize additionally, iodic acid, and then visualize the Schiff reagent to reduce the probability of obtaining false negative results.

A constant problem is known tests mucosa is the need to visually determine and evaluate the results of staining. While such studies are adequate as a preliminary, subjective assessments of the presence or absence of cancer markers, they are only qualitative. They do not give reliable quantitative information about the number and concentration of the markers that were found and which could indicate the degree of development of cancer condition if it Preece is tstuat. In addition, the environment to which is applied the sample, usually a membrane of cellulose, such as paper filter itself may contain compounds capable of participating in reactions that are associated with the appearance of colors. This can give the background, complicating the interpretation of test results and reduce its sensitivity. This requires qualified personnel, trained to interpret the test results.

Based on the presence of a color test for cholesterol

Connection of a high level of cholesterol in the serum of patients prone to the development of atherosclerosis and, consequently, increased hazard of coronary vessels of the heart, stroke and PVD, firmly established, therefore, often desirable to control the level of cholesterol in patients. Usually cholesterol determined in samples of blood. Often on selected blood samples also provide many other diagnostic tests, but most of them should be longer intervals of time than the determination of cholesterol. The invasive nature of the procedure of selection of blood samples for cholesterol deprives many patients desire to check for cholesterol as often as necessary. Accordingly there is a need to test for cholesterol without invasion.

It is estimated that skin which contains about 11% of the total cholesterol of the body, that is largely the result of epidermal education of steroids and diffusion of cholesterol from the blood vessels. It was postulated that the level of cholesterol in the skin may more accurately reflect the degree of atherosclerosis than the amount of cholesterol in serum.

Nikitin, Y.P., Gordenko I.A., Dolgov A.V. and Filimonova T.A. ("cholesterol in the skin and its correlation with the assessment of lipids in the serum of normal subjects and patients with coronary heart disease, Cardiology, 1987, No. 10, p.48-51) and others have shown that there is a close correlation between the content of cholesterol in the arteries and cholesterol in the skin of the patient. This suggests the possibility of developing a test on the skin to determine the level of cholesterol in a patient.

However, the method described Nikitin and others, includes the removal and analysis of skin samples in vitro, which is impractical for clinical use.

In U.S. patent 5489510 and 5587295 (Lopukhin and others) described a diagnostic test without invasion, which is performed on the surface of the skin of the patient and which shows the level of cholesterol in the skin. In the test described in these patents apply reagents to form compounds with affinity to enzymes, bifunctional in nature. Such bifunctional compound a-b includes a binding agent (for example, digitonin), which is first able to selectively form stable complexes with cholesterol in the skin, to give a bifunctional compound in General affinity for cholesterol; and a visualizing agent, for example an enzyme, such as peroxidase, which allows to determine the presence of bifunctional compounds associated with cholesterol of the skin. In the practical use of this test, the complex of the bonding agent and visualizing agent, possibly in conjunction with masticables agent to improve the sensitivity of the test, for example bifunctional compound A-C-b, may be applied on the skin of the palm of the hand of the patient. Multicourse the agent may be a high molecular weight polyfunctional compound such as a polysaccharide or a protein; it serves to separate the visualizing agent and a binder agent in order to minimize steric obstruction reaction binds cholesterol agent. After an appropriate incubation period for communication of complex with cholesterol skin surface is thoroughly washed with clean water to remove unbound reagents. Then the reacted surface is treated with the agent-indicator D with the aim of carrying out the reaction with a visualizing agent for the formation of color. The higher the cholesterol level, the higher the degree of education relations bifunctional compounds with the skin and the higher the degree of occurrence of color.

One of the disadvantages of this test is the need for the visual evaluation of color changes. Since it is assumed that the test can be performed by unskilled personnel, for example a patient, the visual evaluation of color change is subjective and largely qualitative. This can give a meaningful indication of the levels of cholesterol and, therefore, potentially existing problems, but no quantitative measurements of this type, which usually prefers the attending physician. This assessment can easily influence the nature and background color, namely skin.

The objective of the invention is to create a new way of defining and measuring the biochemical or immunological tests, the OS is consistent on education, coloring, not associated with subjective visual assessment. More specifically the objective is to create a way of measuring, quantifiable, the number of identified compounds in the sample of biological material, which was subjected to biochemical or immunological tests, calling the color formation in positive correlation with the number specified defined connection.

The objective of the invention is the creation of a new test liquid and semi-liquid secretions of the body, as well as set for use in this test, suitable for the diagnosis of cancer.

In addition, more specific objective of this invention is to provide a new test of rectal mucous and other secretions, liquid and semi-liquid, including the chair, and mixtures thereof, and set for use in this test, which overcome or at least significantly reduced one or more of the above disadvantages. In the following description, the term "in contact with colon semi-solid substances" are used to denote mucus, stool, and other liquid and semi-solid substances obtained from the rectum or the colon of the patient and their mixtures, which provide suitable for the analysis of the material for use in the method according to this invention.

