GFAP detection method, method for assisting diagnosis of alzheimer 's disease, and kit used therefor

Abstract

Provided is a method for detecting GFAP in a sample by immunoassay, the method including a first step for forming a complex of a first antibody and GFAP, and a second step for forming a complex of a second antibody and GFAP, the first antibody binding to a region consisting of amino acids at positions 111 to 115 in the amino acid sequence of GFAP.

Description

Method for detecting GFAP, method for assisting in the diagnosis of Alzheimer's disease, and kit for use therein

[0001] The present invention relates to a method for detecting GFAP, a method for assisting in the diagnosis of Alzheimer's disease, and a kit for use therein, and more particularly to a method for detecting GFAP in a sample by immunoassay, thereby assisting in the diagnosis of Alzheimer's disease, and a kit for use therein.

[0002] Glial fibrillary acidic protein (GFAP) is known as a biomarker for diagnosing central nervous system damage and diseases, but in recent years it has become clear that it is also useful as a biomarker for diagnosing Alzheimer's disease.

[0003] Alzheimer's disease (Alzheimer's type dementia, AD) is a progressive neurodegenerative disease, and its main symptoms include memory impairment, higher brain dysfunction (aphasia, apraxia, agnosia, constructional apraxia), personality changes, etc. The number of Alzheimer's disease patients is steadily increasing with the aging of the population, and it is currently a serious social problem, but the full picture of the mechanism of onset has not yet been clarified, and no cure has yet been developed.

[0004] On the other hand, since it has become possible to delay the progression of symptoms, there is a demand for earlier diagnosis of Alzheimer's disease in the treatment of the disease. However, current detection methods for diagnosis mainly involve detecting cerebral and hippocampal atrophy and senile plaque (amyloid plaque) deposition using electroencephalography, CT, MRI, PET / SPECT, etc. However, these methods are difficult to apply easily to a wide range of subjects and require special techniques and devices, so they have not yet led to early diagnosis.

[0005] Therefore, it is expected that by detecting the above-mentioned GFAP from a sample (specimen) collected from a subject, it will be possible to relatively easily detect subjects who are suffering from or are likely to suffer from Alzheimer's disease. As a method for detecting GFAP from a sample, detection methods for diagnosing stroke and traumatic brain injury are known. For example, International Publication No. 2018 / 096049 (Patent Document 1), U.S. Patent Application Publication No. 2019 / 0302127 (Patent Document 2), and Japanese Patent Laid-Open No. 2022-66139 (Patent Document 3) describe methods for detecting GFAP in a sample by immunoassay using anti-GFAP antibodies that bind to various epitopes.

[0006] International Publication No. 2018 / 096049 US Patent Application Publication No. 2019 / 0302127 JP 2022-66139 A

[0007] The detection methods described in Patent Documents 1 to 3 are applicable to diseases such as stroke and traumatic brain injury, and in the case of these diseases, a relatively large amount of GFAP is contained in a sample (specimen) such as serum or plasma, so that its detection is relatively easy even by immunoassay. However, in the case of Alzheimer's disease, the amount of GFAP contained in a sample (specimen) collected from a patient is usually so small that it is difficult to detect with high accuracy, so further improvement in the accuracy of GFAP detection by immunoassay is required.

[0008] The present invention has been made in consideration of the problems associated with the above-mentioned prior art, and aims to provide a detection method capable of detecting at least the amount of GFAP in a sample with high accuracy by immunoassay, a method thereby assisting in the diagnosis of Alzheimer's disease, and a kit for use therein.

[0009] In order to achieve the above-mentioned object, the inventors have conducted extensive research and produced and examined multiple anti-GFAP antibodies. As a result, they have discovered that by using a specific anti-GFAP antibody that binds to a region consisting of amino acids 111 to 115 in the amino acid sequence of GFAP, it is possible to detect with high accuracy by immunoassay at least the amount of GFAP contained in a sample, as in Alzheimer's disease, and have thus completed the present invention.

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

[0011] According to the present invention, it is possible to provide a detection method that can detect at least the amount of GFAP in a sample with high accuracy by immunoassay, a method that thereby assists in the diagnosis of Alzheimer's disease, and a kit for use therein.

[0012]

[0023] Figure 1 is a schematic diagram showing the position of each GFAP truncated fragment prepared in (1) of (Test Example 2) within the amino acid sequence of full-length GFAP. (a) shows the results of Western blotting in which the a.a. 1-230, a.a. 1-104, a.a. 72-214, and a.a. 116-214 fragments were reacted with an anti-His tag antibody (Anti-His), and (b) shows the results of Western blotting in which the a.a. 91-160, a.a. 96-160, a.a. 101-160, a.a. 106-160, a.a. 111-160, a.a. 121-170, a.a. 131-180, and a.a. a.a. 1-125, a.a. 1-126, a.a. 1-127, a.a. 1-128, a.a. 1-129, a.a. 1-130, a.a. 1-131, a.a. 1-132, a.a. 1-133, a.a. 1-134, a.a. 1-135, a.a. 1-136, a.a. 1-137, a.a. 1-138, a.a. 1-139, a.a. 1-200, a.a. 1-214, a.a. 1-215, a.a. 1-220, a.a. 1-225, a.a. 1-226, a.a. 1-127, a.a. 1-128, a.a. 1-129, a.a. 1-130, a.a. 1-131, a.a. 1-132, a.a. 1-133, a.a. 1-134, a.a. 1-226, a.a. 1-227, a.a. 1-228, a.a. 1-229, a.a. 1-135, a.a. 1-229, a.a. 1-230, a.a. 1-131, a.a. 1-132, a.a. 1-133, a.a. 1-134, a.a. 1-235, a.a. 1-236, a.a. 1-237, a.a. 1-238, a.a. 1-239, a.a. 1-240, a.a. (a) shows the results of Western blotting in which the 177-230 fragment was reacted with an anti-GST antibody (Anti-GST), and (b) shows the results of Western blotting in which the 177-230 fragment was reacted with GFAP-Ab1. a. a. 91-160, a. a. 96-160, a. a. 101-160, a. a. 106-160, a. a. 111-160, a. a. 121-170, a. a. 131-180, a. a. 141-190, a. a. 151-200, a. a. FIG. 1A shows the results of Western blotting in which the 161-214 fragment was reacted with an anti-GST antibody (Anti-GST), and FIG. 1B shows the results of Western blotting in which the 161-214 fragment was reacted with GFAP-Ab2.a.a.177-230, a.a.178-230, a.a.179-230, a.a.180-230, a.a.181-230, a.a.182-230, a.a.183-230, a.a.184-230, a.a.185-230, a.a.186-230, and a.a.1-125 fragments were reacted with (a) anti-GST antibody (Anti-GST), and (b) GFAP-Ab2. This figure shows the distribution of GFAP levels obtained by measuring GFAP in a cognitively normal group (CU) and an Alzheimer's disease patient group (AD).

[0013] The present invention will be described in detail below based on preferred embodiments thereof.

[0014] <Detection method> The detection method of the present invention is a method for detecting GFAP in a sample by immunoassay, and comprises a first step of forming a complex between a first antibody and GFAP, and a second step of forming a complex between a second antibody and GFAP, wherein the first antibody is an antibody that binds to a region consisting of amino acids 111 to 115 in the amino acid sequence of GFAP.

