Test piece for protein measurement and protein measurement method

Abstract

The present invention addresses the problem of providing: a test piece for protein measurement that exhibits an improved detection sensitivity at the time of measurement and is capable of achieving an optical density with which it is possible to measure a protein in a sample even when many proteins are included in the sample; and a protein measurement method using the test piece for protein measurement. The present invention provides a test piece for protein measurement including a support body and a reagent holding layer provided on the support body, wherein the reagent holding layer contains a dye that changes color in the presence of a protein, a molybdate, and a hydroxy acid having a hydroxy group and a carboxy group, and the hydroxy acid content is 5-40 times the amount of the dye that changes color in the presence of a protein in terms of molar ratio.

Description

Protein measurement test strip and protein measurement method

[0001] The present invention relates to a test strip for measuring protein and a method for measuring protein, which uses a dye that changes color in the presence of protein.

[0002] Tests for quantifying proteins contained in samples derived from various organisms, including humans, require accuracy and high sensitivity for the diagnosis and treatment of a wide variety of diseases. One known test method for quantifying proteins is a method using pyrocatechol violet (also referred to as PV) and ammonium molybdate. The complex formed by pyrocatechol violet and ammonium molybdate exhibits a quantity-dependent change in absorption wavelength upon interaction with proteins. Test strips for detecting and quantifying proteins utilizing this feature are known.

[0003] Patent Document 1 describes a test strip for detecting proteins in urine, which includes an absorbent carrier impregnated with a reagent system consisting of a buffer and a protein error indicator that changes color in the presence of protein, and is characterized in that tartaric acid is used as a cation-sensitive agent in combination with a non-cation-sensitive buffer in an amount that prevents the pH of the reagent from increasing to a level that would cause the protein error indicator to change color even when a significant amount of protein is not present in the urine.

[0004] Patent Document 2 describes a test strip for measuring protein, which comprises an absorbent carrier containing a composition for measuring protein, which comprises (1) molybdate, (2) a dye, (3) a chelating agent that binds to molybdenum, or a metal ion that does not react with the dye but can bind to oxalic acid, citric acid, etc., and (4) a buffer material.

[0005] Patent Document 3 describes a dry analytical element used for measuring proteins, which comprises (A) a porous spreading layer, (B) one or more additional layers containing (i) a dye, (ii) a molybdate, and (iii) a polymer containing acrylamide; and (C) a support.

[0006] Japanese Patent No. 3621209 Japanese Patent Application Publication No. 1983-006170 Japanese Patent Application Publication No. 11-337547

[0007] The test strips described in Patent Documents 1 to 3 use a hydroxy acid having a hydroxy group and a carboxy group to prevent discoloration of dyes such as PV when measuring samples that do not contain protein. However, there is a problem in that detection sensitivity decreases when ΔOD, which is the difference between OD (signal magnitude) when protein is present and OD (signal magnitude) when protein is absent, is small. Furthermore, it is not possible to obtain an optical density (high degree of discoloration) that can measure protein in a sample even when a large amount of protein is contained in the sample, in addition to the concentration at the boundary between normal and abnormal protein, and measurement sensitivity is insufficient.

[0008] An object of the present invention is to provide a test strip for measuring protein, which has improved detection sensitivity during measurement and can provide an optical density that allows measurement of protein in a sample even when the sample contains a large amount of protein.A further object of the present invention is to provide a method for measuring protein using the test strip for measuring protein.