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

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

From the perspective of overcoming the shortcomings of previous diagnostic tests, based on the need for the visual evaluation of color changes, this invention in its broadest aspect uses some specific parameters that can be determined by using a spectrophotometer, but which had not been used for these applications. These colorimetric parameters are examined and analyzed to provide research and diagnosis with high sensitivity and specificity. The color produced in the test or test, measured at different wavelengths; measure the angle of hue and/or chroma to ensure relevant information relating to the presence or absence of defined compounds, illnesses or other conditions that are the target of the test or the test is based on the formation of color. In some cases, the measurement of additional color characteristics, such as brightness or saturation, may further improve the sensitivity of the test or tests.

In accordance with another aspect of the present invention is proposed JV is a method for diagnosing a liquid or semi-solid samples of secretions of the patient's body for evidence of violations in the tissues or organs, of which is distinguished from these glands, which includes the selection of the sample liquid or semi-solid body secretions from the patient, placing at least part of this sample on the substrate, as a rule, white, staining of the sample on the substrate and the manifestation of color-treated sample, determination of the specific color characteristics shown color sample using spectrophotometry and classification of the sample as normal or abnormal depending on the values thus obtained specific characteristics of the color.

In accordance with another aspect of the present invention proposed a system for analysis of liquid and semi-solid samples of body secretions obtained from patients-people, in order to diagnose the presence or absence of abnormalities in patients by using the definition of the specific characteristics of the color obtained in the sample, selected from the corner of the painting, chroma or saturation, and brightness, and this system includes:

white, not containing cellulose substrate with porous "grainy" surface, for receiving and holding the sample during processing;

source galactosidase adapted for applying galactosidase on the surface of the substrate for selective enzymatic oxidation of the sample deposited on this surface;

the source of the Schiff reagent, prisposoblenny for the application of Schiff's reagent for the oxidized sample on the substrate for manifestation suitable for analysis of characteristics;

and means for presenting the sample shown in color in a portable reflectance spectrophotometer is able to detect and give a specific characteristic of the color chosen from the angle of hue, chroma or color saturation and brightness, painted designs on the specified substrate.

In accordance with another more specific aspect of the invention, a method for diagnosing rectal semi-solid samples in contact with a colon, for evidence of abnormalities in a patient, which includes the selection of contact with the semi-colon sample from the patient, the location of at least part of this sample on the substrate, usually white staining pattern on the substrate by galactosialidosis, the manifestation of the color of dyed sample with Schiff's reagent, to determine the specific color characteristics of the color sample shown using spectrophotometry and classification of the sample as normal or abnormal in accordance with the value thus obtained a certain color characteristics.

In accordance with another aspect of the present invention proposed a test kit semi-solid samples in contact with the colon and obtained from patients-people with the aim of diagnosing the presence or absence of rectal abnormalities in PAC is enta, including:

usually white, not containing cellulose substrate for receiving the sample;

source galactosidase;

the source of Schiff's reagent;

and portable reflectance spectrophotometer is able to detect and give a specific characteristic of the color chosen from the angle of hue, chroma or saturation of the color and brightness of the painted designs on the specified substrate.

In accordance with another aspect of the present invention proposed a test in which a liquid or semi-solid reagents applied to the skin of the patient to associate the cholesterol of the skin with subsequent manifestation of color in the reagents, and the degree of manifestation of the color is directly related to the amount of cholesterol in the skin. However, instead of the visual assessment, liquid or semi-solid reagents, in which the apparent color, analyze colorimetrically to determine the degree of manifestation of the painting, where you can at least semiquantitative get the cholesterol level. The selected colorimetric parameters such as the angle of the hue or tint, does not depend on the density of color, intensity, or luminance (L) and simply measure the shade. It largely eliminates the uncertainty introduced by the background color, so this test can be done on the skin surface of the patient. Instrumental colorimetric the cue (spectrophotometric) analysis provides objective numbers which are at least semi-quantitatively, and show the level of cholesterol in a patient.

In accordance with this invention in another aspect, a method for determining cholesterol in the skin of the patient, which includes:

coating the surface of human skin reagent that selectively binds to cholesterol in the skin;

the implementation of the chemical reactions manifestations color thus formed compound cholesterol skin - binding reagent with the formation of a colored complex;

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

Another aspect of the present invention is a kit for determination of cholesterol levels in the skin of the patient-person, including:

the source agent detector capable of binding with cholesterol of the patient's skin with the formation of their compounds on the surface of the skin;

source imaging agent capable of reaction with the coupling agent detector - binding agent with formation of a modified optical complex;

source manifesting agent and means for applying manifesting agent for optically modified complex for the manifestation in him of painting;

and means for holding the air traffic management and representation of the specified optically modified complex in portable reflectance spectrophotometer to determine the specific characteristics of its color, the selected angle of hue, chroma or saturation.

BRIEF DESCRIPTION of DRAWINGS

In the subsequent description of the preferred alternative implementation of the present invention regarding the above-mentioned aspect of the invention related to a method and kit for determination of cholesterol levels in a patient, provides links to the figures of the drawings, where:

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

figure 2 represents the image spectrophotometric reading device when it is used to measure the level of cholesterol in the skin according to this invention 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 bottom or pads, spectrophotometer, shown in figure 2 and figure 3.

