GFAP-containing composition

Abstract

A GFAP-containing composition containing GFAP and at least one surfactant selected from the group consisting of an amphoteric surfactant and an anionic surfactant.

Description

GFAP-containing composition

[0001] The present invention relates to a GFAP-containing composition, and more particularly to a GFAP-containing composition and a method for improving the storage stability of GFAP.

[0002] Glial fibrillary acidic protein (GFAP) is known as a biomarker for diagnosing central nervous system damage and diseases, and in recent years, it has also been shown to function as a biomarker for diagnosing Alzheimer's disease.

[0003] Immunoassay methods are mainly used as a method for detecting GFAP as a biomarker from a specimen collected from a subject. For example, Japanese Patent Application Laid-Open No. 2022-166139 (Patent Document 1) describes a method for detecting GFAP in a specimen (sample) using an anti-GFAP antibody that specifically binds to GFAP.

[0004] In detecting GFAP by immunoassay, for example, anti-GFAP antibody is labeled with a labeling substance, and GFAP in the sample is detected based on the signal derived from the labeling substance bound to GFAP via the anti-GFAP antibody.At this time, the amount of detected signal can be compared with the amount of signal similarly detected using a standard solution containing a known concentration of GFAP, thereby quantifying the amount of GFAP in the sample.Usually, for example, a calibration curve can be prepared from the GFAP concentration in the standard solution and the amount of signal detected in the standard solution, and the GFAP concentration in the sample can be obtained by comparing the amount of signal detected in the sample with the amount of signal detected in the standard solution.

[0005] Regarding GFAP used in the immunoassays and the like, for example, Chinese Patent Application Publication No. 113759130 (Patent Document 2) discloses that a buffer solution containing 4-morpholineethanesulfonic acid, NaCl, ZnCl is used as a storage buffer for freeze-drying GFAP. ２ , MgCl ２ ・6H ２ A buffer containing 0, bovine serum albumin, casein, glycine, polyethylene glycol, and trehalose is described.

[0006] Japanese Patent Publication No. 2022-166139 Chinese Patent Application Publication No. 113759130

[0007] For example, as a standard solution used in quantifying GFAP by immunoassay, an aqueous solution of GFAP dissolved in a buffer solution or the like is usually used, but the present inventors have found that when GFAP is stored in an unfrozen state, particularly as an aqueous solution, the storage stability of GFAP is insufficient, resulting in a decrease in the amount of GFAP detected via binding with an anti-GFAP antibody. This decrease in the amount of GFAP due to storage has a significant effect on the accuracy of quantification, for example, when an aqueous solution of GFAP is used as the standard solution as described above. Therefore, the present inventors have found that a problem exists in that a GFAP composition (particularly an aqueous solution) is required to have a higher level of storage stability of GFAP than conventionally.

[0008] The present invention has been made in view of the problems associated with the above-mentioned prior art, and aims to provide a GFAP-containing composition having excellent storage stability of GFAP, and a method for improving the storage stability of GFAP.

[0009] The present inventors have conducted extensive research to achieve the above-mentioned object and have found that by including at least one surfactant selected from the group consisting of amphoteric surfactants and anionic surfactants as a component of a GFAP-containing composition, a decrease in the amount of GFAP detected with an anti-GFAP antibody can be suppressed at a high level. Furthermore, they have found that such a suppressive effect is superior to that when no surfactant is included or when other surfactants are included, and that this is a specific effect on GFAP that is not exerted by other proteins, and have thus completed the present invention.

[0010] That is, the present invention relates to a GFAP-containing composition and a method for improving the storage stability of GFAP, and more specifically provides the following: [1] A GFAP-containing composition containing GFAP and at least one surfactant selected from the group consisting of amphoteric surfactants and anionic surfactants. [2] The GFAP-containing composition according to [1], which is a GFAP standard solution. [3] The GFAP-containing composition according to [1] or [2], which has a pH of 7.5 to 9.0. [4] The GFAP-containing composition according to any one of [1] to [3], which further contains a nonionic surfactant. [5] The GFAP-containing composition according to any one of [1] to [4], which contains an amphoteric surfactant, and which is a sulfobetaine surfactant having a linear alkyl group having 8 to 25 carbon atoms. [6] The GFAP-containing composition according to any one of [1] to [5], which has a GFAP content of 0.001 to 500 ng / mL. [7] The GFAP-containing composition according to any one of [1] to [6], wherein the content of the surfactant is 0.01 to 5.0 w / v %. [8] The GFAP-containing composition according to any one of [1] to [7], further comprising a protein stabilizing substance. [9] The GFAP-containing composition according to any one of [1] to [8], further comprising a buffering agent.

[10] A method for improving the storage stability of GFAP in a GFAP-containing composition, comprising the step of further incorporating at least one surfactant selected from the group consisting of amphoteric surfactants and anionic surfactants into the GFAP-containing composition.

[0011] According to the present invention, it is possible to provide a GFAP-containing composition having excellent storage stability of GFAP and a method for improving the storage stability of GFAP. In the GFAP-containing composition provided by the present invention, the amount of GFAP detected by an anti-GFAP antibody is stably maintained at an extremely high level, and therefore such a GFAP-containing composition is particularly useful as a GFAP-containing composition of known concentration that serves as a reference when detecting and quantifying GFAP in a sample by immunoassay, i.e., as a GFAP standard solution (calibrator).