[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 of human GFAP (amino acid SEQ ID NO: 1) consisting of all 432 amino acids under accession number P14136. However, the "amino acid sequence of GFAP" according to the present invention is not limited thereto as long as it contains a region consisting of amino acids 111 to 115 (amino acid SEQ ID NO: 2) below (and preferably a region consisting of amino acids 191 to 200 (amino acid SEQ ID NO: 3) below).

[0016] Examples of the "amino acid sequence of GFAP" according to the present invention include an amino acid sequence that has 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 amino acid sequence shown in SEQ ID NO: 1, and that has a region consisting of the amino acids at positions 111 to 115 described below (and preferably a region consisting of the amino acids at positions 191 to 200 described below); and an amino acid sequence in which one or several 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 amino acid sequence shown in SEQ ID NO: 1, and that has a region consisting of the amino acids at positions 111 to 115 described below (and preferably a region consisting of the amino acids at positions 191 to 200 described below).

[0017] The first antibody according to the present invention described below binds to a region consisting of amino acids at positions 111 to 115 in the amino acid sequence of GFAP. The amino acid sequence of this region is shown in SEQ ID NO: 2. In this specification, amino acid positions indicated as "position 111" and "position 115", etc., indicate the number of amino acid residues from the N-terminus in the amino acid sequence of GFAP (typically, the amino acid sequence shown in SEQ ID NO: 1). In the present invention, the region referred to as "a region consisting of amino acids at positions X to Y in the amino acid sequence of GFAP (X, Y: numbers 1 to 432)" encompasses regions consisting of amino acids corresponding to this region.

[0018] In the present invention, an amino acid "corresponding to" a specific amino acid in an amino acid sequence refers to an amino acid that is aligned with the reference amino acid (i.e., for example, the amino acids at positions 111 to 115 of the amino acid sequence set forth in SEQ ID NO: 1) when amino acid sequences are aligned using amino acid sequence analysis software (e.g., GENETYX-MAC, Sequencher, etc.) or ClustalW, etc. (for example, parameters: default values ​​(i.e., initial settings)). Amino acids "corresponding to" a specific amino acid in an amino acid sequence may be amino acids having a chemically similar amino acid side chain (e.g., amino acids having a hydroxy group (serine, threonine), amino acids having an aromatic group (phenylalanine, tyrosine, tryptophan), etc.), but are preferably the same amino acid.

[0019] In the present invention, the test substance "GFAP" is sufficient as long as it has the region consisting of amino acids 111 to 115 of the amino acid sequence of GFAP (and preferably the region consisting of amino acids 191 to 200 described below), and may be the full-length amino acid sequence of GFAP, a fragment containing a portion of the sequence, a multimer containing the full-length and / or fragment, or a complex of these with other proteins, etc.

[0020] [Sample] The "sample" used in the detection method of the present invention is not particularly limited as long as it is a sample in which GFAP as the test substance can be present. For example, various organisms (including cultured cells) and extracts thereof; suspensions of specimens (body fluids such as serum, plasma, whole blood, cerebrospinal fluid, ascites, amniotic fluid, etc.; tissues) collected from humans and non-human animals can be used appropriately depending on the purpose. Examples of non-human animals include mammals such as chimpanzees, monkeys, cows, pigs, horses, sheep, mice, and rabbits. Among these, when detecting GFAP as a test substance to be used as a standard for diagnosing Alzheimer's disease in the medical field or clinical testing field, the sample according to the present invention is generally preferably a specimen collected from a subject (preferably a human) as a diagnostic target, such as serum, plasma, or brain tissue, with plasma or serum being more preferred.

[0021] The sample may be one that has been subjected to processing such as pulverization or freezing, one that has been appropriately diluted or suspended in a diluent, or one that has been appropriately pH-adjusted. Examples of the diluent include water, physiological saline, and known buffer solutions (sodium phosphate buffer, MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, Tricine buffer, Bicine buffer, glycine buffer, etc.), and the diluent may contain proteins such as BSA and serum, metal ions (Zn, ２＋ , Mg ２＋ The sample to be subjected to the method of the present invention is preferably an aqueous sample, and is preferably diluted or suspended as needed with the diluent.

[0022] Furthermore, the detection method of the present invention enables highly accurate detection of GFAP in a sample, regardless of the storage conditions of the sample. Samples to be subjected to immunoassays are typically stored frozen at −80 to −20°C, and preferably thawed only once, or, if not frozen, stored at 2 to 10°C for 6 hours or less. However, the storage conditions of samples to be subjected to the detection method of the present invention are not particularly limited, and may include, for example, samples that have been subjected to the freeze-thaw cycle two or more times, or samples that have been stored at 2 to 30°C for 0 to 48 hours. The detection method of the present invention enables highly accurate detection of GFAP even when the sample has been stored at 2 to 30°C for a long period of time (e.g., 12 hours or more, 24 hours or more, 24 to 48 hours, etc.).

[0023] [Antibody] In the detection method of the present invention, two types of antibodies, a first antibody and a second antibody, are used in combination. In the present invention, the term "antibody" includes not only complete antibodies but also functional fragments thereof. In the present invention, the term "functional fragment" refers to a portion (partial fragment) of a complete antibody that binds to the GFAP, and specific examples include Fab, F(ab')2, Fab', variable region fragment (Fv), disulfide-linked Fv, single-chain Fv (scFv), sc(Fv)2, diabodies, and polymers thereof.

[0024] As used herein, "Fab" refers to a monovalent antigen-binding fragment of an immunoglobulin consisting of one light chain and part of a heavy chain, and can be obtained, for example, by papain digestion of an antibody or by recombinant methods. "F(ab')2" refers to a divalent antigen-binding fragment of an immunoglobulin consisting of both light chains and part of both heavy chains, and can be obtained, for example, by pepsin digestion of an antibody or by recombinant methods. "Fab'" can be obtained, for example, by reduction of F(ab')2, and differs from Fab by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines in the antibody hinge region.

[0025] Furthermore, "variable region fragment (Fv)" refers to the smallest antibody fragment that has a complete antigen-recognition and binding site. Fv is a dimer in which the heavy chain variable region and the light chain variable region are tightly linked by non-covalent bonds. "Single-chain Fv (scFv)" contains the heavy chain variable region and the light chain variable region of an antibody, and these regions are present in a single polypeptide chain. "sc(Fv)2" is a single chain in which two heavy chain variable regions and two light chain variable regions are linked by a linker or the like. "Diabodies" are small antibody fragments that have two antigen-binding sites, and this fragment contains a heavy chain variable region linked to a light chain variable region in the same polypeptide chain, with each region pairing with a complementary region on another chain.

[0026] In the present invention, the term "antibody" includes all classes and subclasses of immunoglobulins, and also includes polyclonal and monoclonal antibodies. A "polyclonal antibody" refers to an antibody preparation containing different antibodies against different epitopes, and a "monoclonal antibody" refers to an antibody (including antibody fragments) obtained from a substantially homogeneous antibody population. The antibody according to the present invention is preferably a monoclonal antibody.

[0027] Furthermore, the antibody according to the present invention is not particularly limited in terms of origin, type, shape, etc. Specific examples include antibodies derived from humans, antibodies derived from non-human animals (e.g., rabbit antibodies, mouse antibodies, rat antibodies, and camel antibodies), chimeric antibodies, humanized antibodies, and functional fragments of these antibodies.