As is evident from the above discussion, the main applicant's invention is the use of colorimetric measurements, in particular the color saturation and/or hue angle for determining the results of examinations and tests, based on the formation of colour - finds application in a number of aspects and embodiments. In affect, the, below are described two aspects of the present invention and the preferred ways of their implementation, separately and under separate headings.

Spectrophotometers, suitable for use in all aspects of this invention are portable, reflection-based devices, provides an accurate measure of color characteristics, such as the angle of hue, brightness and chroma, or saturation, in which an incident beam spectrophotometer is reflected from the dyed sample and gets on the receiving device. They are available for sale. A specific example of a suitable device is a spectrophotometer "Model CA22"distributed X-Rite, Grand Falls, Michigan, U.S.A. He is equipped with appropriate software, so it can connect with computer to ensure accurate reading of the angle of the hue of the colored sample for the test. This spectrophotometer perceives the reflected light with a wavelength of about 400-700 nm, that is, in the most part of the visible spectrum, typically at intervals of about 20 nm.

It is known that color can define and Express terms of hue angle. The concept of "hue angle " is defined and discussed in standard textbooks, such as "Principles of color technology" ("Principles of Color Technology", by Fred W. Billmeyer and Max Saltzman; Ed. John Wiley and Sons) (see especially g is ava 1 and 2), incorporated herein by reference. Hue - the color or tone of the sample that does not depend on its brightness or intensity, and the angle of hue for the color or colors is the determination of the wavelength of its reflected color with its angular position relative to the standard three-dimensional ellipsoidal coordinate system of the General continuum spectrum of visible light. The continuum of visible light (color) is represented by an angle from 0 to 360°and angular values read by the spectrophotometer operating in reflected light is converted into a linear shape with obtaining transformed "hue angle"used in the method according to this invention.

(i) a Test for cancer

It has been unexpectedly found in accordance with this invention that the presence of a whole range of conditions, including the pathology of the intestine, lung, cervix, and others, can be determined by determining the angle of the hue or another particular above-mentioned color characteristics of a color obtained in liquid or semi-solid body secretions from the corresponding tissue or organ of the body. Reaction and spectrophotometric analysis of mucus from the rectum will be used to diagnose colon cancer. Thus, in individuals with colon cancer samples of mucus from the rectum after staining and the degree of color development as it is about what isana, have higher values of hue angle than those who have normal bowel. Thus, the angle of the hue or another specific color characteristic of the colored pattern can be used to distinguish individuals with cancerous lesions of the bowel from those who do not have such lesions. More specifically, it was found that the samples of the cancerous lesions, had a hue angles typically in the range of 375-425°upper quartile measurements for clinical samples. In addition, since the test result is interpreted using a portable spectrophotometer, there is no requirement that the test results were obtained qualified, trained personnel.

Similarly lung cancer and pre-cancerous condition of the lung can be diagnosed by exposing the mucus and phlegm from the lungs similar to staining and spectrophotometric analysis. Cancer and precancerous condition of the cervix can be diagnosed using the same procedures applied to the mucus of the cervix. Seminal secretions, such as semen, can be analyzed in the same way cancer of the reproductive organs, such as testicular cancer. Mucus from the throat can similarly be analyzed for detection and diagnosis of throat cancer. The mucus from the throat and lungs can be obtained with the help of the local procedures such as bronchoscopy or broncho-alveolar washings. Liquid selected from the nipple of the breast, is a fluid body, which likewise can be analyzed by the method according to this invention in a test for breast cancer.

An essential aspect of preferred embodiments of the present invention is the use of a porous membrane made of fiberglass, on which the sample is oxidized and its color is displayed. Such material fiberglass substantially devoid of particles forming the color, so there are no particles that will be subjected to enzymatic oxidation and to participate in subsequent reactions the degree of color development. In accordance with this effectively eliminates the manifestation of the background color, which may distort the diagnostic tests or to prevent them. In addition, this membrane is essentially a pure white color, which further reduces the background "noise"against which to read the results.

An additional characteristic of the membrane from the glass in this aspect of the present invention is the porosity of the surface, which allows for additional distribution of the sample of mucus on it in such a way as to exhibit additional carbohydrate markers in the sample to participate in the oxidation reactions and the expression is Oia color and therefore, to improve the sensitivity of the test method.

Specific preferred examples of the porous membrane made of fiberglass for use in this invention is a material marketed from Laboratory Department Whatman Inc. called "Filter glass microfiber Whatman 934-AH", filter medium made of borosilicate glass having a high capacity and high retention efficiency at high flow rates. It is recommended for use when collecting cells grown in culture, and ways of counting the scintillations in the liquid. This, however, is only an example, and can also be used and other, mostly pure white, not containing carbohydrate substrates fiberglass with surface porosity capable of efficient allocation of surface in contact with a semi-colon sample.