[0012] The present invention provides a GFAP-containing composition (sometimes referred to herein as the "composition of the present invention") containing GFAP and at least one surfactant selected from the group consisting of amphoteric surfactants and anionic surfactants.

[0013] [Composition] The composition of the present invention is preferably an aqueous solution. However, the composition of the present invention also includes a lyophilized composition obtained by lyophilizing the aqueous solution, a mixed composition for preparing the aqueous solution, and a concentrated solution for diluting the aqueous solution by a factor of 2 to 10, etc., prior to use. Unless otherwise specified, the content of a component contained in the composition of the present invention basically includes the content in such a lyophilized composition, mixed composition, or concentrated solution. However, when the content of a component contained in the composition of the present invention is expressed in units such as "ng / mL," "mM," and "w / v %" herein, the content refers to the content when the composition of the present invention is made into the aqueous solution. In this specification, "w / v %" refers to weight / volume percent (g / mL x 100).

[0014] When the composition of the present invention is the freeze-dried composition, the mixed composition, or the concentrated solution, it can be dissolved or diluted with water to a desired GFAP concentration to prepare the aqueous solution. In these cases, when the composition of the present invention does not contain the buffering agent described below or contains only a part of the buffering agent described below, it may be dissolved or diluted with, for example, a buffer solution adjusted so that the concentration of the buffering agent in the resulting composition (aqueous solution) falls within a preferred range, instead of water.

[0015] [GFAP] In the present invention, "GFAP" refers to glial fibrillary acidic protein, which is typically a 49,880 Da protein consisting of 432 amino acids. The amino acid sequence of GFAP can be obtained from known databases such as Uniprot, and a typical amino acid sequence of GFAP according to the present invention is the amino acid sequence consisting of all 432 amino acids of human GFAP (isoform 1) with NCBI accession number NP_002046. However, the "amino acid sequence of GFAP" according to the present invention is not limited to this.

[0016] For example, the "amino acid sequence of GFAP" according to the present invention includes an amino acid sequence having a homology (preferably identity) of 70% or more, preferably 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more to the above-mentioned exemplary amino acid sequence; and also includes an amino acid sequence in which one or more amino acids (70 amino acids or less, preferably 45 amino acids or less, 25 amino acids or less, 20 amino acids or less, 10 amino acids or less, 5 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid) have been substituted, deleted, added, and / or inserted in the above-mentioned exemplary amino acid sequence.

[0017] Furthermore, the "GFAP" contained in the composition of the present invention may be the full-length amino acid sequence of the GFAP, a fragment containing a part of it, a multimer containing the full-length and / or fragment, or a complex of these with other proteins, etc., or may be glycosylated, etc. For example, when the composition of the present invention is used as a standard solution for an immunoassay, it is sufficient that the anti-GFAP antibody used in the immunoassay can specifically bind to it.

[0018] The content of GFAP contained in the composition of the present invention can be, for example, 0.0001 to 5000 ng / mL relative to the total amount of the composition, and can be further adjusted appropriately depending on the purpose of use. However, from the viewpoint that storage stabilization tends to be particularly effective when stored in a non-frozen state as an aqueous solution (preferably a standard solution), the content is more preferably 0.001 to 500 ng / mL, and even more preferably 0.01 to 100 ng / mL.

[0019] [Surfactant] The composition of the present invention contains at least one surfactant selected from the group consisting of amphoteric surfactants and anionic surfactants (sometimes referred to herein as "amphoteric surfactant and / or anionic surfactant"). The composition of the present invention may contain either an amphoteric surfactant or an anionic surfactant alone, or may contain both in combination.

[0020] (Amphoteric surfactant) An "amphoteric surfactant" refers to an ionic surfactant that dissociates to form ions (charged atoms or atomic groups), and that has both an anion and a cation when ionized in an aqueous solution. Depending on the pH of the aqueous solution, an amphoteric surfactant exhibits the properties of an anionic surfactant in the alkaline range and the properties of a cationic surfactant in the acidic range.

[0021] The amphoteric surfactant according to the present invention may be one type or a combination of two or more types, and is not particularly limited. However, for example, an amphoteric surfactant having a hydrophobic alkyl group and a hydrophilic portion containing a quaternary ammonium group in the molecule is preferred. The hydrophobic alkyl group is preferably a linear alkyl group, a cycloalkyl group having one to four rings, or an aromatic hydrocarbon group, and a linear alkyl group is more preferred. Furthermore, the hydrophilic portion preferably contains at least one negatively charged substituent selected from the group consisting of a sulfonate group, a carboxy group, and a phosphatidyl group, and the amphoteric surfactant according to the present invention is more preferably a sulfobetaine surfactant having a sulfonate group. Examples of such amphoteric surfactants include those represented by the following formula (1): R １—CO—NH—R ２ -N ＋ (CH ３ ) ２ - [(CH ２ ) ３ -SO ３ － ](R １ represents an alkyl group or a cycloalkyl group having 1 to 4 rings, and R ２ represents an alkylene group (preferably an alkylene group having 1 to 3 carbon atoms). １ Preferably, the carbon number of the linear alkyl group of the sulfobetaine surfactant is 8 to 25, more preferably 12 to 20, even more preferably 13 to 16, and even more preferably 13 to 15. Specific examples of such amphoteric surfactants include sulfobetaine surfactants having a linear alkyl group with 8 to 25 carbon atoms (preferably 12 to 20, more preferably 13 to 16).

