Albumin measuring test piece and albumin measuring method

Abstract

To provide an albumin measuring test piece and an albumin measuring method, free from the influence of humidity upon measurement.SOLUTION: An albumin measuring test piece including a support, and a reagent holding layer provided on the support, in which the reagent holding layer contains a protein denaturation agent, an SH reagent, bromocresol purple, a nonionic surfactant, and at least one kind of compound represented by specific formulas (1)-(4).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a test strip for measuring albumin and a method for measuring albumin using bromocresol purple. [Background technology]

[0002] Test strips using the bromocresol green method (BCG method) are widely used for measuring serum albumin. Because the BCG method is affected by globulin and develops color in the presence of globulin, test strips using the bromocresol purple method (BCP method), which is not affected by globulin and has high specificity for albumin, are desired.

[0003] Patent Document 1 describes the measurement of glycated proteins using BCP in the presence of one or more selected from deoxycholic acid, deoxycholic acid amide, etc., and / or ascorbic acid oxidase and a buffer having no 4-(2-hydroxyethyl)-1-piperazinyl group. Furthermore, as a test strip that overcomes the adverse effect of being easily affected by coexisting substances in serum, Patent Document 2 discloses a test strip that contains a protein denaturant and an SH reagent to convert reduced albumin to oxidized albumin, which is then reacted with BCP. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2008-278898 A [Patent Document 2] JP 2022-085876 A Summary of the Invention [Problem to be solved by the invention]

[0005] Such a test strip is useful as a test strip that is not easily affected by coexisting substances in serum, but since such a test strip is expected to be measured in various temperature and humidity ranges, it is desirable to minimize the influence of the environmental temperature and humidity of the test strip on the measured value, and the influence of the environmental temperature and humidity temporarily maintained before measurement on the measured value. However, when measuring albumin using a test strip that reacts with bromocresol purple (hereinafter also referred to as BCP), it has been found that the measured value is affected by the environmental temperature and humidity temporarily maintained before measurement, especially by the environmental humidity, and the problem is to prepare an albumin test strip that reacts with BCP and is not affected by the environmental temperature and humidity. That is, the problem to be solved by the present invention is to provide a test strip for measuring albumin that is not affected by humidity during measurement and a method for measuring albumin. [Means for solving the problem]

[0006] As a result of intensive research conducted by the present inventors to solve the above problems, they have found that a test strip for measuring albumin containing a compound having a specific structure is less susceptible to the effects of environmental humidity. The present invention has been completed based on the above findings. According to the present invention, the following inventions are provided.