[0009] As a result of extensive research to solve the above-mentioned problems, the present inventors have found that in a protein measurement test strip having a reagent retention layer containing a dye that changes color in the presence of protein, molybdate, and a hydroxy acid, by setting the molar ratio of the hydroxy acid to the dye that changes color in the presence of protein, the hydroxy acid interacts with the PV / ammonium molybdate complex in the absence of protein, inhibiting complex formation and reducing the absorbance of the complex, while at the same time achieving a sufficient concentration when protein is present in the sample. The present invention was completed based on the above findings. The present invention provides the following:

[0010] <1> A test strip for measuring protein, comprising a support and a reagent holding layer provided on the support, wherein the reagent holding layer contains a dye that changes color in the presence of protein, molybdate, and a hydroxy acid having a hydroxy group and a carboxy group, wherein the content of the hydroxy acid is 5 to 40 times the molar ratio of the dye that changes color in the presence of protein. <2> The test strip for measuring protein according to <1>, wherein the dye that changes color in the presence of protein is pyrogallol red or pyrocatechol violet. <3> The test strip for measuring protein according to <1>, wherein the dye that changes color in the presence of protein is pyrocatechol violet. <4> The test strip for measuring protein according to any one of <1> to <3>, wherein the hydroxy acid is a compound having two or more carboxy groups. <5> The test strip for measuring protein according to any one of <1> to <4>, wherein the hydroxy acid includes at least one of tartaric acid, citric acid, and malic acid. <6> The test strip for measuring protein according to any one of <1> to <5>, wherein the reagent holding layer comprises a water absorption layer and a spreading layer. <7> A method for measuring protein, comprising spotting a protein-containing sample on the test strip for measuring protein according to any one of <1> to <6>, and measuring color development in the reagent holding layer.

[0011] According to the protein measurement test strip and protein measurement method of the present invention, the detection sensitivity during measurement is improved, and an optical density that allows measurement of proteins in a sample can be obtained even when the sample contains a large amount of protein.

[0012] The present invention will be described in detail below. In this specification, the symbol "to" indicates a range that includes the numerical values ​​before and after it as the minimum and maximum values, respectively.

[0013] The present invention provides a test strip for measuring protein, comprising a support and a reagent holding layer provided on the support, wherein the reagent holding layer contains a dye that changes color in the presence of protein, molybdate, and a hydroxy acid having a hydroxy group and a carboxy group, and the content of the hydroxy acid is 5 to 40 times the molar ratio of the dye that changes color in the presence of protein.

[0014] In the present invention, it has been found that in a test strip containing a dye that changes color in the presence of protein and molybdate, by including a hydroxy acid in a molar ratio of 5 to 40 times that of the dye that changes color in the presence of protein, the signal before protein detection (hereinafter referred to as background) is reduced while the signal after protein detection (hereinafter referred to as signal) is maintained, thereby making it possible to improve ΔOD (calculated as signal - background).

[0015] The amount of protein contained in biological samples, particularly urine, is a very useful indicator for easily evaluating the presence or absence of renal dysfunction, tumors in the bladder, ureter, or prostate, and inflammation. The kidneys regulate the amount of protein in the body, filtering out unnecessary proteins and excreting them in the urine. However, as renal function declines, proteins necessary for the body are also excreted by the kidneys, resulting in an increase in the amount of protein in the urine. Detecting this change makes it possible to detect the above-mentioned diseases. The type of protein is not particularly limited, but examples include albumin.

[0016] The test strip of the present invention is a test strip for measuring protein, comprising at least a support and a reagent holding layer provided on the support, wherein the reagent holding layer may be a single layer or may be divided into multiple layers. Preferably, at least one of the reagent holding layers contains a dye that changes color in the presence of protein, molybdate, and a hydroxy acid having a hydroxy group and a carboxy group.

[0017] (Dye that changes color in the presence of protein) As the dye that changes color in the presence of protein, pyrogallol red or pyrocatechol violet is preferred, and pyrocatechol violet is more preferred. The amount of the dye used in the reagent holding layer is 0.005 g / m ２ ~0.5g / m ２ is preferred, and 0.02 g / m ２ ~0.2g / m ２ is more preferred.

[0018] (Molybdate) As the molybdate, ammonium molybdate, sodium molybdate, potassium molybdate, lithium molybdate, calcium molybdate, magnesium molybdate, etc. are preferably used, and ammonium molybdate can be more preferably used. The content of ammonium molybdate in the reagent holding layer is 0.008 g / m ２ ~0.8g / m ２ is preferred, and 0.05 to 0.5 g / m ２ is more preferred.

