Methods for detecting gfap, methods for aiding in the diagnosis of alzheimer's disease, and kits therefor

Abstract

A detection method which is a method of detecting GFAP in a sample by immunoassay, the detection method comprising: a first step of forming a complex of a first antibody and GFAP; and a second step of forming a complex of a second antibody and GFAP, the first antibody being an antibody that binds to a region including amino acids at positions 111 to 115 in the amino acid sequence of GFAP.

Description

Technical Field

[0001] This invention relates to methods for detecting GFAP, methods for assisting in the diagnosis of Alzheimer's disease, and kits for using them, and more specifically, to methods for detecting GFAP in samples by immunoassay, thereby assisting in the diagnosis of Alzheimer's disease, and kits for using them. Background Technology

[0002] GFAP (Glial fibrillary acidic protein) is known as a biomarker for the diagnosis of damage and diseases of the central nervous system. In recent years, it has also been clarified that it is useful as a biomarker for the diagnosis of Alzheimer's disease.

[0003] Alzheimer's disease (Alzheimer's dementia, AD) is a progressive neurodegenerative disease. Its main symptoms include memory impairment, higher brain function impairment (aphasia, apraxia, agnosia, and behavioral aphasia), and personality changes. The number of Alzheimer's patients is increasing with the aging population, and it has become a serious social problem. However, the full picture of its pathogenesis remains unclear, and no cure has yet been developed.

[0004] On the other hand, because the progression of symptoms can be delayed, earlier diagnosis is required in the treatment of Alzheimer's disease. However, current diagnostic methods mainly involve detecting brain and hippocampal atrophy and the deposition of senile plaques (amyloid plaques) through EEG, CT, MRI, PET / SPECT, etc. These methods are difficult to implement easily on a wide range of subjects and require special techniques and equipment, thus failing to achieve early diagnosis.

[0005] Therefore, it is expected that by detecting the aforementioned GFAP in samples collected from subjects, it is possible to relatively easily detect subjects with Alzheimer's disease or those at high risk of having Alzheimer's disease. As a method for detecting GFAP from samples, there are known detection methods for diagnosing stroke or traumatic brain injury. For example, International Patent Publication No. 2018 / 096049 (Patent Document 1), U.S. Patent Application Publication No. 2019 / 0302127 (Patent Document 2), and Japanese Patent Application Publication No. 2022-66139 (Patent Document 3) describe methods for detecting GFAP in samples using anti-GFAP antibodies that bind to various epitopes through immunoassay.

[0006] Existing technical documents Patent documents Patent Document 1: International Publication No. 2018 / 096049 Patent Document 2: U.S. Patent Application Publication No. 2019 / 0302127 Patent Document 3: Japanese Patent Application Publication No. 2022-66139 Summary of the Invention

[0007] The technical problem that the invention aims to solve The detection methods described in Patent Documents 1-3 are applicable to diseases such as stroke or traumatic brain injury. In these cases, the presence of large amounts of GFAP in samples such as serum or plasma makes detection relatively easy, even using immunoassay. However, in the case of Alzheimer's disease, the amount of GFAP in samples collected from patients is typically trace and difficult to detect with high precision. Therefore, further improvements in the precision of GFAP detection using immunoassay are required.

[0008] The present invention was made in view of the technical problems of the prior art described above, and its object is to provide a detection method that can detect GFAP with high precision by immunoassay even when the amount in the sample is small, a method thereby assisting in the diagnosis of Alzheimer's disease, and kits for use therein.

[0009] Technical solutions for solving technical problems To achieve the above objectives, the inventors have made repeated efforts to research and develop multiple anti-GFAP antibodies. As a result, they discovered that by using a specific anti-GFAP antibody that binds to the region containing amino acids 111 to 115 in the amino acid sequence of GFAP, even if the amount of GFAP contained in the sample is small, such as in Alzheimer's disease, it can be detected with high precision by immunoassay, thus completing the present invention.

[0010] That is, the present invention relates to a method for detecting GFAP in a sample by immunoassay, thereby aiding in the diagnosis of Alzheimer's disease, and kits for use therein, and more specifically, provides the following. [1] One detection method is a method for detecting GFAP in a sample through immunoassay. The detection method includes: a first step of forming a complex of a first antibody and GFAP; and a second step of forming a complex of a second antibody and GFAP. The first antibody is an antibody that binds to the region containing amino acids 111 to 115 in the amino acid sequence of GFAP. [2] According to the detection method described in [1], the second antibody is an antibody that binds to the region of the amino acid sequence of GFAP containing amino acids from position 191 to position 200. [3] According to the detection method described in [1] or [2], the first antibody is an antibody that does not bind to the region of the amino acid sequence of GFAP containing amino acids from position 116 to position 214. [4] According to any one of [1] to [3], the pH of the reaction system in the first step is 6.2 to 7.5. [5] According to any one of [1] to [4], the detection method comprises a first step of capturing GFAP with a capture medium containing a first antibody and an insoluble carrier, and a second step of labeling GFAP with a label medium containing a second antibody and a labeling substance; or The first step is to label GFAP with a label containing a first antibody and a labeling substance, and the second step is to capture GFAP with a capture medium containing a second antibody and an insoluble carrier. [6] A method for assisting in the diagnosis of Alzheimer's disease, comprising the step of detecting GFAP in a sample collected from a subject using any one of the detection methods described in [1] to [5]. [7] A diagnostic method for Alzheimer's disease, comprising the step of detecting GFAP in a sample collected from a subject using any one of the detection methods described in [1] to [5]. [8] A kit for detecting GFAP in a sample by immunoassay, comprising: A first antibody that binds to the region of the GFAP amino acid sequence containing amino acids 111 to 115; and A second antibody that binds to a region in the amino acid sequence of GFAP that is different from that of the first antibody. [9] According to the kit described in [8], the second antibody is an antibody that binds to the region of the amino acid sequence of GFAP containing amino acids from position 191 to position 200.

[0020] Invention Effects According to the present invention, a detection method is provided that can detect GFAP with high precision by immunoassay even when the amount in the sample is small, thereby assisting in the diagnosis of Alzheimer's disease, and kits for use therein. Attached Figure Description

[0021] Figure 1 This is a schematic diagram showing the position of each GFAP cleavage fragment prepared in (1) of (Example 2) within the amino acid sequence of the full-length GFAP.

[0022] Figure 2 The graph shows the results of immunoblotting for (a) anti-His tag antibody (Anti-His) reaction and (b) GFAP-Ab1 reaction for the fragments aa1-230, aa1-104, aa72-214, and aa116-214.

[0023] Figure 3 The graphs show the results of immunoblotting for the fragments aa91-160, aa96-160, aa101-160, aa106-160, aa111-160, aa121-170, aa131-180, aa141-190, aa151-200, and aa161-214, respectively: (a) the results of immunoblotting for anti-GST antibody (Anti-GST), and (b) the results of immunoblotting for GFAP-Ab1.

[0024] Figure 4 The graphs show the results of immunoblotting for the fragments aa1-125, aa1-126, aa1-127, aa1-128, aa1-129, aa1-130, aa1-131, aa1-132, aa1-133, aa1-134, and aa177-230, respectively: (a) the results of immunoblotting reacting with anti-GST antibody (Anti-GST), and (b) the results of immunoblotting reacting with GFAP-Ab1.

[0025] Figure 5 The graphs show the results of immunoblotting for the fragments aa91-160, aa96-160, aa101-160, aa106-160, aa111-160, aa121-170, aa131-180, aa141-190, aa151-200, and aa161-214, respectively: (a) the results of immunoblotting for anti-GST antibody (Anti-GST), and (b) the results of immunoblotting for GFAP-Ab2.

[0026] Figure 6The graphs show the results of immunoblotting for the fragments aa177-230, aa178-230, aa179-230, aa180-230, aa181-230, aa182-230, aa183-230, aa184-230, aa185-230, aa186-230, and aa1-125, respectively. (a) The results of immunoblotting for the fragments aa177-230, aa178-230, aa179-230, aa180-230, aa181-230, aa182-230, aa183-230, aa184-230, aa185-230, aa186-230, and aa1-125 are shown.