[0007] <1> A test strip for measuring albumin comprising a support and a reagent holding layer provided on the support, the reagent holding layer containing a protein denaturant, an SH reagent, bromocresol purple, a nonionic surfactant, and at least one compound represented by the following formulas (1) to (4): [ka] In formula (1) and formula (2), R1, R2, R3, and R4 each independently represent a hydroxy group, a halogen, a hydrogen atom, or an alkyl group; R5, R6, and R7 each independently represent a hydroxy group, a halogen, a hydrogen atom, or an alkyl group; In formula (3) and formula (4), R11, R12, R13, R14, and R15 each independently represent a hydroxy group, a halogen, a hydrogen atom, or an alkyl group, and R16, R17, R18, and R19 each independently represent a hydroxy group, a halogen, a hydrogen atom, or an alkyl group. <2> At least one compound represented by formulas (1) to (4) is at least one compound represented by formula (1) or formula (2), in which in formulas (1) and (2), any two of R1, R2, R3, and R4 are hydroxy groups, any one is halogen, and any one is a hydrogen atom or an alkyl group, and any one of R5, R6, and R7 is hydroxy groups, any one is halogen, and any one is a hydrogen atom or an alkyl group. <1> 2. A test strip for measuring albumin according to claim 1. <3> At least one compound represented by formulas (1) to (4) is at least one compound represented by formula (1) or formula (2), in which in formulas (1) and (2), any two of R1, R2, R3, and R4 are hydroxy groups, any two of R5, R6, and R7 are each independently a hydrogen atom or an alkyl group, and any one of R5, R6, and R7 is a hydroxy group, and any two of R5, R6, and R7 are each independently a hydrogen atom or an alkyl group. <1> 2. A test strip for measuring albumin according to claim 1. <4> At least one compound represented by formulas (1) to (4) is at least one compound represented by formula (3) or formula (4), in which in formulas (3) and (4), any two of R11, R12, R13, R14, and R15 are hydroxy groups, any three of R11, R12, R13, R14, and R15 are each independently a hydrogen atom or an alkyl group, and any one of R16, R17, R18, and R19 is a hydroxy group, and any three of R16, R17, R18, and R19 are each independently a hydrogen atom or an alkyl group. <1> 2. A test strip for measuring albumin according to claim 1. <5> the halogen is bromine; <1> , <2> and <4> 2. A test strip for measuring albumin according to any one of the above. <6> In formula (1) and formula (2), R1, R2, R3 and R4 each independently represent a hydroxy group, a halogen or a hydrogen atom, R5, R6 and R7 each independently represent a hydroxy group, a halogen or a hydrogen atom, in formula (3) and formula (4), R11, R12, R13, R14 and R15 each independently represent a hydroxy group, a halogen or a hydrogen atom, and R16, R17, R18 and R19 each independently represent a hydroxy group, a halogen or a hydrogen atom. <1> from <5> 2. A test strip for measuring albumin according to any one of the above. <7> The content of at least one compound represented by formulas (1) to (4) is 0.5% by mass to 15% by mass of the content of bromocresol purple. <1> from <6> 2. A test strip for measuring albumin according to any one of the above. <8> The protein denaturant is sodium dodecyl sulfate; <1> from <7> 2. A test strip for measuring albumin according to any one of the above. <9> The SH reagent is 5,5'-dithiobis(2-nitrobenzoic acid). <1> from <8> 2. A test strip for measuring albumin according to any one of the above. <10> The nonionic surfactant is a nonionic surfactant having a polyoxyethylene group. <1> from <9> 2. A test strip for measuring albumin according to any one of the above. <11> <1> from <10> 2. A method for measuring albumin, comprising: applying an albumin-containing sample to the test strip for measuring albumin described in any one of 1 to 10; and measuring color development in the reagent holding layer. Effect of the Invention

[0008] According to the test strip for measuring albumin and the method for measuring albumin of the present invention, the influence of the environmental temperature and humidity before the measurement, particularly the humidity, can be minimized, and accurate measurement of albumin can be performed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] 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.

[0010] The present invention provides a test strip for measuring albumin comprising a support and a reagent holding layer provided on the support, the reagent holding layer containing a protein denaturant, an SH reagent, bromocresol purple, a nonionic surfactant, and at least one compound represented by the following formulas (1) to (4). [ka] In formula (1) and formula (2), R1, R2, R3, and R4 each independently represent a hydroxy group, a halogen, a hydrogen atom, or an alkyl group; R5, R6, and R7 each independently represent a hydroxy group, a halogen, a hydrogen atom, or an alkyl group; In formula (3) and formula (4), R11, R12, R13, R14, and R15 each independently represent a hydroxy group, a halogen, a hydrogen atom, or an alkyl group, and R16, R17, R18, and R19 each independently represent a hydroxy group, a halogen, a hydrogen atom, or an alkyl group.

[0011] In a first preferred embodiment, at least one compound represented by formulas (1) to (4) is at least one compound represented by formula (1) or formula (2), and in formulas (1) and (2), any two of R1, R2, R3, and R4 are hydroxy groups, any one is halogen, and any one is a hydrogen atom or an alkyl group; and any one of R5, R6, and R7 is hydroxy groups, any one is halogen, and any one is a hydrogen atom or an alkyl group.

[0012] In a second preferred embodiment, at least one compound represented by formulas (1) to (4) is at least one compound represented by formula (1) or formula (2), and in formulas (1) and (2), any two of R1, R2, R3, and R4 are hydroxy groups, any two of R5, R6, and R7 are independently a hydrogen atom or an alkyl group.

[0013] In a third preferred embodiment, at least one compound represented by formulas (1) to (4) is at least one compound represented by formula (3) or formula (4), and in formulas (3) and (4), any two of R11, R12, R13, R14, and R15 are hydroxy groups, any three of them are each independently a hydrogen atom or an alkyl group, and any one of R16, R17, R18, and R19 is a hydroxy group, and any three of them are each independently a hydrogen atom or an alkyl group.