[0028] (First Antibody) The first antibody according to the present invention is an anti-GFAP antibody that binds to a region consisting of amino acids 111 to 115 of the amino acid sequence of GFAP. The amino acid sequence of the region consisting of amino acids 111 to 115 is shown in SEQ ID NO: 2. In the present invention, "binding to" a region consisting of an amino acid sequence means that the antibody binds to at least one of the amino acids contained in the region, and "not binding to" a region consisting of an amino acid sequence means that the antibody does not bind to any of the amino acids contained in the region. The binding of an antibody to the region consisting of amino acids 111 to 115 of the amino acid sequence of GFAP can be confirmed, for example, by the method shown in the Examples below, i.e., by detecting a band due to antibody binding to the region in Western blotting using a polypeptide fragment containing the region consisting of amino acids 111 to 115.

[0029] The first antibody according to the present invention is preferably any one of an antibody that does not bind to a region consisting of amino acids 1 to 104, an antibody that does not bind to a region consisting of amino acids 116 to 214, and an antibody that does not bind to a region consisting of amino acids 177 to 230 in the amino acid sequence of GFAP, and more preferably an antibody that does not bind to any of these regions.

[0030] (Second Antibody) The second antibody of the present invention may be any antibody capable of binding, preferably specifically binding, to GFAP as the test substance. Known anti-GFAP antibodies or commercially available anti-GFAP antibodies may be used as appropriate. Furthermore, it is preferable that the second antibody be a different antibody that binds to a different amino acid sequence than the first and second antibodies of the present invention, but this does not exclude the second antibody being the same antibody as the first antibody. Note that when the antibody contained in the capture body described below is the first antibody and the capture step described below is performed before or simultaneously with the labeling step, or when the antibody contained in the label described below is the first antibody and the labeling step described below is performed before or simultaneously with the capture step, the term "second antibody capable of binding to GFAP" encompasses an antibody capable of binding, preferably specifically binding, to a complex between GFAP and the first antibody. Examples of the manner of binding to the complex between GFAP and the first antibody include binding to the binding site between GFAP and the first antibody.

[0031] Among these, the second antibody according to the present invention is preferably an antibody that binds to a different region from that of the first antibody, and is more preferably an antibody that binds to a region consisting of amino acids at positions 191 to 200 in the amino acid sequence of GFAP. The amino acid sequence of the region consisting of amino acids at positions 191 to 200 is shown in SEQ ID NO: 3. Binding of an antibody to the region consisting of amino acids at positions 191 to 200 in the amino acid sequence of GFAP can be confirmed, for example, by the method shown in the Examples below, i.e., by detecting a band due to antibody binding to such region in Western blotting using a polypeptide fragment including the region consisting of amino acids at positions 191 to 200.

[0032] The second antibody according to the present invention is more preferably any one of an antibody that does not bind to a region consisting of amino acids 1 to 125, an antibody that does not bind to a region consisting of amino acids 111 to 115, and an antibody that does not bind to a region consisting of amino acids 91 to 190 in the amino acid sequence of GFAP, and even more preferably an antibody that does not bind to any of these regions.

[0033] The antibody of the present invention can be produced by a conventionally known method or a method similar thereto, for example, by the hybridoma method or recombinant DNA method. Representative examples of the hybridoma method include the Kohler and Milstein method (Kohler & Milstein, Nature, 256:495 (1975)) and methods similar thereto. Furthermore, methods commonly used for purifying polypeptides can be used to separate and purify the antibody.

[0034] The antibody-producing cells used in the cell fusion step in the hybridoma method are spleen cells, lymph node cells, peripheral blood leukocytes, etc. of animals (e.g., mice, rats, hamsters, rabbits, monkeys, goats) immunized with an antigen (GFAP, a peptide containing the region consisting of amino acids 111 to 115, or cells expressing the same, etc.). It is also possible to use antibody-producing cells obtained by reacting the antigen in a culture medium with the above-mentioned cells or lymphocytes, etc., previously isolated from an unimmunized animal. Various known cell lines can be used as the myeloma cells used in the cell fusion step. The antibody-producing cells and myeloma cells may be derived from different animal species, as long as they are fusible, but are preferably derived from the same animal species. Hybridomas are produced, for example, by cell fusion between spleen cells obtained from a mouse immunized with an antigen and mouse myeloma cells, and then, by screening, hybridomas producing monoclonal antibodies specific to the region consisting of amino acids 111 to 115 can be obtained. The monoclonal antibody can be obtained in a substantially pure and homogeneous form by culturing the hybridoma and isolating and purifying it from the hybridoma or the culture medium, or from the ascites of a mammal to which the hybridoma has been administered.

[0035] In the recombinant DNA method, for example, DNA cloned from the hybridoma is inserted into an appropriate vector, which is then introduced into host cells (e.g., mammalian cell lines such as HEK cells, E. coli, yeast cells, insect cells, plant cells, etc.) to produce the antibody of the present invention as a recombinant antibody (e.g., P. J. Delves, Antibody Production: Essential Techniques, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; Vandamme A. M. et al., Eur. J. Biochem. 192:767-775 (1990)). In this case, the host cells are cultured, and the antibody is separated and purified from within the host cells or from the culture medium, thereby obtaining the antibody in a substantially pure and homogeneous form.

[0036] [Immunoassay] The detection method of the present invention is to contact the test substance (GFAP) in a sample with a first antibody and a second antibody, and detect the GFAP in the sample by immunoassay based on the immune complex formed by antigen-antibody reaction.Immunoassay methods include, but are not limited to, labeled immunoassay using an antibody labeled with a labeling substance, EIA (enzyme immunoassay) using an enzyme as the labeling substance, ELISA and CLEIA (chemiluminescent enzyme immunoassay) which are one form of EIA, RIA (radioimmunoassay) using a radioisotope as the labeling substance, CLIA (chemiluminescent immunoassay) using a chemiluminescent compound as the labeling substance, immunochromatography, and immunoagglutination methods (latex agglutination, gold colloid agglutination, etc.) that measure by detecting agglutination.

[0037] More specifically, the detection method of the present invention comprises a first step of contacting a test substance (GFAP) in a sample with a first antibody and / or a second antibody to form a complex between the first antibody and GFAP, and a second step of forming a complex between the second antibody and GFAP. The first step and the second step may be performed in either order, or may be performed simultaneously, thereby forming complexes between the first antibody and GFAP and the second antibody.

[0038] As the detection method of the present invention, a sandwich method using a first antibody and a second antibody is preferred from the viewpoint that it tends to have higher sensitivity. In the sandwich method, the analyte (GFAP) is captured with a capture body containing an antibody and an insoluble carrier, and a label containing an antibody and a labeling substance is bound to the capture body. After B / F separation (washing), detection according to the type of the labeling substance is performed. Alternatively, as in the immunochromatography method, the labeling substance may be bound to the analyte (GFAP), and while B / F separation is performed, the analyte may be captured with a capture body, and detection according to the type of the labeling substance may be performed.

[0039] In this case, in the detection method of the present invention, either the first antibody or the second antibody may be used as the antibody for the capture body or the antibody for the label. That is, a preferred embodiment of the detection method of the present invention is a method in which: The first step is a step of capturing GFAP with a capture body comprising the first antibody and an insoluble carrier, and the second step is a step of labeling GFAP with a label comprising the second antibody and a labeling substance; or The first step is a step of labeling GFAP with a label comprising the first antibody and a labeling substance, and the second step is a step of capturing GFAP with a capture body comprising the second antibody and an insoluble carrier. Hereinafter, one embodiment of the detection method of the present invention will be described using such a sandwich method as an example, but the detection method of the present invention is not limited thereto.