[0027] Figure 7 This is a graph showing the distribution of GFAP levels obtained by measuring GFAP in cognitively normal individuals (CU) and Alzheimer's disease patients (AD). Detailed Implementation

[0028] The present invention will now be described in detail according to its suitable embodiments.

[0029] <Detection Method> The detection method of the present invention is a method for detecting GFAP in a sample by immunoassay, comprising: The first step involves forming a complex of a first antibody and GFAP; and the second step involves forming a complex of a second antibody and GFAP. The first antibody is an antibody that binds to the region containing amino acids 111 to 115 in the amino acid sequence of GFAP.

[0030] [GFAP] In this invention, "GFAP" refers to glial fibrillary acidic protein, typically a 49,880 Da protein containing 432 amino acids. The amino acid sequence of GFAP can be obtained from known databases such as UniProt. A typical example of the amino acid sequence of GFAP involved in this invention is the amino acid sequence containing all 432 amino acids of human GFAP (amino acid sequence number: 1), accession number P14136. However, the "amino acid sequence of GFAP" involved in this invention is not limited to simply having a region containing amino acids from position 111 to 115 (amino acid sequence number: 2) (and preferably a region containing amino acids from position 191 to 200 (amino acid sequence number: 3)).

[0031] The "amino acid sequence of GFAP" involved in this invention includes, for example, an amino acid sequence having 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 homology (preferably uniformity) with respect to the amino acid sequence shown in Serial No. 1, and having a region containing amino acids from positions 111 to 115 (and preferably a region containing amino acids from positions 191 to 200); and an amino acid sequence in which one or more (up to 70 amino acids, preferably up to 45 amino acids, up to 25 amino acids, up to 20 amino acids, up to 10 amino acids, up to 5 amino acids, up to 3 amino acids, up to 2 amino acids, or 1 amino acid) amino acids are replaced, deleted, added, and / or inserted in the amino acid sequence shown in Serial No. 1, and having a region containing amino acids from positions 111 to 115 (and preferably a region containing amino acids from positions 191 to 200), etc.

[0032] The first antibody involved in this invention binds to a region containing amino acids 111 to 115 of the amino acid sequence of the GFAP. The amino acid sequence of this region is designated as Serial No. 2. It should be noted that, in this specification, the positions of amino acids such as "111th position" and "115th position" indicate the number of amino acid residues in the amino acid sequence of the GFAP (typically, the amino acid sequence shown in Serial No. 1). The region in this invention that "contains amino acids X to Y of the amino acid sequence of the GFAP (X, Y: numbers from 1 to 432)" each contains a region containing the corresponding amino acid.

[0033] In this invention, the amino acid "corresponding" to a specific amino acid in the amino acid sequence refers to the amino acid that is in the same column as the control amino acid (i.e., amino acids 111 to 115 of the amino acid sequence recorded in sequence number 1) when the amino acid sequence is compared using amino acid sequence analysis software (e.g., GENETYX-MAC, Sequencher, etc.) or ClustalW (e.g., parameter: default value (i.e., initial setting value)). The amino acid "corresponding" to the specific amino acid in the amino acid sequence can be an amino acid having the same chemical side chain (e.g., amino acids with hydroxyl groups (serine, threonine), amino acids with aromatic groups (phenylalanine, tyrosine, tryptophan), etc.), but is preferably the same amino acid.

[0034] In this invention, the "GFAP" used as the test substance can be any region containing amino acids from position 111 to position 115 (and preferably a region containing amino acids from position 191 to position 200 as described below) in the amino acid sequence of the GFAP. It can be the full length of the amino acid sequence of the GFAP, or a fragment containing a portion of it, or a polymer containing the full length and / or a fragment, or a complex of them with other proteins, etc.

[0035] [Sample] The "sample" used in the detection method of this invention can be any sample in which GFAP, the test substance, is present, without particular limitation. For example, various organisms (including cultured cells) and their extracts can be used appropriately depending on the purpose; suspensions of samples (serum, plasma, whole blood, bone marrow fluid, ascites, amniotic fluid, etc.; tissues) collected from humans and animals other than humans can be used. Examples of animals other than humans include mammals such as chimpanzees, monkeys, cattle, pigs, horses, sheep, mice, and rabbits. Among these, when detecting GFAP, the test substance used as a diagnostic benchmark for Alzheimer's disease, in the medical or clinical examination field, samples collected from subjects (preferably humans) such as diagnostic subjects are generally preferred, such as serum, plasma, and brain tissue, with plasma or serum being more preferred.

[0036] The sample can be a sample that has undergone pulverization, freezing, or other similar treatments; a sample that has been appropriately diluted or suspended with a diluent; or a sample whose pH has been appropriately adjusted. Examples of diluents include water, physiological saline, and known buffers (sodium phosphate buffer, MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, tricine buffer, bicine buffer, glycine buffer, etc.). Proteins such as BSA and serum, and metal ions (Zn) can also be added. 2+ Mg 2+ Salts (NaCl), etc., are used as samples for the method of the present invention. Aqueous samples are preferred, and samples appropriately diluted or suspended with the diluent as needed are even more preferred.

[0037] Furthermore, the detection method according to the present invention enables high-precision detection of GFAP in a sample regardless of the sample storage conditions. Samples for immunoassay are typically frozen at -80 to -20°C with one thaw cycle, or preferably stored at 2 to 10°C for 6 hours or less without freezing. However, the storage conditions for samples used in the detection method of the present invention are not particularly limited; for example, samples subjected to the aforementioned freeze-thaw cycle more than twice can be used, or samples stored at 2 to 30°C for 0 to 48 hours can be used. According to the detection method of the present invention, GFAP can be detected with high precision even when the sample is stored at 2 to 30°C for a long period (e.g., more than 12 hours, more than 24 hours, 24 to 48 hours, etc.).

[0038] [Antibody] In the detection method of this invention, two antibodies, a first antibody and a second antibody, are used in combination. In this invention, "antibody" includes not only the complete antibody but also its functional fragments. In this invention, a "functional fragment" refers to a portion (partial fragment) of the complete antibody, representing the fragment that binds to the GFAP. Specifically, examples include Fab, F(ab′)2, Fab′, variable region fragments (Fv), disulfide bond Fv, single-chain Fv (scFv), sc(Fv)2, bispecific antibodies (diabody), and their polymers.

[0039] Here, "Fab" refers to a monovalent antigen-binding fragment of an immunoglobulin containing a portion of one light chain and one heavy chain, which can be obtained, for example, by papain digestion and recombination with an antibody. "F(ab′)2" refers to a bivalent antigen-binding fragment of an immunoglobulin containing portions of two light chains and two heavy chains, which can be obtained, for example, by pepsin digestion and recombination with an antibody. "Fab′" can be obtained, for example, by reducing F(ab′)2, and contains one or more cysteine ​​residues of the hinge region of the antibody, differing from Fab by adding a small number of residues to the carboxyl terminus of the CH1 domain of the heavy chain.

[0040] Additionally, a "variable region fragment (Fv)" refers to the smallest antibody fragment with complete antigen recognition and binding sites. An Fv is a dimer strongly linked by non-covalent bonds between a heavy chain variable region and a light chain variable region. A "single-chain Fv (scFv)" contains both the heavy chain and light chain variable regions of the antibody, and these regions exist within a single polypeptide chain. "sc(Fv)2" is a single-chain antibody formed by binding two heavy chain variable regions and two light chain variable regions together using a linker or similar method. A "bispecific antibody" refers to a small antibody fragment with two antigen-binding sites, containing a heavy chain variable region that binds to the light chain variable region within the same polypeptide chain, with each region pairing with a complementary region of the other chain.

[0041] Furthermore, in this invention, "antibody" includes all classes and subclasses of immunoglobulins, and includes both polyclonal and monoclonal antibodies. "Polyclonal antibody" refers to an antibody preparation containing different antibodies targeting different epitopes, and "monoclonal antibody" refers to an antibody (containing antibody fragments) obtained from a substantially homogeneous group of antibodies. Monoclonal antibodies are preferred as the antibodies involved in this invention.

