Composition

Abstract

To provide a technique for suppressing coloring of test reagents containing trehalose in blank samples.SOLUTION: A composition according to an aspect of the present invention contains a reaction component that reacts with a measurement target component contained in a biological sample and trehalose having a purity of 99.9% or greater.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition. [Background technology]

[0002] In clinical testing and biochemical research, components in biological samples are commonly qualitatively and quantitatively determined by optical or electrochemical detection using enzymes. Enzyme reactions are also commonly used to replicate and amplify DNA in biological samples.

[0003] When using the reaction activity of an enzyme, deterioration of the enzyme's reaction activity should be avoided because it can cause deterioration in the performance of reagents that use it. To prevent deterioration of the enzyme's reaction activity, it is effective to dry the aqueous protein solution and store it in a solid state.

[0004] For example, Patent Document 1 discloses a dry reagent for quantitatively analyzing a specific component contained in a liquid sample, which comprises a nicotinamide coenzyme and a smoothing agent for smoothing the dry reagent, and is configured so that an increase or decrease in the nicotinamide coenzyme is measured by a transmittance method using light in the ultraviolet region.

[0005] Furthermore, Patent Document 1 discloses that the smoothing agent is a combination of an alkali and at least one selected from a sugar and a surfactant, and that specific examples of the sugar include glycerin, D-sorbitol, sucrose, and trehalose. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2013-165709 Summary of the Invention [Problem to be solved by the invention]

[0007] The above-mentioned conventional clinical test reagents have a problem in that when they contain trehalose as a sugar, they develop color in the blank sample.

[0008] One aspect of the present invention aims to provide a technique for suppressing color development in a blank sample in a test reagent containing trehalose. [Means for solving the problem]

[0009] In order to solve the above problems, a composition according to one embodiment of the present invention contains trehalose and a reactive component that reacts with a target component to be measured contained in a biological sample, wherein the trehalose has a purity of 99.9% or higher. [Effects of the Invention]

[0010] According to one aspect of the present invention, a technique for suppressing color development in a blank sample in a test reagent containing trehalose can be provided. DETAILED DESCRIPTION OF THE INVENTION

[0011] One embodiment of the present invention will be described in detail below. In this specification, unless otherwise specified, the expression "A to B" representing a range of numerical values ​​means "A or more and B or less."

[0012] [1. Composition] A composition according to one embodiment of the present invention contains trehalose and a reactive component that reacts with a target component to be measured contained in a biological sample, the trehalose having a purity of 99.9% or higher, thereby making it possible to suppress color development in a blank sample in a test reagent containing the composition according to one embodiment of the present invention.

[0013] Suppressing color development in blank samples has the following advantages. First, the trehalose content in a composition according to one embodiment of the present invention can be increased. Trehalose can function as a stabilizing component that suppresses a decrease in the activity of reaction components. Increasing the trehalose content in a composition according to one embodiment of the present invention can further enhance the stabilizing effect of the test reagent. Second, changes in the calibration curve due to the trehalose content in the composition can be suppressed. This reduces the likelihood of differences between lots of test reagents, eliminating the need to consider weighing tolerances. Furthermore, it can suppress deterioration in reagent performance due to color development in blank samples. This allows the measurement range to be expanded to a wider range compared to when blank color development is present, and improves reproducibility due to fewer factors of variation caused by blank color development. A test reagent comprising a composition according to one embodiment of the present invention will be described later.

[0014] Herein, the term "blank sample" refers to a sample that does not contain a component that reacts with the reactive component in the composition according to one embodiment of the present invention. Such a component is also the component that is the target of measurement contained in a biological sample (hereinafter referred to as the "target component").

[0015] As used herein, the term "biological sample" refers to compositions, secretions, organisms such as viruses present in organisms, and attached organic and inorganic substances from organisms such as animals, plants, fish, aquatic organisms, and fungi. More specifically, it refers to body fluids, blood, tissues inside and on the surface of organisms.

[0016] In this specification, the "component to be measured" refers to, for example, inorganic ions such as sodium in body fluids; specific proteins in body fluids; enzymes in blood; various organic compounds in blood; inorganic compounds in blood; cells; epidermis; bones; etc.

[0017] As used herein, "inhibiting color development" means that the color development when a blank sample is measured with a test reagent containing a composition according to an embodiment of the present invention is weaker than the color development when a blank sample is measured with a comparative reagent containing a comparative composition having the same composition as the composition according to an embodiment of the present invention except that it contains 99.7% pure trehalose. 150 mM PBS buffer is used as the blank sample. The color development of the blank sample can be confirmed by measuring the absorbance at 630 nm using a spectrophotometer. The higher the absorbance value at 630 nm, the stronger the color development.