[0022] More specifically, examples of the amphoteric surfactant according to the present invention include N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C12APS), N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C14APS), N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C16APS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), N- Examples include benzyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (NDSB-256), 3-[(3-chloramidopropyl)dimethylammonio]-2-hydroxypropanesulfonate (CHAPSO), N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C10APS), and 3-(N,N-dimethyloctylammonio)propanesulfonic acid (C8APS), and these may be used alone or in combination of two or more. Among these, the amphoteric surfactant according to the present invention is preferably at least one selected from the group consisting of C12APS, C14APS, C16APS, and CHAPS, more preferably C14APS and C16APS, and even more preferably C14APS.

[0023] (Anionic surfactant) An "anionic surfactant" refers to an ionic surfactant that ionizes to form ions (charged atoms or atomic groups), and when ionized in an aqueous solution, the moiety containing the hydrophobic group ionizes to negative ions.

[0024] The anionic surfactant according to the present invention may be one type or a combination of two or more types, and is not particularly limited. Examples thereof include carboxylates, sulfonates, sulfates, phosphates, acyl sarcosines, alkyl ether sulfates, phosphatidylcholines, dialkyl sulfosuccinates, and polyoxyethylene alkyl ethers.

[0025] More specifically, examples of the anionic surfactant according to the present invention include carboxylic acid surfactants such as polyoxyalkylene alkyl ether sulfate, alkyl sulfate, polyoxyalkylene alkyl ether phosphate, sodium laurate, sodium oleate, sodium stearate, lauroamidopropyl betaine, sodium cocoyl glutamate, lauryl glucoside, and decyl glucoside; sodium dodecylbenzenesulfonate, sodium α-olefinsulfonate, α-sulfofatty acid methyl ester salts, sulfosuccinates, sodium lauryl sulfonate, sodium stearyl sulfonate, sodium lauryl ether sulfonate, sodium stearyl ether sulfonate, sodium nonylbenzenesulfonate, and sodium decylbenzenesulfonate. Examples of anionic surfactants include sulfonates such as sodium lauryl sulfoacetate and sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), polyoxyethylene alkyl ether sulfates (AES), sodium lauryl sulfate, sodium stearyl sulfate, sodium lauryl alcohol sulfate, sodium lauryl ether sulfate, and sodium lauryl sulfofatty acid esters; phosphate salts of higher alcohols or their ethylene oxide adducts; acyl sarcosine salts such as N-lauroyl sarcosine (NLS), N-myristeoyl sarcosine (NMS), N-palmitoyl sarcosine (NPS), and N-lauroyl oil sarcosine; and bile acids such as taurocholic acid, cholic acid, glycocholic acid, and deoxycholic acid, and these may be used alone or in combination of two or more. Among these, the anionic surfactant according to the present invention is preferably at least one selected from the group consisting of sulfate ester salts, acyl sarcosine salts, and bile acids, and more preferably at least one selected from the group consisting of SDS, NLS, and taurocholic acid.

[0026] The content of the surfactant (amphoteric surfactant and / or anionic surfactant) in the composition of the present invention (the total content of the surfactants when two or more types of surfactants are used) is preferably 0.001 to 5.0 w / v%, more preferably 0.01 to 5.0 w / v%, even more preferably 0.01 to 2.0 w / v%, and even more preferably 0.03 to 1.0%, relative to the total amount of the composition, from the viewpoint that the content tends to be particularly effective in stabilizing the storage of GFAP when stored in a non-frozen state as an aqueous solution (preferably a standard solution).

[0027] When the surfactant in the composition of the present invention is a combination of an amphoteric surfactant and an anionic surfactant, the mass ratio of the amphoteric surfactant to the anionic surfactant in the composition of the present invention (mass of amphoteric surfactant:mass of anionic surfactant) is not particularly limited, but is preferably, for example, 1:10 to 10:1, more preferably 1:6 to 6:1, and even more preferably 1:3 to 3:1.

[0028] Specific and preferred combinations of an amphoteric surfactant and an anionic surfactant as the surfactant include, for example, a combination of a sulfobetaine surfactant (e.g., C14APS, C16APS) and NLS, a combination of a sulfobetaine surfactant and SDS, and a combination of a sulfobetaine surfactant and taurocholic acid.

[0029] (Nonionic Surfactant) The composition of the present invention preferably further contains a nonionic surfactant. The term "nonionic surfactant" refers to a surfactant that does not ionize in an aqueous solution, i.e., does not become an ion (an atom or atomic group having an electric charge).

[0030] The nonionic surfactant according to the present invention may be one type or a combination of two or more types, and is not particularly limited. Examples include ester types (e.g., esters of polyhydric alcohols and fatty acids), ether types (e.g., ether-type anionic surfactants having a polyoxyethylene structure, more specifically, polyoxyethylene (POE) alkyl ethers, polyoxyethylene (POE) alkylphenyl ethers, polyoxyethylene polyoxypropylene glycols), and ester-ether types (e.g., ethylene oxide adducts of esters of polyhydric alcohols and fatty acids).