[0019] (Hydroxy acid having a hydroxy group and a carboxy group) As the hydroxy acid having a hydroxy group and a carboxy group, it is preferable to use a hydroxy acid having a hydroxy group at the α-position of the carboxy group. As the hydroxy acid having a hydroxy group and a carboxy group, it is also preferable to use a compound having two or more carboxy groups.

[0020] As the hydroxy acid having a hydroxy group and a carboxy group, more preferably, tartaric acid, malic acid, citric acid, glycolic acid, 2-hydroxybutyric acid, isocitric acid, or lactic acid can be used, and even more preferably, at least one of tartaric acid, citric acid, and malic acid can be used. The amount of hydroxy acid used in the reagent holding layer is 0.025 g / m ２ ~2.5g / m ２ is preferred, and 0.1 g / m ２ ~1.0 g / m ２ However, the amount of the hydroxy acid used is such that the ratio of the amount of the hydroxy acid to the coloring matter described below is satisfied.

[0021] (Mass Ratio of Hydroxy Acid to Dye) As a result of extensive investigations, the present invention has been found to be effective when the molar ratio of the amount of hydroxy acid to the amount of dye in the reagent holding layer is 5 to 40, which is sufficient to obtain sufficient absorbance when protein is present in the sample, while inhibiting the formation of a complex between the dye that changes color in the presence of protein and molybdate, thereby reducing the absorbance of the complex. A preferred molar ratio of the amount of hydroxy acid to the amount of dye in the reagent holding layer, which provides the full effect of the present invention, is 10 to 40, more preferably 12 to 36, and even more preferably 12 to 20. If the ratio of hydroxy acid to the dye is less than 5, the effect of inhibiting the formation of the dye / ammonium molybdate complex is expected to be insufficient, resulting in insufficient reduction in absorbance in the absence of protein. On the other hand, if the ratio of hydroxy acid to the dye is greater than 40, even in the presence of protein, the dye / ammonium molybdate complex is expected to be insufficient, resulting in insufficient absorbance.

[0022] The protein measurement test strip of the present invention includes a support and a reagent holding layer provided on the support. The reagent holding layer may include a water-absorbing layer and a spreading layer.

[0023] The spreading layer is a layer that has the function (metering function) of spreading the aqueous liquid sample that has been applied dropwise to the upper surface of the protein measurement test strip laterally without causing any substantial uneven distribution of the components contained in the aqueous liquid sample, and supplying it to the water-absorbing layer containing an absorbent water-soluble polymer at an approximately constant volume per unit area.

[0024] Furthermore, layers such as adhesive layers may be provided between the support and the layer provided thereon, and between each layer provided on the support.

[0025] The water absorption layer and the spreading layer may be provided as separate layers, but they may also be provided as a single layer by simultaneously providing two or more of the above-mentioned functions in the same layer. That is, the water absorption layer and the spreading layer may be provided as a single layer.

[0026] The support is preferably a water-impermeable support. Examples of suitable materials for the water-impermeable support include polymers such as polyethylene terephthalate, bisphenol A polycarbonate, polystyrene, and cellulose esters (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), with polyethylene terephthalate being particularly preferred. The support may be a smooth, flat, transparent support having a thickness of about 50 μm to about 1 mm, preferably about 80 μm to about 300 μm, that transmits electromagnetic radiation having wavelengths in at least a portion of the range from about 200 nm to about 900 nm. A known primer layer or adhesive layer may be provided on the surface of the support to enhance adhesion to the water-absorbing layer.

[0027] The water-absorbing layer is preferably a layer containing a water-soluble polymer that swells and absorbs water upon contact with water, and has the effect of facilitating the spreading of the aqueous liquid sample in the spreading layer when water in the aqueous liquid sample spotted on the spreading layer reaches the upper surface of this layer during an analytical operation.

[0028] The content of the water-soluble polymer in the water-absorbing layer is preferably 10 to 100 g / m ２ and more preferably 20 to 70 g / m ２ is.