[0018] <Trehalose> The trehalose contained in a composition according to one embodiment of the present invention has a purity of 99.9% or higher. This makes it possible to suppress color development in blank samples in test reagents containing trehalose. To further suppress color development in blank samples, the purity of the trehalose contained in a composition according to one embodiment of the present invention is preferably 99.97% or higher. There is no particular upper limit to the purity of trehalose. To further suppress color development in blank samples, a higher purity of trehalose is preferable. However, as long as the purity of trehalose is as described above, the desired effect can be sufficiently achieved, and therefore a purity of 99.99% or lower is acceptable. The purity of trehalose in a composition according to one embodiment of the present invention can be measured by conventional purity measurement methods such as nuclear magnetic resonance analysis (NMR) and high-performance liquid chromatography (HPLC).

[0019] (Trehalose content) From the viewpoint of improving the storage stability of the composition according to one embodiment of the present invention, the composition preferably contains 0.1% by weight or more of trehalose, and more preferably 1% by weight or more, relative to 100% by weight of the composition. Furthermore, since dissolving trehalose at a high concentration makes scattering more likely, from the viewpoint of reducing scattering, the composition preferably contains 50% by weight or less of trehalose, and even more preferably 20% by weight or less, relative to 100% by weight of the composition.

[0020] <Reaction components> The reactive component contained in the composition according to one embodiment of the present invention is a component that reacts with a component to be measured contained in a biological sample, and is a component that selectively uses a specific component in the biological sample as a reaction substrate to cause some kind of chemical reaction to proceed. Even in the presence of multiple components, the reactive component has the property of selectively reacting with a specific component. Specific examples include enzymes, catalysts, antibodies, etc.

[0021] Examples of reaction components commonly used in clinical testing reagents are shown in Table 1. [Table 1]

[0022] The type of reactive component contained in the composition according to one embodiment of the present invention is not particularly limited, and may be determined appropriately depending on the type of component to be measured.

[0023] The reaction components of the composition according to one aspect of the present invention may include an enzyme and, optionally, a substrate and a dye, for reasons explained below.

[0024] (enzyme) Enzymes are proteins, and maintaining their higher-order structure is important for maintaining their activity. The higher-order structure is maintained by the mild aggregation of constituent amino acids due to their hydrophobicity in water, hydrogen bonding between the constituent amino acids, and other factors. When enzymes are stored in a dry state, the water surrounding the enzyme is removed during drying, which removes factors that maintain these structures, causing structural reorganization (denaturation), resulting in a decrease in enzyme activity. Therefore, if a non-volatile compound that replaces the functions of water, such as hydrogen bonding and providing a hydrophilic environment, is present around or inside the protein when the enzyme is dried, the above-mentioned structural reorganization is less likely to occur, and the decrease in enzyme activity can be suppressed. As described above, the composition according to one embodiment of the present invention contains trehalose, which can function as a stabilizing component. Therefore, the composition according to one embodiment of the present invention can suppress the decrease in enzyme activity when the enzyme is stored in a dry state.

[0025] Examples of enzymes contained in the reaction components include glucose oxidase (GOD), glucose dehydrogenase (GDH), glucose-6-phosphate dehydrogenase (G6P), diaphorase (DI), creatinine amidohydrolase (CNH), creatine amidinohydrolase (CRH), sarcosine oxidase (SAO), as well as peroxidase (POD), uricase (UAO), urease (URH), pyruvate oxidase (PYO), hexokinase (HK), etc. The type of enzyme may be determined appropriately depending on the type of component to be measured.

[0026] For example, when the component to be measured is glucose (GLU), the enzyme as a reaction component contained in a composition according to one embodiment of the present invention preferably includes at least one glucose-specific enzyme selected from the group consisting of GOD, GDH, and G6P, and more preferably includes GDH. Here, the "glucose-specific enzyme" refers to an enzyme that specifically reacts with GLU as a reaction substrate. Preferably, the enzyme further includes diaphorase (DI).

[0027] Furthermore, for example, when the component to be measured is creatinine (CREA), the enzymes serving as reaction components preferably include creatinine amidohydrolase (CNH), creatine amidinohydrolase (CRH), sarcosine oxidase (SAO), and peroxidase (POD).

[0028] From the viewpoint of reaction sensitivity, the composition according to one embodiment of the present invention preferably contains the enzyme so as to have a specific activity of 0.001 U / mg or more, and more preferably contains the enzyme so as to have a specific activity of 0.01 U / mg or more. Furthermore, although a high enzyme content in the composition according to one embodiment of the present invention does not cause any problems, from the viewpoint of economy, the composition preferably contains the enzyme so as to have a specific activity of 1,000 U / mg or less, and more preferably contains the enzyme so as to have a specific activity of 100 U / mg or less.

[0029] (substrate) The substrate may be any known substrate, such as oxidized nicotinamide adenine dinucleotide (NAD), adenosine triphosphate (ATP), adenosine diphosphate (ADP), flavin adenine dinucleotide disodium n-hydrate (FAD), oxidized nicotinamide adenine dinucleotide phosphate (NADP), and thionicotinamide-adenine dinucleotide (Thio-NAD+).

[0030] The content of the substrate in the composition according to one embodiment of the present invention may be appropriately set depending on the object to be measured from the viewpoint of reaction sensitivity, but is preferably 0.1% by weight or more, more preferably 1% by weight or more, relative to 100% by weight of the composition. There is no particular upper limit, but since adding more than the reaction equivalent amount is uneconomical, the content is preferably 90% by weight or less, more preferably 80% by weight or less, relative to 100% by weight of the composition.

