Protective agent for drying, biological sample measurement reagent, and analytical method

Abstract

To provide a protective agent for drying that prevents an excipient from deforming and becoming poorly soluble, while suppressing the prolonged dissolution time of a reactive component contained therein that reacts with a specific component in a biological sample.SOLUTION: A protective agent for drying contains a stable component that suppresses a reduction in the activity of a reactive component that reacts with a specific component in a biological sample, and an excipient that suppresses the deformation of the reactive component during drying. Specific examples of the stable component include at least one selected from the group consisting of saccharides configured by including two to four monosaccharides, sugar alcohols, and salts including amino acids and carboxylic acids.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a drying protection agent, a biological sample measurement reagent containing the drying protection agent, and an analytical method using the biological sample measurement reagent. [Background technology]

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

[0003] For example, as shown in the following formula (1), in order to measure glucose in blood, glucose dehydrogenase (GDH), which is a glucose dehydrogenase, is allowed to act in the presence of oxidized nicotinamide adenine dinucleotide (NAD) as a substrate. This produces reduced nicotinamide adenine dinucleotide (NADH) in an amount equimolar to the amount of glucose present, and the amount (concentration) of glucose can be indirectly quantified by measuring its specific absorption wavelength, approximately 340 nm, and its absorbance.

[0004] [ka]

[0005] Enzymes are a type of protein, so they generally degrade at high temperatures, making long-term storage difficult. In particular, in aqueous solutions, reactions with hydroxide ions, hydrogen ions, etc. progress, and degradation progresses due to the cleavage of amino acid bonds in proteins. Degradation also progresses due to changes in the higher-order structure of proteins. In the case of enzymes, these factors reduce their reaction activity. For this reason, aqueous solutions of these substances must be stored in a refrigerator or freezer.

[0006] When using the reaction activity of an enzyme, the above-mentioned deterioration of the reaction activity of the enzyme should be avoided because it causes deterioration of the performance of the reagents using the enzyme. As described above, when using the reaction activity of an enzyme to quantify the concentration of glucose, it should be avoided because it causes errors. In order to prevent the deterioration of the reaction activity of the enzyme, it is effective to dry the aqueous solution of the protein and store it in a solid state. Natural drying, hot air drying, freeze drying, etc. are used for the drying process. However, deterioration often progresses even in a dry state. Therefore, even in a dry state, refrigeration or freezing is often required. Also, decomposition and deterioration progresses gradually even when refrigerated or frozen in a dry state.

[0007] Therefore, when drying proteins such as enzymes, a drying protective agent has been used to further improve the stability during storage. Such a technique has been widely studied, and for example, the following drying protective agents have been used:

[0008] (1) Use of sugars Drying protective agents using sugars are known (see, for example, Patent Documents 1 to 3). Patent Document 1 describes a method for improving the stability of dried biological substances (enzymes, etc.). It describes that the storage stability of the biological substances is improved by drying, for example, by freeze-drying, an aqueous solution of a hydrocarbon compound as a drying protective agent for improving the stability during storage and an inhibitor of the Maillard reaction.

[0009] Examples of hydrocarbon compounds used as drying protective agents include maltitol (4-O-β-D-glucopyranosyl-D-glucitol), lactitol (4-O-β-D-galactopyranosyl-D-glucitol), isomaltulose, palatinit and its constitutional isomers (GPS, α-D-glucopyranosyl-1→6-sorbitol and GPM, α-D-glucopyranosyl-1→6-mannitol), sugar alcohols derived from isomaltulose (palatinose) (6-α-D-glucopyranosyl-mannitol and 6-α-D-glucopyranosyl-sorbitol), sucrose and its corresponding sugar alcohols.

[0010] Patent Document 1 describes that by using trehalose as a drying protective agent, the activity of the restriction enzyme PstI was maintained even after storage for 70 days at 55° C. and for 35 days at 70° C. On the other hand, it describes that monosaccharides and dextran were ineffective as drying protective agents.

[0011] Patent Document 2 describes the use of so-called polysaccharides such as agar, algin, carrageenan, furcellaran, gum ghatti, gum tragacanth, karaya gum, guaran, locust bean gum (= carob bean gum), tamarind seed gum, arabinogalactan, xanthan (gum), etc. as drying protective agents in order to stably store the enzyme phosphatase in a dry state.