[0014] In this specification, the alkyl group may be linear, branched or cyclic, is preferably an alkyl group having 1 to 6 carbon atoms, more preferably an alkyl group having 1 to 4 carbon atoms, and particularly preferably a methyl group. As used herein, halogen includes fluorine, chlorine, bromine, or iodine, preferably bromine.

[0015] Preferably, in formula (1) and formula (2), R1, R2, R3 and R4 each independently represent a hydroxy group, a halogen or a hydrogen atom, and R5, R6 and R7 each independently represent a hydroxy group, a halogen or a hydrogen atom.

[0016] Preferably, in formula (3) and formula (4), R11, R12, R13, R14, and R15 each independently represent a hydroxy group, a halogen, or a hydrogen atom, and R16, R17, R18, and R19 each independently represent a hydroxy group, a halogen, or a hydrogen atom.

[0017] More specifically, the compounds represented by formulas (1) to (4) are preferably compounds having the following structures:

[0018] [ka]

[0019] The compounds represented by formulae (1) to (4) change between open and closed ring structures depending on the pH, and the equilibrium shifts to the open ring structure at low pH. The equilibrium is between the compound represented by formula (1) and the compound represented by formula (2), and between the compound represented by formula (3) and the compound represented by formula (4). The content of the compounds represented by formulas (1) to (4) is not particularly limited within the range in which the effects of the present invention can be exhibited, but it is preferably 0.5% by mass or more, more preferably 0.8% by mass or more, even more preferably 1% by mass or more, particularly preferably 2% by mass or more, and most preferably 3% by mass or more, based on the content of bromocresol purple. The upper limit of the content of the compounds represented by formulas (1) to (4) is preferably 25% by mass or less, more preferably 20% by mass or less, and even more preferably 15% by mass or less, based on the content of bromocresol purple. The content of at least one compound represented by formulas (1) to (4) is preferably 0.5% by mass to 15% by mass of the content of bromocresol purple.

[0020] The compounds represented by formulas (1) to (4) may be commercially available products, or may be synthesized by organic synthesis.

[0021] The reagent holding layer in the present invention contains a protein denaturant, an SH reagent, and a nonionic surfactant in addition to BCP and at least one compound represented by formulas (1) to (4).

[0022] As the protein denaturant, an anionic surfactant is preferable. In one or more embodiments, the anionic surfactant may be an alkyl sulfate surfactant, a polyoxyethylene alkyl phenyl ether sulfate salt, a polyoxyethylene alkyl ether sulfate salt, or an alkyl benzene sulfonate salt. In one or more embodiments, the alkyl sulfate surfactant may be sodium dodecyl sulfate (SDS) or sodium cetyl sulfate. In one or more embodiments, the polyoxyethylene alkyl phenyl ether sulfate salt may be sodium polyoxyethylene alkyl phenyl ether sulfate.

[0023] In one or more embodiments, examples of the polyoxyethylene alkyl ether sulfate include sodium polyoxyethylene alkyl ether sulfate and triethanolamine polyoxyethylene alkyl ether sulfate. In one or more embodiments, examples of the alkylbenzene sulfonate include sodium laurylbenzene sulfonate and sodium cetylbenzene sulfonate. Among these, sodium dodecyl sulfate is particularly preferred. The amount of protein denaturant used in the reagent holding layer is 0.075 g / m 2 ~7.5g / m 2 is preferred.

[0024] Examples of SH reagents include 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 2,2'-dithiobis(5-nitropyridin) (DTNP), 2,2'-dithiodipyridine (2-PDS), 4,4'-dithiodipyridine (4-PDS), and 4,4'-dithiobis(phenyl benzene) (4,4'-dithiobis-(phenyl benzene)). azide):DTBPA), disulfides such as oxidized glutathione, oxidizing agents such as iodine, ferricyanide, iodosobenzoic acid, iodates, chlorites, mercury, zinc, alkylating agents such as iodoacetic acid, chloroacetic acid, iodoacetamide, chloroacetophenone, maleimides such as maleimide derivatives such as N-methylmaleimide, N-ethylmaleimide, N,N'-p-phenylenedimaleimide, thiophthalimide, etc. Among these, disulfides such as DTNB, 2-PDS, 4-PDS, maleimide derivatives such as N-ethylmaleimide, etc. are particularly preferably used. As the SH reagent, DTNB is particularly preferred. These may be used alone or in appropriate combination. The amount of SH reagent used in the reagent holding layer is 0.01 g / m 2 ~1g / m 2 is preferred.