[0031] (dye) As the dye, conventionally known dyes can be used, and examples thereof include tetrazolium salts, leuco dyes, Trinder's reagents, pH indicators, chelating color-developing reagents, and enzyme reaction indicators (GluCANA).

[0032] The tetrazolium salt may be a known substance, for example, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8); 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1); 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-3); 2-benzothiazole-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbomoyl)phenyl]-2H-tetrazolium (WST- 4); 2,2'-Dibenzothiazolyl-5,5'-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3'-(3,3'-dimethoxy-4,4'-biphenylene)ditetrazolium disodium salt (WST-5); 2-(4-nitrophenyl)-5-phenyl-3-[4-(4-sulfophenylazo)-2-sulfophenyl]-2H-tetrazolium monosodium salt (WST-9); 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (NITRO-TB); and others.

[0033] As the leuco dye, a conventionally known substance can be used, and examples thereof include triphenylmethane derivatives, phenothiazine derivatives, and diphenylamine derivatives. More specifically, examples thereof include triphenylmethane derivatives such as 4,4'-benzylidenebis(N,N-dimethylaniline); phenothiazine derivatives such as 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine salt (DA-67), 10-(methylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, and 10-(N-methylcarbamoyl)-3-dimethylamino-7-hydroxy-10H-phenothiazine; diphenylamine derivatives such as 4,4'-bis(dimethylamino)diphenylamine and N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine salt (DA-64); and 1-(ethylaminothiocarbonyl)-2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-diethylaminophenyl)imidazole. The salt is not particularly limited, but examples thereof include sodium salts, potassium salts, and calcium salts.

[0034] The tetrazolium salt is reduced by an electron carrier in the presence of DI to produce a formazan dye, a color-changing dye. The electron carrier can be any known substance capable of reducing the tetrazolium salt to produce a formazan dye, such as reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH). As shown in the following formula (1), NADH is produced by reacting GDH with GLU in the presence of NAD. [ka]

[0035] Therefore, when the component to be measured is GLU, a tetrazolium salt can be suitably used as the dye from the viewpoint of sensitivity.

[0036] Furthermore, the leuco dye is oxidized by the action of peroxidase and hydrogen peroxide (HO) to produce the color dye methylene blue (MB). HO is produced during the reaction between creatinine, the component to be measured, and an enzyme, which is a reactive component in a composition according to one embodiment of the present invention. Therefore, when the component to be measured is CREA, a leuco dye can be preferably used as the dye from the viewpoint of sensitivity.

[0037] The content of the dye in the composition according to one embodiment of the present invention may be appropriately set depending on the object to be measured from the viewpoint of reaction sensitivity, but is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, relative to 100% by weight of the composition. Furthermore, from the viewpoint of reducing the influence of the dye's self-coloring or the coloring of the dye before discoloration on the measured value, the content is preferably 50% by weight or less, more preferably 30% by weight or less, relative to 100% by weight of the composition.

[0038] <Other ingredients> The composition according to one aspect of the present invention may contain, as necessary, components other than those described above, to the extent that the effects of the present invention are not impaired. For example, the composition according to one aspect of the present invention preferably further contains at least one selected from the group consisting of buffer salts and additives.

[0039] (buffer salts) The buffer salt may be a conventionally known substance that has the effect of buffering the pH of the reaction system, such as 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 2-morpholinoethanesulfonic acid (MES), phosphate-buffered saline (PBS), N-[Tris(hydroxymethyl)methyl]glycine (Tricine), N,N-Bis(2-hydroxyethyl)glycine (Bicine), and other so-called "good buffers." The buffer salt may be selected appropriately according to the pH.

[0040] The content of the buffer salt in the composition according to one embodiment of the present invention is not particularly limited, but from the viewpoint of the pH buffering capacity in the reaction solution, it is preferably 0.01% by weight or more, and more preferably 0.1% by weight or more, relative to 100% by weight of the composition. Furthermore, the upper limit of the content of the buffer salt in the composition according to one embodiment of the present invention is not particularly limited, but the amount may be appropriately adjusted so as to avoid adding too much, which would be uneconomical.

[0041] (additives) Examples of additives include stabilizing components other than trehalose, excipients, preservatives, surfactants, synthetic polymers having surface activity, and the like.

[0042] As a stabilizing component other than trehalose, one or more compounds selected from the group consisting of sugars containing 2 to 4 monosaccharides, sugar alcohols, amino acids, and salts containing amino acids and carboxylic acids can be used. Examples of compounds that can be suitably used are as follows:

[0043] Examples of sugars containing two to four monosaccharides include disaccharides such as sucrose (cane sugar), lactulose, lactose, maltose, cellobiose, kojibiose, nigerose, isomaltose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiose, melibiulose, neolactose, galactosucrose, scillabiose, neohesperidose, rutinose, rutinulose, vicianose, xylobiose, and primeverose; trisaccharides such as nigerotriose, maltotriose, melezitose, maltotriulose, raffinose, and kestose; and tetrasaccharides such as nystose, nigerotetraose, and stachyose. Among these, sucrose, lactose, maltose, palatinose, maltotriose, and raffinose are preferably used in terms of stabilizing effect, ease of availability, etc. Sucrose and maltose are more preferably used in terms of their stabilizing effect.

