Biological sample preserving composition, biological sample preserving kit, biological sample preserving method, and method for analyzing biological substance contained in biological sample

Abstract

To provide means that preserves a biological sample over a long period, and that has high safety, is simple, and is suited to analyze biological substances such as protein.SOLUTION: Provided is a composition for preserving a biological sample, containing a water-absorbing polymer. Provided is also a kit for preserving a biological sample, that contains the composition for preserving a biological sample. Further, provided is a method for preserving a biological sample, and a method for analyzing a biological substance of one kind or more contained in a biological sample.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a composition for storing a biological sample, a kit for storing a biological sample, a method for storing a biological sample, and a method for analyzing a biological substance contained in a biological sample.

Background Art

[0002] Compared with biological samples such as blood and urine, saliva can be collected non-invasively and easily. For this reason, saliva has attracted attention as a target for new biomarker analysis. It is known that some of the proteins contained in saliva reflect biological reactions. For example, it has been reported that the amounts of cortisol, immunoglobulin A (IgA), dehydroepiandrosterone (DHEA), and α-amylase in saliva increase during psychological stress (Non-Patent Document 1). In type 2 diabetic patients, it has been reported that the amount of glutathione increases (Non-Patent Document 2). In periodontitis patients, it has been reported that 8-hydroxy-2'-deoxyguanosine (8-OHdG) increases (Non-Patent Document 3).

[0003] In recent years, the development of diagnostic methods for early detection of cancer has also been advanced from specific biomarkers in saliva. In addition, a method for diagnosing the aging level of an individual from the proteins in saliva has also been reported (Non-Patent Document 4). For example, a method for evaluating the aging degree of an individual from the amounts of GREM1 protein and GREM2 protein in saliva has been reported (Patent Document 1). In addition, methods for predicting the aging states of the brain and blood have also been developed using fractalkine, interleukin-1α (IL-1α), tumor necrosis factor-α (TNF-α), and CD30L as indicators (Patent Document 2).

[0004] Thus, in order to prevent and / or improve aging, a method for biochemically evaluating an individual's aging level is beneficial. Since the aging state of the living body changes fluidly, the proteins in saliva must be accurately monitored. However, the proteins in saliva are rapidly degraded by enzymes such as proteases at room temperature. Therefore, it has been difficult to preserve the proteins in saliva for a long time.

[0005] As a method for preserving proteins in saliva, a method of immediately freezing and storing at -20°C after collection is known (Non-Patent Document 5). Also, as a method for storing saliva at room temperature, a method of adding a high concentration of a strong denaturing agent such as guanidine hydrochloride or urea is known (Non-Patent Document 6).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non - Patent Document 4

Non - Patent Document 5

Non - Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0008] As described above, saliva is useful as a biological sample for biochemically evaluating an individual's aging level. Techniques for storing biological samples such as saliva over a long period are known. However, there was room for improvement in these techniques. For example, in the case of the cryopreservation method (Non-Patent Document 5), it was necessary to prepare the equipment environment for storage, and there was a problem that it was disadvantageous in terms of simplicity. Alternatively, in the case of the method of adding a high-concentration denaturing agent (Non-Patent Document 6), there are concerns about the safety of the denaturing agent for the human body. In addition, due to the use of a high-concentration denaturing agent, subsequent analysis becomes difficult, and there was a problem that the analysis method was limited. These problems exist not only for saliva but also for other biological samples such as blood and urine.

[0009] Therefore, an object of the present invention is to provide a means for storing a biological sample over a long period, which is highly safe, simple, and suitable for analyzing biological substances such as proteins.

Means for Solving the Problems

[0010] The present inventors variously studied means for solving the above problems. The present inventors found that by adding a water-absorbing polymer to a biological sample such as saliva, the biological sample can be easily stored over a long period even at room temperature. In addition, the present inventors found that by the above method, biological substances such as proteins and nucleic acids contained in the biological sample can also be stored over a long period and can be stably used for analysis. Based on the above findings, the present inventors completed the present invention.