[0025] Bromocresol Purple (BCP) can be any commercially available product. BCP is used as a coloring agent for measuring albumin. In the present invention, the content of bromocresol purple in the reagent holding layer is 0.78 g / m 2 ~3.81g / m 2 and preferably 0.78 g / m 2 ~2.21g / m 2 It is.

[0026] The nonionic surfactant is preferably a nonionic surfactant having a polyoxyethylene group. More preferably, the nonionic surfactant may be one or more nonionic surfactants selected from the group consisting of polyoxyethylene lauryl ether, polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene tert-octylphenyl ether, and polyoxyethylene isooctylphenyl ether.

[0027] Examples of polyoxyethylene oleyl ether include polyoxyethylene (20) oleyl ether, polyoxyethylene (23) lauryl ether (Brij35), and polyoxyethylene (50) oleyl ether.

[0028] As the nonionic surfactant, Triton X-100, Tween 20, Tween 80, Triton X-405, Emulgen 108, Emulgen 120, Emulgen 150, Emulgen 430, or the like can be used.

[0029] The amount of nonionic surfactant applied to the albumin measurement test strip is 0.01 g / m 2 ~0.4g / m 2 It is preferable that:

[0030] In the present invention, the ratio of the content of the nonionic surfactant to the content of bromocresol purple is 0.01 to 0.3, and preferably 0.01 to 0.2.

[0031] The test strip for measuring albumin of the present invention comprises a support and a reagent holding layer provided on the support. Preferably, the test strip for measuring albumin of the present invention further comprises an intermediate layer between the support and the reagent holding layer. In this case, the test strip for measuring albumin of the present invention comprises the support, the intermediate layer, and the reagent holding layer.

[0032] More preferably, the test strip for measuring albumin of the present invention comprises a support; an intermediate layer; and a spreading layer including a reagent holding layer.

[0033] The intermediate layer is preferably a layer (water absorbing layer) composed mainly of a water-soluble polymer that absorbs water and swells. The spreading layer has the function (metering function) of spreading the aqueous liquid sample applied in a spot on the upper surface of the albumin measurement test strip laterally without causing any substantial uneven distribution of the components contained in the aqueous liquid sample, and supplying the sample to an intermediate layer containing an absorbent water-soluble polymer at an approximately constant volume per unit area.

[0034] 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.

[0035] The intermediate layer and the spreading layer may be provided as separate layers, but they may also be provided as one layer by simultaneously providing two or more of the above-mentioned functions in the same layer. That is, the intermediate layer and the spreading layer may be provided as the same layer.

[0036] The support is preferably a water-impermeable support. The material of the water-impermeable support is preferably a polymer such as polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose ester (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), and polyethylene terephthalate is particularly preferred. The support may be a smooth, flat support having a thickness of about 50 μm to about 1 mm, preferably about 80 μm to about 300 μm, which is transparent and transmits electromagnetic radiation having at least a part of a wavelength range of about 200 nm to about 900 nm. A known undercoat layer or adhesive layer may be provided on the surface of the support to strengthen adhesion to the intermediate layer.

[0037] The intermediate layer is preferably a layer containing a water-soluble polymer capable of swelling and absorbing water upon contact with water, and has the effect of improving 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.

[0038] The content of the water-soluble polymer in the intermediate layer is preferably 10 to 100 g / m 2 and more preferably 20 to 70 g / m 2 It is.

[0039] The water-soluble polymer used in the intermediate layer is preferably a non-protein water-soluble polymer that has the property of swelling and absorbing water when it comes into contact with water. Examples of non-protein water-soluble polymers include polyacrylamide, agarose, acrylamide copolymers such as acrylamide-N-vinylpyrrolidone copolymers described in JP-A-57-50660 and JP-A-58-77664, methallyl alcohol copolymers such as binary or ternary copolymers of methallyl alcohol and acrylamide or its derivatives, acrylic acid or its derivatives, methacrylic acid or its derivatives, or N-vinyl-2-pyrrolidone described in JP-A-62-137564 (methallyl alcohol copolymers can be crosslinked and cured, for example, acrylamide-N-vinyl-2-pyrrolidone-methallyl alcohol ternary copolymers).

[0040] 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.