In a particular way, using a preferred aspect of the present invention, with reference to the definition of colon cancer using enzymatic oxidation reactions for the detection of color first patient a sample is taken for testing. Coated glove finger of the operator is applied grease. The finger is introduced into the anus of the patient and rotates 360°to obtain a representative sample in contact with that is stand-colon semi-solid substances, such as mucus from the rectum. The finger is removed from the rectum, and the sample smear on the surface of the above-described white membrane filter placed on map testing mucus from the rectum with an appropriate coating, the protective layer and identification, and analyze this map.

To analyze the backing card testing mucus from the rectum is removed and the map tests put 50 ál of the standard solution galactosidase. In the standard process incubation lasts 10 minutes. Then map immersed in bidistilled water for 30 seconds, and then put 1 ml of Schiff's reagent for 3 minutes. Then color display by passing the card through a four flush with water for 10 minutes each rinse. Then give the map to dry and make a determination by reading the hue angle using a portable spectrophotometer previously described type. The angle of the colour tone lower than a set value (350° in the case of samples of mucus from the rectum) indicates a normal, healthy nature of the fabric. Values above a set value (370° in the case of mucus from the rectum) indicate a cancerous nature of the fabric.

Samples giving intermediate values, can be subjected to universal oxidation to contribute to the final diagnosis. Based on the knowledge that in any sample only a certain fraction of the vicinal hydroxyl groups on the carbohydrate marker of oxidized enzyme with the formation of coloring, you can then oxidize any remaining such groups are powerful oxidizing agent, such as periodate, and then again to show the color and definition. If there is a large difference compared to the initial results, this indicates that this sample should be classified as if he gave first a better result. If it turns out only a small difference't a noticeable difference, then this sample is safer to group samples, which gave lower results.

From the values of hue angle, which simply read on the spectrophotometer in this way, the operator can, without any subjective interpretation to determine whether to take a sample from a patient having a healthy, normal colon, or infected with a cancer of a thick gut, with a significantly reduced probability of erroneous positive or false negative definitions compared with previous methods of diagnosis. Apply the same standard screening stage, staining, incubation, and the degree of color development and the same standard reagents, so that the new method according to this invention can be adopted existing diagnostic laboratories with minimal disruption and economic costs.

Very similar methods are used in related and other mucus samples from other organs and tissues of the body. Enzymatic oxidation galactosidase followed by reaction with Schiff's reagent is preferred as the method of carrying out the reaction the degree of color development for analysis of color in this invention. However, this invention is not limited. You can use any method that allows selective reaction, leading to the manifestation of color characteristics in the development of cancer. For example, for the manifestation of color you can use the direct reaction of the sample with Schiff's reagent without stage enzymatic reaction. In all cases spectrophotometric analysis revealed colors to determine the objective of setting tone or tone color, such as hue angle used in this invention, such parameters of color, has been found to correlate with the presence and degree of development of various types of cancer.

(ii) Test for cholesterol, conducted without invasion

In a preferred method of this aspect of the present invention using a liquid or semi-solid biochemical reagents, are in color, which is a measure of cholesterol in the skin of the patient, and subjected to manifest thus color spectrophotometric analysis. In accordance with this invention, the exact nature and essence, or tint thus obtained is Veta, characterized by, for example, the hue angle correlates with the amount of the formed relatively complex and, therefore, with the cholesterol in the skin. This measurement specifications color is objective and at least semi-quantitative. Accordingly, it is held regardless of the background colour, which does not affect this measurement to any significant degree.

Is usually convenient to put all reagents in the appropriate sequence to the skin surface of the patient, to carry out the act of painting on the surface of the skin, and then spectrophotometrically to explore cause the color complex, while he remains on the skin. The entire test can be performed in less than five minutes. The surface of the skin selected for the test must be largely free from the sebaceous glands, such as cancer bring holsteinsgade secret, which can distort the results. The soles of the feet and the palm of the hand are appropriate areas of the skin, and the palm of the hand is the most convenient for use in this test.

The kit includes a means for retaining and submitting forms a color complex for analysis using a portable spectrophotometer. These tools can be a container in the form of a stick ckage strips with one or more window, passing through it so that the reagents contained in the Windows, could communicate with the patient's skin. The design of the container will be determined largely by the physical characteristics of the spectrophotometer. Instead of using a container for inert reagents thixotropic agent may be added to the reagents to limit their distribution on the surface of the skin and to prevent mixing of the reagents for the test, control reagents, applied to the surrounding skin.

As in the case of other tests and trials on the basis of color, who are subject to this invention, a hallmark conducted without invasion test for cholesterol in accordance with this aspect of the invention is that the angle of the hue of the colored complex associated with cholesterol of the skin correlates with cholesterol in the skin.

Suitable chemical agents for use in accordance with this aspect of the invention are generally those described in the aforementioned patents Lopukhin and others, descriptions of which are incorporated here by reference. Their exact choice is not essential or restrictive characteristic of the present invention; their use in combination with each other should lead to chemical manifestation of the colors by the formation of ties with holes what Erin skin. The term "education connection" is used here in its broad sense of the course of chemical reactions with the addition of one chemical species to another, as well as specific interaction type "capture" (on the basis of affinity), which often occurs in biochemical systems.