[0011] That is, the gist of the present invention is as follows. (Embodiment 1) A composition for storing a biological sample, containing a water-absorbing polymer. (Embodiment 2) The composition according to Embodiment 1, wherein the water-absorbing polymer is an acrylic acid-based water-soluble polymer. (Embodiment 3) The composition according to Embodiment 2, wherein the acrylic acid-based water-soluble polymer is an acrylic acid / sodium acrylate copolymer or a sodium acrylate polymer. (Embodiment 4) The composition according to Embodiment 3, wherein the degree of neutralization of the acrylic acid / sodium acrylate copolymer or the sodium acrylate polymer is 70% or more. (Embodiment 5) The composition according to any one of Embodiments 1 to 4, wherein the biological sample is a body fluid. (Embodiment 6) The composition according to any one of Embodiments 1 to 5, wherein the biological sample is saliva, urine or blood. (Embodiment 7) The composition according to any one of Embodiments 1 to 6, wherein the biological sample contains one or more biological substances selected from the group consisting of proteins and nucleic acids. (Embodiment 8) A kit for storing a biological sample, comprising the composition for storing a biological sample according to any one of Embodiments 1 to 7. (Embodiment 9) A method for storing a biological sample, comprising: a mixing step of mixing the composition for storing a biological sample according to any one of Embodiments 1 to 7 with a biological sample collected from a living body; and a storing step of storing the mixture obtained in the mixing step. (Embodiment 10) A method for analyzing one or more biological substances contained in a biological sample, comprising: a mixing step of mixing the composition for storing a biological sample according to any one of Embodiments 1 to 7 with a biological sample collected from a living body; and an elution step of eluting one or more biological substances contained in the biological sample by adding a metal salt to the mixture obtained in the mixing step. [Advantages of the Invention]

[0012] According to the present invention, it is possible to provide a means for storing a biological sample over a long period of time, which is highly safe, simple, and suitable for analyzing biological substances such as proteins. Problems, configurations, and effects other than those described above will be clarified by the description of the following embodiments. [Brief Description of the Drawings]

[0013]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0014] Hereinafter, preferred embodiments of the present invention will be described in detail.

[0015] <1. Composition for Preserving Biological Samples> One aspect of the present invention relates to a composition for preserving a biological sample.

[0016] In each aspect of the present invention, a biological sample means a sample such as a body fluid collected from a living body, which contains one or more biological substances. In each aspect of the present invention, a living body is usually an individual of a human or non-human mammal (e.g., a warm-blooded animal such as a pig, dog, cow, rat, mouse, guinea pig, rabbit, sheep, cat, monkey, mandrill or chimpanzee) or a part thereof (e.g., a cell, tissue or organ), particularly a human individual or a part thereof (e.g., a cell, tissue or organ). In each aspect of the present invention, a biological sample is usually one or more body fluids selected from the group consisting of saliva, urine, blood, sputum, throat swab fluid, nasal swab fluid, oral (buccal) mucosal swab fluid, lacrimal gland secretion, vaginal fluid, semen, cervical secretion and sweat, which are derived from the living body exemplified above, and particularly, saliva, urine or blood. Alternatively, the biological sample may be hair or skin, or a tape strip peeled off after being attached thereto. The one or more biological substances are usually one or more substances selected from the group consisting of proteins, nucleic acids and endocrine signaling substances (hormones) contained in the biological sample exemplified above, and particularly, one or more substances selected from the group consisting of proteins and nucleic acids. Examples of the protein include various proteins known as biomarkers, such as IgA, α-amylase, glutathione, GREM1, GREM2, fractalkine, IL-1α, TNF-α and CD30L. Examples of the nucleic acid include, for example, RNA and DNA, particularly various RNAs and DNAs known as biomarkers. By applying each aspect of the present invention to the biological sample exemplified above, the biological sample and the one or more biological substances contained therein can be stored safely and simply for a long period of time.