[0041] The intermediate layer may contain various components that do not adversely affect the performance of the BCP. An example of such a component is a nonionic surfactant. Specific examples of nonionic surfactants include those that can be contained in the reagent holding layer. By containing a nonionic surfactant in the intermediate layer, water in the aqueous liquid sample is more easily absorbed substantially uniformly into the intermediate layer during the analytical operation, and liquid contact with the spreading layer is rapid and substantially uniform. The intermediate layer may also contain a buffering agent, which will be described later.

[0042] The intermediate layer may contain a crosslinking agent (also called a curing agent or hardener). As the crosslinking agent, various inorganic and organic crosslinking agents well known in the field of organic polymer chemistry may be used. 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 intermediate layer may be selected depending on the coating amount of the intermediate layer to be crosslinked and hardened and the degree of hardening, but generally it is about 50 mg / m2 in terms of coating amount. 2 ~about 5000mg / m 2 , preferably about 100 mg / m 2 ~about 2000mg / m 2 The range is.

[0043] 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, etc.) described in JP-A-60-222769, spreading layers made of organic polymer fiber pulp-containing paper described in JP-A-57-148250, and spreading layers made of organic polymer fiber pulp-containing paper 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 continuous microvoid-containing porous layer in which the membrane filter (brush polymer layer) described in JP-A-55-90859, polymer microbeads, glass microbeads, and diatomaceous earth are held in a water-soluble polymer binder, and a continuous microvoid-containing porous layer (three-dimensional lattice-like granular structure layer) in which the polymer microbeads described in JP-A-55-90859 are bonded in a point-contact manner with a polymer adhesive that does not swell in water. As the spreading layer, a knitted spreading layer (for example, tricot knitted fabric, double tricot knitted fabric, Milanese knitted fabric, etc.) is preferred.

[0044] The spreading layer includes a reagent holding layer containing a protein denaturant, an SH reagent, bromocresol purple, a nonionic surfactant, and a compound represented by formula (1). The reagent holding layer may further include a pH adjuster and a water-soluble polymer.

[0045] Examples of the water-soluble polymer that can be contained in the reagent holding layer include acrylics (polyacrylamide, etc.), celluloses (carboxymethyl cellulose, hydroxymethyl cellulose, etc.), etc. As the water-soluble polymer, preferably, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, etc. can be used.

[0046] The coating weight of the water-soluble polymer in the reagent holding layer is approximately 0.1 g / m 2 From about 30g / m 2 , preferably about 0.5 g / m 2 From about 20g / m 2 The range is.

[0047] The liquid pH of the reagent holding layer is preferably 4.5 to 5.8, more preferably 4.6 to 5.7, and even more preferably 4.7 to 5.6. The reaction between albumin and bromocresol purple increases in amount as the pH approaches 7, but the coloring (background) of the bromocresol purple body also increases, so the above range is considered to be preferable. The liquid pH of the reagent holding layer refers to the pH of the coating liquid used to form the reagent holding layer. As the pH adjuster for adjusting the pH of the reagent holding layer to the above-mentioned pH range, succinic acid, disodium succinate, and sodium hydroxide are preferable, and succinic acid is more preferable.

[0048] The intermediate layer and the spreading layer may contain a buffering agent.

[0049] As the buffer, at least one organic acid selected from the group consisting of hydroxycarboxylic acids and dicarboxylic acids is used. Examples of hydroxycarboxylic acids include malic acid, lactic acid, citric acid, and tartaric acid. Examples of dicarboxylic acids include malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, and 3,3-dimethylglutaric acid. Among the above, succinic acid is preferred.

[0050] The buffer content is 1m 2 The range is from about 30 milliequivalents to about 500 milliequivalents, preferably from about 50 milliequivalents to about 300 milliequivalents per layer. The buffer is preferably dispersed in a water-soluble polymer together with other reagents and dispersed and held in one or more layers of the intermediate layer, the reagent holding layer and the spreading layer.