[0029] The water-soluble polymer used in the water-absorbing layer is preferably a non-proteinaceous water-soluble polymer that swells and absorbs water upon contact with water. Examples of non-proteinaceous water-soluble polymers include polyacrylamide, agarose, acrylamide copolymers such as the acrylamide-N-vinylpyrrolidone copolymers described in JP-A-57-50660 and JP-A-58-77664, polyvinyl alcohol, methallyl alcohol copolymers such as binary or ternary copolymers of methallyl alcohol and acrylamide or a derivative thereof, acrylic acid or a derivative thereof, methacrylic acid or a derivative thereof, or N-vinyl-2-pyrrolidone (methallyl alcohol copolymers can be crosslinked and cured, for example, acrylamide-N-vinyl-2-pyrrolidone-methallyl alcohol ternary copolymers), and polyvinyl alcohol.

[0030] Among these non-proteinaceous water-soluble polymers, acrylamide copolymers such as acrylamide-N-vinylpyrrolidone copolymer, or methallyl alcohol-containing copolymers such as acrylamide-N-vinyl-2-pyrrolidone-methallyl alcohol ternary copolymer are preferred.

[0031] The water-absorbing layer can contain various components that do not adversely affect the dye's performance. Examples of such components include nonionic surfactants. Specific examples of nonionic surfactants include those that can be contained in the reagent holding layer. The inclusion of a nonionic surfactant in the water-absorbing layer facilitates substantially uniform absorption of water from the aqueous liquid sample into the water-absorbing layer during the analytical procedure, and also ensures rapid and substantially uniform liquid contact with the spreading layer.

[0032] The water-absorbing layer may contain a crosslinking agent (also called a curing agent or hardener). Various inorganic and organic crosslinking agents well known in the field of organic polymer chemistry can be used as the crosslinking agent. An example of an organic crosslinking agent for polyvinyl alcohol is dimethylurea, and an example of an organic crosslinking agent for methallyl alcohol-containing polymers is formaldehyde. The content of the crosslinking agent in the water-absorbing layer can be selected depending on the coating amount and the degree of curing of the water-absorbing layer to be crosslinked and cured, but generally, the coating amount is about 50 mg / m. ２ ~Approx. 5000mg / m ２ , preferably about 100 mg / m ２ ~Approx. 2000mg / m ２ The range is.

[0033] Examples of the spreading layer include woven spreading layers (e.g., plain weave fabrics such as broadcloth and poplin) described in JP-A-55-164356 and JP-A-57-66359, knitted spreading layers (e.g., tricot knitted fabric, double tricot knitted fabric, Milanese knitted fabric) described in JP-A-60-222769, spreading layers made of paper containing organic polymer fiber pulp described in JP-A-57-148250, and spreading layers made of paper containing organic polymer fiber pulp described in JP-B-53-21677 and U.S. Pat. No. 3,992,158. Examples of the non-fibrous isotropic porous spreading layer that can be used include a membrane filter (brush polymer layer) described in [Japanese Patent Laid-Open Publication No. 2003-102094], a porous layer containing continuous microvoids in which polymer microbeads, glass microbeads, or diatomaceous earth are held in a water-soluble polymer binder, and a non-fibrous isotropic porous spreading layer comprising a porous layer containing continuous microvoids (a three-dimensional lattice-like granular structure layer) in which polymer microbeads described in Japanese Patent Laid-Open Publication No. 55-90859 are bonded in a point-contact manner with a polymer adhesive that does not swell in water. Knitted spreading layers (e.g., tricot knitted fabric, double tricot knitted fabric, Milanese knitted fabric, etc.) are preferred as spreading layers.

[0034] The spreading layer contains a dye that changes color in the presence of protein, molybdate, and a hydroxy acid having a hydroxy group and a carboxy group.

[0035] Examples of water-soluble polymers that can be contained in the spreading layer include acrylic polymers (such as polyacrylamide), cellulose polymers (such as carboxymethyl cellulose and hydroxymethyl cellulose), etc. As the water-soluble polymer, preferably used are polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, etc.