[0012] Patent document 3 describes the use of α-cyclodextrin, a cyclic sugar, certain sugars, carboxylic acids such as citric acid and trisodium ethylenediaminetetraacetate, and bovine serum albumin as drying protective agents to stabilize p-hydroxybenzoate hydroxylase in a dry state.

[0013] (2) Use of amino acids, amino acid derivatives, and their salts Also, protective agents for drying using amino acids, amino acid derivatives, and salts thereof are known (see, for example, Non-Patent Document 1, Patent Documents 4 and 5). Non-Patent Document 1 describes the use of a salt of a basic amino acid and a carboxylic acid compound (such as a salt of arginine and citric acid) as a protective agent for drying to suppress the decrease in activity of lactate dehydrogenase during freeze-drying.

[0014] Patent Document 4 describes that for approximately 30 types of enzymes used in clinical test reagents, by coexisting poly-γ-L-glutamic acid in an aqueous solution of the enzyme, 95% or more of the enzyme activity can be maintained for a period of 7 days at 35° C. Although there is no experimental data on storage in a dry state, it describes that the method can also be applied to storage in a dry state.

[0015] Patent Document 5 describes a compound in which the amino group of arginine is acylated as a drying protective agent for drying and preserving enzymes and the like by freeze-drying.

[0016] (3) Use of ionic liquids Also, a drying protection agent using an ionic liquid is known (see, for example, Patent Document 6). Patent Document 6 describes that an ionic liquid consisting of a quaternary ammonium salt and a carboxylic acid contributes to improving the storage stability of an enzyme. Specific compounds described include an ionic liquid consisting of triethanolamine and citric acid, and an ionic liquid consisting of tris(hydroxymethyl)aminomethane and citric acid. Enzymes such as urease and catalase are dissolved in the ionic liquid and stored at 25°C or 40°C for up to 90 days, after which their activity is evaluated.

[0017] (4) Mechanism of protein stabilization Non-Patent Document 2 describes the mechanism by which proteins such as enzymes are stabilized when sugar is used as a drying protective agent. That is, when an aqueous protein solution containing sugar is dried, the protein molecules are surrounded by sugar molecules. At this time, hydrogen bonds are formed between the protein and the sugar molecules. Since the hydroxyl groups of the sugar are replaced by water molecules, the three-dimensional structure of the protein becomes almost the same as in the aqueous solution. As the drying progresses further, the sugar becomes a solid and surrounds the protein. In addition, hydrogen bonds with the protein inhibit the molecular motion of the protein. In order to prevent the structure of the protein from being destroyed when changing from a liquid to a solid state, the sugar must not crystallize, but must become an amorphous solid. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] Special Publication No. 10-505591 [Patent Document 2] Special Publication No. 2007-519409 [Patent Document 3] JP 2008-206491 A [Patent Document 4] JP 2015-8707 A [Patent Document 5] WO 2008 / 68885 [Patent Document 6] JP 2018-29590 A [Non-patent literature]

[0019] [Non-Patent Document 1] Chemical & Pharmaceutical Bulletin 2009, Vol. 57, pp. 43-48 [Non-Patent Document 2] Journal of the Japanese Society for Food Technology, 2020, Vol. 21, pp. 95-111 Summary of the Invention [Problem to be solved by the invention]

[0020] Since the drying protection agent is stored in a refrigerator or freezer, it is necessary to improve the storage stability after freezing. In particular, with the recent global warming, it is required to improve the storage stability in a relatively high temperature environment.

[0021] However, as shown in, for example, the above-mentioned Patent Document 6, although the conventional drying protection agents have a certain stabilizing effect compared to glycerin, aqueous albumin solution, etc., none of them can maintain 90% or more of the initial activity after 90 days of storage at 40°C.

[0022] According to the study by the present inventors, it was found that when a reactive component that reacts with a specific component in a biological sample, i.e., an enzyme, etc., is stored at high temperature using the above-mentioned drying protective agent, the drying protective agent gradually deforms and generates a poorly soluble solid containing the enzyme, etc. When such a poorly soluble solid is generated, it takes a long time to dissolve the enzyme, etc. in water, etc., in order to use the enzyme, etc. For example, when the enzyme, etc. is used to quantify a specific component in a biological sample, the enzyme, etc. is gradually released from the poorly soluble solid, so that the concentration of the dissolved enzyme changes over time. Therefore, the reaction does not proceed at the intended speed, and an error occurs in the quantitative value. Such a defect can be a serious defect in a quantitative reagent containing an enzyme, etc., especially in the use of clinical testing.