[0051] Methods for incorporating a reagent holding layer 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, isopropyl alcohol, etc.; dialkyl ketones such as acetone, methyl ethyl ketone, etc.; dialkyl ethers such as dimethyl ether; aliphatic cyclic ethers such as tetrahydrofuran, dioxane, etc.; acetonitrile; hexane; β-methoxyethanol; ethylene glycol, etc.) containing a protein denaturant, SH reagent, bromocresol purple, and a nonionic surfactant is applied or sprayed substantially uniformly onto the spreading layer by a known method and then dried, as well as a method in which a material for the spreading layer is immersed in a solution containing a protein denaturant, SH reagent, bromocresol purple, and a nonionic surfactant, and then laminated and integrated on top of the intermediate layer (water-soluble polymer binder layer) in a dry or semi-dry state.

[0052] The albumin measuring test strip of the present invention can be prepared by a method known to those skilled in the art. For example, a coating solution prepared as an intermediate layer coating solution is applied to a support and dried to prepare a dry film with a thickness of about 40 μm, and then a woven fabric of a spreading layer is attached to the support. Then, a coating solution prepared as a reagent holding layer solution is applied from the woven fabric side of the spreading layer and dried to prepare an albumin measuring test strip. The albumin measuring test strip is cut into square pieces with sides of about 15 mm to about 30 mm or small pieces of the same shape or similar size, and is preferably used as a chemical analysis slide by placing it in a slide frame described in JP-B-57-28331, JP-A-56-142454, JP-A-57-63452, JP-A-58-32350, JP-T-58-501144, etc., from the viewpoints of various aspects such as production, packaging, transportation, storage, and measurement operation. Depending on the intended use, the tape may be in the form of a long strip stored in a cassette or magazine, or in small pieces affixed to or stored in an apertured card.

[0053] According to the present invention, there is provided a method for measuring albumin, which comprises spotting an albumin-containing sample on the albumin measurement test strip of the present invention and measuring the color development in the reagent holding layer.

[0054] For example, about 5 μL to about 30 μL, preferably about 8 μL to about 15 μL, of an aqueous liquid sample such as whole blood, plasma, serum, lymph, or urine is spotted on 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, and detectable changes such as color change and color development in the albumin measurement test strip are measured by reflection photometry from the support side, and the content of albumin in the liquid sample can be determined by the principle 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 albumin-BCP complex, and the content of albumin in the liquid sample can be determined by the principle of colorimetric measurement using a previously prepared calibration curve. By keeping the amount of the aqueous liquid sample to be spotted, the incubation time, and the temperature constant, quantitative analysis of albumin can be performed with high accuracy. Measurement can be performed very easily using chemical analyzers described in JP-A-60-125543, JP-A-60-220862, JP-A-61-294367, JP-A-58-161867, etc., to perform highly accurate quantitative analysis.

[0055] Next, the present invention will be described with reference to examples, but the present invention is not limited to these. EXAMPLES

[0056] <Comparative Example 1> An aqueous solution having the following composition was applied to a gelatin-subbed, colorless, transparent, smooth polyethylene terephthalate film of 180 μm in thickness and then dried. (Middle class) Polymer AAm-VP-MAOH 44.0g / m 2 Succinic acid 3g / m 2

[0057] The pH of the aqueous solution was adjusted to 5.0. The polymer AAm-VP-MAOH represents a polyacrylamide-N-vinyl-2-pyrrolidone-methallyl alcohol ternary copolymer (monomer molar ratio 57:38:5 (manufactured by Fujifilm Corporation)) (hereinafter the same).

[0058] Next, apply approximately 30 g / m 2 After wetting the entire surface by supplying water at a supply rate of 1000 g / m², a tricot knit fabric made of 50 denier polyethylene terephthalate spun yarn knitted at 36 gauge was laminated on the fabric under light pressure and then dried.

[0059] Next, an aqueous solution (pH 4.7) of ethanol (30% by weight) having the following composition was applied so that each component was present in the amount shown below, and then dried to prepare an integral multilayer analytical element.

[0060] (Reagent retention layer) Succinic acid 3.5g / m 2 Polyvinylpyrrolidone 8g / m 2 BCP (Tokyo Chemical Industry Co., Ltd.) 1.7g / m 2 Surfactant Briji35 0.3g / m 2 Protein denaturant (Component A) Sodium dodecyl sulfate (SDS) 0.76g / m 2 SH Reagent (Component B) 5,5'-Dithiobis(2-nitrobenzoic acid) 0.1g / m 2

[0061] The surfactant used here was a non-ionic surfactant, polyoxyethylene (23) lauryl ether (Brij35, Fujifilm Wako Pure Chemical Industries, Ltd.) The pH of the coating solution used to form the reagent holding layer was adjusted to 4.8 with succinic acid.