Thus, the binding agent is chosen from the group of substances capable of selectively form stable complexes with the free cholesterol of the skin, in order to give to all a bifunctional compound in which it is included, the affinity for cholesterol. It can form a stable complex by direct reaction with cholesterol before or after it is chemically join visualizing agent directly or via multicourse agent C.

Representative classes of compounds suitable as binding cholesterol agent And include:

steroid glycosides, containing as aglycone cyclopentanoperhydrophenanthrene fragment rows of furostanol or spirostanol, and oligosaccharide fragment comprising from 3 to 10 monosaccharide residues with linear or branched structures (Hinta P.H. "Structure and biological activity of steroid glycosides series spirostana and furostana", Kishinev, shtiintsa, 1987, p.142), specific preferred examples of which are fungoides, D, E, F, G, and I, dioscin, Romsey C, D and E, anothony, digitonin and Tomatin;

triterpene glycosides containing aglycon series of alpha - or beta-Amarela, lupane, hopane, dammarane, lanostane or cholestane, and oligosaccharides containing sacharine remains a branched or linear structure (Dekanosidze G., f.chirva V.Y., Sergienko, T., Uvarov, D., "Investigation of triterpene glycosides", Tbilisi, back to text, 1982);

hydrophobic proteins able to selectively form a complex compound with cholesterol (Himov A.N., Titova, GV, Kozhevnikov HA, Biochemistry, 1982, CH, No. 2, s.226-232; Himov A.N., Kozhevnikov HA, N. Klueva. and other Questions Honey. Chemistry, 1984, CH, No. 3, pp.86-90; Titov, GV, Helleva N.N., Kozhevnikov HE and others, Biochemistry, 1980, Vol.45, No.1, p.51-55);

protein toxins, able to selectively form complex compounds with cholesterol. Get them from bacteria, marine organisms, insects or snakes (Dalin MV, fish N.G. "Protein toxins of microorganisms, Moscow, Medicine, 1980); or

polyene antibiotics, able to selectively form complex compounds with cholesterol (I.J.Katzenstein, A.M.Spielvogel, A.W.Norman, J. Antibiot, 27, 12, 1874, pp.943-951; Jong Shan Shyng, Wang Hsi-Hua, Clin. J. Environ., 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, substrate which is cholesterol, and which are not is have high affinity. All the above publications are included here by reference.

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

Visualizing agent is usually an enzyme, as especially useful are the reaction of the enzyme/substrate, resulting in color change. Specific examples of suitable enzymes include acetylcholinesterase, tyrosinase, glucose-6-phosphatedehydrogenase, glucose oxidase, glucoamylase, beta-D-galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin and pyrophosphatase. The preferred choice is a peroxidase, such as horseradish peroxidase (HRP).

Use mostcourageous agent improves the technical implementation of the method and makes it easier to obtain the final desired compound a-C-b, which can be a color, while the functional activity of the agents a and b is maintained. Most preferred complexes a-C-b are those in which use of steroid glycosides, containing as aglycone cyclopentanoperhydrophenanthrene fragment from the ranks of furostanol or spirostanol and fragments of oligosaccharide comprising from 2 to 10 monosaccharide residues with linear or branched structures, such as digitonin as having affinity for cholesterol forming mos the IR agent A. Especially, it is desirable to use multicourse the agent, if the linking cholesterol agent And selected digitonin, and as an imaging agent selected HRP, because HRP is a relatively large molecule, which, if it were directly connected with digitonin could sterically hinder the reaction of digitonin with cholesterol skin. As mostcourageous agent for such purposes it is preferable to use high molecular weight polyfunctional compounds. Their use allows a wide range to adjust the ratio of agents a and C in the final complex. Such high molecular weight polyfunctional multicourse agents can be different polysaccharides, proteins or synthetic polymers, that is, any suitable high molecular weight compounds containing the functional group of the primary amine, carboxyl, hydroxyl, aldehyde, galodamadruga, mixed anhydrides, aminoether, azide, hydroxide, maleimide, isocyanate or epoxy group. The most preferred high molecular weight polyfunctional multicourse agents are copolymers of acrylic acid or maleic acid or maleic anhydride and N-vinylpyrrolidone. You can also use asymmetric bifunctional connected to the I with a low molecular weight, such as brazian, trichlorotriazine or 2-amino-4,6-sodium dichloro-3-triazine.

Agent-indicator D typically contains a substrate of the enzyme used as a visualizing agent, and additional connections needed to make the reaction between the enzyme and substrate visible. A concrete example of such an agent-indicator D when using the enzyme peroxidase as a visualizing agent is an agent containing hydrogen peroxide, N,N-diethyl-p-familienzelt together with appropriate stabilizers. Agent-indicator D is chosen in combination with the imaging agent of a number known in the field of connections that will be given for the formation of color in combination with the selected enzyme.