[0023] In particular, it has been found that such problems occur when the product is stored for a long period of time at a temperature exceeding 37° C. Therefore, it has been desired to suppress the formation of poorly soluble solids in the drying protection agent during storage.

[0024] It is therefore an object of the present invention to provide a drying protectant that can be stored for long periods at temperatures above 37°C. [Means for solving the problem]

[0025] Therefore, the present inventors have conducted extensive research to solve this problem and have found that the problem can be solved by the following means.

[0026] That is, the present invention provides: (1) A drying protectant comprising a stabilizing component that suppresses a decrease in activity of a reactive component that reacts with a specific component in a biological sample, and an excipient that suppresses deformation of the reactive component when dried, Another invention is (2) The dry protection agent according to (1), wherein the stabilizing component includes at least one selected from the group consisting of saccharides containing 2 to 4 monosaccharides, and salts containing amino acids and carboxylic acids; (3) The protective agent for drying according to (1) or (2), wherein the excipient is at least one selected from the group consisting of polysaccharides containing at least 6 monosaccharides, where n is the number of the monosaccharides, and containing 3×n or more hydroxyl groups, and polymers having an amino acid skeleton; (4) The dry protection agent according to (3), wherein the polysaccharide is a cyclic polysaccharide.

[0027] Another invention is (5) A biological sample measurement reagent, which further comprises the reaction component in addition to the drying protection agent according to any one of (1) to (4).

[0028] Another invention is (6) An analytical method for analyzing a biological sample using a biological sample measurement reagent containing a drying protection agent, which is stored at a temperature range higher than 37°C, characterized in that the drying protection agent comprises a stabilizing component that suppresses a decrease in activity of a reactive component that reacts with a specific component in the biological sample, and an excipient that suppresses deformation of the reactive component when dried,

[0029] Another invention is (7) The analytical method according to (6), characterized in that the excipient is a biological sample measurement reagent comprising at least six or more monosaccharides, where n is the number of monosaccharides, and comprises at least one selected from the group consisting of polysaccharides containing 3×n or more hydroxyl groups, and polymers having an amino acid backbone. Effect of the Invention

[0030] The present invention includes a stabilizing component that suppresses the decrease in activity of a reactive component that reacts with a specific component in a biological sample, and an excipient that suppresses deformation of the reactive component when dried. Therefore, according to the present invention, the enzyme activity is maintained even after storage at a temperature range higher than 37°C, and the excipient can suppress deformation of the stabilizing component during storage. That is, according to the present invention, the excipient is deformed and becomes insoluble, and the dissolution of the reactive component that reacts with a specific component in a biological sample contained therein can be suppressed from taking a long time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention will now be described in detail. [Protective agent for drying] The drying protection agent according to one embodiment of the present invention is composed of two main components. One is a stabilizing component (also referred to as a stabilizing component in the present invention) that suppresses a decrease in the activity of a reactive component that reacts with a specific component in a biological sample. The other is an excipient (also referred to as an excipient in the present invention) that suppresses deformation of the reactive component when dried.

[0032] In the present invention, a biological sample refers to compositions and secretions of organisms such as animals, plants, fish, aquatic organisms, fungi, etc., organisms such as viruses present in the organism, and attached organic and inorganic substances. More specifically, it refers to the body fluids, blood, internal and external tissues of organisms. In the present invention, specific components in a biological sample refer 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.

[0033] In the present invention, a reactive component that reacts with a specific component in a biological sample (also referred to as a reactive component in the present invention) is a component that selectively uses a specific component in the biological sample as a reaction substrate and causes some kind of chemical reaction to proceed. Even when multiple components are mixed, it has the property of selectively causing a reaction with a specific component. Specific examples include enzymes, catalysts, antibodies, etc.