[0042] Furthermore, the source, type, and shape of the antibody involved in this invention are not particularly limited. Specifically, examples include antibodies derived from humans, antibodies derived from non-human animals (e.g., rabbit antibodies, mouse antibodies, rat antibodies, camel antibodies), chimeric antibodies, humanized antibodies, and functional fragments of these antibodies.

[0043] (First Antibody) The first antibody involved in this invention is an anti-GFAP antibody that binds to a region containing amino acids 111 to 115 of the amino acid sequence of the GFAP. The amino acid sequence containing the region containing amino acids 111 to 115 is designated as Serial No. 2. In this invention, "binding" to a region containing an amino acid sequence means that the antibody binds to at least any of the amino acids contained in that region, and "not binding" to a region containing an amino acid sequence means that the antibody does not bind to any of the amino acids contained in that region. Binding of the antibody to the region containing amino acids 111 to 115 of the amino acid sequence of the GFAP can be confirmed, for example, by the method shown in the examples described later: that is, in an immunoblot using a polypeptide fragment containing the region containing amino acids 111 to 115, the antibody binds to that region to detect a band.

[0044] As the first antibody of the present invention, it is further preferred to be any one of the following: an antibody that does not bind to the region containing amino acids from position 1 to position 104 in the amino acid sequence of the GFAP; an antibody that does not bind to the region containing amino acids from position 116 to position 214 in the amino acid sequence of the GFAP; and an antibody that does not bind to the region containing amino acids from position 177 to position 230 in the amino acid sequence of the GFAP. More preferably, it is an antibody that does not bind to any of these regions in the amino acid sequence of the GFAP.

[0045] (Second antibody) As the second antibody involved in this invention, any antibody capable of binding to GFAP, the test substance, preferably specifically, is acceptable. Well-known anti-GFAP antibodies or commercially available anti-GFAP antibodies can be used appropriately. Furthermore, the first and second antibodies involved in this invention are preferably different antibodies that bind to different amino acid sequences, but this does not preclude the second antibody from being the same as the first antibody. It should be noted that when the antibody included in the described trap is the first antibody and the described trapping step is performed before or simultaneously with the labeling step, or when the antibody included in the described label is the first antibody and the described labeling step is performed before or simultaneously with the trapping step, the "second antibody capable of binding to GFAP" includes an antibody capable of binding to the complex of GFAP and the first antibody, preferably specifically. As a method of binding to the complex of GFAP and the first antibody, for example, binding to the binding sites of GFAP and the first antibody can be cited.

[0046] The second antibody involved in this invention is preferably an antibody that binds to a region different from that of the first antibody, and more preferably an antibody that binds to a region in the amino acid sequence of the GFAP containing amino acids from positions 191 to 200. The amino acid sequence containing the region of amino acids from positions 191 to 200 is designated as Serial Number 3. The binding of the antibody to the region in the amino acid sequence of the GFAP containing amino acids from positions 191 to 200 can be confirmed, for example, by the method shown in the examples described later: that is, in an immunoblot using a polypeptide fragment containing the region containing amino acids from positions 191 to 200, the antibody binds to this region to detect a band.

[0047] As the second antibody involved in this invention, it is further preferred to be any one of the following: an antibody that does not bind to the region containing amino acids from position 1 to position 125 in the amino acid sequence of the GFAP; an antibody that does not bind to the region containing amino acids from position 111 to position 115 in the amino acid sequence of the GFAP; and an antibody that does not bind to the region containing amino acids from position 91 to position 190 in the amino acid sequence of the GFAP. More preferably, it is an antibody that does not bind to any of these regions.

[0048] The antibodies involved in this invention can be produced using existing known methods or methods based thereon, such as hybridoma methods or recombinant DNA methods. Representative examples of hybridoma methods include the method of Kohler and Milstein (Kohler & Milstein, Nature, 256:495 (1975)) and methods based thereon. Furthermore, the antibodies can be isolated and purified using methods commonly used in peptide purification.

[0049] The antibody-producing cells used in the cell fusion step of the hybridoma method are spleen cells, lymph node cells, peripheral blood leukocytes, etc., from animals (e.g., mice, rats, hamsters, rabbits, monkeys, goats) immunized with an antigen (GFAP, a peptide containing the region containing amino acids from positions 111 to 115, or cells expressing it). Antibody-producing cells obtained by acting the antigen in a culture medium on the aforementioned cells or lymphocytes pre-isolated from unimmunized animals can also be used. Various known cell lines can be used as myeloma cells in the cell fusion step. The antibody-producing cells and myeloma cells can be from different animal species, but are preferably from the same animal species, provided they can fuse. Hybridomas are generated, for example, by cell fusion between spleen cells obtained from mice immunized with an antigen and mouse myeloma cells. Subsequent screening yields hybridomas that produce monoclonal antibodies specific to the region containing amino acids from positions 111 to 115. Monoclonal antibodies can be cultured into hybridomas, isolated and purified from within the hybridoma or from the culture medium, and obtained in a substantially pure and homogeneous form. Alternatively, they can be obtained from the ascites fluid of mammals that have been administered the hybridoma.

[0050] In the recombinant DNA method, for example, DNA cloned from the hybridoma is integrated into a suitable vector and introduced into a host cell (e.g., mammalian cell lines such as HEK cells, Escherichia coli, yeast cells, insect cells, plant cells, etc.), so that the antibody of the present invention is produced as a recombinant antibody (e.g., PJ Delves, Antibody Production: Essential Techniques, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS, Vandamme AM et al., Eur. J. Biochem. 192: 767-775 (1990)). At this time, the aforementioned host cells can be cultured, and the antibody can be isolated and purified from the host cells or culture medium to obtain a substantially pure and homogeneous form.

[0051] [Immune Analysis] The detection method of the present invention involves contacting the test substance (GFAP) in the sample with a first antibody and a second antibody, and detecting the GFAP in the sample by immunoassay based on the immune complex formed by the antigen-antibody reaction. Examples of immunoassay methods include, but are not limited to, labeling immunoassay using antibodies labeled with a labeled substance, EIA (enzyme immunoassay) using an enzyme as the labeled substance, ELISA (chemiluminescent enzyme immunoassay) as a type of EIA, CLEIA (chemiluminescent enzyme immunoassay), RIA (radioimmunoassay) using a radioactive isotope as the labeled substance, CLIA (chemiluminescent immunoassay) using a chemiluminescent compound as the labeled substance, immunochromatography, and immunoaggregation methods (latex aggregation method, colloidal gold aggregation method, etc.) that detect aggregation.

[0052] More specifically, the detection method of the present invention includes: a first step of contacting the test substance (GFAP) in the sample with a first antibody and / or a second antibody to form a complex of the first antibody and GFAP; and a second step of forming a complex of the second antibody and GFAP. The first step and the second step can be performed either first or simultaneously, thereby forming the complex of the first antibody and the complex of the second antibody and GFAP.

[0053] From the viewpoint of higher sensitivity, a sandwich method using a first antibody and a second antibody is preferred as the detection method of the present invention. In the sandwich method, the test substance (GFAP) is captured by a trap containing an antibody and an insoluble carrier, and then bound to a label containing an antibody and a labeled substance. After B / F separation (washing), detection is performed according to the type of the labeled substance. Alternatively, as in immunochromatography, the label can be bound to the test substance (GFAP), and B / F separation can be performed while capturing it using a trap, followed by detection according to the type of labeled substance.

[0054] In this case, in the detection method of the present invention, either the first antibody or the second antibody can be used as the antibody for capturing the target, or as the antibody for labeling the target. That is, as a preferred embodiment of the detection method of the present invention, the following method can be cited: The first step is to capture GFAP using a capture medium containing a first antibody and an insoluble carrier, and the second step is to label GFAP using a label medium containing a second antibody and a labeling substance; or The first step is to label GFAP with a label containing a first antibody and a labeling substance, and the second step is to capture GFAP with a capture medium containing a second antibody and an insoluble carrier.