Offered a kit for carrying out the test according to this invention. This kit contains the necessary reagents in appropriate closed containers, such as vials or bottles with dropper, container or other means of restraint, in which it is possible to conduct the reaction of formation of color on the patient's skin in such a way as to prevent the spread of reagents on too large a surface, and of which the resulting color may be submitted for research and measurement tools for defining and obtaining data on the specific nature of the statistics of a painting, such as the angle of hue, such as portable reflectance spectrometer. This container is usually a sticky tape provided with one or more cut out Windows, a source provided with a protective layer on the back side to protect the adhesive layer. Preferably the container has at least two or three Windows, so that during the test you can conduct controlled experiments. In order to facilitate the proper conduct of the experiments at the test and control experiments, these Windows are usually made different in appearance from each other, for example they have different forms.

Figure 1 shows such a container for use in this invention in the form of strips 10 for the test, having a rectangular shape. This strip includes a porous base 10 with the layer 12 is compatible with the skin adhesive material temporarily protected peelable film 14. The first Central window 16 for testing purposes, having a round cross-section, passes through the porous substrate strips and through the adhesive layer 12. The second window 18 for the positive control, having a rhombic cross-section, and a third window 20 for the negative control, having a square cross-section, similarly done in an expanded bottom strips 10 on each side of the Central window 16. Various on the form view window help the operator in carrying out the AI tests, contributing choosing the right window for the relevant purposes.

The test is carried out preferably on the skin of the palm of the hand of the patient. Container with reagents having the kind of adhesive tape, for a time is placed on the skin so that the bottom of the window in contact with the skin. The reagents are falling in the window occurs in the Windows color and then the color is determined by the spectrophotometer without removing the strip from the skin. For this purpose apply the specially designed spectrophotometer, which is another aspect of the present invention. The spectrophotometer passes the received data to a computer for analysis. The spectrophotometer is designed in such a way as to ensure proper placement on top of the test cell.

Accordingly, in this aspect of the present invention proposed a spectrophotometer adapted for transmitting signals, the measured color in the reflected light from the test sample on the computer, and this spectrophotometer has a body located in the housing means of light radiation, the lower part of the body with a hole through which you can direct the light emitted by the tool, and the recess on the bottom surface of the specified lower part, adapted so that the cover strip to test with Windows placed on the skin surface of the patient, on whom I ensure accurate compliance with the provisions of the spectrophotometer and determined samples in the window specified strips with Windows for the test. Preferably the lower part of the spectrophotometer attached to the housing on hinges so that it can appropriately be set to the correct registration of the strip for the test in the open position, and then attach to the body of the spectrophotometer for measurement. Preferably also, so that this flip hinged at the bottom part and the housing of the spectrophotometer was equipped with electric contacts, acting as a switch to turn the light source of the spectrophotometer when folding the bottom part attached to the body of the spectrophotometer.

In figure 2, 3 and 4 schematically depicts a spectrophotometer. He has a body 22 with an electrical connection (not shown) with a computer programmed accordingly, for the analysis of the results obtained by the reader. Has a bottom portion 24, at position 26 is attached in a hinged connection to the housing 22. The lower portion 24 has a hole in position 28. Width of the bottom surface of the lower part 24 passes the groove 30, which protrudes from the bottom surface up. The groove is closed on one side of the bottom. The width of the groove 30 is made so that it accurately and strictly complies with the strip 10 to perform the test, depicted in figure 4. When should carry out the measurement, the end strips for PR is doing test 10 corresponds to the closed end of the groove, and this in combination with the placement of the strips to test 10 within the width of the groove 30 provides an exact correspondence box 16, where test is performed with a beam of light that should be emitted from the housing 22 the reader through the hole 28. The spectrophotometer contains the appropriate detector device for selection of sample box 16 of the reflected signals and send them for analysis and interpretation of the computer. The housing 22 and lower portion 24 provided corresponding to each other of the electrical contacts 32, 34, which closes the switch when the lower part 24 is attached to the housing 22, including the light for the measurement.

Next will be described a specific test procedure in the form of a specific but not limiting example of the practice of diagnostic test according to this invention.

The components of the kit include a bottle with dropper containing solution of the detector (the connection of digitonin and horseradish peroxidase in aqueous buffer solution containing bromonitrodioxane and methylisothiazolone with a concentration of less than 0.01% as stabilizers, 1.5 ml), with brightly coloured cover (green); the bottle with dropper with a more concentrated solution of the reagents of the detector, in the same buffer solution and stabilized less than 0.01% bromonitrodioxane and methylisothiazolone as POS is the positive control (1.5 ml), with brightly coloured cover (red); the bottle with dropper containing the indicator reagent solution (4,0 ml 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide with 5% N,N-dimethylformamide as a stabilizer) for reaction with the detector and the RS-reagents, which are connected with cholesterol in the skin, with the formation of a bluish-green color with a brightly coloured cover (blue); foam substrate, as shown in figure 1, which can be submitted reagents, alcohol swabs, and the corresponding user manual. These chemical reagents are stable when stored in the refrigerator at 2-8°for a long time. The system also includes a spectrophotometer, as shown in figure 2 and 3, coupled with an appropriately programmed computer, and records for calibration for use with spectrophotometer. Set in as it comes, does not include a spectrophotometer, except, apparently, the case of the initial sale; the same spectrophotometer is used again filled with subsequently set.