[0031] More specific examples of the nonionic surfactant according to the present invention include sorbitan fatty acid esters; polyoxyalkylene sorbitan mono-fatty acid esters such as Tween (Tween 20, Tween 40, Tween 80, etc., manufactured by Sigma-Aldrich); polyoxyalkylene alkylphenyl ethers such as poly(oxyethylene)octylphenyl ether (e.g., Triton X (Triton X-100, Triton X-305, Triton X-405, Triton X-705, etc., manufactured by Sigma-Aldrich)); secondary ethoxylated alcohols such as Tergitol (Tergitol 15-s-9, Tergitol 15-s-7, etc., manufactured by Sigma-Aldrich); polyoxyalkylene castor oil; and polyoxyalkylene hydrogenated castor oil. These surfactants may be used alone or in combination of two or more. Among these, the nonionic surfactant according to the present invention is preferably at least one selected from the group consisting of polyoxyalkylene sorbitan mono-fatty acid esters and secondary ethoxylated alcohols, and more preferably at least one selected from the group consisting of polyoxyalkylene sorbitan mono-fatty acid esters.

[0032] When the composition of the present invention contains a nonionic surfactant, the content of the nonionic surfactant in the composition (when there are two or more types of nonionic surfactants, the total content of the nonionic surfactants) relative to the total amount of the composition is preferably 0.01 to 5.0 w / v%, more preferably 0.05 to 2.0 w / v%, even more preferably 0.1 to 1.0 w / v%, and even more preferably 0.1 to 0.5 w / v%, from the viewpoint that the content tends to be particularly effective in stabilizing the storage of GFAP when stored in a non-frozen state as an aqueous solution (preferably a standard solution).

[0033] Furthermore, when the composition of the present invention contains a nonionic surfactant, the mass ratio of the nonionic surfactant to the amphoteric surfactant and / or anionic surfactant in the composition of the present invention (mass of nonionic surfactant:mass of amphoteric surfactant and / or anionic surfactant) is not particularly limited, but is, for example, preferably 1:10 to 10:1, more preferably 1:6 to 6:1, and even more preferably 1:3 to 3:1.

[0034] Furthermore, specific and preferred combinations of the above-mentioned amphoteric surfactants and / or anionic surfactants with nonionic surfactants include, for example, a combination of a sulfobetaine surfactant (e.g., C14APS, C16APS) with a polyoxyalkylene sorbitan mono-fatty acid ester (e.g., Tween 20), a combination of a sulfobetaine surfactant (e.g., C14APS, C16APS), NLS, and a polyoxyalkylene sorbitan mono-fatty acid ester (e.g., Tween 20), and the like.

[0035] (Cationic Surfactant) The composition of the present invention may contain a cationic surfactant, but preferably does not substantially contain one. A "cationic surfactant" refers to an ionic surfactant that ionizes to form ions (charged atoms or atomic groups), and a surfactant in which a moiety containing a hydrophobic group ionizes to a positive ion when ionized in an aqueous solution.

[0036] Examples of the cationic surfactant include alkyltrimethylammonium chloride, alkyldimethylbenzalkonium chloride, alkylamine, etc. When the composition of the present invention contains a cationic surfactant, the content of the cationic surfactant in the composition (when there are two or more types of cationic surfactants, the total content of these) is preferably 0.05 w / v % or less, and more preferably 0.01 w / v % or less, based on the total amount of the composition.

[0037] [Buffering Agent] The composition of the present invention preferably has an aqueous solution having a pH in the alkaline region, more specifically, a pH of preferably 7.1 to 9.5, more preferably 7.5 to 9.0, and even more preferably 7.7 to 8.5. Therefore, the composition of the present invention preferably further contains a buffering agent, the main purpose of which is to adjust the pH to the above-mentioned preferred range.

[0038] Examples of the buffering agent include lactic acid, acetic acid, hydrochloric acid, succinic acid, phthalic acid, phosphoric acid, boric acid, citric acid, maleic acid, carbonic acid, ammonia, Tris (tris(hydroxymethyl)aminomethane), Bis-Tris (bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane), MES (2-morpholinoethanesulfonic acid), ADA (N-2(acetamido)iminodiacetic acid), PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)), ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), BES ( Examples of suitable glycerides include N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid), MOPS (3-morpholinopropane-1-sulfonic acid), Tricine (N-[tris(hydroxymethyl)methyl]glycine), and Bicine (N,N-bis(2-hydroxyethyl)glycine), and these may be used alone or in combination of two or more. Among these, when the composition of the present invention is used as a standard solution and an enzyme is used as a labeling substance in an immunoassay, Tris is preferred from the viewpoint of not inhibiting the enzymatic activity.

[0039] The composition of the present invention is preferably an aqueous solution containing water, but the buffer may also be contained in the composition of the present invention in the form of a buffer such as Tris-HCl buffer (Tris-hydrochloric acid buffer), sodium phosphate buffer, etc., which serves as a part or all of the water.

[0040] When the composition of the present invention contains the buffer, the content of the buffer in the composition (when two or more buffers are used, the total content of the buffers) is preferably an amount such that the pH of the composition in aqueous solution becomes 7.1 to 9.5, preferably 7.5 to 9.0, and more preferably 7.7 to 8.5. The specific amount cannot be generalized because it is adjusted appropriately depending on the type and pH of the buffer, but for example, in the case of Tris, the amount is preferably 10 to 500 mM, more preferably 20 to 100 mM, relative to the total amount of the composition in aqueous solution.

[0041] [Others] The composition of the present invention may also contain salts, protein stabilizing substances, water-soluble polymers, chelating agents, preservatives (NaN ３ , Proclin 950 (manufactured by Sigma-Aldrich), antibiotics, etc.