[0044] Suitable sugar alcohols include glycerol, erythritol, threitol, arabinitol, xylitol, sorbitol, lactitol, mannitol, and maltitol. In the composition according to one embodiment of the present invention, there are no particular limitations on the amino acids used as stabilizing components, but preferred amino acids include basic amino acids such as arginine, histidine, and lysine.

[0045] Inorganic salts of amino acids can also be suitably used, for example, inorganic salts of acidic amino acids such as sodium aspartate, potassium aspartate, sodium glutamate, potassium glutamate, etc.

[0046] Although the salt containing an amino acid and a carboxylic acid can be used without any particular limitation, a preferred combination is a salt containing a basic amino acid and a non-volatile carboxylic acid, as this prevents the carboxylic acid from evaporating and allows the effect of the stabilizing component to be maintained for a long time.

[0047] Examples of basic amino acids include arginine, histidine, and lysine.

[0048] Examples of carboxylic acids include acetic acid, lactic acid, butyric acid, octanoic acid, and the like, which have one carboxyl group in the molecule; dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, phthalic acid, isophthalic acid, terephthalic acid, fumaric acid, malic acid, and tartaric acid, which have two carboxyl groups in the molecule; and tricarboxylic acids such as citric acid, isocitric acid, and oxalosuccinic acid. Among these, dicarboxylic acids and tricarboxylic acids are more preferably used because they can prevent decomposition of the salt due to volatilization of the carboxylic acid when forming a salt with an amino acid. Preferred carboxylic acids include malic acid, tartaric acid, and citric acid, taking into account their effectiveness as stabilizing components and ease of availability.

[0049] These stabilizing components other than trehalose may be used alone or in combination of two or more.

[0050] The content of the stabilizing component other than trehalose in the composition according to one embodiment of the present invention is not particularly limited, but from the viewpoint of the stability effect, it is preferably 0.1% by weight or more, more preferably 1% by weight or more, relative to 100% by weight of the composition. Furthermore, from the viewpoint of the solubility of the stabilizing component, it is preferably 90% by weight or less, more preferably 80% by weight or less, relative to 100% by weight of the composition.

[0051] The excipient is preferably a substance that does not interfere with the target reaction and that undergoes minimal deformation even at high temperatures when mixed with the stabilizing component. Specifically, the composition according to one embodiment of the present invention may contain the following excipients: polysaccharides; cyclic sugars; proteins such as albumin; glucose; etc. The stabilizing component itself may also function as an excipient.

[0052] The excipient is composed of at least six or more monosaccharides, and includes at least one selected from the group consisting of polysaccharides (referred to as the polysaccharides of the present invention) containing 3×n or more hydroxyl groups, where n is the number of monosaccharides (n is an integer), and polymers having an amino acid skeleton.

[0053] Any polysaccharide containing a certain number of hydroxyl groups can be used as an excipient, as this increases solubility in water. Examples of such polysaccharides include linear polysaccharides such as cellulose, chitin, starch, glycogen, agarose, carrageenan, dextran, and dextrin (k, 3xk), as well as cyclic polysaccharide cyclodextrins such as α-cyclodextrin and its methylated or hydroxypropylated derivatives, β-cyclodextrin and its methylated or hydroxypropylated derivatives, and γ-cyclodextrin and its methylated or hydroxypropylated derivatives.

[0054] Among these, polysaccharides that are composed of at least six or more monosaccharides and contain 3×n or more hydroxyl groups, where n is the number of monosaccharides (n is an integer), are preferred.

[0055] Specific examples of suitable polysaccharides that can be used as excipients include linear polysaccharides such as starch, glycogen, dextran, and dextrin, as well as cyclic polysaccharides such as cyclodextrins, including α-cyclodextrin and its derivatives such as methylated or hydroxypropylated derivatives, β-cyclodextrin and its derivatives such as methylated or hydroxypropylated derivatives, and γ-cyclodextrin and its derivatives such as methylated or hydroxypropylated derivatives. More preferred examples include dextran and cyclodextrins, which are even more preferred because they are easily soluble in water.

[0056] A polymer having an amino acid skeleton that can be used as an excipient is a polymer in which multiple single amino acids are linearly bonded, such as polylysine formed by bonding L-lysine, poly-L-arginine formed by bonding arginine, and its hydrochloride.

[0057] These excipients may be used alone or in combination of two or more. The stability of the enzyme can also be improved by combining the excipient with an inorganic salt such as sodium chloride or potassium chloride.

[0058] The content of the excipient in the composition according to one embodiment of the present invention is not particularly limited, but from the viewpoint of the effect of the excipient, it is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, relative to 100% by weight of the composition, and from the viewpoint of the solubility of the excipient, it is preferably 90% by weight or less, more preferably 80% by weight or less, relative to 100% by weight of the composition.