[0036] The coating amount of the water-soluble polymer in the spreading layer is preferably 0.1 g / m ２ to 30 g / m ２ and more preferably 0.3 g / m ２ to 20 g / m ２ and more preferably 0.5 g / m ２ to 15 g / m ２ is.

[0037] The liquid pH of the spreading layer is preferably 1.0 to 5.0, more preferably 1.5 to 4.5, and even more preferably 2.0 to 4.0. The liquid pH of the spreading layer means the pH of the coating liquid used to form the spreading layer.

[0038] Methods for incorporating a reagent into the spreading layer include a method in which an aqueous solution, an aqueous-organic solvent mixed solvent solution, or an organic solvent solution (examples of organic solvents: aliphatic alcohols such as methanol, ethanol, and isopropyl alcohol; dialkyl ketones such as acetone and methyl ethyl ketone; dialkyl ethers such as dimethyl ether; aliphatic cyclic ethers such as tetrahydrofuran and dioxane; acetonitrile; hexane; β-methoxyethanol; ethylene glycol; etc.) containing a dye that changes color in the presence of protein, molybdate, and a hydroxy acid having a hydroxy group and a carboxy group is applied or sprayed substantially uniformly onto the spreading layer by a known method and dried; and a method in which a material for the spreading layer is immersed in a solution containing a dye that changes color in the presence of protein, molybdate, and a hydroxy acid having a hydroxy group and a carboxy group, and then layered in a dry or semi-dry state on a water-absorbing layer (water-soluble polymer binder layer) to form an integrated layer.

[0039] The protein measurement test strip of the present invention can be prepared by methods known to those skilled in the art. For example, a coating solution formulated as a water-absorbing layer coating solution is applied to a support and dried to form a dry film approximately 40 μm thick, and then a woven fabric for the spreading layer is attached to the resulting film. A coating solution formulated as a reagent holding layer solution is then applied to the woven fabric side of the spreading layer and dried to produce a protein measurement test strip. The protein measurement test strip is cut into square pieces with sides of approximately 15 mm to approximately 30 mm or similar shapes, and placed in a slide frame as described in JP-B-57-28331, JP-A-56-142454, JP-A-57-63452, JP-A-58-32350, JP-T-58-501144, etc., and used as a chemical analysis slide, which is preferred from the viewpoints of various aspects such as production, packaging, transportation, storage, and measurement operations. Depending on the intended use, the tape may be stored in a cassette or magazine as a long tape, or may be attached to or stored in a card with an opening.

[0040] According to the present invention, there is provided a method for measuring a protein, which comprises spotting a protein-containing sample on the protein measurement test strip of the present invention and measuring the color development in the reagent holding layer.

[0041] For example, an aqueous liquid sample such as whole blood, plasma, serum, lymph, or urine, in a range of about 5 μL to about 30 μL, preferably about 8 μL to about 15 μL, is applied to the spreading layer and incubated at a substantially constant temperature in the range of about 20°C to about 40°C, preferably at a substantially constant temperature around 37°C, for about 1 to about 10 minutes, preferably about 2 to about 7 minutes. A detectable change, such as a color change or color development, within the protein measurement test strip is measured by reflection photometry from the support side, and the protein content in the liquid sample can be determined based on the principles of colorimetric measurement. In the present invention, the optical density of the spreading layer is measured by reflection photometry using light at or near the absorption maximum wavelength of the protein-dye bond, and the protein content in the liquid sample can be determined based on the principles of colorimetric measurement using a pre-prepared calibration curve. By maintaining a constant amount of aqueous liquid sample applied, incubation time, and temperature, quantitative protein analysis can be performed with high accuracy. Measurement can be performed with extremely simple procedures using chemical analyzers described in Japanese Patent Laid-Open Nos. 60-125543, 60-220862, 61-294367, and 58-161867, and highly accurate quantitative analysis can be carried out.