[0055] The following description uses the sandwich method as an example to illustrate one aspect of the detection method of the present invention, but the detection method of the present invention is not limited thereto.

[0056] (capture body) The capture body involved in this invention is a complex comprising a first antibody or a second antibody and an insoluble carrier, wherein the first antibody or the second antibody is directly or indirectly bound to and supported on the insoluble carrier.

[0057] The "insoluble carrier" contained in the capture body of the present invention is non-water-soluble and mainly carries a first antibody or a second antibody, functioning as a carrier for immobilization. It should be noted that in the present invention, "non-water-soluble" means insoluble in water at room temperature and pressure (the solubility relative to water is less than 0.001 g / mL, preferably less than 0.0001 g / mL, and the same applies below).

[0058] The material used as such an insoluble carrier can be any material suitable for use as an insoluble carrier in known immunoassays, without particular limitation. Examples include at least one material selected from polymers (polystyrene, (meth)acrylates, polymethyl methacrylate, polyimide, nylon, etc.), gelatin, cellulose, nitrocellulose, glass, latex, silica, metals (gold, platinum, etc.), and metal compounds (iron oxide, cobalt oxide, nickel ferrite, etc.). Alternatively, the material for the insoluble carrier can be a composite material, such as an organic-inorganic composite material comprising at least one organic polymer selected from the aforementioned polymers, gelatin, cellulose, and latex, and at least one metal compound selected from iron oxide (spinel ferrite, etc.), cobalt oxide, and nickel ferrite.

[0059] Furthermore, in this invention, the shape of the insoluble carrier is not particularly limited; for example, it can be any of the following: a plate, fiber, membrane, or particles. From the viewpoint of reaction efficiency, particles are preferred, and from the viewpoint of automation and shorter reaction time, magnetic particles are more preferred. As such an insoluble carrier, conventionally known insoluble carriers and commercially available insoluble carriers can be appropriately used.

[0060] In the capture body of the present invention, there is no particular limitation on the content of the first antibody or the second antibody, but in order to further improve the detectability of the test substance, it is preferable to set the number of first antibody or second antibody molecules that bind to the insoluble carrier 1 molecule as much as possible. For example, the mass of the first antibody or the second antibody relative to 100 parts by mass of the insoluble carrier is preferably 0.005 to 0.05 parts by mass, more preferably 0.01 to 0.04 parts by mass.

[0061] The capture body of the present invention can be manufactured by immobilizing a first antibody or a second antibody by binding it to the insoluble carrier. As a manufacturing method, depending on the type of the insoluble carrier and the first or second antibody, a known method or a method based thereon can be appropriately employed, allowing the first or second antibody to bind directly or indirectly to the insoluble carrier.

[0062] As a direct binding method, examples include: endowing the insoluble carrier and / or antibody (first antibody or second antibody) with active groups (e.g., thiol, maleimide, succinimide), or using a substance having these active groups as the insoluble carrier and / or antibody, and binding by covalent bonds based on these active groups. As the insoluble carrier and antibody endowed with the active groups, commercially available products such as insoluble carriers and second antibodies can be used, or the active groups can be introduced onto the surface of the insoluble carrier and / or antibody under appropriate reaction conditions. Alternatively, as a method for indirectly binding the insoluble carrier to the first antibody or second antibody, examples include binding via polyhistidine, polyethylene glycol, oligopeptides, linker molecules, etc. Alternatively, one side can be modified and a substance that captures the modified portion can be added to the other side, allowing them to bind. For example, one side can be biotinylated and the other avidinized, using an avidin-biotin binding method, or an indirect binding method using a secondary antibody, protein G, protein A, etc. The ratio of the insoluble carrier to the first or second antibody in this manufacturing method can be appropriately selected to achieve a preferred range of the contents of each of the aforementioned traps. Furthermore, as such a trap, commercially available substances such as antibody-binding particles using a second antibody can be appropriately used.

[0063] (Tag body) The labeling agent involved in this invention is a complex comprising a first antibody or a second antibody and a labeling agent, which is a conjugate formed by the direct or indirect binding of the first antibody or the second antibody and the labeling substance. Furthermore, it may further comprise a water-soluble carrier carrying the first antibody or the second antibody and the labeling substance. When the antibody contained in the trapping agent is a first antibody, the antibody contained in the labeling agent is preferably a second antibody; when the antibody contained in the trapping agent is a second antibody, the antibody contained in the labeling agent is preferably a first antibody.

[0064] The "labeling substance" contained in the labeling body of the present invention mainly functions as a label for detecting test substances and can be used without particular limitation as a labeling substance in known immunoassays.

[0065] Examples of labeling substances involved in this invention include: enzymes; radioactive isotopes (iodine, tritium, carbon, etc.); luminescent substances such as acridinium derivatives; fluorescent substances such as europium; fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE); low molecular weight labeling substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); gold particles; latex; dinitrobenzene (DNP); and digoxigenin (DIG). These can be a single substance or a combination of two or more. Among these, enzymes are preferred as the labeling substance involved in this invention. Examples of enzymes used in enzyme immunoassays include various enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP). β -Galactosidase ( β -gal), glucose oxidase, and luciferase. When using enzymes as the labeling substances, various detections can be performed based on the substrates, such as chromogenic substrates, fluorescent substrates, and chemiluminescent substrates. As such labeling substances and substrates, existing known substances and commercially available substances can be appropriately used.

[0066] It should be noted that, in the labeling agents involved in this invention, in addition to the method of forming a complex with GFAP by binding the labeling substance to a first antibody or a second antibody, there is also a method in which a complex with GFAP is formed without binding the labeling substance to the first antibody or the second antibody, and then a secondary antibody, protein G, protein A, etc., bound to the labeling substance is bound to the first antibody or the second antibody. Here, "secondary antibody" refers to an antibody that exhibits reactivity to an antibody that directly binds to an antigen (primary antibody, i.e., the first antibody or the second antibody). Furthermore, as a similar method, there are also methods such as pre-binding avidin or biotin to the first antibody or the second antibody, and then binding a labeling substance bound to biotin or avidin after forming its complex with GFAP.

[0067] In the labeling agent of the present invention, the molar ratio of the labeling substance to the first antibody or the second antibody is not particularly limited and can be appropriately adjusted according to the combination of their types, the ease of binding with the test substance, etc. For example, the first antibody or the second antibody is preferably 0.01 to 10,000 moles relative to 1 mole of the labeling substance, more preferably 0.05 to 10 moles.

[0068] The marker according to the present invention can be manufactured by binding the marker substance to a first antibody or a second antibody. As a manufacturing method, depending on the type of the marker substance and the first or second antibody, a known method or a method based thereon can be appropriately employed to allow the marker substance to bind directly or indirectly to the first or second antibody. Examples of such binding methods include the binding of the insoluble carrier to the first or second antibody. The ratio of the marker substance to the first or second antibody in this manufacturing method can be appropriately selected to achieve a preferred range of content in the marker. Furthermore, as such a marker, for example, any commercially available marker, such as an enzyme-labeled antibody using a second antibody, can be appropriately used.

[0069] (Capture process) When the detection method of the present invention is a sandwich method, it includes: a capture step in which, before the labeling step described below, the sample is contacted with the trapping body, and if the sample contains a test substance, the test substance is captured by the trapping body through the binding of the test substance with a first antibody or a second antibody, forming a complex of the trapping body and the test substance, i.e., a trapping body-test substance complex (hereinafter referred to as "first complex" as appropriate); or a capture step in which, after or simultaneously with the labeling step described below, the second complex obtained in the labeling step described below is contacted with the trapping body, forming a complex of the labeling body-test substance-trapping body (hereinafter referred to as "third complex" as appropriate). From the viewpoint of removing impurities other than the test substance contained in the sample, and from the viewpoint of further improving detection accuracy by performing multiple washing steps, such a capture step is more preferably included before the labeling step described below.