[0040] (Capture body) The capture body of the present invention is a complex containing a first antibody or a second antibody and an insoluble carrier, and is a conjugate in which the first antibody or the second antibody is directly or indirectly bound to and supported on the insoluble carrier.

[0041] The "insoluble carrier" contained in the capture body of the present invention is water-insoluble and functions as a carrier that mainly supports and immobilizes the first antibody or the second antibody. In the present invention, "water-insoluble" means insoluble in water (solubility in water is 0.001 g / mL or less, preferably 0.0001 g / mL or less, the same applies hereinafter) at room temperature and normal pressure.

[0042] The material of such an insoluble carrier can be any material used as an insoluble carrier in known immunoassays, and is not particularly limited. For example, at least one selected from the group consisting of high molecular weight polymers (polystyrene, (meth)acrylic acid esters, 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.) can be mentioned. The material of the insoluble carrier can also be a composite of these materials, for example, an organic-inorganic composite composed of at least one organic polymer selected from the group consisting of high molecular weight polymers, gelatin, cellulose, and latex, and at least one metal compound selected from the group consisting of iron oxide (spinel ferrite, etc.), cobalt oxide, and nickel ferrite.

[0043] In the present invention, the shape of the insoluble carrier is not particularly limited, and may be, for example, a plate, a fiber, a membrane, a particle, etc., but from the viewpoint of reaction efficiency, particles are preferable, and from the viewpoint of automation and shortening the reaction time, magnetic particles are more preferable. As such an insoluble carrier, conventionally known carriers can be used as appropriate, and commercially available carriers can also be used as appropriate.

[0044] In the capture body of the present invention, the content of the first antibody or the second antibody is not particularly limited, but in order to further improve the detectability of the test substance, it is preferable to set the number of molecules of the first antibody or the second antibody bound to one molecule of the insoluble carrier so that it is as large as possible. For example, the mass of the first antibody or the second antibody per 100 parts by mass of the insoluble carrier is preferably 0.005 to 0.05 parts by mass, and more preferably 0.01 to 0.04 parts by mass.

[0045] The capture body according to the present invention can be produced by binding and immobilizing the first or second antibody to the insoluble carrier. As a production method, a conventionally known method or a method similar thereto can be appropriately adopted depending on the type of the insoluble carrier and the first or second antibody, and the first or second antibody may be bound directly or indirectly to the insoluble carrier.

[0046] Examples of the direct binding method include a method in which an active group (e.g., a thiol group, a maleimide group, or a succinimide group) is attached to the insoluble carrier and / or the antibody (the first antibody or the second antibody), or an insoluble carrier and / or an antibody having such an active group is used, and the insoluble carrier and / or the antibody are bound by a covalent bond via the active group. The insoluble carrier and the antibody to which the active group is attached may be a commercially available insoluble carrier or second antibody, or may be prepared by introducing the active group onto the surface of the insoluble carrier and / or the antibody under appropriate reaction conditions. Examples of the insoluble carrier and the first antibody or the second antibody to which the insoluble carrier and the antibody are indirectly bound include a method in which the insoluble carrier and the first antibody or the second antibody are bound via polyhistidine, polyethylene glycol, an oligopeptide, a linker molecule, or the like. Alternatively, one of the antibodies may be modified in some way and the other may be attached with a substance that captures the modified portion, and the two may be linked via these. For example, one antibody may be biotinylated and the other avidinylated, and a binding method based on avidin-biotin binding may be employed, or an indirect binding method using a secondary antibody, protein G, protein A, or the like may be employed. The ratio of the insoluble carrier to the first antibody or second antibody used in such a production method can be appropriately selected so as to achieve a preferred range for each content in the above-mentioned capture body. Furthermore, as such a capture body, for example, commercially available second antibodies, such as antibody-bound particles, may be used as appropriate.

[0047] (Labeled Body) The labeled body according to the present invention is a complex comprising a first antibody or a second antibody and a labeled body, and is a conjugate in which the first antibody or the second antibody is directly or indirectly bound to a labeled substance. The labeled body may further comprise a water-soluble carrier or the like that supports the first antibody or the second antibody and the labeled substance. When the antibody contained in the capture body is the first antibody, it is preferable that the antibody contained in the labeled body is the second antibody, and when the antibody contained in the capture body is the second antibody, it is preferable that the antibody contained in the labeled body is the first antibody.

[0048] The "labeling substance" contained in the labeled body of the present invention functions mainly as a label for detecting the test substance, and any substance used as a labeling substance in known immunoassays can be used without particular limitation.

[0049] Examples of labeling substances according to the present invention include enzymes; radioisotopes (isotopes such as iodine, tritium, and carbon); 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; dinitrophenyl phosphate (DNP); and digoxigenin (DIG). These may be used alone or in combination of two or more. Among these, enzymes are preferred as labeling substances according to the present invention. Examples of the enzyme include various enzymes conventionally used in enzyme immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, and luciferase. When an enzyme is used as the labeling substance, various detections can be performed depending on the substrate by using a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, etc. As such labeling substances and substrates, conventionally known substances can be used as appropriate, and commercially available substances can also be used as appropriate.

[0050] The labeled body according to the present invention includes not only an embodiment in which the labeled substance is bound to a first or second antibody to form a complex with GFAP, but also an embodiment in which the first or second antibody is not bound to a labeled substance but a complex with GFAP is formed, and then a secondary antibody, protein G, protein A, or the like bound to the labeled substance is bound to the first or second antibody. Here, the term "secondary antibody" refers to an antibody that exhibits reactivity with an antibody that directly binds to an antigen (the primary antibody, i.e., the first or second antibody). Similar embodiments also include an embodiment in which avidin or biotin is bound to the first or second antibody, a complex is formed between the first or second antibody and GFAP, and then a labeled substance bound to biotin or avidin is bound to the first or second antibody.

[0051] In the labeled form according to 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 adjusted as appropriate depending on the combination of these types, ease of binding to the test substance, and the like. For example, the ratio of the first antibody or the second antibody to 1 mole of the labeling substance is preferably 0.01 to 10,000 moles, and more preferably 0.05 to 10 moles.

[0052] The labeled substance of the present invention can be produced by conjugating the labeled substance with a first antibody or a second antibody. Such a production method can be appropriately selected from conventionally known methods or methods based thereon, depending on the types of the labeled substance and the first or second antibody. The labeled substance and the first or second antibody may be directly or indirectly conjugated to each other. Examples of such conjugation methods include those described above as methods for conjugating the insoluble carrier with the first or second antibody. The ratio of the labeled substance to the first or second antibody used in such a production method can be appropriately selected so as to achieve the preferred range of content in the labeled substance. Furthermore, commercially available second antibodies, such as enzyme-labeled antibodies, may be used as appropriate.