[0034] The drying protection agent according to the present invention can be suitably applied in the fields of biochemistry and molecular biology. The specific component in the biological sample varies depending on the reactive component of the present invention, and various components are used. Specific examples of biological samples include human, animal, and fish blood, plasma obtained by removing red blood cells from the blood, and serum, which contain various inorganic ions, low molecular weight organic substances, and proteins such as enzymes and antibodies, and are generally used in the field of clinical testing because they are useful for diagnosing diseases by performing qualitative and quantitative analysis of these. In addition, bacteria and viruses contained in human and animal saliva can also be cited as examples of the biological sample, and are similarly useful for diagnosing diseases. The specific component is a component that is the subject of the qualitative and quantitative analysis, and various inorganic ions, low molecular weight organic substances, proteins, etc. correspond to this. For example, when the reactive component is glucose oxidase, which is a type of enzyme, the enzyme specifically uses glucose as a reactive substrate and causes its oxidation reaction to proceed, so the specific component is glucose. As another example, when aspartate aminotransferase, a type of enzyme whose measurement is recognized as being significant in clinical tests, is used as the reaction component, the characteristic component is aspartic acid, since the enzyme specifically promotes the oxidation reaction of aspartic acid.

[0035] In addition, examples of reaction components of the present invention that are generally used in clinical test reagents are shown in Table 1.

[0036] [Table 1]

[0037] In the present invention, the stabilizing component is described in detail below.

[0038] Here, an enzyme will be taken as an example of a reaction component of the present invention. Enzymes are proteins, and maintaining their higher-order structure is important for maintaining activity. The higher-order structure is maintained by a mild aggregate structure of the constituent amino acids due to hydrophobicity in water, hydrogen bonds between the constituent amino acids, and the like. When an enzyme is stored in a dry state, the water surrounding the enzyme is removed during drying, and the factors that maintain the structure are removed, causing structural recombination (denaturation), resulting in a decrease in the activity.

[0039] If non-volatile compounds that replace the functions of water, such as hydrogen bonding and providing a hydrophilic environment, are present around or inside the protein during the drying process, the above-mentioned structural rearrangements are less likely to occur, and the decrease in activity can be suppressed.

[0040] The stabilizing component may be any component that reacts with a specific component in a biological sample and suppresses a decrease in the activity of the reactive component.Specifically, the stabilizing component may be one or more compounds selected from the group consisting of sugars containing 2 to 4 monosaccharides, sugar alcohols, amino acids, amino acids, and salts containing carboxylic acids.Examples of compounds that can be suitably used are as follows.

[0041] Examples of sugars containing 2 to 4 monosaccharides include disaccharides such as trehalose, 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, trehalose, sucrose, lactose, maltose, palatinose, maltotriose, and raffinose can be preferably used in terms of stabilizing effect, ease of availability, etc. Trehalose, sucrose, and maltose can be more preferably used in terms of their stabilizing effect.

[0042] Examples of sugar alcohols that can be suitably used include glycerol, erythritol, threitol, arabinitol, xylitol, sorbitol, lactitol, mannitol, and maltitol.

[0043] There are no particular limitations on the amino acids used as the stabilizing component of the present invention, but examples of amino acids that can be suitably used include basic amino acids such as arginine, histidine, and lysine.

[0044] 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.

[0045] The salt of the amino acid and the carboxylic acid of the present invention can be used without any particular limitation, but a preferred combination is a salt of the basic amino acid and the non-volatile carboxylic acid of the present invention, which can prevent the carboxylic acid from escaping due to volatilization and can maintain the effect of the stabilizing component for a long time.

[0046] Examples of basic amino acids include arginine, histidine, and lysine.

[0047] Examples of carboxylic acids include acetic acid, lactic acid, butyric acid, octanoic acid, etc., 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 can be used more preferably because they can prevent the decomposition of the salt due to the volatilization of the carboxylic acid when forming a salt with an amino acid. Examples of carboxylic acids that are preferably used include malic acid, tartaric acid, and citric acid, taking into account their effect as a stabilizing component and ease of availability.

[0048] In the present invention, an excipient that suppresses deformation of the reactive component of the present invention upon drying (hereinafter referred to as the excipient of the present invention) is used to suppress deformation of the stable component of the present invention contained in a composition containing the drying protection agent of the present invention during long-term storage.

[0049] The drying protectant of the present invention is expected to be applied to a qualitative dry reagent or a quantitative dry reagent that contains a protein such as an enzyme and is stored in a dry state. In such a reagent, it is necessary that it can be easily dissolved in water or the like when used in order to control the operability and reaction time. In the case of a composition that contains a protein such as an enzyme only in the stable component of the present invention without using the excipient of the present invention and is dried, the stable component of the present invention may gradually deform during long-term storage and form a poorly soluble solid. This causes a situation in which the enzyme reaction starts with a part of the enzyme reaction remaining in the solidified stable component, even though it is necessary to instantly dissolve the enzyme in water and start the enzyme reaction by dissolving it in water. In other words, the reaction proceeds without the required concentration and amount of enzyme being present in water, which results in normal qualitative and quantitative analysis being impossible, which is undesirable.