[0059] Suitable surfactants include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants, as long as they are necessary for the reaction and do not affect the reaction. Nonionic surfactants can be used without particular limitations, as long as they can be mixed with the stabilizing component. Examples of commonly available surfactants include: polyoxyethylene lauryl ether sugar polyoxyethylene fatty acid ethers; sorbitan fatty acid esters such as sorbitan monolaurate and sorbitan monooleate; 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS); Triton-X; and SDS. Because surfactants can inhibit enzymatic reactions, they are generally added in smaller amounts than polysaccharides.

[0060] The surfactant may be used alone or in combination of two or more. The content of the surfactant in the composition according to one embodiment of the present invention is not particularly limited, but from the viewpoint of obtaining the effects of the surfactant, it is preferably 0.01% by weight or more, and more preferably 0.05% by weight or more, relative to 100% by weight of the composition. Furthermore, in consideration of the influence on the enzyme reaction, it is preferably 10% by weight or less, and more preferably 5% by weight or less, relative to 100% by weight of the composition.

[0061] Preservatives that can be suitably used include sodium azide, Proclin, and the like, as long as they are necessary for the reaction and do not affect the reaction.

[0062] The preservative may be used alone or in combination of two or more. The content of the preservative in the composition according to one embodiment of the present invention is not particularly limited, but from the viewpoint of obtaining the effect of the preservative, it is preferably 0.01 wt% or more, and more preferably 0.05 wt% or more, relative to 100 wt% of the composition. Furthermore, considering that the preservative itself becomes toxic at high concentrations, it is preferably 1 wt% or less, and more preferably 0.1 wt% or less, relative to 100 wt% of the composition.

[0063] (moisture) The composition according to one embodiment of the present invention may be in a liquid state or a solid form. The "liquid state" refers to a state in which a solution containing the components of the composition according to one embodiment of the present invention is not dried, and the composition contains bound water, free water, and bulk water. In contrast, the "solid form" refers to a state in which a solution containing the components of the composition according to one embodiment of the present invention is dried.

[0064] When the composition according to one embodiment of the present invention is in a solid form, the composition according to one embodiment of the present invention may contain water as needed. The water content when the composition according to one embodiment of the present invention is in a solid form can be adjusted to a desired range by adjusting the drying conditions when a solution containing the components of the composition according to one embodiment of the present invention is dried to form the solid form.

[0065] Here, in this specification, the term "moisture" refers to both bound water and free water present in a solid composition according to an embodiment of the present invention. When the water content of a solid composition obtained by drying a solution containing the components of the composition according to an embodiment of the present invention is greater than 1% by weight, it can be said that bound water remains in the composition. The water content in the solid composition according to an embodiment of the present invention and the water content in the dry reagent described below can be measured by the following method of measuring infrared (IR) spectra. (Measurement method) (1) Use calcium chloride desiccant to maintain a relative humidity of 10% or less around the IR measurement unit (single reflection attenuated total reflection (ATR) ATR method: diamond crystal). (2) 0.5 μL of the sample to be measured is dropped onto the measuring part of the IR and allowed to dry. (3) Measure the IR spectrum over time to quantify the water content. The measurement conditions are as follows: measurement range 500 to 4000 cm -1 , resolution cm -1 The number of times of accumulation is 4. The resulting 1640 cm -1 The remaining amount of water is calculated from the area of ​​the peak at 1640cm. -1 and a calibration curve of the peak area of

[0066] A composition according to one embodiment of the present invention can be used as a test reagent using dry chemistry technology (hereinafter also referred to as a "dry reagent"). However, considering application to slide-reagent-type dry chemistry technology, it is preferable that the composition according to one embodiment of the present invention is in a solid form and contains bound water. Therefore, from the viewpoint of retaining bound water in the composition according to one embodiment of the present invention, the water content in the composition is preferably 1% by weight or more, more preferably more than 1% by weight, and even more preferably 3% by weight or more, based on 100% by weight of the composition. Furthermore, from the viewpoint of making the composition according to one embodiment of the present invention in a solid form, the water content in the composition is preferably 30% by weight or less, more preferably 10% by weight or less, based on 100% by weight of the composition.

[0067] Dry chemistry refers to a dry testing method in which a target component in a biological sample is detected using a test strip in which a layer of a reagent composition containing a reactive component that reacts with the target component contained in the biological sample is formed on a support such as a slide. Test reagents using dry chemistry technology are useful for point-of-care testing (POCT) performed at the patient's bedside.

[0068] Furthermore, the composition according to one embodiment of the present invention has improved storage stability due to the inclusion of trehalose. Therefore, it is preferable that the water content in the composition be within a range that does not impair the effects of trehalose. More specifically, it is preferable that the water content in the composition according to one embodiment of the present invention be within the above-mentioned range, and that the weight of the water be 5 times or less the weight of the trehalose contained in the composition.

[0069] A solid composition having a water content within the above range can be produced by air-drying a solution containing the components of the composition according to one embodiment of the present invention.