First spectrophotometer appropriately calibrated by inserting the calibration plate in its clamping device and closing the bottom cover to illuminate the calibration plate and to obtain data for calibration of the spectrophotometer and the computer is Ter. Given the signal that the calibration is successfully completed.

The arm of the patient is thoroughly washed with soap and water, rinse and well dried. Then the outer side of the palm of the patient is carefully cleaned with a swab with alcohol with sufficient force to ensure thorough cleaning. After the hand is dry, remove the protective film 14 with strips to 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 it on the table, and tightly presses to ensure reliable attachment of the substrate to the palm.

Then add reagents in the appropriate box for reagents. One drop (42 μl) solution of the detector add in a circular window for test 16, one drop of the solution for positive control, add in a diamond-shaped window 18, and the third, a square window 20 at this stage the liquid is added. Added the solution was incubated for 1 minute; while the patient holds the arm on which the test is run, motionless. The patient then turns over his hand and presses the substrate of foamed material to a paper towel to remove fluid from the Windows. Visually check that the substrate and the Windows for the test were completely dry. The patient then leaves the hand on a flat surface with the palm up.

Undertake a visual inspection of the window a positive control 18 and the window negative control 20. If the liquid in the box to the negative control colourless, and the liquid in the window of a positive control is painted, then the test is carried out correctly. The color formed when the positive control, no quantitative measurement is not performed. This colour is obtained at a very high concentration of the reagent solution, which gives the color of even very small amounts of cholesterol on the skin; it is simply an indicator of the health of reagents and is used for other control purposes.

Remaining on the strip for the test liquid is removed and the strip for the test are removed from the palm, followed by purification of the palm swab moistened with alcohol.

Currently used spectrophotometer preferably measures absorbing the Yu ability reflected from the test sample light and converts it by an appropriate algorithm in the value of hue angle. In the specific case of obtaining a color by the reaction of horseradish peroxidase - TMB described above, suitable conditions of measurement is the absorption at 450 nm And450nm. Experimentally determined optical density absorptive capacity at 450 nm to associate it with the hue angle by ratio:

h°(C)=490,45·And450 nm+57,124

This ratio is determined by measuring the optical density of the number of serially diluted reaction samples at 450 nm, the angle measurement of the color tone of the same samples and build on the results of the regression curve to determine dependencies. A similar dependence can be obtained experimentally in the same way and for other selected tests, during which the formed coating made using various pairs, enzyme - substrate, giving a variety of colors that allow you to convert readings of optical density in the value of the angle of hue.

Although the above were the specific diagnostic tests and kits for their conduct, the expert can understand that a large number of color-based tests and analyses can be improved by using new measurements described here, based on the angle of the hue or color saturation, for example:

(i) obtaining quantitative results is the ATA when the solid phase immunological tests, such as determining the spot, lateral flow tests and tests with a penetrating sample (the sample is applied to the membrane with absorbing device for it; then an analyte is captured and identified by adding a labeled detector, such as an antibody labeled with an enzyme or gold);

(ii) obtaining quantitative results in test-based microparticles, using colored beads;

(iii) alternatively, densitometry in the analysis of stained gels;

(iv) to obtain quantitative results when the analysis is spot on Western y.

The method according to this invention provides a simple approach to quantify these results, thereby complementing the information obtained during tests, the possibility of applying the results of the tests are more advanced methods of statistical analysis.

Accordingly, variations of the present invention should not be regarded as a departure from the essence and scope of the claims, and all such modifications as obvious to the specialist, should be included in the scope of the claims.

1. The method of analysis of a sample of biological material for biochemical or immunological tests on any designated substance, comprising the following operations:

the treatment of the sample, PR is which produces a color, corresponding to the amount of analyte in the sample;

the measurement of the angle of the hue or color saturation of the resulting color by means of suitable spectrophotometer or other equivalent device, and

analysis of measurements of the angle of the hue 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 the biological material is a concentrated liquid or semi-solid secrets of the body, taken from the patient-person to diagnose the presence of deviations from the norm, and specified the designated substance consists, essentially, in this sample, markers, indicating the presence of cancer, and measured the angle of the hue or color saturation is used to classify the sample as being normal or abnormal.

3. The method according to claim 2, where these secrets are mucus from the lungs, the mucus from the throat, the mucus from the cervix of the uterus, seminal fluid or liquid selected from the nipple of the breast.

4. The method according to claim 2 or 3, where the specified pattern to be applied on the white overall basis, and the specified processing to form color involves the reaction with the enzyme.

5. The method of analysis of a sample of biological material according to claim 1, where

specified brezec biological material is in contact with colon semi-solid sample, selected patients to diagnose for the presence of anomalies;

the specified designated substance consists essentially of markers, indicating the presence of anomalies;

the operation of the treatment sample includes applying this sample, white in General, the substrate and receiving the color of this sample by enzymatic reactions and

the measured angle of the hue or color saturation is used to classify the sample as being 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 glass.