[0050] By drying the excipient of the present invention together with the stabilizing component of the present invention, the deformation of the stabilizing component of the present invention can be prevented, and the above-mentioned problems can be suppressed. It is believed that the excipient of the present invention, when mixed with the stabilizing component of the present invention, suppresses the physical deformation of the stabilizing component and suppresses the transition of the amorphous portion to a crystalline state. It is believed that this maintains the porous structure formed during drying, resulting in the effect of maintaining solubility.

[0051] The excipient of the present invention is preferably a substance that does not interfere with the target reaction and does not deform much even at high temperatures when mixed with the stable component of the present invention. That is, the substances that can be used as the excipient of the present invention are as follows: polysaccharides, cyclic saccharides, surfactants, synthetic polymers having surface activity, etc.

[0052] The excipient of the present invention is composed of at least six or more monosaccharides, and contains at least one type selected from the group consisting of polysaccharides (referred to as the polysaccharide 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] The polysaccharide of the present invention may be any polysaccharide containing a certain number of hydroxyl groups. This is because it can increase the solubility in water. Examples of the polysaccharide include linear polysaccharides such as cellulose, chitin, starch, glycogen, agarose, carrageenan, dextran, and dextrin (k, 3×k), as well as cyclodextrins, which are cyclic polysaccharides, such as 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.

[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 of the present invention include linear polysaccharides such as starch, glycogen, dextran, dextrin, etc., and cyclodextrins, which are cyclic polysaccharides, such as α-cyclodextrin and its derivatives such as methylated or hydroxypropylated derivatives, β-cyclodextrin and its derivatives such as methylated or hydroxypropylated derivatives, γ-cyclodextrin and its derivatives such as methylated or hydroxypropylated derivatives, etc. More preferred examples include dextran and cyclodextrins, which are even more preferred because they are easily dissolved in water.

[0056] The polymer having an amino acid skeleton of the present invention refers to a polymer in which a single amino acid is linearly bonded in a plurality of units, specifically, specific examples thereof include polylysine formed by bonding L-lysine, poly-L-arginine formed by bonding arginine, and its hydrochloride.

[0057] The nonionic surfactant usable as the excipient of the present invention can be used without any particular limitation as long as it can be mixed with the stabilizing component of the present invention. Examples of commonly available surfactants are as follows. That is, polyoxyethylene fatty acid ethers of polyoxyethylene lauryl ether sugar, sorbitan fatty acid esters such as sorbitan monolaurate and sorbitan monooleate can be mentioned. Since surfactants may inhibit enzyme reactions, etc., they are generally added in amounts less than polysaccharides, etc.

[0058] These excipients may be used alone or in combination with one another. In addition, physical strength and water solubility can be improved by combining with inorganic salts such as sodium chloride and potassium chloride.

[0059] [Biological sample measurement reagents] Using the drying protective agent of the present invention, a biological sample measurement reagent can be constructed in a dry state. Specifically, the biological sample measurement reagent of the present invention can be constructed to include a main component (also referred to as a basic composition in the present invention) containing a reactive component, the above-mentioned stable component, and the above-mentioned excipient. The thus-constructed biological sample measurement dry reagent (also referred to as a dry reagent in the present invention) has the advantage of being excellent in long-term storage stability, being stored without refrigeration, and being able to immediately perform qualitative and quantitative measurements by dissolving it in a blood sample when necessary. This will be described in detail below.

[0060] 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.

[0061] These tests often involve mixing a test reagent with blood or the like (serum, plasma, cerebrospinal fluid) in an aqueous solution, causing the components of the biochemical test reagent to react with the components of the blood or the like, and measuring the change in absorbance due to a color reaction or the like.

[0062] Enzymes are often used as components of the reagent. For example, in a reagent for measuring glucose in serum, the amount of NADPH produced by the following reaction is quantified using the change in absorbance at 340 nm, which is the absorption wavelength specific to NADPH.