[0070] There are no particular limitations on the method for contacting the sample or second composite with the trapping body; any existing known method or method based thereon can be appropriately employed. Examples include injecting the aqueous sample or second composite into the insoluble carrier when it is a plate, and mixing a particulate liquid containing the insoluble carrier with the aqueous sample or second composite when the insoluble carrier is granules. As a dispersion medium for the particulate liquid, examples of dispersion media listed as diluents can be included.

[0071] (Marking process) When the detection method of the present invention is a sandwich method, it includes: before the above-mentioned capture step, contacting the sample with the label, and if the sample contains a test substance, forming a complex of the label and the test substance (referred to as a "second complex" in this specification, depending on the circumstances), i.e., a label-test substance complex, through the binding of the test substance with a first antibody or a second antibody, or a labeling step including: after or simultaneously with the above-mentioned capture step, contacting the first complex obtained in the capture step with the label to form a label-test substance-capture complex (third complex).

[0072] There are no particular limitations on the method for contacting the sample or first composite with the marker; any existing known method or method based thereon can be appropriately employed. For example, a method of adding a labeled liquid to the aqueous sample or first composite can be cited. As a solvent for the labeled liquid, solvents listed as diluents can be cited as examples.

[0073] In the reaction system of the trapping agent and the test substance in the trapping step, and in the reaction system of the labeling agent and the test substance in the labeling step, the content (final concentration) of the trapping agent and the labeling agent in each reaction system is not particularly limited, and can be appropriately adjusted according to the type and concentration of the sample, trapping agent, and labeling agent, and therefore is not particularly limited. Furthermore, the conditions of each reaction system are not particularly limited, and can be appropriately adjusted. For example, the reaction can be carried out at room temperature to 45°C, preferably 20 to 37°C, pH 6 to 9, preferably pH 7 to 8, for about 5 seconds to 10 minutes, preferably 30 seconds to 8 minutes. It should be noted that in this invention, "reaction system" refers to an aqueous system, preferably an aqueous solution containing these components used to react the first antibody or the second antibody with GFAP.

[0074] However, in the detection method of the present invention, in the first step of forming the complex of the first antibody and GFAP, the pH of the reaction system (or the pH of the aqueous solvent of the first antibody (or the label or trap containing it) (corresponding to the particle dilution in Test Examples 3 and 4 of the following embodiments) is preferably 6.0 to 9.0, more preferably 6.2 to 7.5, and even more preferably 6.3 to 6.7 or 7.0 to 7.4. That is, when the detection method of the present invention is a sandwich method, if the trap contains the first antibody, it is preferable that the pH of the trapping step meets the above conditions; if the label contains the first antibody, it is preferable that the pH of the labeling step meets the above conditions. In addition, in this case, the pH of the second step of forming the complex of the second antibody and GFAP is also more preferably the same condition.

[0075] In the reaction system in which the first or second antibody reacts with GFAP, in addition to GFAP, the labeled and / or trapping bodies, and their aqueous solvents (sample dilution, particle dispersion medium, labeled solution solvent, etc.), the following components may also be included: pH buffer (Tris(tris(hydroxymethyl)aminomethane), MOPS(3-morpholinopropane-1-sulfonic acid), surfactants (N-tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate, Tween-20, Tween-80, etc.), salts (NaCl, etc.), sugars (sucrose, etc.), chelating agents (EDTA-2Na, etc.), proteins (BSA, etc.), and other components (dextran, dextran salts, etc.). Furthermore, the sample dilution, particle dispersion, and labeled solution may also serve as buffers for the reaction system containing these components.

[0076] (Cleaning process) When the detection method of the present invention is a sandwich method, it is preferable to further include a cleaning step to remove impurities not captured by the capturing body. If the capturing step is included before the marking step, it is more preferable to include a cleaning step between the capturing step and the marking step to remove impurities not captured by the capturing body, i.e., components other than the first complex. Furthermore, in this case, it is more preferable to further include a cleaning step after the marking step to remove impurities not captured by the capturing body, i.e., components other than the third complex contained in the reaction system.

[0077] There are no particular limitations on the method for removing the impurities, and existing known methods or methods based thereon can be appropriately used. For example, if the insoluble carrier is a plate, a method for removing the liquid phase (supernatant) from the plate can be used; if the insoluble carrier is particles, a method for removing the liquid phase (supernatant) by recovering the particles from the reaction buffer through centrifugation and magnetic attraction can be used. Furthermore, in the washing process, the injection and removal of the washing solution can be repeated as needed. Examples of the washing solution include known neutral (preferably pH 6-9) buffer solutions (sodium phosphate buffer, MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, tricine buffer, bicine buffer, glycine buffer, etc.). Additionally, substances obtained by adding stabilizing proteins such as BSA, surfactants, etc., can also be used.

[0078] (Inspection process) In the detection method of the present invention, GFAP is detected by detecting the complex of the test substance (GFAP) with the first antibody and the second antibody (the third complex in the sandwich method described above). In the case of the sandwich method described above, GFAP is detected by detecting the signal from the labeled substance that binds to the GFAP. The "signal" can be, depending on the type of labeled substance, for example, color development, reflected light, luminescence, fluorescence, radiation generated by radioactive isotopes, etc. In addition to signals that can be confirmed by the naked eye, it also includes signals that can be confirmed by a detection method or apparatus corresponding to the type of signal.

[0079] When using an enzyme as the labeling substance, it is preferable to perform the capture and washing steps before the labeling step and the washing step after the labeling step to remove impurities not captured by the capture body. Then, a chromogenic substrate, fluorescent substrate, chemiluminescent substrate, etc., corresponding to the type of enzyme, is added as a substrate to allow the reaction to proceed, thereby enabling the detection of signals (fluorescence, luminescence, color development, etc.) corresponding to the substrate. Such substrates and reaction conditions can be appropriately adjusted according to the type of enzyme, etc. As the detection method of the present invention, the detected signal count can be directly used as the value corresponding to the amount of GFAP in the sample, or, as needed, the amount of GFAP can be quantified by comparing the signal value of GFAP with that in a standard sample of known concentration.

[0080] <Methods to aid in the diagnosis of Alzheimer's disease> This invention also provides a method for assisting in the diagnosis of Alzheimer's disease (hereinafter referred to as a "diagnostic aid method") and a method for diagnosing Alzheimer's disease (hereinafter referred to as a "diagnostic method"), wherein the method for assisting in the diagnosis of Alzheimer's disease includes a step of detecting GFAP in a sample collected from a subject using the detection method of the present invention described above, and the method for diagnosing Alzheimer's disease includes a step of detecting GFAP in a sample collected from a subject using the detection method of the present invention described above. Furthermore, the diagnostic aid method of the present invention can also be manifested as: a method for a physician to detect GFAP for diagnosing Alzheimer's disease, and a method for informing the physician of the presence or absence of the detected GFAP. The detection method, including its preferred embodiments, is as described above. According to the detection method of the present invention, even if the amount of GFAP in the sample is small, it can be detected with high precision, and therefore it is suitable as a detection method for diagnosing Alzheimer's disease where the concentration in the sample is low compared to brain injury. However, this does not preclude the use of the detection method of the present invention as an aid in the diagnosis of brain injury (stroke, traumatic brain injury, etc.).

[0081] In the diagnostic auxiliary method and diagnostic method of the present invention, the subject is preferably a human being, and the sample is preferably plasma or serum.

[0082] As a diagnostic method of the present invention, it is preferable to further include a step of diagnosing the subject as having Alzheimer's disease or a high probability of having it, using the presence or amount of GFAP as an indicator. In the diagnostic aid method and diagnostic method of the present invention, as long as a small amount of GFAP is detected from the sample by the detection method, the subject from which the sample originates can be determined, screened, or identified as having Alzheimer's disease or a high probability of having it. However, it is preferable to determine, screen, or identify based on the amount of GFAP detected. For example, the amount of GFAP detected by the detection method can be compared with a predetermined threshold value, making subjects with a value above the threshold have Alzheimer's disease or a high probability of having it. Furthermore, the present invention can also provide a screening method that includes the detection step and includes a step of screening subjects with Alzheimer's disease or a high probability of having it, using the presence or amount of GFAP as an indicator.