[0042] The salts refer to salts added to the composition as salts other than those derived from the buffer, and include, for example, sodium chloride and potassium chloride, and may be one of these or a combination of two of them. When the composition of the present invention contains the salts, the content thereof (the total content when two or more salts are contained) is, for example, preferably 50 to 300 mM, and more preferably 100 to 200 mM, relative to the total amount of the composition when made into an aqueous solution.

[0043] Examples of the protein stabilizing substance include sugars and proteins other than GFAP, and the substance may be one of these or a combination of two or more of these.

[0044] Examples of the sugars include monosaccharides, disaccharides, trisaccharides, polysaccharides, and sugar alcohols. More specifically, examples include glucose, sucrose, maltose, sorbitol, mannitol, xylitol, trehalose, cyclodextrin, and the like. These sugars may be used alone or in combination of two or more. Among these, disaccharides such as sucrose are preferred as the sugar. When the composition of the present invention contains the sugars, the content thereof (or the total content thereof when two or more types are used) is, for example, preferably 0.5 to 10 w / v %, more preferably 1.0 to 5.0 w / v %, and even more preferably 1.5 to 2.5 w / v %, relative to the total amount of the composition when prepared as an aqueous solution.

[0045] Examples of the protein other than GFAP include casein, sodium caseinate, bovine serum albumin (BSA), fetal bovine serum (FBS), equine serum albumin, and equine serum, and the composition of the present invention may contain one or a combination of two or more of these proteins. When the composition of the present invention contains the protein, the content of the protein (the total content of two or more proteins) is preferably 0.5 to 10 w / v %, and more preferably 1.0 to 5.0 w / v %, relative to the total amount of the composition when it is prepared as an aqueous solution.

[0046] Examples of the water-soluble polymer include PVP (polyvinylpyrrolidone), PVA (polyvinyl alcohol), PEG (polyethylene glycol), dextran, dextran salts, and hydrophilic polymers (e.g., Lipidure, manufactured by NOF Corporation), and the water-soluble polymer may be one of these or a combination of two or more of these. When the composition of the present invention contains the water-soluble polymer, the content thereof (when two or more types are used, the total content thereof) is, for example, preferably 0.01 to 2.0 w / v %, and more preferably 0.1 to 1.0 w / v %, relative to the total amount of the composition when made into an aqueous solution.

[0047] Examples of the chelating agent include citrates (such as sodium citrate), ethylenediaminetetraacetic acid (EDTA) salts (such as dipotassium ethylenediaminetetraacetic acid (EDTA-2K) and disodium ethylenediaminetetraacetic acid (EDTA-2Na)), diaminopropanol tetraacetate, and ethylenediaminedipropionate, and the composition may contain one of these or a combination of two or more of them. When the composition of the present invention contains the chelating agent, the content thereof (the total content when two or more types are used) is, for example, preferably 0.1 to 10 mM, and more preferably 0.5 to 5.0 mM, relative to the total amount of the composition when made into an aqueous solution.

[0048] [Storage Stability] The composition of the present invention has excellent storage stability for GFAP. More specifically, the composition of the present invention can maintain a high level of GFAP detected with an anti-GFAP antibody, and even when stored as an aqueous solution in a non-frozen state (e.g., 37°C) such as 4°C or 37°C for several days (e.g., 3 days), the amount of GFAP detected with the anti-GFAP antibody can be maintained at, for example, 85% or more, more preferably 87% or more, even more preferably 88% or more, or 90% or more, relative to the amount detected when stored at -80°C (100%).

[0049] The anti-GFAP antibody is not particularly limited as long as it can specifically bind to the GFAP according to the present invention. It may be a complete antibody or a functional fragment thereof (e.g., Fab, F(ab')2, Fab', variable region fragment (Fv), disulfide-linked Fv, single-chain Fv (scFv), sc(Fv)2, diabody, or a polymer thereof). Furthermore, in the present invention, "antibody" includes all classes and subclasses of immunoglobulins, and may be a polyclonal or monoclonal antibody. The origin, type, shape, etc. of the antibody are also not particularly limited. Such antibodies can be prepared by conventionally known methods (e.g., hybridoma method or recombinant DNA method) or methods similar thereto, and may be commercially available.

[0050] [Standard Solution] The composition of the present invention has excellent storage stability of GFAP, and therefore can be suitably used as a standard solution (GFAP standard solution) containing a specified concentration of GFAP, more preferably as a standard solution for immunoassays requiring strict accuracy of GFAP amount. The immunoassay is not particularly limited, and examples thereof include EIA (enzyme-linked immunosorbent assay) using an enzyme as a labeling substance, ELISA and CLEIA (chemiluminescent enzyme immunoassay), which are embodiments of EIA, labeled immunoassay using an antigen or antibody labeled with a labeling substance, RIA (radioimmunoassay) using a radioisotope as a labeling substance, CLIA (chemiluminescent immunoassay) using a chemiluminescent compound as a labeling substance, immunochromatography, and immunoagglutination methods (latex agglutination, gold colloid agglutination, etc.) that measure by detecting agglutination. In addition, it may be a non-competitive method or a competitive method. The immunoassay may be, for example, a sandwich immunoassay using an enzyme as a labeling substance, but is not limited thereto.

[0051] When the composition of the present invention is used as a standard solution, the standard solution is an aqueous solution.When the composition of the present invention is the above-mentioned freeze-dried composition, mixed composition, or concentrated solution, it can be dissolved or diluted with water (or optionally a buffer solution) to obtain a desired GFAP concentration to obtain a standard solution according to the purpose.In addition, the standard solution can be further diluted to obtain a desired GFAP concentration according to the purpose.