[0063] (Enzyme reaction by hexokinase) Glucose + ATP → glucose-6-phosphate + ADP (The reaction proceeds in the presence of Mg ions)

[0064] (Enzyme reaction catalyzed by glucose-6-phosphate dehydrogenase) Glucose-6-phosphate + ADP → D-gluconolactone-6-phosphate +NADPH + H+

[0065] This reaction can be utilized to construct a dry reagent for measuring glucose in a serum sample. The dry reagent for measuring a biological sample can be constructed by placing the necessary amounts of ATP, hexokinase, pH buffer, and Mg salt used in the above reaction in a measurement cell in a dry state. Furthermore, by adding the stable component of the present invention to the basic composition of the dry reagent in order to subtract the storage stability of the enzyme, and by adding the excipient of the present invention to prevent the stable component of the present invention from being deformed, the dry reagent can be constructed such that the entire amount is instantly dissolved when a serum sample is added and the reaction starts at a predetermined concentration even after long-term storage.

[0066] The basic composition of the dry reagent for biological sample testing, the stabilizing component of the present invention, and the excipient of the present invention must be mixed at the molecular level. That is, it is important that each molecule of each component of the basic composition after drying is surrounded by the molecules of the stabilizing component of the present invention as much as possible, even if not completely. In order to dry in such a state, it is necessary to mix the basic composition, the stabilizing component of the present invention, and the excipient of the present invention in the same solvent in advance before drying to prepare a uniform solution, and then dry it.

[0067] Since the basic compositions are generally used in aqueous solutions, the solvent is generally water. The concentrations of the basic compositions before drying are generally the concentrations when they are designed as aqueous solution reagents.

[0068] The concentration of the stable component of the present invention and the excipient of the present invention before drying is preferably equal to or higher than the concentration of the enzyme contained in the basic composition. Also, in order to prevent each compound of the basic composition from escaping from the container by the drying operation, it is necessary to make them occupy a certain volume. The preferred concentration range is 0.01% by mass to 50% by mass, the more preferred concentration range is 0.05% by mass to 20% by mass, and the even more preferred concentration range is 0.1% by mass to 10% by mass.

[0069] The ratio of the stabilizing component of the present invention and the excipient of the present invention to the whole after drying is 50% by mass or more, preferably 80% by mass or more, and more preferably 90% by mass or more when the stabilizing component of the present invention and the excipient of the present invention are present in large excess, so that the reactive components of the present invention are surrounded by the stabilizing component of the present invention alone and can be stabilized without agglomerating with each other.

[0070] The ratio of the stabilizing ingredient of the present invention to the excipient of the present invention can be used without any particular limitation, but the preferred range is that the calculated value of (mass of the stabilizing ingredient of the present invention ÷ mass of the excipient of the present invention) is preferably in the range of 10 to 100,000 in the case of a surfactant, more preferably in the range of 100 to 100,000, and in other cases, it is 0.01 to 10, more preferably in the range of 0.01 to 5.

[0071] The method for drying the solution containing the basic composition of the dry reagent for measuring biological samples, the stable component of the present invention, and the excipient of the present invention is not particularly limited, and any known method can be used. Preferred methods include natural drying, hot air drying, spray hot air drying, and freeze drying. Since enzymes are often contained in the dry reagent for measuring biological samples, freeze drying and natural drying are more preferably used.

[0072] The dry reagent for measuring a biological sample prepared by the above method is generally stored at -20°C to 100°C, more preferably 0°C to 80°C, and even more preferably 40°C to 60°C.

[0073] [Analysis method] In the present invention, a dry reagent for measuring a biological sample, which contains the drying protection agent of the present invention, can be stored at a temperature range higher than 37°C, and then the stored dry reagent for measuring a biological sample can be used to analyze the biological sample (also referred to as the analytical method in the present invention).

[0074] Conventionally, the dry reagent for measuring biological samples used in the analysis was stored in a refrigerator or freezer, and immediately before the measurement, it was heated to 37°C, which is the optimal reaction temperature for the enzymes contained in the reagent, before the measurement. This required several minutes to several tens of minutes before the measurement could begin, and it was desired to shorten this time. By using the analysis method of the present invention, the time from taking the biological sample measuring reagent out of the storage place to using it for the measurement can be significantly shortened. In addition, when the reagent is heated to 37°C after being stored in a refrigerator, the reagent may crack and fall out of the container, making it impossible to perform the required measurement. In addition, a reagent capable of measuring biological samples containing the reactive components of the present invention such as enzymes and antibodies and capable of being stored at room temperature has been developed and is commercially available in recent years, but the upper limit of the storage temperature is about 30°C, and it is not suitable for the recent increase in summer temperatures. EXAMPLES

[0075] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0076] (Examples 1-3) Storage and stabilization of dried enzymes The storage stability of the dried enzyme was examined by the following steps: (i) drying the enzyme, and (ii) measuring the change in enzyme activity over time.