[0053] (Capture Step) When the detection method of the present invention is a sandwich method, the method includes a capture step, prior to the labeling step described below, in which the sample is contacted with the capture body, and if a analyte is present in the sample, the capture body captures the analyte via binding between the analyte and the first antibody or the second antibody, thereby forming a complex between the capture body and the analyte, i.e., a capture body-analyte complex (sometimes referred to as a "first complex" herein), or a capture step, following or simultaneously with the labeling step described below, in which the capture body is contacted with a second complex obtained in the labeling step described below, thereby forming a label-analyte-capture body complex (sometimes referred to as a "third complex" herein). It is more preferable that such a capture step be included prior to the labeling step described below, from the viewpoint of removing contaminants other than the analyte contained in the sample and further improving detection accuracy by performing a washing step multiple times.

[0054] The method for contacting the sample or the second complex with the capture body is not particularly limited, and any conventionally known method or a method based thereon can be used as appropriate. For example, if the insoluble carrier is a plate, the aqueous sample or the second complex can be injected into the plate, or if the insoluble carrier is particles, the aqueous sample or the second complex can be mixed with a particle solution containing the insoluble carrier. Examples of the dispersion medium for the particle solution include those listed as the diluent.

[0055] (Labeling Step) When the detection method of the present invention is a sandwich method, the method includes a labeling step prior to the capture step in which the sample is contacted with the label, and if a test substance is present in the sample, a complex between the label and the test substance (sometimes referred to herein as a "second complex"), i.e., a label-test substance complex, is formed via binding between the test substance and the first antibody or the second antibody; alternatively, the method includes a labeling step in which, after or simultaneously with the capture step, the first complex obtained in the capture step is contacted with the label, and a label-test substance-capture substance complex (third complex) is formed.

[0056] The method for contacting the sample or the first complex with the labeled entity is not particularly limited, and any conventionally known method or a method based thereon can be appropriately adopted, for example, a method in which a labeled body fluid is added to the aqueous sample or the first complex. Examples of the solvent for the labeled body fluid include those exemplified as the diluent.

[0057] In the reaction system between the capturer and the test substance in the capture step, and the reaction system between the label and the test substance in the labeling step, the contents (final concentrations) of the capturer and label in each reaction system are not particularly limited and are adjusted appropriately depending on the type and concentration of the sample, capturer, and label, etc. Furthermore, the conditions for each reaction system are also not particularly limited and can be adjusted appropriately. For example, the reaction can be carried out at room temperature to 45°C, preferably 20 to 37°C, at a pH of about 6 to 9, preferably 7 to 8, for about 5 seconds to 10 minutes, preferably 30 seconds to 8 minutes. In the present invention, the "reaction system" is preferably an aqueous system, and is preferably an aqueous solution containing the first antibody or second antibody for reacting them with GFAP.

[0058] However, in the detection method of the present invention, in the first step of forming a complex between the first antibody and GFAP, the pH of the reaction system (or the pH of the aqueous solvent for the first antibody (or a label or capture body containing the first antibody) (corresponding to the particle dilution solution in Test Examples 3 and 4 of the Examples below)) 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 first antibody is contained in the capture body, the pH of the capture step preferably satisfies the above-mentioned condition, and if the first antibody is contained in the label, the pH of the labeling step preferably satisfies the above-mentioned condition. In this case, it is more preferable that the pH of the second step of forming a complex between the second antibody and GFAP is also under the same condition.

[0059] The reaction system in which the first antibody or the second antibody is reacted with GFAP may further contain, in addition to GFAP, a label and / or a capturer, and an aqueous solvent therefor (such as a sample diluent, a dispersion medium for a particle liquid, or a solvent for a labeled body fluid), a pH buffer (such as Tris (tris(hydroxymethyl)aminomethane), MOPS (3-morpholinopropane-1-sulfonic acid)), a surfactant (such as N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, Tween-20, or Tween-80), salts (such as NaCl), sugars (such as sucrose), a chelating agent (such as EDTA-2Na), a protein (such as BSA), or other components (such as dextran or a dextran salt). Furthermore, the sample diluent, particle liquid, or labeled body fluid may also serve as a buffer for the reaction system containing these components.

[0060] (Washing step) When the detection method of the present invention is a sandwich method, it is preferable to further include a washing step for removing contaminants not captured by the capture body. When the capture step is included before the labeling step, it is more preferable to include a washing step between the capture step and the labeling step to remove contaminants not captured by the capture body, i.e., components other than the first complex. In this case, it is also more preferable to include a washing step after the labeling step to remove contaminants not captured by the capture body, i.e., components other than the third complex contained in the reaction system.

[0061] The method for removing the impurities is not particularly limited, and any conventionally known method or a method based thereon can be used as appropriate. For example, when the insoluble carrier is a plate, a method of removing the liquid phase (supernatant) from the plate can be used. When the insoluble carrier is a particle, a method of recovering the particles from the reaction buffer by centrifugation or magnetic collection and then removing the liquid phase (supernatant) can be used. Furthermore, after the washing step, injection and removal of a washing solution can be repeated as necessary. Examples of the washing solution include known neutral (preferably pH 6 to 9) buffers (such as sodium phosphate buffer, MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, tricine buffer, bicine buffer, and glycine buffer), and these may also contain stabilizing proteins such as BSA, surfactants, and the like.

[0062] (Detection step) In the detection method of the present invention, GFAP is detected by detecting a complex between the test substance (GFAP) and the first and second antibodies (the third complex in the sandwich method) (detection step). When the detection method of the present invention is the sandwich method, GFAP is detected by detecting a signal derived from the labeling substance of the label bound to GFAP. The "signal" may be, depending on the type of labeling substance, for example, color development (color development), reflected light, luminescence, fluorescence, radiation from a radioisotope, etc., and includes signals that can be confirmed with the naked eye as well as signals that can be confirmed by a detection method or device depending on the type of signal.

[0063] When an enzyme is used as the labeling substance, preferably, the capture step and washing step are performed before the labeling step, and a washing step is performed after the labeling step. After removing impurities not captured by the capturer, a chromogenic substrate, fluorescent substrate, chemiluminescent substrate, etc. depending on the type of enzyme is added as a substrate and reacted, thereby detecting a signal (fluorescence, luminescence, color (color development), etc.) depending on the substrate. Such substrates and reaction conditions can be appropriately adjusted depending on the type of enzyme, etc. In the detection method of the present invention, the value of the detected signal amount (counts) may be directly taken as a value corresponding to the amount of GFAP in the sample, or, if necessary, the amount of GFAP may be quantified by comparing it with the signal value in a standard sample with a known GFAP concentration.

[0064] <Method for assisting in the diagnosis of Alzheimer's disease> The present invention also provides a method for assisting in the diagnosis of Alzheimer's disease (sometimes referred to herein simply as a "diagnostic assisting method"), which comprises the step of detecting GFAP in a sample collected from a subject using the detection method of the present invention, and a method for diagnosing Alzheimer's disease (sometimes referred to herein simply as a "diagnostic method"), which comprises the step of detecting GFAP in a sample collected from a subject using the detection method of the present invention. The diagnostic assisting method of the present invention can also be described as a method for detecting GFAP for a physician's diagnosis of Alzheimer's disease, or a method for informing a physician of the presence or absence of detection. The detection method, including its preferred embodiments, is as described above. Since the detection method of the present invention can detect the amount of GFAP in a sample with at least high accuracy, it is suitable as a detection method used in the diagnosis of Alzheimer's disease, which has a lower concentration in a sample than brain injury. However, this does not exclude the use of the detection method of the present invention to assist in the diagnosis of brain injury (stroke, traumatic brain injury, etc.).