[0062] The above integrated multilayer analytical element was cut into a chip of 12 mm × 13 mm square, and placed in a slide frame (described in JP-A-57-63452) to prepare albumin measurement test piece 1, which was then sealed in a moisture-proof bag in a low humidity (20% relative humidity (RH)) environment.

[0063] <Example 1> An aqueous solution having the following composition was applied to a gelatin-subbed, colorless, transparent, smooth polyethylene terephthalate film of 180 μm in thickness and then dried. (Middle class) Polymer AAm-VP-MAOH 44.0g / m 2 Succinic acid 3g / m 2

[0064] The pH of the aqueous solution was adjusted to 5.0. The polymer AAm-VP-MAOH represents a polyacrylamide-N-vinyl-2-pyrrolidone-methallyl alcohol ternary copolymer (monomer molar ratio 57:38:5 (manufactured by Fujifilm Corporation)) (hereinafter the same).

[0065] Next, apply approximately 30 g / m 2 After wetting the entire surface by supplying water at a supply rate of 1000 g / m², a tricot knit fabric made of 50 denier polyethylene terephthalate spun yarn knitted at 36 gauge was laminated on the fabric under light pressure and then dried.

[0066] Next, an aqueous solution (pH 4.7) of ethanol (30% by weight) having the following composition was applied so that each component was present in the amount shown below, and then dried to prepare an integral multilayer analytical element.

[0067] (Reagent retention layer) Succinic acid 3.5g / m 2 Polyvinylpyrrolidone 8g / m 2 BCP + Bromopyrogallol Red 1.7g / m 2 Surfactant Briji35 0.3g / m 2 Protein denaturant (Component A) Sodium dodecyl sulfate (SDS) 0.76g / m 2 SH Reagent (Component B) 5,5'-Dithiobis(2-nitrobenzoic acid) 0.1g / m 2

[0068] The BCP+bromopyrogallol red used was prepared so as to contain 5 mass% bromopyrogallol red (Bromopyrogaiiol Red (BPR): manufactured by Tokyo Chemical Industry Co., Ltd.) in terms of the BCP amount ratio relative to the BCP (manufactured by Tokyo Chemical Industry Co., Ltd.) used in Comparative Example 1.

[0069] The surfactant used here was a non-ionic surfactant, polyoxyethylene (23) lauryl ether (Brij35, Fujifilm Wako Pure Chemical Industries, Ltd.) The pH of the coating solution used to form the reagent holding layer was adjusted to 4.8 with succinic acid.

[0070] The above integrated multilayer analytical element was cut into a chip of 12 mm x 13 mm square, placed in a slide frame (described in JP-A-57-63452) to prepare albumin measurement test piece 2, and sealed in a moisture-proof bag in a low humidity (20% RH) environment.

[0071] <Example 2> A test piece 3 was prepared in the same manner as in Example 1, except that pyrogallol red (Pyrogallol Red (PR): manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of bromopyrogallol red used in Example 1, and the test piece 3 was sealed in a moisture-proof bag in a low humidity (20% RH) environment.

[0072] <Example 3> A test piece 4 was prepared in the same manner as in Example 1, except that pyrocatechol violet (PV: manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of bromopyrogallol red used in Example 1, and the test piece 4 was sealed in a moisture-proof bag in a low humidity (20% RH) environment.

[0073] <Example 4> Test piece 5 was prepared in the same manner as in Example 3, except that the pyrocategol violet used in Example 3 was contained at 10 wt % instead of 5 wt %, and was sealed in a moisture-proof bag in a low humidity (20% RH) environment.

[0074] <Example 5> Test piece 6 was prepared in the same manner as in Example 3, except that the pyrocategol violet used in Example 3 was contained at 1 wt % instead of 5 wt %, and the test piece 6 was sealed in a moisture-proof bag in a low humidity (20% RH) environment.

[0075] <Evaluation of albumin content> A test solution containing 4 g / dL of albumin (ALB) was prepared, and the albumin test pieces of Comparative Example 1 and Examples 1 to 5 were removed from the moisture-proof bags. Immediately afterwards, 10 μL of the test solution was applied to the test pieces, and the amount of albumin was measured using Fuji Dry Chem 7000 (manufactured by Fuji Film Corporation). The measurement results were evaluated as the average value of three repeated measurements.