[0077] (i) Drying of the enzyme 1 mL of each aqueous solution containing the enzyme, the stable component of the present invention, and the excipient of the present invention shown in Table 2 was prepared. 100 μL of this aqueous solution was dispensed into three 1.5 mL polypropylene tubes. The tubes were placed in a freezer at -80°C for 1 hour to freeze the aqueous solution. The tubes were removed from the freezer and placed on the drying shelf of a shelf-type freeze dryer (Tokyo Rikakikai Co., Ltd. FDL-1000). The shelf temperature was changed as follows under a reduced pressure of 20 Pa or less to dry the tubes, and then the tubes were removed from the drying shelf and the tube lids were tightened. The tubes were placed in an aluminum pouch containing a desiccant (silica gel), the aluminum pouch was sealed, and the tubes were stored at 40°C, 60°C, and 80°C. -40℃ 20 minutes → -10℃ 1 hour → 20℃ 5 hours

[0078] [Table 2]

[0079] (ii) Measurement of changes in urease enzyme activity over time The aluminum pouch containing the tube was removed from the thermostatic bath and left at room temperature for 30 minutes to allow the temperature inside the tube to return to room temperature. The aluminum pouch was opened and the tube was removed. 100 μL of water was added to the tube to dissolve the contents. 30 μL of this aqueous solution and 170 μL of a 50% ethanol aqueous solution of 0.1% bromocresol green were dispensed into a 96-well microplate. After incubation at 37°C for 5 minutes, 20 μL of a 15 mg / mL solution of urea nitrogen, which is the substrate, was added, and the absorbance at 610 nm was measured every 15 seconds immediately after that. The difference between the absorbance after 10 minutes and the absorbance immediately after the start of the measurement was calculated as the change in absorbance.

[0080] The change in absorbance immediately after the start of the storage test at each temperature was taken as 100%, and the change in absorbance after storage for a specified number of days was calculated as a percentage, which was taken as the enzyme activity. The results of the storage tests at various temperatures and for various days are shown in Table 3.

[0081] Comparative Example 1 The time course of change in the enzyme activity of urease was measured in the same manner as in Example 1, except that the excipient of the present invention was not used. The results are also shown in Table 3.

[0082] Comparative Example 2 The change over time in the enzyme activity of urease was measured in the same manner as in Example 2, except that the stabilizing component of the present invention was not used. The results are also shown in Table 3.

[0083] [Table 3]

[0084] In Examples 1 to 3, 90% or more of the activity was maintained even after 60 days or more at temperatures of 40° C., 60° C., and 80° C. In contrast, in Comparative Example 1, since a stabilizing component was contained, 90% or more of the activity was maintained after 30 days, but after 60 days, the activity decreased to 70% or less. Furthermore, in Comparative Example 2, the activity decreased to 50% or less after 30 days, indicating degradation of the enzyme.

[0085] (Examples 4-6) Dry reagent for measuring biological samples 1 mL of an aqueous solution containing the composition shown in Table 4 was prepared. 100 μL of this aqueous solution was dispensed into three 1.5 mL polypropylene tubes. The tubes were placed in a freezer at -80°C for 1 hour to freeze the aqueous solution. The tubes were removed from the freezer and placed on the drying shelf of a shelf-type freeze dryer (Tokyo Rikakikai Co., Ltd. FDL-1000). The shelf temperature was changed as follows under reduced pressure of 30 Pa or less to dry the solution, and then the tubes were removed from the drying shelf and the tube lids were tightened. In this way, a dry reagent for measuring a biological sample in a dry state was prepared in the tube. The tubes were placed in an aluminum pouch containing a desiccant (silica gel), the aluminum pouch was sealed, and the tubes were stored at room temperature, 40°C, 60°C, and 80°C. -40℃ 20 minutes → -10℃ 1 hour → 20℃ 5 hours

[0086] [Table 4]

[0087] (ii) Measurement of changes in the performance of dry reagents for measuring biological samples over time The aluminum pouch containing the tube was removed from the thermostatic bath and left at room temperature for 30 minutes to allow the temperature inside the tube to return to room temperature. The aluminum pouch was opened and the tube was removed. 200 μL of aqueous solution (containing urea nitrogen 10 mg / dL, glucose 100 mg / dL, and cholesterol 180 mg / dL) was added to the tube to dissolve the contents. Immediately after that, the aqueous solution was dispensed into a 96-well microplate. After incubation at 37° C. for 5 minutes, the absorbance was measured every 15 seconds (measured at 610 nm in Example 4, Comparative Example 3, and Comparative Example 4, 340 nm in Example 5, and 500 nm in Example 6). The difference between the absorbance after 10 minutes and the absorbance immediately after the start of measurement was calculated as the absorbance change.