[0083] The "critical value" is a predetermined value used as a benchmark for judgment based on the amount of GFAP, and represents the boundary value used to distinguish between the positive and negative groups. Such a critical value is appropriately set according to the purpose of diagnosis, the subjects, the nature of the sample, dilution conditions, etc., and therefore is not particularly limited. For example, by setting the critical value to a relatively low value, the detection sensitivity can be improved, that is, a wider range of subjects suspected of having Alzheimer's disease can be collected to some extent; on the other hand, by setting the critical value to a relatively high value, the detection accuracy can be further improved.

[0084] According to the diagnostic aid and diagnostic methods of the present invention, information on patients with Alzheimer's disease or those at high risk of having Alzheimer's disease can be provided with high precision, distinguishing them from healthy individuals, thus enabling early intervention and treatment. Furthermore, for patients screened as having a high risk of Alzheimer's disease, examinations can be performed using methods such as brainwave analysis, CT scans, MRI, and PET / SPECT to detect brain and hippocampal atrophy and the deposition of senile plaques (amyloid plaques).

[0085] Furthermore, the present invention provides a method for determining GFAP by detecting the presence or amount of a region containing amino acids from positions 115 to 191 of GFAP in a sample, and a method for assisting in the diagnosis of Alzheimer's disease. Moreover, the present invention provides a method for using the presence or amount of a region containing amino acids from positions 115 to 191 of GFAP in a sample as an indicator for determining GFAP, and as an indicator for diagnosing Alzheimer's disease.

[0086] <Reagent Kit> The present invention also provides a kit for use in the detection method, diagnostic aid method, or diagnostic method of the present invention described above, comprising a first antibody and a second antibody that binds to a region of the amino acid sequence of GFAP that is different from the first antibody. These first and second antibodies are preferably disposed in the manner described above. Furthermore, these first and second antibodies are more preferably included in the kit of the present invention as the trapping or labeling agents, and these trapping and labeling agents are also preferably disposed in the manner described above. Moreover, they can each be independently in solid (powder) form, or in liquid form dissolved or suspended in a solution (the diluent, the dispersion medium of the particulate liquid, the solvent of the labeled liquid, etc.).

[0087] As a kit of the present invention, it may further include, for example, at least one selected from the following: standard samples (at various concentrations), control samples, the diluent, the washing solution, the dispersion medium of the particulate liquid, the solvent of the labeled body fluid, the buffer for the reaction system, the substrate, the enzyme-substrate reaction buffer, and the enzyme-substrate reaction stop buffer. Furthermore, if the labeled substance contains unbound antibodies, it is also possible to combine a substance that binds the labeled substance to a substance that binds to the antibody (e.g., a secondary antibody, protein G, protein A, etc.). In addition, the kit of the present invention may also include an instruction manual for the kit.

[0088] Example The present invention will now be described in more detail with reference to the embodiments, but the present invention is not limited to the embodiments described below. It should be noted that in the following test examples, unless otherwise specified, "%" indicates a weight / volume (w / v) percentage (g / 100mL).

[0089] (Experimental Example 1) Obtaining Anti-GFAP Antibody (1) Mouse immunization In 7-8 week old female BALB / c and ICR mice, intraperitoneal administration of 50 μ An emulsion containing g / body of recombinant GFAP (LSBio: a recombinant protein containing full-length human GFAP) and an equal volume of Freund's complete adjuvant. Subsequently, 50 g / body of the emulsion was administered intraperitoneally every 2 weeks. μ The emulsion of recombinant GFAP and an equal amount of Freund's incomplete adjuvant was repeated 2 to 5 times until an increase in antibody titer was confirmed.

[0090] (2) Preparation of hybridomas from anti-GFAP antibodies Mice with confirmed adequate antibody titer elevation were administered 50 mg intraperitoneally. μRecombinant GFAP solution was administered at a concentration of g / body. Three to four days after drug administration, the spleen of mice was removed. The removed spleen cells were fused with P3U1 myeloma cells pre-cultured in RPMI 1640 via electroporation to create fused cells. The culture supernatant from 7 to 14 days post-fusion was immobilized with GFAP and added to a washed plate for a first reaction. Following three washes with PBS-T, HRP-labeled anti-mouse immunoglobulin antibody (Dako) was added for a second reaction. After three washes with PBS-T, ABTS or TMB was added for a colorimetric reaction. The colorimetric reaction was stopped by adding 1.5M oxalic acid aqueous solution or 0.5M H2SO4, and the absorbance was measured at 405nm or 450nm. Clones of fused cells showing a high response to GFAP (strong color development) were selected. Using limited dilutions of the clones, multiple monoclonal antibodies were used to generate hybridomas.

[0091] (3) Purification of anti-GFAP antibody Hybridomas were cultured in serum-free medium for 1–2 weeks, and the culture supernatant was recovered. The recovered culture supernatant was passed through a Protein A column to allow antibody binding. After washing with Washing Buffer 1 (20 mM PB (phosphate buffer), 2 M NaCl, 5% sucrose, pH 7.3) and Washing Buffer 2 (50 mM acetate buffer, 5% sucrose, pH 5.0), the antibody was eluted with Elution Buffer (50 mM acetate buffer, 5% sucrose, pH 3.8), and neutralized with Neutralization Buffer (1 M Tris-HCl, pH 8.5). The neutralized antibody solution was dialyzed overnight with Preservation Buffer (PBS, 5% sucrose, pH 7.2), and concentrated using an ultrafiltration column to obtain purified anti-GFAP antibody. The purified anti-GFAP antibodies obtained by the above method included the following five anti-GFAP antibodies from the experimental examples: GFAP-Ab1, GFAP-Ab2, GFAP-Ab3, GFAP-Ab4, and GFAP-Ab5.

[0092] (Experimental Example 2) Identification of Epitopes (1) Preparation of GFAP fragments The nucleotide sequence encoding the full-length human GFAP was appropriately fragmented to create the amino acid sequence of the full-length GFAP (aa1-432), which encodes the amino acid sequence containing... Figure 1At the ends of the nucleotide sequences of the amino acid fragments shown, GFAP cleavage fragment coding sequences with added restriction enzyme sites were constructed. Using the corresponding restriction enzymes, these GFAP cleavage fragment coding sequences were introduced into GST expression vectors or His-tagged expression vectors to create GFAP cleavage fragment expression vectors with GST fused to the N-terminus or His tags fused to the C-terminus. The constructed expression vectors were then introduced into *E. coli* DH5α for plasmid amplification. The resulting plasmids were then introduced into *E. coli* BL21(DE3) and cultured overnight in LB medium with shaking. The bacterial culture was then diluted 100-fold with LB medium and cultured with shaking for 3–6 hours. Expression was then induced with 0.4 mM IPTG and cultured overnight with shaking. The bacterial cells were recovered by centrifugation at 15000 rpm at room temperature for 1 minute. PBS (1 / 10 the volume of LB medium) was added to the bacterial cells, and the suspension was sonicated for 15 minutes. The ultrasonically disrupted fluid was centrifuged at 15,000 rpm at room temperature for 10 minutes, and the supernatant was collected to prepare GFAP fragment (GFAP fragment fused with GST or GFAP fragment fused with His tag) solution.

[0093] (2) Identification of epitopes based on immunoblotting Add half the amount of 2×SDS-PAGE sample buffer to each GFAP fragment solution prepared in (1) above, and heat at 96°C for 5 minutes to modify. Add 10 μL of the heat-modified liquid to an SDS-PAGE gel (MiniPROTEANT Glucon Precast Gel, 4-20%, Biorad). μ L(50 μ Lculture fractions were subjected to electrophoresis at a constant voltage of 200V for 30 minutes. The electrophoresed gel was transferred to a PVDF membrane using a Trans-Blot Turbo system (Biorad). The PVDF membrane was then blocked by immersing it in PBS containing 1% skim milk. The blocked PVDF membrane was then immersed in PBS containing 1% skim milk. μ The PVDF membrane was subjected to a first reaction by shaking any of the following: g / mL anti-GST antibody, anti-His-tagged antibody, or the anti-GFAP antibody prepared in Example 1 above, in PBS containing 1% BSA. Next, the PVDF membrane was washed three times with PBS-T for 5 minutes each time, and then immersed in POD-labeled anti-mouse antibody solution diluted 2000 times with PBS containing 1% BSA for a second reaction. The PVDF membrane was then washed three times with PBS-T for 5 minutes each time, and a luminescent substrate (ECLSelect, GE Healthcare) was added to the PVDF membrane. After removing excess liquid, luminescence was confirmed using a LAS500 (GE Healthcare).