[0070] Furthermore, when the composition according to an embodiment of the present invention is in a liquid state, in consideration of the effect on reactivity in the liquid, the water content in the composition is preferably 50 wt % or more, and more preferably 70 wt % or more, relative to 100 wt % of the composition. Furthermore, since there is no problem as long as the necessary amounts of components other than water contained in the composition according to an embodiment of the present invention are dissolved in the water, there is no particular upper limit to the water content in the composition.

[0071] (Examples of combinations of enzymes, substrates, and dyes) The combination of the reaction components and the optional substrate and dye contained in the composition according to one embodiment of the present invention may be determined appropriately depending on the type of component to be measured and the reaction type to be used.

[0072] An example will be described in which the component to be measured is GLU. The composition according to embodiment 1 of the present invention contains GOD and POD as enzymes, and a leuco dye and Trinder's reagent as dyes. The composition according to embodiment 1 of the present invention does not contain a substrate. The composition according to embodiment 1 of the present invention can be preferably used to form a GLU reagent for measuring GLU in a serum sample using a conventionally known colorimetric method.

[0073] The composition according to the second embodiment of the present invention contains GDH and DI as reaction components, NAD as a substrate, and WST-4 as a dye. The composition according to the second embodiment of the present invention can be preferably used to construct a GLU reagent using dry chemistry technology to measure GLU in a serum sample using a conventionally known colorimetric method.

[0074] The composition according to the third embodiment of the present invention contains G6P and HK as reaction components and ATP and NAD as substrates. The composition according to the third embodiment of the present invention does not contain a dye, since it is sufficient to measure the wavelength of NADH. The composition according to the third embodiment of the present invention can be preferably used to form a GLU reagent using dry chemistry technology to measure GLU in a serum sample using a conventionally known colorimetric method.

[0075] Next, an example will be described in which the component to be measured is CREA. A composition according to a fourth embodiment of the present invention contains CNH, CRH, SAO, and POD as reactive components, and DA-67 as a dye. The composition according to the fourth embodiment of the present invention can be preferably used to form a CREA reagent using dry chemistry technology to measure CREA in a serum sample using a conventionally known colorimetric method.

[0076] <Uses of the composition> By appropriately selecting the reaction components and optional substrate and dye types according to the purpose, a composition according to one embodiment of the present invention can be used in liquid reagent applications and various dry reagent applications using dry chemistry technology. The composition according to one embodiment of the present invention can be used, for example, as a reagent for biochemistry items such as a GLU reagent composition, a CREA reagent composition, a BUN reagent composition, an ALT reagent composition, and an AST reagent composition. The effect of the composition according to one embodiment of the present invention, which is to suppress color development in blank samples, can be more significantly achieved when the composition according to one embodiment of the present invention is used as a GLU reagent composition.

[0077] Furthermore, the composition according to one aspect of the present invention can be applied to slide reagent-type dry chemistry techniques, and therefore the composition according to one aspect of the present invention can be used as a slide reagent composition.

[0078] <Method of producing the composition> An example of a method for producing a composition according to one embodiment of the present invention will be described. The composition according to one embodiment of the present invention can be produced by a production method including the following step (i): (i) A step of mixing each component of the composition according to one embodiment of the present invention in the same solvent to prepare a homogeneous solution.

[0079] According to this production method, the reaction components and trehalose can be thoroughly mixed. As a result, trehalose can be present around or inside the molecules of the reaction components. As described above, trehalose can substitute for the functions of water, such as hydrogen bonding and providing a hydrophilic environment. Therefore, the presence of trehalose around or inside the reaction components makes it difficult for structural recombination of the reaction components to occur, thereby preventing a decrease in the activity of the reaction components.

[0080] The method for producing a composition according to one embodiment of the present invention may further include the following step (ii): (ii) A step of drying the solution obtained in the step (i). By carrying out the step (ii) following the step (i), a composition according to one embodiment of the present invention in a solid form can be produced.

[0081] Each step will be explained below. (Step (i)) The main components of the composition according to one embodiment of the present invention, including the reactive components, are generally used in aqueous solutions. Therefore, the solvent is generally water. The method for mixing the components of the composition according to one embodiment of the present invention in step (i) is not particularly limited, and known methods can be used. Examples include vortex mixing and ultrasonic dispersion.

[0082] (Step (ii)) The method for drying the solution obtained in step (i) is not particularly limited, and known methods can be used. Examples include air drying, hot air drying, spray hot air drying, and freeze drying. As an example, when the reaction component contains an enzyme, freeze drying and air drying are preferably used as methods for drying the solution obtained in step (i). As another example, when a composition according to one embodiment of the present invention is applied to slide reagent-type dry chemistry technology, it is preferable that bound water remains in the dried product, and therefore air drying is preferably used as a method for drying the solution obtained in step (i).

[0083] The method for air-drying the composition according to one embodiment of the present invention is not particularly limited, and any known method can be used. For example, the composition according to one embodiment of the present invention can be placed in a desiccator together with a drying protection agent at room temperature (15°C to 40°C) and dried overnight in a low-humidity environment. Here, the "low-humidity environment" refers to an environment with a relative humidity of 30% RH or less at room temperature.