[0065] In the diagnostic auxiliary method and diagnostic method of the present invention, the subject is preferably a human, and the sample is preferably plasma or serum.

[0066] The diagnostic method of the present invention preferably further includes a step of diagnosing the subject as having or having a high probability of having Alzheimer's disease using the presence or absence of GFAP or the amount of GFAP as an indicator. In the diagnosis related to the diagnostic auxiliary method and diagnostic method of the present invention, if even a small amount of GFAP is detected in the sample by the detection method, the subject from whom the sample was derived may be determined, selected, or differentiated as having or having a high probability of having Alzheimer's disease. However, it is preferable to determine, select, or differentiate based on the amount of GFAP detected. For example, the amount of GFAP detected by the detection method may be compared with a predetermined cutoff value, and subjects with a value higher than the cutoff value may be determined to have or have a high probability of having Alzheimer's disease. The present invention can also provide a screening method that includes the detection step and a step of selecting subjects as having or having a high probability of having Alzheimer's disease using the presence or absence of GFAP or the amount of GFAP as an indicator.

[0067] The "cutoff value" is a predetermined value that serves as a standard for determining the amount of GFAP, and refers to a boundary value for determining whether a group is positive or negative. Such a cutoff value is not particularly limited, and is set appropriately depending on the purpose of diagnosis, the properties of the subject or sample, dilution conditions, etc. For example, by setting the cutoff value to a relatively low value, the detection sensitivity can be increased, i.e., subjects suspected of having Alzheimer's disease can be collected to a certain extent. On the other hand, by setting the cutoff value to a relatively high value, the detection accuracy can be increased.

[0068] According to the diagnostic assistance method and diagnostic method of the present invention, it is possible to provide information on patients who are affected by Alzheimer's disease or who are likely to be affected by Alzheimer's disease, distinguishing them from a group of healthy individuals with high accuracy, thereby enabling early therapeutic intervention, etc. Furthermore, patients who have been selected as having a high probability of being affected by Alzheimer's disease can also be subjected to further tests using electroencephalography, CT, MRI, PET / SPECT, etc. to detect atrophy of the cerebrum or hippocampus and deposition of senile plaques (amyloid plaques).

[0069] The present invention also provides a method for measuring GFAP and a method for assisting in the diagnosis of Alzheimer's disease by detecting the presence or amount of a region containing amino acids 115 to 191 of GFAP in a sample. Furthermore, the present invention provides a method for using the presence or amount of a region containing amino acids 115 to 191 of GFAP in a sample as an index for measuring GFAP and for diagnosing Alzheimer's disease.

[0070] <Kit> The present invention also provides a kit for use in the detection method, diagnostic auxiliary method, and diagnostic method of the present invention, comprising a first antibody and a second antibody that binds to a region of the amino acid sequence of GFAP that differs from that of the first antibody. These first and second antibodies are as described above, including preferred embodiments thereof. It is further preferred that these first and second antibodies are included in the kit of the present invention as the capture bodies or labels, and these capture bodies and labels are as described above, including preferred embodiments thereof. Furthermore, these antibodies may each independently be in the form of a solid (powder) or a liquid dissolved or suspended in a solution (such as the diluent, the dispersion medium of the particle liquid, or the solvent of the labeled body fluid).

[0071] The kit of the present invention may further include, for example, at least one selected from the group consisting of a standard sample (each concentration), a control sample, the diluent, the washing solution, a dispersion medium for the particle liquid, a solvent for the labeled body fluid, a buffer for the reaction system, the substrate, a buffer for the enzyme-substrate reaction, and a buffer for stopping the enzyme-substrate reaction. Furthermore, when the kit includes an antibody to which the labeled substance is not bound, it is also possible to combine a substance (e.g., a secondary antibody, protein G, protein A, etc.) that binds to the antibody with the labeled substance bound thereto. Furthermore, the kit of the present invention may further include instructions for use of the kit.

[0072] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples. In each of the following test examples, "%" indicates weight / volume (w / v) percentage (g / 100 mL) unless otherwise specified.

[0073] (Test Example 1) Obtaining anti-GFAP antibodies (1) Mouse immunization Seven to eight-week-old BALB / c mice and ICR mice (female) were intraperitoneally administered an equal volume emulsion of 50 μg / body of recombinant GFAP (LS Bio: recombinant protein containing human full-length GFAP) and Freund's complete adjuvant. Thereafter, every two weeks, an equal volume emulsion of 50 μg / body of recombinant GFAP and Freund's incomplete adjuvant was intraperitoneally administered 2 to 5 times until an increase in antibody titer was confirmed.

[0074] (2) Preparation of anti-GFAP antibody-producing hybridomas Mice in which a sufficient increase in antibody titer was confirmed were intraperitoneally administered with 50 μg / body of recombinant GFAP solution. Three to four days after administration, the mouse spleens were removed, and the removed splenocytes were fused by electroporation with myeloma cells P3U1, which had been previously cultured in RPMI 1640, to produce fusion cells. Seven to 14 days after fusion, the culture supernatant was added to a washed plate on which GFAP had been immobilized, and a primary reaction was carried out. After washing three times with PBS-T, an HRP-labeled anti-mouse immunoglobulin antibody (Dako) was added, and a secondary reaction was carried out. After washing three times with PBS-T, ABTS or TMB was added, and a color reaction was carried out. A 1.5 M oxalic acid solution or 0.5 M H ２ SO ４ The color reaction was stopped by adding 100 mg of 10 ...

[0075] (3) Purification of anti-GFAP antibodies. Each hybridoma was cultured in serum-free medium for 1 to 2 weeks, and the culture supernatant was collected. The collected culture supernatant was passed through a Protein A column to bind the antibody. After washing with wash solution 1 (20 mM phosphate buffer (PB), 2 M NaCl, 5% sucrose, pH 7.3) and wash solution 2 (50 mM acetate buffer, 5% sucrose, pH 5.0), the antibody was eluted with elution solution (50 mM acetate buffer, 5% sucrose, pH 3.8) and neutralized by adding neutralizing solution (1 M Tris-HCl, pH 8.5). The neutralized antibody solution was dialyzed overnight against a storage solution (PBS, 5% sucrose, pH 7.2) and concentrated using an ultrafiltration column to obtain purified anti-GFAP antibodies. Of the purified anti-GFAP antibodies obtained by the above methods, five types of anti-GFAP antibodies were used in the following test examples: GFAP-Ab1, GFAP-Ab2, GFAP-Ab3, GFAP-Ab4, and GFAP-Ab5.

[0076] (Test Example 2) Identification of Epitopes (1) Preparation of GFAP Cleavage Fragments The nucleotide sequence encoding human full-length GFAP was appropriately fragmented, and restriction enzyme sites were added to the termini of the nucleotide sequences encoding fragments consisting of the amino acid ranges shown in Figure 1 of the amino acid sequence of full-length GFAP (a.a. 1-432) to prepare GFAP cleavage fragment coding sequences. These GFAP cleavage fragment coding sequences were introduced into a GST expression vector or a His tag expression vector using the corresponding restriction enzymes to prepare GFAP cleavage fragment expression vectors fused to GST at the N-terminus or GFAP cleavage fragment expression vectors fused to a His tag at the C-terminus. Each of the prepared expression vectors was transfected into Escherichia coli DH5α, and the plasmids were amplified. The resulting plasmids were introduced into Escherichia coli BL21 (DE3) and cultured overnight with shaking in LB medium. The bacterial solution was then diluted 100-fold with LB medium and cultured with shaking for 3 to 6 hours. Next, expression was induced with 0.4 mM IPTG, and the cells were further cultured overnight with shaking. The cells were collected by centrifugation at 15,000 rpm at room temperature for 1 minute. PBS was added to the cells in an amount 1 / 10 of the amount of LB medium, and the suspension was sonicated for 15 minutes. The sonicated solution was centrifuged at 15,000 rpm at room temperature for 10 minutes, and the supernatant was collected and used as a solution of GFAP cleavage fragments (GST-fused GFAP cleavage fragments or His-tag-fused GFAP cleavage fragments).