[0094] In the GFAP cleavage fragments (His-tagged fusion fragments) prepared in (1) above, for fragments aa1-230, aa1-104, aa72-214, and aa116-214, the results of the anti-His-tagged antibody (Anti-His) reaction in a single reaction are shown below. Figure 2 (a) will show the results of the GFAP-Ab1 reaction in the anti-GFAP antibody. Figure 2 (b)

[0095] Additionally, for the GFAP cleavage fragments (GST fusion fragments), specifically fragments aa91-160, aa96-160, aa101-160, aa106-160, aa111-160, aa121-170, aa131-180, aa141-190, aa151-200, and aa161-214, the results of the anti-GST antibody (Anti-GST) reaction in a single reaction are shown below. Figure 3 (a) will show the result of the GFAP-Ab1 reaction in the anti-GFAP antibody. Figure 3 (b)

[0096] Further, in the GFAP cleavage fragments (GST fusion fragments), for fragments aa1-125, aa1-126, aa1-127, aa1-128, aa1-129, aa1-130, aa1-131, aa1-132, aa1-133, aa1-134, and aa177-230, in a single reaction, the results of the anti-GST antibody (Anti-GST) reaction are respectively shown in the figure. Figure 4 (a) shows the result of the GFAP-Ab1 reaction in the anti-GFAP antibody. Figure 4 (b)

[0097] like Figure 2 As shown, GFAP-Ab1 binds in the aa72-214 segment, but not in the aa1-104 and aa116-214 segments. Additionally, as... Figures 3-4 As shown, GFAP-Ab1 binds, for example, in the aa111-160 fragment, but not in the aa121-214 range. Based on these results, it is confirmed that GFAP-Ab1 binds at least in the aa111-115 range.

[0098] In the GFAP cleavage fragments (GST fusion fragments) prepared in (1) above, for fragments aa91-160, aa96-160, aa101-160, aa106-160, aa111-160, aa121-170, aa131-180, aa141-190, aa151-200, and aa161-214, in a single reaction, the results of the anti-GST antibody (Anti-GST) reaction are shown below. Figure 5 (a) will show the result of the GFAP-Ab2 reaction in the anti-GFAP antibody. Figure 5 (b)

[0099] Additionally, for the GFAP cleavage fragments (GST fusion fragments), specifically fragments aa177-230, aa178-230, aa179-230, aa180-230, aa181-230, aa182-230, aa183-230, aa184-230, aa185-230, aa186-230, and aa1-125, the results of the anti-GST antibody (Anti-GST) reaction in a single reaction will be shown in [the table / document / etc.]. Figure 6 (a) shows the results of the reaction using the GFAP-Ab2 in the anti-GFAP antibody. Figure 6 (b)

[0100] like Figures 5-6 As shown, GFAP-Ab2 binds, for example, in the aa151-200 segment, but not in the aa141-190 and aa1-125 segments. Based on these results, it is confirmed that GFAP-Ab2 binds at least in the aa191-200 range. Similarly, it is confirmed that GFAP-Ab3, GFAP-Ab4, and GFAP-Ab5 all bind in the aa257-377 range.

[0101] (Experimental Example 3) Determination of GFAP using sandwich immunoassay 1 Following conventional methods, the capture organisms were immobilized on magnetic particles using anti-GFAP antibody to prepare magnetic particle-immobilized anti-GFAP antibody. This was added to a particle dilution buffer (containing 50 mM MOPS, 150 mM NaCl, 1 mM EDTA-2Na, 2% BSA, 2% sucrose, and 0.05% dextran sulfate (pH 7.2)) at a concentration of 0.025% to prepare the capture organism fluid. Additionally, following conventional methods, the labeled anti-GFAP antibody was prepared into Fab′ by pepsin digestion and reduction, followed by alkaline phosphatase labeling to prepare labeled anti-GFAP antibody. This labeled anti-GFAP antibody was then diluted with a labeling buffer (containing 50 mM MOPS, 150 mM NaCl, 2% BSA, 2% sucrose, 1.5% Pluronic F-108, 0.1% Tween-80, and 1.0% PVP (pH 7.2)) at a concentration of 0.5%. μ The solution was diluted at a concentration of g / mL to prepare a labeled solution. The anti-GFAP antibody prepared above (Example 1) was used as the capture antibody and the labeling antibody, respectively, in the combinations shown in the "Capture Antibody" and "Label Antibody" columns of Table 1 below.

[0102] GFAP was measured using a Lumipulse G1200 (manufactured by Fuji Rubio Co., Ltd.). First, 100... μ L sample and 20 μ L of the immunoreaction solution (a solution containing 50 mM MOPS, 150 mM NaCl, 1 mM EDTA-2Na, 1.5% N-tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate, 1.5% Tween-80, and 1.0% Tergitol™ 15-s-9 (Thermo Fisher, pH 7.2)) was mixed. Then, it was mixed with 150 mM of the capture fluid. μ The mixture was stirred at 37°C for 10 minutes (capture process). After the reaction, the magnetic beads were magnetized and cleaned using Lumipulse cleaning solution (manufactured by Fuji Rebio Co., Ltd.) (cleaning process). Then, 150... μ L-labeled body fluid was reacted at 37°C for 10 minutes (labeling step). Next, the magnetic beads were magnetized, cleaned with Lumipulse cleaning solution (cleaning step), and then 50... μL contains Lumipulse substrate solution (manufactured by Fuji Rebio Co., Ltd.) containing AMPPD (3-(2′-spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)phenyl-1,2-dioxane disodium salt), and reacts at 37°C for 5 minutes. The amount of light emitted, with maximum absorption at a wavelength of 463 nm, is measured as a result of the decomposition of AMPPD by alkaline phosphatase trapped by magnetic beads. The results are output as the luminescence intensity (count) of the substrate (AMPPD). As samples, diluted samples (diluted samples, GFAP concentration: 0–1000 [pg / mL]) diluted with human plasma and plasma samples from Alzheimer's disease patients (samples A–C) are used. Two samples are measured for each combination, and the average value is calculated. In addition, regarding the results of diluted samples, the ratio of the count values ​​at a GFAP concentration of 0 pg / mL to that at 10 pg / mL (10 / 0) ​​and the ratio of the count values ​​at a GFAP concentration of 10 pg / mL to that at 1000 pg / mL (1000 / 10) were also calculated. The results are shown in Table 1 below.

[0103] [Table 1]

[0104] As shown in Table 1, when using GFAP-Ab1 (Ab1, Example) as the capture antibody and GFAP-Ab2 (Ab2) as the label antibody, the count values ​​are sufficiently high compared to other antibody combinations. Furthermore, it was confirmed that the ratios of count values ​​between different GFAP concentrations (10 / 0, 1000 / 10) were significantly higher than with other antibody combinations, and the count values ​​increased correctly depending on the GFAP concentration, demonstrating significantly high detection accuracy even at low GFAP concentrations.

[0105] (Experimental Example 4) Determination of GFAP using sandwich immunoassay 2 (1) Anti-GFAP antibodies were obtained in the same manner as in Experimental Example 1 above, and epitopes were identified in the same manner as in Experimental Example 2 above. In the same manner as GFAP-Ab1, antibodies that were confirmed to bind in the range of aa111-115 and not to the aa116-214 fragment were used as GFAP-Ab6 for the determination of GFAP.