[0042] Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

[0043] Example 1 The following amounts of each component were applied to a colorless, transparent, smooth polyethylene terephthalate film and then dried.

[0044] The pH of the aqueous solution was adjusted to 3.05.

[0045] Next, about 12 mg / (10 mm) was applied to the film. ２ After wetting the entire surface with water at a supply rate of 1000 rpm, a tricot knit fabric made of polyethylene terephthalate spun yarn equivalent to 50 denier knitted at 36 gauge was laminated on the fabric under light pressure, and the fabric was dried.

[0046] Next, an aqueous solution (pH 2.5) having the following composition was applied to the substrate in the amounts of the following components and dried to prepare an integral multilayer analytical element.

[0047]

[0048] The tartaric acid was applied at a molar ratio of 6.03 to PV. The integrated multilayer analytical element was cut into chips measuring 12 mm x 13 mm, which were then placed in slide frames (described in JP-A-57-63452) to prepare test strips for measuring protein 6 (Example 1) according to the present invention, and the chips were sealed in moisture-proof bags in a low-humidity (20% RH) environment.

[0049] Examples 2 to 11 Test strips for protein measurement 6 to 15 (Examples 2 to 11) were prepared in the same manner as in Example 1, except that in test strip for protein measurement 1 prepared in Example 1, the type and content of hydroxy acid were changed as shown in Table 1, and the test strips were sealed in moisture-proof bags in a low humidity (20% RH) environment.

[0050] <Comparative Examples 1 to 4> Test strips for protein measurement 1 to 4 (Comparative Examples 1 to 4) were prepared in the same manner as in Example 1, except that in the test strip for protein measurement 1 prepared in Example 1, the type and content of hydroxy acid were changed as shown in Table 1, and the test strips were sealed in moisture-proof bags in a low humidity (20% RH) environment.

[0051] <Evaluation of Protein Amount> Albumin, the most common protein found in biological samples, was used for evaluation. The evaluation concentration was 400 mg / 100 mL, which is the upper limit of the quantifiable concentration range required for general protein measurement test strips. A test solution containing 400 mg / 100 mL of human serum albumin (HSA) was prepared, and protein measurement test strips 1 to 15 prepared in Comparative Examples 1 to 4 and Examples 1 to 11 were removed from their moisture-proof bags. Immediately afterwards, 10 μL of the test solution was applied to the test strip, and the albumin amount was measured using a Fuji Dry Chem 7000 (manufactured by Fujifilm Corporation). ΔOD, which is the difference between the OD in the presence and absence of protein, was calculated. The measurement results were evaluated as the average of three repeated measurements. The evaluation results are shown in Table 1.

[0052]

[0053] <Evaluation Results> It was found that ΔOD was greatest when the content of hydroxy acid relative to PV was 6.03 to 36.03 times in molar ratio.

Claims

1. A test piece for measuring protein comprising a support and a reagent holding layer provided on the support, the reagent holding layer containing a dye that changes color in the presence of protein, molybdate, and a hydroxy acid having a hydroxy group and a carboxy group, the hydroxy acid content being 5 to 40 times the molar ratio of the dye that changes color in the presence of protein.

2. The test strip for measuring protein according to claim 1, wherein the dye that changes color in the presence of protein is pyrogallol red or pyrocatechol violet.

3. The protein measurement test strip according to claim 1, wherein the dye that changes color in the presence of protein is pyrocatechol violet.

4. A test strip for measuring a protein according to any one of claims 1 to 3, wherein the hydroxy acid is a compound having two or more carboxy groups.

5. The test strip for measuring a protein according to any one of claims 1 to 3, wherein the hydroxy acid includes at least one of tartaric acid, citric acid and malic acid.

6. A test strip for measuring protein according to any one of claims 1 to 3, wherein the reagent holding layer comprises a water-absorbing layer and a spreading layer.

7. A method for measuring protein, comprising spotting a protein-containing sample on a protein measurement test strip described in any one of claims 1 to 3, and measuring the color development in the reagent holding layer.

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