[0088] The change in absorbance immediately after the start of the storage test was considered to correspond to the respective concentrations used, and each change in absorbance was converted into a concentration. The results of the storage tests at various temperatures and for various days are shown in Table 5.

[0089] Comparative Example 3 The concentration was calculated from the change in absorbance when 10 mg / dL of urea nitrogen was measured in the same manner as in Example 3, except that the excipient of the present invention was not used. The results are also shown in Table 5.

[0090] Comparative Example 4 The concentration was calculated from the change in absorbance when 10 mg / dL of urea nitrogen was measured in the same manner as in Example 3, except that the stabilizing component of the present invention was not used. The results are also shown in Table 5.

[0091] [Table 5]

[0092] In Examples 4 to 6, the activity maintained 70% or more of the quantified value immediately after the start even after 60 days or more at temperatures of 40° C., 60° C., and 80° C. In contrast, significant deterioration was observed in Comparative Examples 3 and 4, demonstrating that the performance of the dry reagent for measuring biological samples can be maintained even at high temperatures by using the drying protection agent of the present invention.

[0093] Example 7 Ten dry reagents for biological sample measurement were prepared in the same manner as in Example 4 and stored at 40° C. for 60 days. After that, without returning them to room temperature, the same operation as in Example 4-6(ii) Measurement of change over time in performance of dry reagent for biological sample measurement was performed. The results are shown in Table 6.

[0094] Comparative Example 5 Ten dry reagents for biological sample measurement were prepared in the same manner as in Example 4 and stored at 4° C. for 60 days. They were then returned to room temperature and subjected to the same operation as in Example 4-6(ii) Measurement of change over time in performance of dry reagent for biological sample measurement. The results are also shown in Table 6.

[0095] In Example 7, all of the measurements performed using 10 dry reagents for measuring biological samples gave a quantitative value of 10 mg / dL, which is the known concentration. However, in Comparative Example 5, one measurement gave a value that was significantly different from the known concentration. This was thought to be because the dry reagent for measuring biological samples was stored at a low temperature of 4°C for a long time, causing the reagent to shrink, and when it was heated to 37°C during the subsequent measurement, it cracked and came out of the tube.

[0096] [Table 6]

Claims

1. a stabilizing component that suppresses a decrease in activity of a reactive component that reacts with a specific component in a biological sample; An excipient that suppresses deformation of the reaction components when dried; A drying protectant comprising:

2. The stabilizing component is a sugar comprising 2 to 4 monosaccharides; Sugar alcohols, The dry protection agent according to claim 1 , further comprising at least one member selected from the group consisting of an amino acid and a salt containing a carboxylic acid.

3. The excipient contains at least six monosaccharides, and when the number of the monosaccharides is n, The dryness protecting agent according to claim 1 or 2, comprising at least one member selected from the group consisting of polysaccharides containing 3×n or more hydroxyl groups, polymers having an amino acid skeleton, and nonionic surfactants.

4. The drying protectant of claim 3, wherein the polysaccharide is a cyclic polysaccharide.

5. A biological sample measurement reagent comprising the drying protectant according to any one of claims 1 to 4, and further comprising the reaction component.

6. After storing the biological sample measurement reagent containing the drying protectant at a temperature range higher than 37° C., An analytical method for analyzing a biological sample using the stored biological sample measurement reagent, comprising: The drying protection agent comprises a stabilizing component that suppresses a decrease in activity of a reactive component that reacts with a specific component in a biological sample, and an excipient that suppresses deformation of the reactive component when dried. The analytical method according to claim 1, wherein the drying protectant comprises:

7. The excipient contains at least six monosaccharides, and when the number of the monosaccharides is n, A polysaccharide containing 3×n or more hydroxyl groups and a polymer having an amino acid skeleton; The analytical method according to claim 6, wherein the biological sample measurement reagent comprises at least one selected from the group consisting of:

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