HK1053702B - Spectrophotometric measurement in color-based biochemical and immunologigal assays - Google Patents

Description

Spectral measurement in color-based biochemical and immunological assays

Technical Field

The present invention relates to color-based biochemical and immunological assays and tests in which an analytical or test sample is subjected to spectral measurement of color characteristics, particularly hue angle and/or chroma. Assays using such measurements have proven useful in a wide range of drug testing and screening procedures and diagnostic methods to produce quantitative or semi-quantitative results.

Background

As described in detail below, many diagnostic tests rely on visual inspection and evaluation of the color produced by treating a sample of biological material with an agent that produces a color that is positively correlated with the amount of analyte, i.e., the specific compound tested (e.g., cholesterol) or a specific molecular marker present in the sample that is indicative of a disease (e.g., cancer).

Assays requiring visual inspection and assessment of color change are convenient and generally suitable for preliminary subjective assessment of the presence of target compounds or disease markers, but they are not essentially quantitative.

I have surprisingly found that measuring hue angle and/or chroma and related characteristics using reflectance spectrophotometry yields an at least semi-quantitative analysis. From this measurement of one or more defined characteristics of the colour of the analysed sample, a number of valuable information can be obtained as to the presence or absence of progressive diseases (such as cancer) and the stage of development of these diseases.

Color-based cancer test

It is known from us patent 5,162,202 to examine rectal mucus in human patients for the detection of rectal cancer and carcinoma of the large intestine. The mucus was collected on a filter. The cellulosic filter membranes were pretreated by soaking with a solution of galactose oxidase in phosphate buffer and then freeze-dried. In application, the fibrous filter membrane is wetted and then contacted with the filter membrane loaded with mucus sample for 1-2 hours. The filter membrane loaded with mucus was then washed, reacted with basic fuchsin for 15 minutes, washed and dried. Decolorization of fuchsin indicates the presence of carbohydrate markers in the mucus for cancerous or pre-cancerous conditions. Such tests are time consuming and cumbersome and do not have high sensitivity and may therefore give false negative results.

U.S. Pat. No. 5,348,860 to Shamsuddin (20/9 1994) discloses an improved rectal mucus test. In this method, a mucus sample is collected, immobilized on a filter, and treated with galactose oxidase to oxidize any ortho galactose moieties in the sample to ortho aldehyde moieties. This oxidative transition can be visualized with Schiff's reagent. This is a faster method. Samples that were negative in this way could be further oxidised with periodic acid and then visualised with Schiff reagent to reduce the likelihood of a false negative result.

A continuing problem with known mucus tests is the need to visually inspect and evaluate the staining results. While this test is sufficient as a preliminary subjective assessment of the presence or absence of cancer markers, it is still only qualitative. Such tests do not give reliable quantitative information about the amount and concentration of the marker (which is found and, if present, is indicative of the progression of the cancer). In addition, the medium in which the sample is cultured is usually a fibrous membrane (e.g., filter paper), which itself may contain a compound participating in the color reaction. This creates a "background" that complicates interpretation of the assay results, thereby reducing the sensitivity of the assay. This requires a skilled technician who is highly experienced in interpreting the results of the test.

Color-based analysis of cholesterol content

It has been firmly recognized that high serum cholesterol levels in patients are associated with a propensity to develop atherosclerosis and an increased subsequent onset of coronary heart disease, stroke and PVD, and therefore frequent monitoring of the cholesterol levels in patients is required. Typically, the cholesterol content is determined using an extracted blood sample. Many other clinical trials have also been conducted with extracted blood samples, but most of these trials require longer time intervals to be conducted than cholesterol analysis. Many patients are reluctant to receive frequent cholesterol monitoring on demand due to the invasive nature of the blood collection procedure used for cholesterol analysis. There is therefore a need for a non-invasive cholesterol test.

It is estimated that skin contains about 11% of total body cholesterol, mainly from epidermal steroids and cholesterol that diffuses out of blood vessels. It has been hypothesized that levels of skin cholesterol may be more accurate than levels of serum cholesterol in reflecting the degree of atherosclerosis.

Nikitin, YP, Gordenko, i.a., Dolgov, a.v., and Filimonova, t.a., "cholesterol level in the skin and its correlation with lipid coefficients in serum of healthy persons and patients with ischemic heart disease", Cardiology 1987II, No.10, pages 48-51, and other documents show a close correlation between cholesterol level in arterial walls and cholesterol level in skin of patients. This indicates that it is possible to develop skin tests to determine the cholesterol level of a patient. The method described by Nikitin et al, which involves in vitro excision and analysis of skin samples, is impractical for clinical use.