[0017] The present inventors have found that by adding a water-absorbing polymer to a biological sample such as saliva, the biological sample can be easily stored for a long period of time even at room temperature. Therefore, the composition of this aspect contains a water-absorbing polymer.

[0018] In the composition of the present aspect, the water-absorbing polymer is preferably at least one water-soluble polymer selected from the group consisting of acrylic acid-based water-soluble polymers, polyvinylpyrrolidone, vinyl acetate copolymers, and maleic anhydride copolymers, more preferably a water-soluble polymer that is an acrylic acid-based water-soluble polymer or polyvinylpyrrolidone, and even more preferably an acrylic acid-based water-soluble polymer. The water-absorbing polymer exemplified above is highly safe compared to the modifiers used in the conventional preservation methods. Also, when the composition of the present aspect is mixed with a biological sample, the water-absorbing polymer exemplified above is considered to stabilize the biological substance by excluding the water molecules around the biological substance contained in the biological sample. Therefore, by using the composition of the present aspect containing the water-absorbing polymer exemplified above, the biological sample can be stored safely and simply for a long time.

[0019] In an embodiment where the water-absorbing polymer is an acrylic acid-based water-soluble polymer, the acrylic acid-based water-soluble polymer is preferably an acrylic acid / sodium acrylate copolymer or a sodium acrylate polymer, and more preferably an acrylic acid / sodium acrylate copolymer or a sodium acrylate polymer having a structure represented by any of the following formulas (a) to (d). The degree of neutralization of the acrylic acid / sodium acrylate copolymer or the sodium acrylate polymer is preferably 30% or more, more preferably 50% or more, even more preferably 70% or more, and particularly preferably in the range of 70% to 100%. If the degree of neutralization is less than the lower limit value, there is a possibility that the biological substance contained in the biological sample cannot be stably stored. Therefore, by using the composition of the present aspect containing the acrylic acid-based water-soluble polymer exemplified above, the biological sample can be stored safely and simply for a long time.

[0020]

Chemical formula

[0021] In the composition of this embodiment, the content of the water-absorbing polymer is preferably 10% by mass or more, more preferably in the range of 30 to 100% by mass, based on the total mass of the composition. If the content of the water-absorbing polymer is less than the lower limit value, there is a possibility that the biological substances contained in the biological sample cannot be stably preserved. Therefore, by using the composition of this embodiment containing the acrylic acid-based water-soluble polymer exemplified above, the biological sample can be safely and simply preserved over a long period of time.

[0022] The composition of this embodiment may contain one or more other components in addition to the water-absorbing polymer. Examples of the one or more other components include a medium (e.g., a solvent such as water or a water-miscible organic solvent or a solution such as physiological saline), an excipient, a coloring agent, a binder, a vehicle, a solubilizing agent, a preservative, a stabilizer, a swelling agent, a lubricant, a surfactant, an emulsifier, an oily liquid, a suspending agent, a buffer, and an antioxidant.

[0023] The composition of this embodiment can be provided in any form such as a powder, a tablet, a capsule, a liquid, a slurry, or a suspension. In the case of an embodiment where the biological sample to which the composition of this embodiment is applied is the body fluid exemplified above, the composition of this embodiment is preferably in the form of a powder substantially free of a medium. In this embodiment, by adding the composition of this embodiment in the form of a powder to a biological sample that is a body fluid, the composition of this embodiment and the biological sample can be quickly mixed.

[0024] By using the composition of this embodiment having the above characteristics, the biological sample can be safely and simply preserved over a long period of time.