[0077] (2) Identification of epitopes by Western blotting Half the volume of 2x SDS-PAGE sample buffer was added to each GFAP cleavage fragment solution prepared in (1) above, and the mixture was heat-denatured at 96°C for 5 minutes. 10 μL (50 μL culture equivalent) of the heat-denatured solution was added to an SDS-PAGE gel (Mini PROTEAN TGX Precast Gel, 4-20%, Bio Rad) and electrophoresed at a constant voltage of 200 V for 30 minutes. The gel after electrophoresis was transferred to a PVDF membrane using a Transblot Turbo transfer system (Bio Rad), and the PVDF membrane was blocked by immersing it in PBS containing 1% skim milk. After blocking, the PVDF membrane was immersed in 1% BSA-containing PBS containing 1 μg / mL of either an anti-GST antibody, an anti-His tag antibody, or the anti-GFAP antibody prepared in Test Example 1 above, and shaken to carry out the primary reaction. The PVDF membrane was then washed three times for 5 minutes with PBS-T, and then immersed in a POD-labeled anti-mouse antibody solution diluted 2000-fold with 1% BSA-containing PBS, and shaken to carry out the secondary reaction. The PVDF membrane was then washed three times for 5 minutes with PBS-T, and a luminescent substrate (ECL Select, GE Healthcare) was added to the PVDF membrane. After removing excess solution, luminescence was confirmed using an LAS500 (GE Healthcare).

[0078] Of the GFAP cleavage fragments (His tag fusion fragments) prepared in (1) above, the a.a.1-230, a.a.1-104, a.a.72-214, and a.a.116-214 fragments were reacted with an anti-His tag antibody (Anti-His) in a primary reaction, and the results are shown in Figure 2(a) and Figure 2(b) respectively, where the results are shown for the reaction of the anti-GFAP antibody GFAP-Ab1.

[0079] Furthermore, among the GFAP cleavage fragments (GST fusion fragments), the following fragments were reacted in a primary reaction with an anti-GST antibody (Anti-GST): a.a.91-160, a.a.96-160, a.a.101-160, a.a.106-160, a.a.111-160, a.a.121-170, a.a.131-180, a.a.141-190, a.a.151-200, and a.a.161-214. The results are shown in Figure 3(a) and Figure 3(b) , respectively.

[0080] Furthermore, among the GFAP cleavage fragments (GST fusion fragments), the a.a.1-125, a.a.1-126, a.a.1-127, a.a.1-128, a.a.1-129, a.a.1-130, a.a.1-131, a.a.1-132, a.a.1-133, a.a.1-134, and a.a.177-230 fragments were reacted with anti-GST antibody (Anti-GST) in a primary reaction, and the results are shown in Figure 4(a) and Figure 4(b) respectively, where the results are shown when GFAP-Ab1, one of the anti-GFAP antibodies, was reacted.

[0081] As shown in Figure 2, GFAP-Ab1 bound to the aa 72-214 fragment, but did not bind to the aa 1-104 fragment or the aa 116-214 fragment. Furthermore, as shown in Figures 3 and 4, GFAP-Ab1 bound to, for example, the aa 111-160 fragment, but did not bind to the aa 121-214 range. These results confirmed that GFAP-Ab1 binds at least within the aa 111-115 range.

[0082] Of the GFAP cleavage fragments (GST fusion fragments) prepared in (1) above, the a.a.91-160, a.a.96-160, a.a.101-160, a.a.106-160, a.a.111-160, a.a.121-170, a.a.131-180, a.a.141-190, a.a.151-200, and a.a.161-214 fragments were reacted with anti-GST antibody (Anti-GST) in a primary reaction, and the results are shown in Figure 5(a) and Figure 5(b) , respectively, when reacted with GFAP-Ab2, one of the anti-GFAP antibodies.

[0083] Furthermore, among the GFAP cleavage fragments (GST fusion fragments), the following fragments were reacted in a primary reaction with an anti-GST antibody (Anti-GST): a.a.177-230, a.a.178-230, a.a.179-230, a.a.180-230, a.a.181-230, a.a.182-230, a.a.183-230, a.a.184-230, a.a.185-230, a.a.186-230, and a.a.1-125. The results are shown in Figure 6(a) and Figure 6(b) , respectively.

[0084] As shown in Figures 5 and 6, GFAP-Ab2 bound to, for example, the aa 151-200 fragment, but did not bind to the aa 141-190 fragment or the aa 1-125 fragment. These results confirmed that GFAP-Ab2 binds at least within the aa 191-200 range. Similarly, GFAP-Ab3, GFAP-Ab4, and GFAP-Ab5 were all confirmed to bind within the aa 257-377 range.

[0085] Test Example 3 Measurement of GFAP by Sandwich Immunoassay 1 According to a standard method, an anti-GFAP antibody for capture was immobilized on magnetic particles to prepare an anti-GFAP antibody immobilized on magnetic particles, and this was added to a particle diluent (a solution (pH 7.2) containing 50 mM MOPS, 150 mM NaCl, 1 mM EDTA-2Na, 2% BSA, 2% sucrose, and 0.05% dextran sodium sulfate) to a concentration of 0.025%, to prepare a capture solution. Furthermore, according to standard methods, the anti-GFAP antibody for labeling was digested with pepsin and reduced to prepare Fab', which was then labeled with alkaline phosphatase to prepare a labeled anti-GFAP antibody. This was then diluted with label diluent (a solution (pH 7.2) containing 50 mM MOPS, 150 mM NaCl, 2% BSA, 2% sucrose, 1.5% Pluronic F-108, 0.1% Tween-80, and 1.0% PVP) to a concentration of 0.5 μg / mL to prepare a labeled body fluid. The anti-GFAP antibody for capture and the anti-GFAP antibody for labeling were the anti-GFAP antibodies prepared in the above (Test Example 1) in the combinations shown in the "Capture body" and "Labeled body" columns in Table 1 below.