U.S. Pat. Nos. 5,489,510 and 5,587,295 to Lopukhin et al describe a non-invasive diagnostic method performed on the skin of a patient that is indicative of skin cholesterol levels. In the methods described in these patents, the reagents (which are bifunctional in nature) are provided in the form of affinity-enzymatic compounds. These bifunctional compounds A-B comprise a binding agent A which forms a stable complex with cholesterol on the skin with resolution, thus allowing the entire bifunctional compound to have an affinity for cholesterol (e.g. digitonin); also included is a visualization agent B, for example an enzyme such as peroxidase, which can detect bifunctional compounds that bind to skin cholesterol. In performing the test, the complex of binding agent A and visualization agent B, possibly together with bridging agent C to enhance the sensitivity of the test, i.e., bifunctional conjugate A-C-B, may be placed on the skin of the palm of the patient's hand. The bridging agent C is a suitable high molecular weight multifunctional compound such as a polysaccharide or protein that acts to separate the visual agent from the binding agent to minimize steric hindrance of the cholesterol-binding agent reaction. After a suitable incubation time to ensure that the complex binds to cholesterol in the skin, the palm area is thoroughly washed with clear water to remove unbound reagents. The bound site is then treated with indicator D and developed by reaction with visual agent B. The higher the content of cholesterol, the greater the degree of binding of the bifunctional compound to the skin and thus the higher the degree of color development.

The cholesterol assay based on the aforementioned Lopukhin et al patent has been successfully studied commercially and has entered commercial practice. It includes providing a kit containing reagents and a color chart or readout meter. The vial contains most of the reagent and after removal of the protective cover, the user places it on the test site on the palm of the hand. After incubation, the user applies the indicator and visually observes the color change occurring next to the color chart or with a reading meter.

One drawback of this test is the need to visually observe the color change. Although this test is convenient and can be performed by inexperienced persons such as patients, the visual inspection of the resulting color change is subjective and essentially non-quantitative. Such tests may give valuable indications of cholesterol levels and potential disease problems, but are not a quantitative determination of the type of method typically required by physicians. This method is susceptible to the characteristics and color of the background (i.e., skin).

It is a general object of the present invention to provide a novel method of determining and measuring color-based biochemical or immunological assay results that does not rely on subjective visual inspection. It is another more specific object of the invention to provide a method for quantitatively measuring the amount of a target compound in a biological sample that has been subjected to biochemical or immunological analysis to produce a color that is positively correlated with the target compound.

It is another object of the present invention to provide a novel assay method for body secretions, liquids, and semisolids for use in cancer diagnosis and a kit for use therein.

It is a further object of the present invention to provide a novel method of testing rectal mucus and other secretions, liquids and semi-solids including feces and mixtures thereof and kits for use therewith which overcome or at least substantially reduce one or more of the above-mentioned disadvantages. In the following description, the term "semi-solid contained in the colon" refers to mucus, stool and other liquids or semi-solids obtained from the rectum or colon of a patient and mixtures thereof, which can be used as analyzable material in the method of the present invention.

It is another object of the present invention to provide a novel non-invasive diagnostic method for cholesterol.

It is another and more specific object of the present invention to provide a cholesterol assay method that provides at least semi-quantitative results.

Summary of The Invention

To overcome the disadvantages of the prior art diagnostic tests based on the visual requirement for color change, the present invention uses in its broadest aspects certain specific parameters that are measurable by a spectrophotometer, but which are not previously used by the present invention. Detection and analysis of these colorimetric parameters can provide assays and diagnostics with high sensitivity and specificity. Measuring the color developed in the assay or test at various wavelengths and measuring the hue angle and/or chroma provides valuable information on the presence or absence of the target compound, disease condition or other condition, which is the goal of the color-based assay or test. In some cases, measurement of a relevant color characteristic, such as lightness or saturation, may further improve the sensitivity of the assay or assay.

In another aspect of the invention, there is provided a method of diagnosing a liquid or semi-solid sample secreted by a patient's body in order to identify evidence of abnormalities in a tissue or organ secreting such material, said method comprising collecting a sample of the liquid or semi-solid secretion from the patient, placing at least a portion of the sample on a generally white substrate, staining the sample on the substrate, allowing it to develop a color, measuring a defined color characteristic of the developed sample with a spectrophotometer, and identifying the sample as normal or abnormal according to the value of the particular color characteristic obtained.

In another aspect of the present invention, there is provided a system for analyzing a liquid or semi-solid secretion sample from a patient, measuring specific color characteristics exhibited in the sample to diagnose the presence or absence of abnormalities in the patient, the color characteristics selected from hue angle, chroma or saturation and lightness, said system comprising:

a white non-fibrous substrate having a porous 'creped' surface for placement of a sample during development;

a source of galactose oxidase for placing galactose oxidase on a surface of a substrate for selective enzymatic oxidation of a sample thereon;

a source of Schiff reagent for adding Schiff reagent to the oxidised sample on the substrate to effect development of the analyte;

placing the developed sample in a portable reflectance spectrophotometer device capable of measuring and recording specific color characteristics selected from hue angle, chroma or saturation and lightness of a dyed sample on the substrate.