[0084] Examples of desiccation protection agents include conventionally known substances used to dry and protect objects to be dried. Examples of such substances include calcium chloride and synthetic zeolite (molecular sieve). Here, "protection" by a desiccation protection agent means protecting the object from moisture absorption. Therefore, a desiccant capable of adsorbing moisture down to a low humidity range (around 10% RH) is preferred.

[0085] [2. Testing Reagents] The present invention also encompasses a test reagent using a composition according to an embodiment of the present invention. The test reagent according to an embodiment of the present invention may be a liquid reagent or a dry reagent.

[0086] <Dry reagent> A dry reagent according to one aspect of the present invention includes a composition according to one aspect of the present invention in a solid form, a drying protection agent that dries and protects the composition, and a packaging material that packages the composition and the drying protection agent, thereby making it possible to provide a dry reagent that suppresses color development in a blank sample.

[0087] (composition) The composition of the solid composition according to one embodiment of the present invention has already been described, and therefore will not be described again here. In the dry reagent according to one embodiment of the present invention, the composition according to one embodiment of the present invention is provided in a dried state on a support. Examples of the support include optically transparent materials such as polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), and styrene-acrylonitrile resin (ABS resin).

[0088] (Drying protection agent) The drying protection agent has been explained above in the section "Method for producing the composition," and therefore the explanation will not be repeated here.

[0089] (packaging material) Examples of packaging materials include aluminum-deposited resin films and film-like desiccants for packaging.

[0090] The shape and size of the packaging material may be such that the composition according to one embodiment of the present invention and the drying protectant can be packaged therein.

[0091] From the viewpoint of preventing moisture absorption, it is preferable to hermetically package the composition according to an embodiment of the present invention and the desiccant protective agent in a packaging material. For example, the composition according to an embodiment of the present invention and the desiccant protective agent can be hermetically packaged in the packaging material by a method of heat-sealing the opening of an aluminum pouch by thermocompression bonding.

[0092] <Liquid reagent> A liquid reagent according to one aspect of the present invention includes the composition according to one aspect of the present invention in a liquid state, thereby making it possible to provide a liquid reagent in which color development in a blank sample is suppressed.

[0093] The dry reagent according to one embodiment of the present invention has the advantage that it can be dissolved in a blood sample when needed, thereby enabling immediate qualitative and quantitative measurements, as will be described in detail below.

[0094] In the field of clinical testing, there is a series of tests (measurements) called biochemical tests, which quantify inorganic substances, low-molecular-weight organic compounds, proteins, enzymes, etc. contained in body fluids such as blood.

[0095] These tests often involve mixing a test reagent with blood or other fluid (serum, plasma, cerebrospinal fluid) in an aqueous solution, causing the components of the test reagent to react with those of the blood, and measuring the change in absorbance due to a color reaction or other such reaction.

[0096] Enzymes are also often used as components of test reagents. For example, reagents for measuring glucose in serum quantify the amount of NADPH produced by the following reaction using the change in absorbance at 340 nm, the wavelength characteristic of NADPH.

[0097] (enzyme reaction by hexokinase) Glucose + ATP → glucose-6-phosphate + ADP (The reaction proceeds in the presence of Mg ions) (enzymatic reaction catalyzed by glucose-6-phosphate dehydrogenase) Glucose-6-phosphate + ADP → D-gluconolactone-6-phosphate + NADPH + H + This reaction can be utilized to construct a dry reagent for measuring glucose in a serum sample. The composition according to one embodiment of the present invention is prepared so as to contain the necessary amounts of ATP, hexokinase, pH buffer, and Mg salt used in the reaction, and the composition is placed in a measurement cell in a dry state, thereby constructing the dry reagent according to one embodiment of the present invention.

[0098] The composition according to one embodiment of the present invention contains trehalose with a purity of 99.9% or higher, thereby suppressing color development in blank samples. Furthermore, the composition according to one embodiment of the present invention has excellent storage stability due to the inclusion of trehalose. Therefore, a dry reagent containing such a composition according to one embodiment of the present invention has excellent long-term storage stability, can be stored at room temperature or higher, and can be dissolved in a blood sample when needed to perform immediate qualitative and quantitative measurements.

[0099] Conventional biological sample measurement test reagents, which require refrigerated or frozen storage, are heated to 37°C, the optimum reaction temperature for enzymes contained in the test reagent, just before measurement, and therefore require several minutes to several tens of minutes before measurement can begin, and shortening this time has been desired. Furthermore, when conventional biological sample measurement test reagents are dry reagents, there are cases where the dry reagents crack when heated to 37°C, causing them to fall out of their containers, making it impossible to perform the required measurement.

[0100] In contrast, the test reagent according to one embodiment of the present invention can be stored at room temperature or higher, eliminating the need for a heating process. Therefore, the test reagent according to one embodiment of the present invention can significantly reduce the time from removal from storage to measurement, and is less susceptible to defects such as cracks that would prevent measurement in the case of dry reagents. Therefore, the test reagent according to one embodiment of the present invention has improved usability compared to conventional test reagents for measuring biological samples.