[0106] (2) The GFAP assay was performed in the same manner as described above (Example 3), using GFAP-Ab6 as the capture antibody (capture body) and GFAP-Ab2 as the labeling antibody (labeling body). Three samples were measured for each combination, and the average values ​​were calculated. Furthermore, regarding the results of sample dilution, the ratio of the count values ​​at a GFAP concentration of 0 pg / mL to that at 10 pg / mL (10 / 0) ​​and the ratio of the count values ​​at a GFAP concentration of 10 pg / mL to that at 1000 pg / mL (1000 / 10) were also calculated. The results are shown in Table 2 below.

[0107] [Table 2]

[0108] As shown in Table 2, when using GFAP-Ab6 (Ab6, Example) as the capture antibody and GFAP-Ab2 (Ab2) as the label antibody, similarly to when using GFAP-Ab1 as the capture antibody and GFAP-Ab2 as the label antibody (Table 1), the count values ​​and the count ratios (10 / 0, 1000 / 10) were both high, confirming that low concentrations of GFAP can be detected with significantly high accuracy using these antibodies.

[0109] (Experimental Example 5) Study on Measurement Conditions Two particle diluents with different pH values ​​were used: A (a solution containing 50 mM MOPS, 150 mM NaCl, 1 mM EDTA-2Na, 2% BSA, and 0.3% N-tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (pH 6.5)) or B (a solution containing 50 mM Tris, 150 mM NaCl, 1 mM EDTA-2Na, 2% BSA, 0.05% Tween-20, and 0.05% dextran sulfate (pH 7.2)). Otherwise, the GFAP content in each sample was determined in the same manner as described in Example 3. The results were output as substrate luminescence intensity (counts). The assay involved measuring two samples for each combination and calculating the average value. For diluted samples, the ratio of the count values ​​at 0 pg / mL to those at 10 pg / mL (10 / 0) ​​and the ratio of the count values ​​at 10 pg / mL to those at 1000 pg / mL (1000 / 10) were also calculated. The results, along with the combination of anti-GFAP antibody for the trap (trapping body) and anti-GFAP antibody for the label (label), and the pH of the particle dilution solution (dilution pH), are shown in Table 3 below.

[0110] [Table 3]

[0111] As shown in Table 3, high count values ​​and high accuracy were obtained when using particle diluents of any pH (dilution pH). However, when using particle diluent A with pH 6.5 (pH of the reaction system in the capture process: 6.5), a trend of higher count values ​​and higher accuracy (larger ratio of count values) was observed compared to using particle diluent B with pH 7.2 (pH of the reaction system in the capture process: 7.2).

[0112] (Experimental Example 6) Measurement of GFAP using multiple Alzheimer's disease patient samples Plasma samples from 17 cognitively normal individuals and 16 Alzheimer's disease patients were used as samples. Otherwise, GFAP was measured in the same manner as described above (Experimental Example 3). GFAP-Ab1 was used as the capture antibody; GFAP-Ab2 was used as the labeling antibody. Furthermore, diluted samples with known GFAP concentrations were also measured, and a standard curve was generated. Based on this, the GFAP level (pg / mL) in each sample was calculated. The distribution of GFAP levels in the cognitively normal population (CU) and the Alzheimer's disease patient population (AD) is shown in the figure. Figure 7 .

[0113] like Figure 7 As shown, the Wilcoxon assay confirmed a significant difference in GFAP levels between cognitively normal individuals (CU) and Alzheimer's disease patients (AD) (p < 0.0001). Therefore, it is confirmed that the detection method of the present invention using GFAP-Ab1 can accurately distinguish between cognitively normal individuals (CU) and Alzheimer's disease patients (AD).

[0114] (Experimental Example 7) Determination of GFAP using preserved samples As samples, diluted samples of GFAP diluted with human plasma (diluted sample, GFAP concentration: 0–1000 [pg / mL]) and samples of GFAP added to human plasma were prepared and stored under any of the following conditions: -80°C freezing (control), -80°C freezing and thawing repeated twice (FT 2 times), standing at 4°C for 1 day (4°C, 1 day), and standing at room temperature (approximately 25°C) for 1 day (room temperature, 1 day). Anti-GFAP antibodies prepared above (Example 1) were used as the trapping agent and the labeling agent, respectively, in the combinations shown in the "Trapping Agent" and "Labeling Agent" columns of Table 4 below.

[0115] Dilute 2 with PBS μ The trapping agents (g / mL) were added to the detection plate with anti-GFAP antibody. After immobilization, Tris buffer containing 1% BSA and 1% casein was added for blocking. Separately, the labeled anti-GFAP antibody was biotin-labeled as biotin-labeled anti-GFAP antibody. The blocked detection plate was washed three times with PBS-T, and each sample was added with Tris buffer containing 1% BSA and 1% casein for a first reaction. Next, the plate was washed three times with PBS-T, and biotin-labeled anti-GFAP antibody diluted with Tris buffer containing 1% BSA and 1% casein was added for a second reaction. Next, the plate was washed three times with PBS-T, and alkaline phosphatase-labeled streptavidin solution diluted with Tris buffer containing 1% BSA and 1% casein was added for a third reaction. Finally, after washing three times with PBS-T, AMPPD was added to react, and the luminescence intensity of light with maximum absorption at 463 nm was measured. The results were output as the luminescence intensity (count) of the substrate (AMPPD). The results are shown in Table 4 below.

[0116] [Table 4]

[0117] As shown in Table 4, it was confirmed that by using GFAP-Ab1 (Ab1, Example), even combinations of antibodies in the same table resulted in sufficiently high count values. Furthermore, the count values ​​increased correctly depending on the concentration of GFAP, enabling high-precision detection. In addition, for samples stored under the following conditions—freezing and thawing twice at -80°C (FT 2 times), standing at 4°C for 1 day (4°C, 1 day), and standing at room temperature for 1 day (room temperature, 1 day)—the count values ​​were as sufficiently high as those of samples frozen at -80°C (control). However, it was confirmed that the decrease in count values ​​relative to the control was less in the case of the combination of GFAP-Ab1 (Ab1) and GFAP-Ab2 (Ab2) compared to the combination of GFAP-Ab3 (Ab3), indicating a trend towards being less affected by sample storage conditions.

[0118] Industrial applicability As explained above, according to the present invention, it is possible to provide a detection method that can detect GFAP with high precision by immunoassay even when the amount in the sample is small, thereby assisting in the diagnosis of Alzheimer's disease, and kits for use therein.

Claims

1. A detection method, characterized in that, It is a method for detecting GFAP in samples through immunoassay. The detection method includes: a first step of forming a complex of a first antibody and GFAP; and a second step of forming a complex of a second antibody and GFAP. The first antibody is an antibody that binds to the region containing amino acids 111 to 115 in the amino acid sequence of GFAP.

2. The detection method according to claim 1, wherein, The second antibody is an antibody that binds to the region of the GFAP amino acid sequence containing amino acids from position 191 to position 200.

3. The detection method according to claim 1, wherein, The first antibody is an antibody that does not bind to the region of the GFAP amino acid sequence containing amino acids from position 116 to position 214.

4. The detection method according to claim 1, wherein, The pH of the reaction system in the first step is 6.2 to 7.

5.

5. The detection method according to claim 1, wherein, The first step is to capture GFAP using a capture medium containing a first antibody and an insoluble carrier, and the second step is to label GFAP using a label medium containing a second antibody and a labeling substance; or The first step is to label GFAP with a label containing a first antibody and a labeling substance, and the second step is to capture GFAP with a capture medium containing a second antibody and an insoluble carrier.

6. A method for assisting in the diagnosis of Alzheimer's disease, characterized in that, The procedure includes detecting GFAP in a sample collected from a subject using the detection method according to any one of claims 1 to 5.

7. A kit for detecting GFAP in a sample by immunoassay, characterized in that, Include: A first antibody that binds to the region of the GFAP amino acid sequence containing amino acids 111 to 115; and A second antibody that binds to a region in the amino acid sequence of GFAP that is different from that of the first antibody.

8. The kit according to claim 7, wherein, The second antibody is an antibody that binds to the region containing amino acids 191 to 200 in the amino acid sequence of GFAP.

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