In a further aspect of the invention there is provided a method of diagnosing a semi-solid sample exposed to the colorectal region of a patient to provide evidence of abnormality in the patient, said method comprising collecting a semi-solid sample exposed to the colon from a patient, placing at least a portion of the sample on a white substrate, staining the sample on the substrate with galactose oxidase, developing the stained sample with Schiff's reagent, measuring the colour characteristic of the developed sample with a spectrophotometer, and identifying the sample as normal or abnormal by the value of the particular colour characteristic obtained.

In another aspect of the present invention, there is provided a kit for analyzing a liquid or semi-solid secretion sample from a patient to diagnose the presence or absence of a rectal abnormality in the patient, said kit comprising:

a generally white, non-cellulosic substrate for receiving a sample;

a source of galactose oxidase;

a source of Schiff reagent;

a portable reflectance spectrophotometer capable of measuring and recording a specified color characteristic selected from hue angle, chroma or saturation and lightness of a stained sample on said substrate.

Another aspect of the invention provides a test method in which a liquid or semi-solid reagent is placed on the skin of a patient to bind skin cholesterol and then the reagent is developed in color to a degree directly related to the amount of cholesterol in the skin. The degree of development is measured by analyzing the developed liquid or semi-solid reagent by colorimetric means rather than by visual inspection, so that the cholesterol content can be obtained at least semi-quantitatively. The colorimetric parameters selected, such as hue angle or tint, are independent of color density, light intensity or lightness (L) and are merely a measure of tint. This substantially eliminates the uncertainty introduced by the background color of the skin, so that such tests can be performed on the surface of the patient's skin. Instrumental colorimetric (spectrophotometric) analysis can yield at least semi-quantitative objective data indicative of the cholesterol level of the patient.

In another aspect of the present invention, there is provided a method of measuring the cholesterol level of skin of a patient, the method comprising:

applying to the skin of the patient an agent that selectively binds skin cholesterol;

allowing the formed skin cholesterol-binding agent to undergo a chromogenic chemical reaction to form a colored complex;

the colored complex thus formed is subjected to spectrophotometric analysis to read a predetermined characteristic of the color of the colored complex.

Another aspect of the present invention provides a kit for measuring skin cholesterol levels in a patient, said kit comprising:

a source of a detector agent capable of binding to cholesterol on the skin of the patient to form a conjugate on the skin;

a source of a target test agent capable of reacting with the detector-binding agent conjugate to form an optically altered complex;

a source of a colour developing agent and means for applying the colour developing agent to the optically altered complex to develop the colour;

means for receiving and providing said optically altered compound onto a portable reflectance spectrophotometer for determining a color characteristic selected from hue angle, chroma or saturation.

Brief Description of Drawings

In the following description of a preferred embodiment of the invention, which relates to the parts of the method and kit described above in relation to measuring the cholesterol level in the skin of a patient, reference will be made to the accompanying drawings. In the drawings:

FIG. 1 is a schematic representation of a test strip of the present invention for measuring cholesterol levels in a patient's skin;

FIG. 2 is a schematic of a spectrophotometric reader of the present invention for measuring skin cholesterol levels in an open state;

FIG. 3 is a view similar to FIG. 2, but with the spectrophotometer in an off state;

fig. 4 is a detailed view of the spectrophotometer mount shown in fig. 2 and 3.

Description of The Preferred Embodiment

As mentioned above, the applicant's basic invention, i.e. the measurement of the chromaticity and/or hue angle using colorimetric measurements, in particular color-based analysis, can find application in many ways and examples. These two aspects of the invention and some of the preferred embodiments are described in detail below, in separate headings.

Spectrophotometers suitable for use in all aspects of the present invention are portable, reflection-based, and allow for the accurate measurement of color characteristics (e.g., hue angle, lightness and chroma or saturation) as the incident light from the spectrophotometer is reflected from a stained sample back to the receiver of the instrument. Such instruments are commercially available. A preferred example of a suitable instrument is a "model CA22 spectrophotometer" from X-Rite, Grand Falls, Michigan, U.S. A. Suitable software attached thereto may be connected to a computer to give an accurate reading of the hue angle of the dyed sample tested. The spectrophotometer accepts reflections of the majority of the visible spectrum in the wavelength range of about 400-700nm, suitably spaced at about 20 nm.

It is known that colors can be defined and represented in hue angles. The definition and discussion of the concept of "color angle" can be found in standard textbooks, such as the "principles of color technology" by Fred w. billmeyer and Max Saltzman, published by John Wileyand Sons (see in particular chapters 1 and 2 thereof), which are incorporated herein by reference. "hue" is the color or shade of a sample independent of its lightness or light intensity, and the "hue angle" of a color or shade is defined as its reflected wavelength in angular coordinates over a standard three-dimensional elliptical continuum of the complete spectrum of visible light. The continuous spectrum of visible light (color) is represented on a scale ranging from 0 to 360 ° in angle. The angle values read on the reflectance spectrophotometer are converted to a linear form, resulting in a converted "color angle" for use in the method of the invention.