[0101] The use of the test reagent according to one embodiment of the present invention is not particularly limited, and it can be used, for example, for clinical testing and research in the field of biochemistry.

[0102] [3. Analysis method] The present invention also encompasses a method for analyzing a biological sample using a test reagent according to one embodiment of the present invention. By analyzing a biological sample using a test reagent according to one embodiment of the present invention, color development in a blank sample can be suppressed. Furthermore, this method has the advantage of significantly shortening the time from removal from storage to measurement, and is less susceptible to defects such as cracks that would prevent measurement if the reagent is dry.

[0103] 〔summary〕 A composition according to a first aspect of the present invention comprises trehalose and a reactive component that reacts with a target component to be measured contained in a biological sample, wherein the trehalose has a purity of 99.9% or higher.

[0104] In the composition according to aspect 2 of the present invention, in the above-mentioned aspect 1, the content of trehalose is preferably 0.1 wt % or more and 50 wt % or less.

[0105] In the composition according to aspect 3 of the present invention, in the above aspect 1 or 2, the reaction components may include an enzyme.

[0106] A composition according to aspect 4 of the present invention, in accordance with aspect 3 above, may comprise at least one glucose-specific enzyme selected from the group consisting of glucose oxidase, glucose dehydrogenase, and glucose-6-phosphate dehydrogenase.

[0107] In the composition according to aspect 5 of the present invention, in the above aspect 3 or 4, the enzyme may comprise glucose dehydrogenase.

[0108] The composition according to the sixth aspect of the present invention, in any one of the first to fifth aspects above, may further contain at least one selected from the group consisting of a substrate, a dye, a buffer salt, and an additive.

[0109] In the composition according to aspect 7 of the present invention, in the above-mentioned aspect 6, the dye may be a tetrazolium salt.

[0110] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in the embodiments are also included in the technical scope of the present invention. [Example]

[0111] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0112] (R1 reagent raw material) GDH (manufactured by Toyobo Co., Ltd.) DI (manufactured by Toyobo Co., Ltd.) pH 7.0 HEPES buffer (Dojindo Laboratories) Trehalose 999 (Hayashibara Co., Ltd.) Trehalose (Fujifilm Wako Pure Chemical Industries, Ltd.) Mannitol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[0113] (R2 reagent raw material) β-NAD (manufactured by Oriental Yeast Co., Ltd.) ·WST-4 (manufactured by Dojindo Chemical Research Institute Co., Ltd.) ·pH7.0 HEPES (manufactured by Dojindo Laboratories Co., Ltd.) Trehalose 999 (Hayashibara Co., Ltd.) Trehalose (Fujifilm Wako Pure Chemical Industries, Ltd.) Mannitol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[0114] (Blank sample) As a blank sample, 150 mM PBS buffer was used.

[0115] [Experimental Procedure] (1) A GLU reagent for measuring GLU in serum samples was prepared using the following reaction. [ka] More specifically, reagents R1 and R2 were prepared in the blending ratios shown in Tables 2 and 3, respectively. (2) 100 μL of R1 reagent, 100 μL of R2 reagent, and 4 μL of blank sample were mixed. (3) Five minutes after mixing, the absorbance at 630 nm was measured using a spectrophotometer (TECAN, Infinite PRO).

[0116] [Table 2]

[0117] [Table 3]

[0118] [result] The results are shown in Table 4. [Table 4]

[0119] Comparison of Example 1 with Comparative Examples 1 and 3 showed that the use of high-purity trehalose makes it possible to suppress color development in the blank sample.

[0120] Furthermore, mannitol, a sugar commonly added to test reagents, causes almost no color development in blank samples at normal concentrations (1% by weight), but color development in blank samples occurs when the added concentration is increased (Comparative Examples 3 and 4). In contrast, a comparison of Example 2 with Comparative Examples 2 and 4 showed that the use of high-purity trehalose makes it possible to suppress color development in blank samples, even when the added concentration of trehalose is increased. [Industrial Applicability]

[0121] The present invention can be used for liquid reagents and test reagents for dry chemistry techniques in general.

Claims

1. a reactive component that reacts with a target component to be measured contained in a biological sample; Trehalose and Contains The trehalose has a purity of 99.9% or more. composition.

2. The trehalose content is 0.1% by weight or more and 50% by weight or less. The composition of claim 1.

3. The reaction components include an enzyme. The composition according to claim 1 or 2.

4. The enzyme comprises at least one glucose-specific enzyme selected from the group consisting of glucose oxidase, glucose dehydrogenase, and glucose-6-phosphate dehydrogenase. The composition of claim 3.

5. The enzyme includes glucose dehydrogenase. The composition of claim 3.

6. Further containing at least one selected from the group consisting of substrates, dyes, buffer salts and additives; The composition according to claim 1 or 2.

7. The dye is a tetrazolium salt. The composition of claim 6.

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