[0025] <2. Kit> Another aspect of the present invention relates to a kit. The kit of this aspect includes a composition for preserving a biological sample according to one aspect of the present invention. The kit of this aspect is preferably for preserving a biological sample or analyzing a biological substance. The kit for preserving a biological sample of this aspect can be used to implement the method for preserving a biological sample according to one aspect of the present invention described below. The kit for analyzing a biological substance of this aspect can be used to implement the method for analyzing a biological substance according to one aspect of the present invention described below.

[0026] In addition to the composition for preserving a biological sample according to one aspect of the present invention, the kit of this aspect may include a sampling device and instructions. The sampling device includes, for example, devices for sampling biological samples such as sponges, straws, cotton swabs, lancets, cylinders, tubes, and caps. The instructions include an instruction manual explaining the procedure of the method for preserving a biological sample or the method for analyzing a biological substance according to one aspect of the present invention described below, and a consent form explicitly indicating consent to the use of the method. By using the kit for preserving a biological sample of this aspect based on the description of the instructions, the biological sample can be safely and conveniently preserved over a long period. Also, by using the kit for analyzing a biological substance of this aspect based on the description of the instructions, one or more biological samples contained in the preserved biological sample can be analyzed with high precision.

[0027] <3. Method for Preserving Biological Samples> Another aspect of the present invention relates to a method for preserving a biological sample. A flowchart showing each step in an embodiment of the method of this aspect is shown in FIG. 1. As shown in FIG. 1, the method of this aspect includes a mixing step (S1-1) and a preservation step (S1-2). Hereinafter, each step will be described in detail.

[0028] [3-1. Mixing Step] This step includes mixing the composition for preserving a biological sample according to one aspect of the present invention with the biological sample collected from a living body.

[0029] In this process, the composition of one aspect of the present invention is such that the final concentration of the water-absorbing polymer contained in the mixture obtained in this process is usually in the range of 0.1 to 30 mass / volume% with respect to the total volume of the mixture, preferably in the range of 0.3 to 10 mass / volume%, more preferably in the range of 0.5 to 5 mass / volume%, and is mixed with the biological sample in such an amount. When the final concentration of the water-absorbing polymer is less than the lower limit value, it may not be possible to stably preserve the biological substances contained in the biological sample. When the final concentration of the water-absorbing polymer exceeds the upper limit value, it may not be possible to efficiently elute the biological substances contained in the biological sample. Therefore, by carrying out this process so that the final concentration of the water-absorbing polymer contained in the mixture is within the range exemplified above, the biological sample can be safely and simply preserved over a long period of time.

[0030] [3-2. Storage step] This process includes storing the mixture obtained in the mixing process described above.

[0031] In this process, the temperature at which the mixture is stored is not particularly limited. This process can be carried out under any conditions of a temperature below normal temperature or a temperature exceeding normal temperature. In one embodiment, the temperature at which the mixture is stored in this process is a temperature below normal temperature, preferably a temperature of 30°C or lower, and more preferably in the range of 0 to 30°C. In another embodiment, the temperature at which the mixture is stored in this process is a temperature exceeding normal temperature, preferably a temperature exceeding 30°C, more preferably a temperature exceeding 30°C and 60°C or lower, and even more preferably a temperature exceeding 30°C and 50°C or lower. For example, in this process, by storing the mixture at a temperature exceeding normal temperature, the biological sample can be safely and simply preserved over a long period of time even under high temperature conditions (for example, temperature conditions of 40°C or higher, particularly 50°C or higher) where preservation was difficult with the conventional preservation methods.

[0032] In this project, the period for storing the mixture depends on the storage temperature described above, but usually it is within 6 months, for example, within 10 weeks, and in particular, within 5 weeks. By implementing the method of this aspect under the conditions described above, a biological sample can be stored safely and simply for a long time.