(i) Cancer test

It has surprisingly been found in the present invention that by measuring the hue angle developed from liquid or semi-solid secretions of appropriate human tissues or organs or other defined color characteristics as described above, the presence or absence of a wide range of disorders can be determined including bowel disorders, pulmonary disorders, uterine disorders, and the like. Rectal mucus response and spectrophotometric analysis can be used to diagnose colon cancer. Thus, a rectal mucus sample from a person with rectal cancer will, after said staining and developing, have a higher hue angle value than that obtained for normal bowel. The hue angle or other defined color characteristic of the stained sample can thus be used to distinguish cancerous lesions with bowel from those without the lesion. In particular, it has been found that the color angle of cancer lesion samples is typically in the 375- & 425 ° range, which is the top quartile of clinical sample determination. In addition, since the test results are analyzed with a portable spectrophotometer, a technician is not required to read the test results.

In a similar manner, lung cancer and pre-lung cancer symptoms can be diagnosed by subjecting lung mucus or sputum to similar color development and spectrophotometric analysis. Such tests on cervical mucus can diagnose cervical cancer and precancerous conditions. Seminal vesicle secretions, such as semen, can also be similarly analyzed for cancer of reproductive organs, such as the testes. Throat mucus can also be similarly analyzed to detect and diagnose laryngeal cancer. Mucus from the throat and lungs can be obtained by known methods, such as bronchoscopy or bronchioloalveolar irrigation. Nipple aspirates are bodily fluids that can be used to similarly test breast cancer with the methods of the invention.

Another significant aspect of the preferred embodiments of the present invention is the use of a porous glass fiber membrane upon which the sample can be oxidized and developed. This glass fiber material produces substantially no residues of dyeing and therefore no residues which can participate in the subsequent color development reaction when subjected to enzymatic oxidation. Thus, background coloration that may confound or interfere with diagnostic tests is effectively eliminated. In addition, the color of such a film is substantially pure white, thereby further reducing background "interference" in reading the results.

Another property of the fiberglass membrane used in the present invention is its surface porosity, so that the mucus sample thereon can be better spread, thereby exposing additional carbohydrate markers in the sample to participate in oxidation and color reactions, thereby improving the sensitivity of the assay method.

Another preferred example of a glass fiber porous membrane for use in the present invention is a borosilicate glass membrane media commercially available from Whatman inc. Larboratroy Division under the trade designation "Whatman 934-AH glass fiber membrane" which has high loading capacity and high retention efficiency at high flow rates. It is recommended to use it for cell harvesting and liquid scintillation counting methods. However, this is merely an example and other substantially pure white glass fibers, carbohydrate-free, semi-solid samples with surface porosity suitable for contacting the colon, may be used for the surface-spread substrate.

In a particular step with a preferred aspect of the invention, for colon cancer detection and visualization with enzymatic oxidation, the sample to be tested is taken from the patient. The lubricant is applied to the gloved fingers of the operator. A finger is inserted into the rectum of the patient and rotated 360 ° to obtain a representative semi-solid (e.g., rectal mucus) sample that contacts the colon. After removing the finger from the rectum, the sample was spread on the surface of a white filter, mounted on a rectal mucus test card with appropriate coverings, protections and identifiers, and the card was analyzed.

For analysis, the backing was removed from the rectal mucus test card and 50 μ l of a standard galactose oxidase solution was added to the test card. Incubate for 10 minutes using standard methods. The card was then soaked in double distilled water for 30 seconds and then 1ml of Schiff reagent was added for 3 minutes. The cards were then rinsed in water for 10 minutes 4 times for color development. The card is then dried and the hue angle is read using a portable spectrophotometer of the type described above to determine the score. A hue angle score below a predetermined value (350 for a straight intestinal mucus sample) is indicative of a normal healthy tissue source. A score above a predetermined value (370 for rectal mucus) indicates a source of cancerous tissue.

While samples giving intermediate values may also be subjected to comprehensive oxidation to aid in the final diagnosis. Based on the knowledge that only a fraction of the vicinal hydroxyl groups on the carbohydrate labels in any sample can be enzymatically oxidized to develop color, all remaining groups can be oxidized with a strong oxidizing agent (e.g., periodate), and then developed again and tested. If the difference from the initial result is large, it indicates that the sample should be classified as having a high initial value. Samples can be safely classified as having a lower score if there is little or no significant difference.

By simply reading the color angle value from the spectrophotometer by the method, an operator can determine whether the colon of a person from which a sample is taken is healthy and normal or has cancer without subjective judgment and analysis, and compared with the existing diagnosis method, the method greatly reduces the probability of false positive and false negative. The novel method of the invention can be employed in diagnostic laboratories with minimal interference and economic expense, taking the same standard sampling, staining, culturing and chromogenic steps and using the same standard reagents.