9. The method according to claim 5 or 6, where the substrate is, in essence, is pure white.

10. The method according to any of pp.5, 6, or 8, where specified in contact with colon semi-solid sample is essentially rectal mucus.

11. The method according to claim 7, where the specified contact with colon semi-solid sample is essentially rectal mucus.

12. The method according to claim 9, where the specified contact with colon semi-solid sample is essentially rectal mucus.

13. The method according to claim 1, where the specified sample of biological material is the skin surface of the patient is and, as indicated detectable substance is cholesterol skin.

14. Set for analysis of liquid or semi-solid samples of the secrets of the body obtained from patients-people for diagnosing the presence or absence of abnormality in a patient, by determining the angle of the hue or color saturation for the color shown on the model, and this system includes

white, not containing cellulose substrate with porous "grainy" surface for receiving and holding the sample during manifestations;

source galactosidase adapted for applying galactosidase on the surface of the substrate for holding it selective enzymatic oxidation of the sample;

the source of the Schiff reagent adapted for the application of Schiff's reagent for the oxidized sample on the substrate for the manifestation of it is suitable for the analysis of painting; and

device for the presentation of the sample shown in color on a portable reflectance spectrophotometer capable of determining the angle of the hue and color saturation, characterized by the color of the painted designs on the specified substrate.

15. The test kit contact with the semi-colon samples obtained from patients-people for diagnosing the presence or absence of rectal anomalies in a patient, including the surrounding

in General white, not containing cellulose substrate for making sample;

the source of the Schiff reagent and

portable reflectance spectrophotometer capable of determining and displaying information and data on a corner hue and color saturation characterizing color-treated samples on the specified substrate.

16. Set on 15, where the specified substrate is glass.

17. The method for determining cholesterol in the skin of the patient, including

drawing on the skin surface of the patient reagent that selectively binds to cholesterol in the skin;

chemical reactions are manifestations of the named color with the reagent associated with cholesterol of the skin, with the formation of the colored complex and

holding spectrophotometric analysis of the thus obtained colored complex to obtain the values of the angle of the hue or color saturation, characterizing the level of cholesterol in the skin.

18. The method according to 17, where the specified reagent that selectively binds to cholesterol in the skin, selected from the group consisting of

(i) steroid glycosides, containing as aglycone cyclopentanoperhydrophenanthrene fragment number of furostanol or spirostanol, and fragments of oligosaccharide comprising from 3 to 10 monosaccharide residues of the line is passed or branched structures,

(ii) triterpene glycosides containing aglycon series of alpha - or beta-Amarela, lupane, hopane, dammarane, lanostane or cholestane, and oligosaccharides containing sacharine residues with branched or linear structure,

(iii) hydrophobic proteins able to selectively form a complex compound with cholesterol,

(iv) protein toxins, able to selectively form complex compounds with cholesterol,

(v) polyene antibiotics, able to selectively form complex compounds with cholesterol, and

(vi) enzymes, a substrate which is cholesterol and which exhibit high affinity for cholesterol, and

in which the specified education of the colored complex is achieved by processing the specified coupling agent on the surface of the skin first imaging agent, then the agent indicator.

19. The method according to p where education specified colored complex is achieved by sequential processing of the specified binds cholesterol agent on the surface of the skin masticables agent, imaging agent and agent-indicator.

20. The method according to p or 19, where the specified binds cholesterol agent is digitonin.

21. The method according to p or 19, where the specified imaging agents who ω is the enzyme peroxidase, and the specified agent-indicator contains hydrogen peroxide and N,N-diethyl-p-familienzelt together with appropriate stabilizers.

22. The method according to claim 20, where the specified imaging agent is the enzyme peroxidase, and the specified agent-indicator contains hydrogen peroxide and N,N-diethyl-p-familienzelt together with appropriate stabilizers.

23. Kit for determination of cholesterol in the skin of patients-people, including

the source agent detector, capable of binding cholesterol of the patient's skin with the formation of the skin associated combinations;

source imaging agent that can form a relationship with an associated combination of agent-detector - binding agent with the formation of the optically modified complex;

source manifesting agent and means for applying manifesting agent on the modified optical system for manifestation in it of color and

device for keeping the specified optically modified complex and to represent it in a portable reflectance spectrophotometer for determining the angle of the hue or saturation of color that characterize the apparent color.

24. Set in item 23, where said device to hold the optically modified complex and represent it in the spectrophotometer includes the container in the form of adhesive to the skin strips, having at least one window, through it, to hold the specified window reagents in contact with the patient's skin.



 

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FIELD: pharmacology.

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6 cl, 8 tbl, 6 ex, 1 dwg

FIELD: bioassay methods.

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8 cl, 1 dwg, 3 tbl

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2 ex, 1 tbl

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3 tbl, 2 ex

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1 tbl

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1 tbl

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2 ex, 2 tbl

FIELD: veterinary science.

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EFFECT: higher accuracy and efficiency of diagnostics.

1 ex, 1 tbl

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4 cl, 1 tbl, 3 ex

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EFFECT: higher accuracy of diagnostics.

3 ex

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