[0052] [Method for improving the storage stability of GFAP] The present invention also provides a method for improving the storage stability of GFAP in a GFAP-containing composition containing GFAP, the method comprising the step of further adding at least one surfactant selected from the group consisting of amphoteric surfactants and anionic surfactants to the GFAP-containing composition (sometimes referred to in this specification as the "method for improving the storage stability of the present invention").

[0053] In the method for improving storage stability of the present invention, the storage stability of GFAP, amphoteric surfactant, anionic surfactant, and GFAP, including their preferred embodiments, is as described above for the composition of the present invention.

[0054] Furthermore, the GFAP-containing composition containing the amphoteric surfactant and / or anionic surfactant is preferably an aqueous solution, and may further contain, in addition to GFAP, one or more of the above-mentioned other components (i.e., salts, protein stabilizers, water-soluble polymers, chelating agents, preservatives, etc.). The content of GFAP and the content of each of the above-mentioned other components in such a GFAP-containing composition are as described above for the respective contents of the composition of the present invention, including their preferred embodiments, except that "content in the composition of the present invention" is read as "content in the composition after the amphoteric surfactant and / or anionic surfactant are added," i.e., "relative to the total amount of the composition" is read as "relative to the total amount of the composition after the amphoteric surfactant and / or anionic surfactant are added." The pH of the GFAP-containing composition is as described above for the pH of the composition of the present invention, including its preferred embodiments, except that "pH of the composition of the present invention" is read as "pH of the composition after the amphoteric surfactant and / or anionic surfactant are added."

[0055] Methods for further incorporating an amphoteric surfactant and / or anionic surfactant into the GFAP-containing composition include, but are not limited to, a method in which a composition containing an amphoteric surfactant and / or anionic surfactant (e.g., the storage solution described below) is prepared in advance and GFAP is added to the composition, a method in which an amphoteric surfactant and / or anionic surfactant is added to a composition containing GFAP to form the composition of the present invention, and a method in which a composition containing GFAP and a composition containing an amphoteric surfactant and / or anionic surfactant are mixed to form the composition of the present invention.

[0056] Furthermore, the amount of amphoteric surfactant and / or anionic surfactant to be contained in the GFAP-containing composition is preferably such that the "content in the composition after containing the amphoteric surfactant and / or anionic surfactant" is the amount contained in the composition of the present invention, and such content, including its preferred embodiments, is as described above for the composition of the present invention.

[0057] [Preservation Solution] The present invention also provides a preservation solution for preserving GFAP, which is an aqueous solution containing at least one surfactant selected from the group consisting of amphoteric surfactants and anionic surfactants. The preservation solution is as described above for the composition of the present invention, including its preferred embodiments, except that it does not contain GFAP and the "content in the composition of the present invention" of each component can be read as "content in the composition after inclusion of GFAP."

[0058] The preservation solution of the present invention can be used to prepare the composition of the present invention described above, and can be used, for example, as a solution for preparing the standard solution, or as a preservation solution for GFAP collected from a specimen or GFAP produced as a recombinant form, etc.

[0059] The present invention also provides a kit for preserving GFAP, e.g., a kit for preparing the standard solution, which comprises the preservation solution. The kit of the present invention may further comprise at least one selected from the group consisting of GFAP; a diluent (water, a known buffer, etc.) for the concentrated preservation solution; an anti-GFAP antibody for immunoassay, a reaction buffer, a labeling substance, an insoluble carrier (e.g., particles), a substrate, a diluent for these; and instructions for use of the kit.

[0060] The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. In each of the following test examples, "%" indicates weight / volume percent (w / v%: g / mL x 100) unless otherwise specified.

[0061] (Storage Stability Test) The compositions prepared in the following Examples and Comparative Examples were subjected to a storage stability test by the following method: First, the prepared compositions were each dispensed into polypropylene tubes and stored at −80° C. or 37° C. for 3 days.

[0062] Next, GFAP in each composition after storage was measured by a two-step sandwich immunoassay (CLEIA) using a Lumipulse G1200 (Fujirebio Inc.). Specifically, first, two types of anti-GFAP antibodies established by the hybridoma method using mice immunized with recombinant GFAP (a recombinant protein containing human full-length GFAP (NP_002046)) were used as capture and labeling antibodies to prepare capture and labeling body solutions, respectively. The capture body solution was prepared by immobilizing the capture antibody on magnetic particles to prepare magnetic particle-immobilized anti-GFAP antibodies according to standard methods, and adding them to a particle diluent (a composition containing 50 mM MOPS, 150 mM NaCl, 1 mM EDTA-2Na, 2% BSA, and 2% sucrose; pH 7.2) to a concentration of 0.025%. The labeled body fluid was prepared by labeling the labeling antibody with alkaline phosphatase in a standard manner to obtain an ALP-labeled anti-GFAP antibody, which was then added to a label diluent (a composition containing 50 mM Tris, 150 mM NaCl, 2% BSA, 2% Sucrose, and 1.5% Pluronic F-108; pH 7.2) to a concentration of 0.5 μg / mL.