Other mucus samples from human organs and tissues may also be tested in a substantially similar manner. Enzymatic oxidation of galactose oxidase and subsequent reaction with Schiff reagent is a preferred chromogenic reaction step according to the present invention. The invention is not so limited. Any procedure that gives a selective response to the color development characteristics of cancer may be used. For example, direct reaction of the sample with Schiff's reagent (without an enzymatic reaction step) may also be used for color development. In all cases, the use of spectrophotometrically analyzing the displayed color to measure the shade of the color or an objective parameter of the hue, such as hue angle, constitutes a use of the invention. These color parameters described above have been found to be associated with the presence and progression of various cancers.

(ii) Non-invasive cholesterol assay

In a preferred procedure in this aspect of the invention, a liquid or semi-solid biochemical reagent is used to display a color that is dependent on the cholesterol content of the patient's skin, and the displayed color is analyzed spectrophotometrically. In the present invention, the precise nature and character of the displayed color (as characterized by hue angle) is related to the amount of bound complex formed and thus to the cholesterol content of the skin. This measurement of color properties is objective and at least semi-quantitative. Thus, its reading is independent of the color of the background skin, i.e., the background skin color has no significant effect on it.

It is convenient to apply all of the agents to the skin surface of the patient, usually in the correct order, develop the colour on the skin surface and then examine it with a spectrophotometer while the developed complex remains on the skin. The entire test can be completed in 5 minutes. The area of skin selected for the test should be an area substantially free of sebaceous glands, which would provide serum containing cholesterol, which would interfere with the test results. The sole and palm of the foot are such suitable skin areas, with the palm being the most convenient for the test of the present invention.

The kit includes a means for containing and providing a chromogenic complex for analysis with a portable spectrophotometer. Suitably, the container is in the form of a tape which is capable of adhering to the skin and which is provided with one or more through holes so that the agent contained in the holes may be brought into contact with the skin of the patient. The design of the container is determined primarily by the physical characteristics of the spectrophotometer. Alternatively, instead of a container for the reagent, a thixotropic agent may be added to the reagent to limit spreading of the reagent on the skin surface and prevent mixing of the test reagent with the control reagent (applied to the adjacent skin site).

The invention is also applicable to other color-based assays and analyses, another aspect of the invention is a non-invasive cholesterol assay, where the hue angle of the color complex bound to skin cholesterol is correlated to skin cholesterol content.

Suitable chemical agents that can be used in the present invention are those described in the aforementioned Lopukhin et al patent, the contents of which are incorporated herein by reference in their entirety. The precise choice of these agents is not a necessary or limiting feature of the invention, provided that these agents, when used in combination with each other, develop color due to binding to skin cholesterol. The term "binding" as used herein refers to the interaction (typically occurring in biochemical systems) of the broad meaning of chemical reaction adsorption and specific affinity type "engagement" that will attach one chemical species to another.

Thus, the binder a is selected from substances that form a stable complex with free cholesterol on the skin with discrimination, so that a fully bifunctional compound with affinity for cholesterol can be obtained. It can react directly with cholesterol to form a stable complex either directly or after it is chemically bound to the visual agent B via the bridging agent C.

Compounds suitable for use in cholesterol binding agent a, representative classes of which include:

steroid glycosides, which contain as aglycone a fragment of the furosterol (furostanole) or spirosterol (spirostanole) series of cyclopentanoperhydrophenane and an oligosaccharide fragment of 3 to 10 monosaccharide residues with a linear or branched structure (Hinta p.h. "structure and biological activity of spirostane and furostane series of steroid glycosides", hishinnev, Stinza, 1987, page 142), of which particularly preferred examples are funcoside C, D, E, F, G and I, diosgenin, rososide C, D and E, lanotigonine, digitoside and lycoside;

triterpene glycosides, which contain aglycones of the alpha-or beta-liposantalol (amyryl), lupane, horane (hopane), dammarane (dommarane), lanostane (linostane) or holostane (holostane) series and oligosaccharides containing branched or linear sugar residues (deknnosidize g.e., Chirva v.y., sergienkot.v., uva n.l. "studies on triterpene glycosides", Tbilisi, meiereneba, 1982);

hydrophobic proteins that form complex compounds with cholesterol with discrimination (Himov A.N., TitovaG.V., Kozhenikov H.A., Biochemister, 1982, Vol.47, No.2, p.226-232); himov a.n., Hozhevinkov h.a., Klyueva n.n., et al, voprosty meal, hhimi, 1984, volume 30, No.3, pages 86-90; titova G.V., Hilyueva N.N., Hozhevnikov H.A. et al, Biochemistry, 1980, Vol.45, No.1, p.51-55);

a protein toxin which distinguishably forms a complex compound with cholesterol. They can be extracted from bacteria, marine microorganisms, insects and snakes (Dalin m.v., Fish n.g., "protein toxins of microorganisms", Moscow, Medicine, 1980); or

Polyene antibiotics, which form complex compounds with cholesterol with resolution (I.J. Katzenstein, A.M. Spielvogel, A.W. Norman, J.Antibiol., 27, 12, 1974, pp 943-951; Jong Shan Shyng, Wang His-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);

a high affinity enzyme whose substrate is cholesterol and which has a high affinity for cholesterol. All publications mentioned above are incorporated herein by reference.