[0076] <Evaluation of environmental dependency> In the process of evaluating the amount of albumin, the test pieces of Comparative Example 1 and Examples 1 to 5 were taken out of the moisture-proof bag, and then stored in an environment of 32°C and 80% RH for 1 hour, and the albumin amount was measured in the same manner to evaluate the environmental dependency. The results were shown as the degree of change as the ratio of the albumin value evaluated after storing in an environment of 32°C and 80% RH for 1 hour to the albumin value evaluated immediately after taking out of the moisture-proof bag. The results are shown in Table 1.

[0077] [Table 1]

[0078] From Table 1, it was confirmed that by using BPR, PR or PV, an albumin test strip that is less susceptible to the effects of the environmental temperature and humidity before measurement can be prepared.

Claims

1. A test strip for measuring albumin comprising a support and a reagent holding layer provided on the support, the reagent holding layer containing a protein denaturant, an SH reagent, bromocresol purple, a nonionic surfactant, and at least one compound represented by the following formulas (1) to (4): 【Chemistry 1】 In formula (1) and formula (2), R1, R2, R3, and R4 each independently represent a hydroxy group, a halogen, a hydrogen atom, or an alkyl group; R5, R6, and R7 each independently represent a hydroxy group, a halogen, a hydrogen atom, or an alkyl group; In formula (3) and formula (4), R11, R12, R13, R14, and R15 each independently represent a hydroxy group, a halogen, a hydrogen atom, or an alkyl group, and R16, R17, R18, and R19 each independently represent a hydroxy group, a halogen, a hydrogen atom, or an alkyl group.

2. The test strip for measuring albumin according to claim 1, wherein the at least one compound represented by any one of formulas (1) to (4) is at least one compound represented by formula (1) or formula (2), and in formulas (1) and (2), any two of R1, R2, R3, and R4 are hydroxy groups, any one is a halogen, and any one is a hydrogen atom or an alkyl group, and any one of R5, R6, and R7 is a hydroxy group, any one is a halogen, and any one is a hydrogen atom or an alkyl group.

3. The test strip for measuring albumin according to claim 1, wherein the at least one compound represented by any one of formulas (1) to (4) is at least one compound represented by formula (1) or formula (2), and in formulas (1) and (2), any two of R1, R2, R3, and R4 are hydroxy groups, any two of R5, R6, and R7 are independently a hydrogen atom or an alkyl group.

4. The test strip for measuring albumin according to claim 1, wherein the at least one compound represented by any one of formulas (1) to (4) is at least one compound represented by formula (3) or formula (4), and in formulas (3) and (4), among R11, R12, R13, R14, and R15, any two are hydroxy groups and any three are each independently a hydrogen atom or an alkyl group, and among R16, R17, R18, and R19, any one is a hydroxy group and any three are each independently a hydrogen atom or an alkyl group.

5. 5. The test strip for measuring albumin according to claim 1, 2 or 4, wherein the halogen is bromine.

6. 5. The test strip for measuring albumin according to claim 1, wherein in formula (1) and formula (2), R1, R2, R3 and R4 each independently represent a hydroxy group, a halogen or a hydrogen atom, R5, R6 and R7 each independently represent a hydroxy group, a halogen or a hydrogen atom, and in formula (3) and formula (4), R11, R12, R13, R14 and R15 each independently represent a hydroxy group, a halogen or a hydrogen atom, and R16, R17, R18 and R19 each independently represent a hydroxy group, a halogen or a hydrogen atom.

7. 5. The test strip for measuring albumin according to claim 1, wherein the content of at least one compound represented by any one of formulas (1) to (4) is 0.5% by mass to 15% by mass of the content of bromocresol purple.

8. 5. The test strip for measuring albumin according to claim 1, wherein the protein denaturing agent is sodium dodecyl sulfate.

9. 5. The test strip for measuring albumin according to claim 1, wherein the SH reagent is 5,5'-dithiobis(2-nitrobenzoic acid).

10. 5. The test strip for measuring albumin according to claim 1, wherein the nonionic surfactant is a nonionic surfactant having a polyoxyethylene group.

11. A method for measuring albumin, comprising spotting an albumin-containing sample on the test strip for measuring albumin according to claim 1 , and measuring color development in the reagent holding layer.

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