[0063] 0.02 mL of an immune reaction solution (aqueous solution of 50 mM MOPS, 150 mM NaCl, 1 mM EDTA-2Na, 1.5% N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, 1.5% Tween 80, and 1.0% Tergitol 15-s-9 (Sigma-Aldrich); pH 7.2) was mixed with 0.1 mL of each composition after storage. This was mixed with 0.15 mL of the captured body fluid and reacted at 37°C for 10 minutes. After reaction with the captured body fluid, the magnetic particles were collected and washed with Lumipulse washing solution (Fujirebio), after which 0.15 mL of the labeled body fluid was added and reacted at 37°C for 10 minutes. After reaction with the labeled body fluid, the magnetic particles were magnetically collected and washed with Lumipulse wash solution. Then, 0.2 mL of Lumipulse substrate solution (Fujirebio) containing AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane disodium salt) was added, and the mixture was allowed to react at 37°C for 5 minutes. After reaction with the substrate solution, the amount of light emitted, which had a maximum absorption at a wavelength of 463 nm when AMPPD was decomposed by the catalytic action of alkaline phosphatase bound to the magnetic particles, was measured. The amount of GFAP in each composition was output as the luminescence intensity (counts) of the substrate (AMPPD). Two or three samples were measured per condition, and the average value was calculated. Furthermore, for each composition, the results after storage at -80°C for 3 days were used as the control (Cont.), and the percentage of the results after storage at 37°C for 3 days (relative to Cont. [%]) was also calculated.

[0064] Comparative Example 1: GFAP concentrations of 0, 100, 1000, and 5000 pg / mL were prepared using a storage solution (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA-2Na, 2.0% BSA, 0.1% NaN ３ GFAP (recombinant protein containing human full-length GFAP (NP_002046)) was dissolved (or not added) in an aqueous solution of NP_002046 (pH 7.2) to prepare compositions with each GFAP concentration in Comparative Example 1. The storage stability test described above was performed on each composition. The results are shown in Table 1 below. The count average shown in Table 1 is the average of the count values ​​of two samples.

[0065]

[0066] As shown in Table 1, at any GFAP concentration between 100 and 5000 pg / mL, when the composition was stored at 37°C for 3 days, the amount of GFAP in the composition decreased by approximately 18% compared to when the composition was stored at -80°C for 3 days, confirming that the storage stability of GFAP in the composition was low.

[0067] Example 1 Compositions with various GFAP concentrations were prepared in the same manner as in Comparative Example 1, except that the composition of the preservation solution was as shown in Table 2 below: 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA-2Na, 2.0% sucrose, 3.0% BSA, 0.30% N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C14APS), and 0.10% Proclin 950 (Sigma-Aldrich) aqueous solution; pH 8.0, and GFAP was dissolved therein to a concentration of 5000 or 0 pg / mL (or was not added). The above-described storage stability test was performed on each of the prepared compositions. The results for the composition with a GFAP concentration of 5000 pg / mL are shown in Table 2 below, along with the composition of the preservation solution. The content of each component in the preservation solution is the content in the composition after dissolving GFAP in the solution (the same applies hereinafter). As shown in Table 1, in the composition with a GFAP concentration of 0 pg / mL, there was almost no difference in the count value between after storage at -80°C and after storage at 37°C.

[0068] (Comparative Examples 2 to 4) Compositions with various GFAP concentrations were prepared in the same manner as in Example 1, except that polyoxyethylene (20) sorbitan monolaurate (Tween 20 (Sigma-Aldrich)), polyoxyethylene (80) sorbitan monolaurate (Tween 80 (Sigma-Aldrich)), or Tergitol 15-s-9 (Sigma-Aldrich) was used instead of C14APS. The above-mentioned storage stability test was carried out for each of the prepared compositions. The results for each composition with a GFAP concentration of 5000 pg / mL are shown in Table 2 below, along with the composition of each of the preservative solutions. Note that, for each composition with a GFAP concentration of 0 pg / mL, there was almost no difference in count values ​​between storage at −80°C and storage at 37°C, as in Table 1.

[0069]

[0070] As shown in Table 2, in the compositions of Comparative Examples 2 to 4, when stored at 37°C, the amount of GFAP decreased to about the same level as in Comparative Example 1 compared to when stored at -80°C, indicating poor storage stability of GFAP in the composition. On the other hand, in the composition containing C14APS of Example 1, the decrease in count value was significantly smaller than in the compositions containing other surfactants (Comparative Examples 2 to 4), even when stored at 37°C, confirming excellent storage stability of GFAP in the composition.

[0071] (Examples 2 to 4) Compositions with various GFAP concentrations were prepared in the same manner as in Example 1, except that the preservation solution was an aqueous solution having the composition shown in Table 3 below. The above-described storage stability test was performed for each prepared composition. For reference, a composition prepared with the same composition as in Example 1 was also simultaneously subjected to the storage stability test. The results for each composition with a GFAP concentration of 5000 pg / mL are shown in Table 3 below, along with the composition of the preservation solution. In Table 3, "PVP" represents polyvinylpyrrolidone (Polyvinylpyrrolidone K30 (Fujifilm Wako Pure Chemical Industries, Ltd.)), "NLS" represents N-lauroyl sarcosine, and the other components represent the same components as those listed in Table 2. For all compositions with a GFAP concentration of 0 pg / mL, there was almost no difference in count values ​​between storage at -80°C and storage at 37°C, as in Table 1.