The best choice for cholesterol binding agent a is digitonin.

The visual agent B is typically an enzyme, since the color change produced by the enzyme/substrate reaction is particularly useful. Preferred examples of such enzymes include acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, beta-D-galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin and pyrophosphatase. Peroxidase is the best choice, such as horseradish peroxidase (HRP).

The use of the bridging agent C enhances the performance of the process and facilitates the production of the desired A-C-B complex from which the color is developed while retaining the reactivity of the functional groups of the agents A and B. The most preferred A-C-B complexes are those using steroid glycosides (cyclopentane phenanthrene fragments containing the furosterol or spirosterol series as aglycones and oligosaccharide fragments of 2-10 monosaccharide residues with linear or branched structures such as digitonin) as cholesterol affinity binding agent A. When digitonin is selected as the cholesterol binding agent a and HRP is selected as the visual agent, it is desirable to use the bridging agent C because HRP, which has a relatively large molecular weight, may sterically hinder the reaction of digitonin with skin cholesterol when it is directly bound to digitonin. As the bridging agent C for this purpose, a high molecular weight multifunctional compound is preferably used. Their use allows a wide range of control over the ratio of reagents A and C in the final complex. Such high molecular weight multifunctional bridging agents C can be various polysaccharides, proteins or synthetic polymers, i.e. any suitable high molecular weight compound containing primary amine, carboxyl, hydroxyl, aldehyde, halide, mixed anhydride, imino ether, azide, hydroxyl, maleimide, isocyanate or epoxy functional groups. Acrylic acid or maleic anhydride and N-vinylpyrrolidone are the most preferred high molecular weight multifunctional bridging agents C. Unsymmetrical low molecular weight bifunctional compounds such as cyanogen bromide (bromocyanine), trichlorotriazine or 2-amino-4, 6-dichloro-3-triazine may also be used.

Indicator D typically contains the substrate of the enzyme used as visual indicator B, as well as other compounds required to make the reaction of the enzyme with its substrate visible. When peroxidase is used as visual indicator B, preferred examples of these indicators D are reagents containing hydrogen peroxide, a compound of N, N-diethyl-p-cyclohexadienylidene sulfate together with a suitable stabilizer. Indicator D and visual agent B are selected from compounds known in the art that produce a color in reaction with the selected enzyme.

A kit is provided for carrying out the assay of the invention. The kit comprises the desired reagents in a suitable sealed package (e.g., a vial or vial with a dropper), a container or other holding device in which a color reaction on the skin of the patient can be conducted while preventing the reagents from spreading over an excessive area and from which the developed color can be provided to a device for recording specified color characteristics such as hue angle, such as a portable reflectance spectrophotometer, for testing and measurement. The container is suitably an adhesive tape having one or more through holes and initially provided with a protective backing to protect the adhesive. Preferably, the container has at least 2 or 3 wells, so that the control experiment can be performed simultaneously with the test experiment. To facilitate the correct performance of the test and control experiments, the wells are made so that the eyes appear clearly distinguishable from each other, for example, in shape.

Figure 1 of the accompanying drawings shows a container for use in the present invention which is in the form of a rectangular test strip 10. The tape comprises a foam pad 10, a skin compatible adhesive layer 12 temporarily protected by a release sheet 14. The first central hole 16, of circular cross-section, used for the test, penetrated the foam pad and penetrated the adhesive layer 12. A second hole 18 of diamond cross-section for positive control purposes and a third hole of square cross-section for negative control purposes are similarly provided in the foam pad of the strip 10, one on each side of the central hole 16. The different shapes of these holes facilitate the trial for the operator, since the operator can correctly select the hole for his purpose.

The test is preferably performed on the skin of the palm of the patient's hand. The container for the reagent in the form of an adhesive tape is temporarily adhered to the skin, leaving the open bottom of the hole in contact with the skin. Reagents were added to the wells, color developed in the wells, and the color was then read spectrophotometrically without removing the tape from the skin. For this purpose, a specially designed spectrophotometer is used which constitutes a further aspect of the invention. Such a spectrophotometer inputs the readings into a computer for analysis. This spectrophotometer is designed to ensure proper alignment over the test wells.