[0033] <4. Method for Analyzing Biological Substances> After storing a biological sample using the composition of one aspect of the present invention, the inventors have found that by adding a metal salt to the mixture of the composition and the biological sample, one or more biological substances contained in the mixture can be easily and stably eluted. Therefore, another aspect of the present invention relates to a method for analyzing biological substances. A flowchart showing each step in one embodiment of the method of this aspect is shown in FIG. 2. As shown in FIG. 2, the method of this aspect includes a mixing step (S2-1) and an elution step (S2-2). The method of this aspect optionally includes an analysis step (S2-3). Hereinafter, each step will be described in detail.

[0034] [4-1. Mixing Step] This step includes mixing the composition for storing a biological sample of one aspect of the present invention with a biological sample collected from a living body. This step can be carried out under substantially the same conditions as the mixing step in the method for storing a biological sample of one aspect of the present invention described above.

[0035] [4-2. Elution Step] This step includes eluting one or more biological substances contained in the biological sample by adding a metal salt to the mixture obtained in the mixing step described above.

[0036] The metal salts used in this process are preferably salts of metal cations such as sodium, potassium, calcium, magnesium, zinc, aluminum, barium, iron or manganese, and inorganic acid or organic acid anions, more preferably salts of metal cations such as sodium, potassium, calcium, magnesium, zinc, aluminum or barium, and anions such as hydrochloric acid, sulfuric acid, nitric acid or acetic acid, still more preferably sodium chloride (NaCl), calcium chloride (CaCl2), magnesium chloride (MgCl2), magnesium sulfate (MgSO4), zinc sulfate (ZnSO4), potassium alum (AlK(SO4)2), or barium sulfate (BaSO4), and particularly preferably CaCl2 or MgCl2. When this process is carried out using salts of metal cations such as potassium, calcium, magnesium, zinc, aluminum or barium and the anions exemplified above, the water-absorbing polymer aggregates and one or more kinds of biomaterials are eluted, so that one or more kinds of biomaterials can be efficiently recovered. Further, when this process is carried out using salts of the metal cations exemplified above and hydrochloric acid anions, one or more kinds of biomaterials can be analyzed with high precision in the analysis process described below.

[0037] [4-3. Analysis Step] This step includes analyzing one or more kinds of biomaterials obtained in the elution step described above.

[0038] In this step, the one or more kinds of biomaterials to be analyzed are usually the one or more kinds of substances exemplified above, and particularly the proteins or nucleic acids exemplified above known as biomarkers.

[0039] In this project, various analysis means commonly used in the art can be applied to analyze one or more biological substances. Examples of the analysis means include immunoassay methods, instrumental analysis methods, sequence analysis methods, and the like. For example, when one or more biological substances are proteins, measurement methods such as dot blot or Western blot, or instrumental analysis methods such as liquid chromatography, mass spectrometry (MS), or combinations thereof (e.g., liquid chromatography-mass spectrometry (LC-MS)) can be applied. For example, when one or more biological substances are nucleic acids, immunoassay methods such as Southern blot or Northern blot, instrumental analysis methods such as microspectrophotometry or MS, base sequence analysis, microarray, or the like can be applied. By carrying out this project using the means exemplified above, one or more biological substances can be analyzed with high precision.

[0040] As described in detail above, by implementing the method for preserving a biological sample of one aspect of the present invention using the composition of one aspect of the present invention, a biological sample such as saliva can be safely and simply preserved over a long period. Further, by implementing the method for analyzing one or more biological substances of one aspect of the present invention using the composition of one aspect of the present invention, one or more biological substances contained in the biological sample can be easily and stably recovered and applied to various analysis means. Therefore, according to each aspect of the present invention, a biological sample such as saliva can be safely and simply preserved over a long period, and using the preserved biological sample, one or more biological substances such as proteins or nucleic acids of biomarkers contained in the biological sample can be analyzed with high precision.

Examples

[0041] Hereinafter, the present invention will be described more specifically using examples. However, the technical scope of the present invention is not limited to these examples.