[0072]

[0073] As shown in Table 3, the composition containing C14APS of Example 1 showed a significantly small decrease in count value even when stored at 37°C, as in Table 2. Furthermore, when the composition further contained Tween 20, a nonionic surfactant, or NLS, an anionic surfactant, in addition to C14APS, the decrease in count value when stored at 37°C was even smaller.

[0074] (Examples 5 to 7) Compositions with various GFAP concentrations were prepared in the same manner as in Example 1, except that the preservation solution was an aqueous solution having the composition shown in Table 4 below. The above-mentioned storage stability test was carried out for each of the prepared compositions. For reference, a composition prepared with the same composition as in Example 1 was also simultaneously subjected to the storage stability test. The results for each composition with a GFAP concentration of 5000 pg / mL are shown in Table 4 below, along with the composition of the preservation solution. Note that, for all compositions with a GFAP concentration of 0 pg / mL, there was almost no difference in count value between storage at -80°C and storage at 37°C, as in Table 1.

[0075]

[0076] As shown in Table 4, the composition containing C14APS of Example 1 showed a significantly small decrease in count value even when stored at 37° C., as in Tables 2 and 3. Furthermore, when the composition contained anionic surfactants NLS, SDS, or taurocholic acid instead of C14APS, the decrease in count value when stored at 37° C. was also small, confirming that GFAP has excellent storage stability in the composition.

[0077] (Examples 8 to 9, Comparative Example 5) Compositions with various GFAP concentrations were prepared in the same manner as in Example 1, except that the preservation solution was an aqueous solution having the composition shown in Table 5 below. The above-mentioned storage stability test was carried out for each of the prepared compositions. The results for each composition with a GFAP concentration of 5000 pg / mL are shown in Table 5 below, along with the composition of each of the preservation solutions. In Table 5, "C16APS" represents N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate. Note that, for all compositions with a GFAP concentration of 0 pg / mL, there was almost no difference in count value between storage at -80°C and storage at 37°C, as in Table 1.

[0078]

[0079] As shown in Table 5, the composition containing C16APS in Example 9 also showed a significantly small decrease in count value even when stored at 37°C, as with the composition containing C14APS in Example 8 and Table 3, confirming that GFAP has excellent storage stability in the composition.

[0080] (Example 10, Comparative Example 6) A composition (Example 10) with a GFAP concentration of 5000 pg / mL was prepared in the same manner as in Example 1, except that the preservation solution was an aqueous solution with the composition shown in Table 6 below, and the above-mentioned storage stability test was performed on the prepared GFAP-containing composition. Furthermore, a BNP-containing composition (Comparative Example 6) was prepared in the same manner as in Example 10, except that BNP (brain natriuretic peptide) was dissolved in the preservation solution to a concentration of 5000 pg / mL instead of GFAP. The prepared BNP composition was tested in the same manner as the above-mentioned storage stability test, except that anti-BNP antibodies were used as the capture antibody and labeling antibody instead of anti-GFAP antibodies. The results for each composition are shown in Table 6 below, along with the composition of the preservation solution. In Table 6, "Lipidure 502" is a hydrophilic polymer manufactured by NOF Corporation.

[0081]

[0082] As shown in Table 6, the GFAP-containing composition of Example 10 containing C14APS and NLS showed a significantly smaller decrease in count value when stored at 37°C, as in Tables 3 and 5. On the other hand, despite the use of a storage solution of the same composition, the BNP-containing composition of Comparative Example 6 showed a 30% decrease in the amount of BNP in the composition when stored at 37°C for 3 days compared to when stored at -80°C for 3 days, confirming that BNP was not maintained stably. Thus, it was confirmed that the high level of storage stability achieved in the present invention by incorporating the surfactant (amphoteric surfactant and / or anionic surfactant) is an effect specific to the GFAP-containing composition.

[0083] As described above, the present invention makes it possible to provide a GFAP-containing composition having excellent storage stability of GFAP and a method for improving the storage stability of GFAP. In the GFAP-containing composition provided by the present invention, the amount of GFAP detected by an anti-GFAP antibody is stably maintained at an extremely high level, and therefore such a GFAP-containing composition is particularly useful as a GFAP-containing composition of known concentration that serves as a reference when detecting and quantifying GFAP in a sample by immunoassay, i.e., as a GFAP standard solution (calibrator).

Claims

1. A GFAP-containing composition comprising GFAP and at least one surfactant selected from the group consisting of amphoteric surfactants and anionic surfactants.

2. The GFAP-containing composition according to claim 1, which is a GFAP standard solution.

3. The GFAP-containing composition of claim 1, having a pH of 7.5 to 9.

0.

4. The GFAP-containing composition of claim 1, further comprising a non-ionic surfactant.

5. The GFAP-containing composition according to claim 1, which contains the amphoteric surfactant, and the amphoteric surfactant is a sulfobetaine-type surfactant having a linear alkyl group having 8 to 25 carbon atoms.

6. The GFAP-containing composition according to claim 1, wherein the GFAP content is 0.001 to 500 ng / mL.

7. The GFAP-containing composition according to claim 1, wherein the content of the surfactant is 0.01 to 5.0 w / v %.

8. The GFAP-containing composition of claim 1, further comprising a protein stabilizing substance.

9. The GFAP-containing composition of claim 1, further comprising a buffering agent.

10. A method for improving the storage stability of GFAP in a GFAP-containing composition, comprising the step of further adding at least one surfactant selected from the group consisting of amphoteric surfactants and anionic surfactants to the GFAP-containing composition.

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