Accordingly, in one aspect the present invention provides a spectrophotometer adapted to input a signal of a colour reflectance reading of a test sample into a computer, the spectrophotometer comprising: a housing; a light emitter in the housing; a lower portion of the opening of the housing through which the light emitter projects; a recess in the lower surface of the lower portion adapted to fit over a perforated test strip applied to the skin surface of a patient so as to align the spectrophotometer with a test sample in a hole of the perforated test strip. Preferably, the lower portion of the spectrophotometer is hinged to the housing so that when the lower portion is opened it conveniently fits correctly with the test strip and then the lower portion is closed onto the housing of the spectrophotometer for measurement. Preferably contacts are provided on both the lower portion of the hinge of the spectrophotometer and the housing to act as a switch so that the light of the spectrophotometer is turned on when the lower portion and housing are closed.

Fig. 2, 3 and 4 schematically show such a spectrophotometer. It has a housing 22 and is fitted with electrical wiring (not shown) to a suitably programmed computer for analysing the results of the readings. Lower portion 24 is hinged to housing 22 at 26. The lower portion 24 is perforated at 28. A groove 30 extends the width of the bottom surface of the lower portion 24 and projects upwardly from the lowermost surface. The groove is closed at one side of the lower part. The width of the groove 30 is designed to be precise to fit closely to the test strip 10 in fig. 4. When reading, the one end of the test strip is fitted to the closed end of the slot, and the fit of the test strip 10 across the width of the slot 30, allows the test wells 16 to be accurately aligned with the light beam emitted from the housing 22 of the reader through the aperture 28. The spectrophotometer is provided with suitable detection means for receiving the reflected signal from the sample in the well 16 and transmitting the signal to a computer for analysis and reading. The housing 22 and lower portion 24 have contacts 32, 34, respectively, which act as switches to turn on the light for measurement when the lower portion 24 is closed on the housing 22.

A specific test procedure will now be described, using an illustrative (non-limiting) example of a diagnostic assay of the present invention.

The kit comprises: dropper bottles containing detection solution (aqueous buffer of digitonin horseradish peroxidase conjugate, 1.5ml containing 0.01% bromonitrodioxane and methylisothiazolinone as preservatives) with colored lid (green) for easy differentiation; a buffer-like dropper bottle containing a higher concentration of detector reagent, and preservatives (less than 0.01% bromonitrodioxane and methylisothiazolinone) as a positive control, with a colored lid (red) to facilitate differentiation; an indicator dropper bottle containing a solution of a detector reagent that binds skin cholesterol and a PC reagent that produces a blue-green color (4.0ml of 3, 3 ', 5, 5' -tetramethylbenzidine, TMD, hydrogen peroxide solution, containing 5% N, N-dimethylformamide as a preservative), with a readily distinguishable cap (blue); a foam pad as shown in FIG. 1, to which reagents may be added; alcohol sponges and instructions for use. The chemical agents can be stored stably for a long time in a refrigerator at 2-8 ℃. The kit may also include a spectrophotometer as shown in figures 2 and 3 in conjunction with a suitably programmed computer and a calibration plate for use with the spectrophotometer. The kit typically does not include a spectrophotometer unless originally sold with it. The same spectrophotometer can be reused with those drop vials that are later "recharged".

Initially, the calibration plate is inserted into the spectrophotometer's fixture, closed, illuminated, and the calibration readings are sent back to the spectrophotometer and computer. Eventually receiving a signal that calibration has been successfully completed.

The patient's hands are washed and rinsed with soap and water and then thoroughly dried. The outer surface of the palm of the patient is thoroughly cleaned with alcohol sponge, and the patient needs to be thoroughly cleaned with sufficient strength. After allowing the hands to dry, the peel-off protective sheet 14 on the test strip 10 is peeled off and the test strip is then adhered to the cleaned skin area of the palm of the patient's hand. The patient places their hand on a piece of paper towel placed on a table and presses down hard to ensure that the test strip sticks firmly to the palm.

Each reagent is then added to each corresponding reagent well of the test strip foam pad. A drop (42. mu.l) of test fluid is added to a round cross-section test well 16, and a drop of positive control fluid is added to a diamond cross-section well 18, but this time no fluid is added to the third square well 20. The added two solutions were allowed to incubate for 1 minute, at which time the patient was left with their hands to receive the test. The patient then reverses the palm and presses the foam pad against the paper towel, removing the liquid from the hole. Visual inspection ensured that the mat and test wells had completely dried. The patient then rests his hands on a flat surface with the palms facing up.

Subsequently, a drop of indicator was added to all three wells, including the previously unused square section well, and the reaction was allowed to proceed for 2 minutes while leaving the patient in hand. Immediately thereafter, the reader 22 is placed in position over the test well 16, and a reading of the colour displayed in the test well is closed and read, transferred to a spectrophotometer and analysed by a computer to obtain a colour angle value.

The positive control wells 18 and the negative control wells 20 were visually observed. The test is valid if the liquid in the negative control well is colorless and the liquid in the positive control well is colored. The color exhibited by the positive control was not quantitatively measured. This color is exhibited from a high concentration reagent solution, with very little cholesterol on the skin, indicating only the effectiveness of the reagent, etc. (for control purposes).