[0042] <Experiment 1: Examination of the method for preserving saliva and extracting proteins contained in the preserved saliva> The subject rinsed their mouth three times with tap water. Subsequently, saliva that was naturally secreted from the subject was collected for 5 minutes. The collected saliva was stored using any one of the following methods (A) to (D). (A) The saliva was immediately frozen and stored at -80 °C in a freezer after collection. (B) Urea was added to and stirred in the saliva so that the final concentration became 8 M, and then the saliva was stored at room temperature. (C) Sodium dodecyl sulfate (SDS) was added to and stirred in the saliva so that the final concentration became 4 mass / volume%, and then the saliva was stored at room temperature. (D) Any one of the following polymers (1) to (6) was added to and stirred in the saliva so that the final concentration became 0.5 mass / volume%, and then the mixture containing the saliva was gelled. The obtained mixture was stored at room temperature for 4 weeks.

[0043] [Chemical formula] Polymer (1): Carboxyvinyl polymer; Polymer (2): Polyvinylpyrrolidone; Polymer (3): Acrylic acid-sodium acrylate copolymer (neutralization degree 35%); Polymer (4): Acrylic acid-sodium acrylate copolymer (neutralization degree 50%); Polymer (5): Acrylic acid-sodium acrylate copolymer (neutralization degree 70%); Polymer (6): Sodium acrylate polymer (neutralization degree 100%).

[0044] Polymer (1) is a water-soluble thickener, Polymer (2) is a nonionic water-soluble polymer, and Polymers (3) to (6) are acrylic acid-based water-soluble polymers.

[0045] In order to analyze the proteins and nucleic acids contained in saliva using a mixture containing saliva gelated with a polymer and stored obtained by method (D), it is necessary to perform a step of solubilizing the gel contained in the mixture after storage again, and a step of removing the exogenous polymer added to the saliva from the mixture. Therefore, conditions for aggregating and precipitating the polymer contained in the mixture obtained by method (D) and removing it from the mixture were examined. 10 μL of an aqueous solution of each metal salt of 3 M sodium chloride (NaCl), calcium chloride (CaCl2), magnesium chloride (MgCl2), magnesium sulfate (MgSO4), zinc sulfate (ZnSO4), potassium alum (AlK(SO4)2), or barium sulfate (BaSO4) was dropped onto the upper part of the mixture containing saliva gelated with the acrylic acid·sodium acrylate copolymer (neutralization degree 70%) (polymer (5)). Fig. 3 shows the appearance at the time when the aqueous metal salt solution was dropped onto the upper part of the mixture containing saliva gelated with polymer (5). Table 1 shows the evaluation results of the appearance at the time when the aqueous metal salt solution was dropped onto the upper part of the mixture containing saliva gelated with polymer (5).

[0046]

Table 1

[0047] As shown in Fig. 3 and Table 1, the gel was solubilized immediately after any of the aqueous metal salt solutions was dropped. When the aqueous NaCl solution was dropped, although the gel was solubilized, aggregation of the polymer was not confirmed and removal of the polymer was impossible. When an aqueous solution of each metal salt of CaCl2, MgCl2, MgSO4, ZnSO4, AlK(SO4)2, or BaSO4 was dropped, the gel was solubilized and aggregation of the polymer was confirmed. Among them, when an aqueous solution of each metal salt of CaCl2, MgCl2, or MgSO4 was dropped, it was confirmed that the polymer aggregated with the highest efficiency.