The remaining liquid on the test strip was discarded, the test strip was removed from the palm, and the palm was then washed with an alcohol sponge.

The spectrophotometer used in the present invention should be able to measure the absorbance of light reflected by a test sample and convert it into a value of color angle by appropriate calculation. When color is developed from the horseradish peroxidase-TMD reaction described above, absorbance at 450nm, i.e., A_450nmIs a suitable measure. The following experimentally obtained relationship between optical density at 450nm and hue angle is known:

h ° (degree) 490.45 × A_450nm+57.124

The above relationship was determined by measuring the optical density at 450nm of a series of diluted reaction samples and measuring the hue angle of the same samples and plotting the results as a regression curve. Similar relationships can also be calculated for selected other chromogenic assays (using different enzyme-substrate pairs, exhibiting different colors) in the same manner to convert the recorded optical density to a measurement of hue angle.

While preferred diagnostic tests and kits for performing the same have been described above, those skilled in the art will appreciate that many color-based tests and analyses can be performed using such measurements based on hue angles or chromaticities disclosed herein, for example:

(i) quantification of solid phase immunoassay results, such as dot blot, immunochromatographic assays and flow-through assays (applying the sample to a membrane having a wicking means behind the membrane to capture the target analyte and adding a labeled detector such as an enzyme or gold labeled antibody) to the label;

(ii) quantification of fine particle based assays with colored beads

(iii) Alternative densitometry in stained gel analysis;

(iv) quantification of Western blot analysis.

The method of the present invention provides a simple way to quantify these results and collect supplemental information from the analysis, allowing for a more sophisticated approach to statistical analysis of the test results.

Variations to the invention are not to be regarded as being outside the spirit and scope of the invention, and all modifications that would be obvious to one skilled in the art are intended to be within the scope of the claims.

Claims (14)

1. A method of analyzing a sample of biological material in a biochemical or immunological assay for an analyte, said method comprising the steps of:

treating the sample to display a color that correlates with the amount of analyte in the sample;

spectrophotometrically measuring the hue angle or chroma of the reflectance spectrum of the displayed color;

the measurements of hue angle or chroma are analyzed to determine the presence or concentration of an analyte in the sample.

2. The method of claim 1, wherein the sample of biological material is comprised of liquid and semi-solid secretions taken from a patient in need of evidence of a diagnostic abnormality, and the analyte is comprised substantially of a marker in the sample indicative of the abnormality, and the sample is classified as normal or abnormal based on the obtained hue angle or chroma measurements.

3. The method of claim 2, wherein the sample is lung mucus, throat mucus, neck mucus, semen or nipple aspirate.

4. The method of claim 2 or 3, wherein the sample is placed on a white substrate and the chromogenic treatment comprises an enzymatic reaction.

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

the biological material sample is a semi-solid sample of the contacted colon collected from a patient in need of evidence of a diagnostic abnormality;

said analyte consisting essentially of a marker indicative of the presence or absence of an abnormality;

the step of treating the sample comprises placing the sample on a white substrate and allowing the sample to develop color by an enzymatic reaction;

the obtained measurement values of hue angle or chroma are used to classify the sample as normal or abnormal.

6. The method of claim 2, wherein the marker is a carbohydrate marker.

7. The method of claim 5, wherein the marker is a carbohydrate marker.

8. The method of claim 5, wherein the substrate is non-fibrous.

9. The method of claim 8, wherein the substrate is fiberglass.

10. A method according to any one of claims 5 to 9, wherein the semi-solid sample contacting the colon consists essentially of rectal mucus.

11. The method of claim 1, wherein the analyte is skin cholesterol.

12. A system for analyzing a liquid or semi-solid secretion sample collected from a patient in need of evidence of a diagnostic abnormality by determination of the hue angle or chroma of the color displayed in the sample, said system comprising:

a white non-fibrous substrate with a porous "creped" surface for receiving and holding the sample during development;

a galactose oxidase source suitable for the selective enzymatic oxidation of a sample thereon by applying galactose oxidase to a surface of a substrate.

A source of Schiff reagent adapted to apply Schiff reagent to the oxidised sample on the substrate to allow colour development of the analyte therein;

means for providing the developed sample to a portable spectrophotometer capable of measuring and recording the hue angle and chroma characteristic of the color of the sample dyed on the substrate;

a calibration plate for use with said spectrophotometer; and

a computer programmed to analyze the results.

13. A kit for analyzing a semi-solid sample contacting the colon taken from a patient in need of diagnosis for the presence of a rectal abnormality, said kit comprising:

a white non-fibrous substrate for receiving a sample;

a source of Schiff reagent;

a portable reflectance spectrophotometer capable of measuring and recording hue angle and chroma characterizing the color of a stained sample on said substrate.

14. A kit according to claim 13, wherein the substrate is glass fibre.