[0048] The mixture with the aqueous metal salt solution dropped in was centrifuged at 3,500 rpm for 15 minutes. The aggregates of the polymer contained in the mixture were precipitated, and the supernatant was recovered from the mixture. The recovered supernatant was heated at 100 °C for 5 minutes and then centrifuged at 3,500 rpm for 15 minutes. The obtained supernatant was reductively treated to obtain a sample. Using a dot blotter (manufactured by SCIE-PLAS), 10 μL of each sample was aspirated and adsorbed onto a membrane (manufactured by MERCK). The membrane was washed with PBS(-) and blocked with PVDF Blocking Reagent for Can Get Signal (registered trademark) (manufactured by TOYOBO). The membrane was washed with PBS(-). The washed membrane was immersed in Can Get Signal Solution 1 (manufactured by TOYOBO) with the primary antibody added and treated at 4 °C for 12 hours. As the primary antibody, an anti-IL-1a antibody (manufactured by protein tech) was used. After treatment with the primary antibody, the membrane was washed with PBS(-). It was immersed in Can Get Signal Solution 2 (manufactured by TOYOBO) with the secondary antibody added and treated at room temperature for 1 hour. As the secondary antibody, a Peroxidase F(ab')2 Fragment Rabbit Anti-Rat IgG (H+L) antibody (manufactured by Jackson Immuno Research) was used. After treatment with the secondary antibody, the membrane was washed with PBS(-). The membrane was treated with a chromogenic reagent (ECL Prime Western Blotting Detection Reagents, manufactured by cytiva). Using a WSE-6200H LuminoGraph II (manufactured by ATTO), the luminescence intensity of the dots on the membrane was measured. Western blotting was also performed using the same antibodies and reagents. The results of dot blotting and Western blotting of the samples prepared from the mixtures with each aqueous metal salt solution dropped in are shown in Table 2.

[0049] [Table 2]

[0050] As shown in Table 2, protein expression was confirmed by both dot blot and Western blot in samples prepared from the mixtures to which any of the metal salt aqueous solutions were added. On the other hand, in samples prepared from the mixtures to which aqueous solutions of metal salts of MgSO4, ZnSO4, and AlK(SO4)2 containing sulfate ions were added, signal enhancement by the metal salts was confirmed in the analysis by dot blot. From these results, although combinations of metal salts and analytical methods unsuitable for protein analysis were also confirmed, it was shown that any of the metal salts tested were useful for the extraction of proteins from mixtures containing polymer-gelled saliva. Also, in the solubilization of gels, polymer removal, and protein analysis, it was shown that CaCl2 and MgCl2 were the most suitable among the metal salts tested.

[0051] <Experiment 2: Comparison of Protein Preservation Efficiency> Regarding saliva or its mixture preserved using any of methods (A) to (D) of Experiment 1, the expression level of the protein contained therein was analyzed, and the preservation efficiency by each method was compared. Saliva preserved using method (A), (B), or (C) was centrifuged at 3,500 rpm for 15 minutes to collect the supernatant. The collected supernatant was heated at 100°C for 5 minutes and then centrifuged at 3,500 rpm for 15 minutes. The obtained supernatant was reductively treated to obtain a sample. Regarding the mixture of saliva preserved using method (D) (polymer addition), in Experiment 1, a sample was obtained according to the procedure using CaCl2 as the metal salt. In the dot blot described in Experiment 1, the target protein contained in the sample was quantified according to the procedure using anti-fractalkine antibody (manufactured by Gene Tex), anti-IL-1a antibody (manufactured by protein tech), anti-TNF-α antibody (manufactured by Santa Cruz Biotechnology), or anti-GREM2 antibody (manufactured by Genetex) as the primary antibody. Table 3 shows the expression levels of the target proteins contained in saliva or its mixture preserved using any of methods (A) to (D). The values in the table indicate the relative concentration (%) of the target protein in the sample prepared from saliva preserved according to each preservation method, calculated with the concentration of the standard solution of the target protein cryopreserved at -80°C taken as 100%.

[0052]

Table 3

[0053] As shown in Table 3, for any of the target proteins, when a water-soluble polymer (Polymers (2) to (6)) was used in Method (D), a protein preservation effect was confirmed. In particular, when an acrylic acid-based water-soluble polymer (Polymers (3) to (6)) was used, a higher protein preservation effect was confirmed. Furthermore, when an acrylic acid / sodium acrylate copolymer or sodium acrylate polymer (Polymers (5) and (6)) with a neutralization degree of 70% or more was used, it was confirmed that an extremely high preservation effect comparable to that of -80°C frozen storage in Method (A) was exhibited. Also, in Method (D), a similar preservation effect was confirmed when the mixture was stored under high-temperature conditions (60°C or lower) (data not shown). From these results, it was shown that acrylic acid-based water-soluble polymers (Polymers (3) to (6)) are effective in protein preservation.

[0054] <Experiment 3: Comparison of Nucleic Acid Preservation Efficiency> For saliva or its mixture preserved using any of Methods (A) to (D) in Experiment 1, the nucleic acid contained therein was quantified, and the preservation efficiency by each method was compared. Saliva preserved using Method (A), (B), or (C) was centrifuged at 3,500 rpm for 15 minutes to recover the supernatant and obtain a sample. For the mixture of saliva preserved using Method (D) (polymer addition), according to the procedure described in Experiment 1, the mixture to which an aqueous metal salt solution of CaCl2 was added dropwise was centrifuged at 3,500 rpm for 15 minutes to recover the supernatant and obtain a sample. Using a NanoDrop micro-spectrophotometer (manufactured by ThermoFisher), the RNA concentration and DNA concentration contained in each sample were calculated. The RNA concentration and DNA concentration (ng / μL) contained in saliva or its mixture preserved using any of Methods (A) to (D) are shown in Table 4.

[0055]

Table 4

[0056] As shown in Table 4, for both RNA and DNA, when water-soluble polymers (Polymers (2) to (6)) were used in Method (D), a nucleic acid preservation effect was confirmed. In particular, when acrylic acid-based water-soluble polymers (Polymers (3) to (6)) were used, a higher nucleic acid preservation effect was confirmed. Furthermore, when a copolymer of acrylic acid and sodium acrylate or a sodium acrylate polymer (Polymers (5) and (6)) with a neutralization degree of 70% or more was used, it was confirmed that an extremely high preservation effect comparable to that of -80°C frozen storage in Method (A) was exhibited. From these results, it was shown that acrylic acid-based water-soluble polymers (Polymers (3) to (6)) are effective in nucleic acid preservation.

[0057] Note that the present invention is not limited to the above-described examples and includes various modifications. For example, the above-described examples have been described in detail for easy understanding of the present invention and are not necessarily limited to those having all the configurations described. Also, it is possible to add, delete, and / or substitute some of the configurations of each example with other configurations.

Claims

1. A composition for storing a biological sample, comprising a water-absorbing polymer.

2. The composition according to claim 1, wherein the water-absorbing polymer is an acrylic acid-based water-soluble polymer.

3. The composition according to claim 2, wherein the acrylic acid-based water-soluble polymer is an acrylic acid / sodium acrylate copolymer or a sodium acrylate polymer.

4. The composition according to claim 3, wherein the neutralization degree of the acrylic acid / sodium acrylate copolymer or the sodium acrylate polymer is 70% or more.

5. The composition according to claim 1, wherein the biological sample is a body fluid.

6. The composition according to claim 1, wherein the biological sample is saliva, urine or blood.

7. The composition according to claim 1, wherein the biological sample contains one or more biological substances selected from the group consisting of proteins and nucleic acids.

8. A kit for storing a biological sample, comprising the composition for storing a biological sample according to any one of claims 1 to 7.

9. A method for storing a biological sample, comprising: a mixing step of mixing the composition for storing a biological sample according to any one of claims 1 to 7 with a biological sample collected from a living body; and a storing step of storing the mixture obtained in the mixing step.

10. A method for analyzing one or more biological substances contained in a biological sample, comprising: a mixing step of mixing the composition for storing a biological sample according to any one of claims 1 to 7 with a biological sample collected from a living body; and an eluting step of eluting one or more biological substances contained in the biological sample by adding a metal salt to the mixture obtained in the mixing step.

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