[0086] GFAP was measured using a Lumipulse G1200 (Fujirebio). First, 100 μL of sample was mixed with 20 μL of immune reaction solution (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, 1.0% Tergitol™ 15-s-9 (Thermo Fisher) solution (pH 7.2)). This was then mixed with 150 μL of capture fluid and reacted at 37°C for 10 minutes (capture step). After the reaction, the magnetic beads were collected and washed with Lumipulse washing solution (Fujirebio) (washing step). Next, 150 μL of labeled body fluid was added, and the mixture was allowed to react for 10 minutes at 37°C (labeling step). The magnetic beads were then collected and washed with Lumipulse washing solution (washing step), after which 50 μL 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 for 5 minutes at 37°C. The amount of light emitted by the decomposition of AMPPD by the catalytic action of alkaline phosphatase captured on the magnetic beads, with a maximum absorption at a wavelength of 463 nm, was measured. The measurement results were output as the luminescence intensity (counts) of the substrate (AMPPD). The specimens used were diluted samples (GFAP concentration: 0-1000 [pg / mL]) prepared by diluting GFAP (including human full-length GFAP; the same applies hereinafter) with human plasma, and plasma specimens from Alzheimer's disease patients (Samples A-C). Two samples per combination were measured, and the average values ​​were calculated. For the diluted samples, the ratio of count values ​​at GFAP concentrations of 0 pg / mL to 10 pg / mL (10 / 0) ​​and the ratio of count values ​​at GFAP concentrations of 10 pg / mL to 1000 pg / mL (1000 / 10) were also calculated. The results are shown in Table 1 below.

[0087]

[0088] As shown in Table 1, when GFAP-Ab1 (Ab1, Example) was used as the capture antibody (Capture body) and GFAP-Ab2 (Ab2) was used as the labeled antibody (Labeled body), the count value was sufficiently high compared to other antibody combinations. Furthermore, the ratio of the count values ​​between each concentration of GFAP (10 / 0, 1000 / 10) was significantly higher compared to other antibody combinations, and the count value increased correctly depending on the GFAP concentration, confirming that the detection accuracy was remarkably high even at low GFAP concentrations.

[0089] (Test Example 4) Measurement of GFAP by Sandwich Immunoassay 2 (1) Anti-GFAP antibodies were obtained in the same manner as in Test Example 1 above, and the epitope was identified in the same manner as in Test Example 2 above. Among them, an antibody that was confirmed to bind within the aa 111-115 range, like GFAP-Ab1, but not to the aa 116-214 fragment, was designated GFAP-Ab6 and used for measuring GFAP.

[0090] (2) GFAP measurement was performed in the same manner as described above (Test Example 3), except that the anti-GFAP antibody for capture (Capture body) was GFAP-Ab6 and the anti-GFAP antibody for labeling (Labeled body) was GFAP-Ab2. Three samples were measured for each combination, and the average values ​​were calculated. For the results of the diluted samples, the ratio of the count values ​​when the GFAP concentration was 0 pg / mL to that when it was 10 pg / mL (10 / 0) ​​and the ratio of the count values ​​when the GFAP concentration was 10 pg / mL to that when it was 1000 pg / mL (1000 / 10) were also calculated. The results are shown in Table 2 below.

[0091]

[0092] As shown in Table 2, when GFAP-Ab6 (Ab6, Example) was used as the capture antibody (Capture body) and GFAP-Ab2 (Ab2) was used as the labeling antibody (Labeled body), both the count value and the count value ratio (10 / 0, 1000 / 10) were high, as in the case when GFAP-Ab1 was used as the capture antibody (Capture body) and GFAP-Ab2 was used as the labeling antibody (Labeled body) (Table 1), confirming that these antibodies also enable detection of low concentrations of GFAP with significantly high accuracy.

[0093] Test Example 5: Investigation of Measurement Conditions GFAP in each sample was measured in the same manner as in Test Example 3 above, except that particle diluents 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-ammonio-1-propanesulfonate (pH 6.5)) and particle diluent B (a solution containing 50 mM Tris, 150 mM NaCl, 1 mM EDTA-2Na, 2% BSA, 0.05% Tween-20, and 0.05% dextran sodium sulfate (pH 7.2)) with two different pH values ​​were used as particle diluents. Measurement results were output as the luminescence intensity (counts) of the substrate. For each combination, two samples were measured and the average values ​​were calculated. For the diluted samples, the ratio of the count values ​​when the GFAP concentration was 0 pg / mL to 10 pg / mL (10 / 0) ​​and the ratio of the count values ​​when the GFAP concentration was 10 pg / mL to 1000 pg / mL (1000 / 10) were also calculated. The results are shown in Table 3 below, along with the combinations of anti-GFAP antibodies for capture (Capture body) and anti-GFAP antibodies for label (Labeled body) and the pH of the particle dilution solution (Dilution pH).

[0094]

[0095] As shown in Table 3, when particle dilutions of any pH (Dilution pH) were used, sufficiently high count values ​​and highly accurate results were obtained. However, it was confirmed that when particle dilution A with a pH of 6.5 was used as the particle dilution (pH of the reaction system in the capture step: 6.5), the count values ​​tended to be higher and the accuracy higher (the ratio of count values ​​was larger) than when particle dilution B with a pH of 7.2 was used (pH of the reaction system in the capture step: 7.2).

[0096] (Test Example 6) Measurement of GFAP Using Multiple Alzheimer's Disease Patient Samples GFAP was measured in the same manner as in Test Example 3 above, except that plasma samples from 17 cognitively normal individuals and plasma samples from 16 Alzheimer's disease patients were used as samples. GFAP-Ab1 was used as the anti-GFAP antibody for capture, and GFAP-Ab2 was used as the anti-GFAP antibody for labeling. Diluted samples with known GFAP concentrations were also measured in the same manner to create a calibration curve, from which the amount of GFAP (pg / mL) in each sample was calculated. The distribution of GFAP levels in the cognitively normal group (CU) and the Alzheimer's disease patient group (AD) is shown in Figure 7.

[0097] 7, a Wilcoxon test revealed a significant difference in the GFAP levels between the cognitively normal group (CU) and the Alzheimer's disease group (AD) (p<0.0001). Therefore, it was confirmed that the cognitively normal group (CU) and the Alzheimer's disease group (AD) can be distinguished with high accuracy by detecting GFAP in a sample using the detection method of the present invention using GFAP-Ab1.

[0098] (Test Example 7) Measurement of GFAP Using Preserved Samples Samples were prepared by diluting GFAP with human plasma (Diluted sample, GFAP concentration: 0-1000 [pg / mL]), and by adding GFAP to human plasma and storing it under one of the following storage conditions: frozen at -80°C (control), frozen and thawed twice at -80°C (FT 2 times), left at 4°C for 1 day (4°C, 1 day), or left at room temperature (approximately 25°C) for 1 day (rt, 1 day). The anti-GFAP antibodies used as capture and label were the anti-GFAP antibodies prepared in Test Example 1 above, in the combinations shown in the "Capture body" and "Labeled body" columns in Table 4 below.

[0099] A 2 μg / mL anti-GFAP antibody for capture diluted with PBS was added to an assay plate and immobilized. Blocking was then performed by adding a Tris buffer containing 1% BSA and 1% casein. The anti-GFAP antibody for labeling was labeled with biotin to produce a biotin-labeled anti-GFAP antibody. After blocking, the assay plate was washed three times with PBS-T, and each sample mixed with a Tris buffer containing 1% BSA and 1% casein was added to the plate for a primary reaction. The plate was then washed three times with PBS-T, and a biotin-labeled anti-GFAP antibody diluted with a Tris buffer containing 1% BSA and 1% casein was added to the assay plate for a secondary reaction. The plate was then washed three times with PBS-T, and an alkaline phosphatase-labeled streptavidin solution diluted with a Tris buffer containing 1% BSA and 1% casein was added to the plate for a tertiary reaction. After washing three times with PBS-T, AMPPD was added and reacted, and the amount of light emitted with maximum absorption at a wavelength of 463 nm was measured. The measurement results were output as the luminescence intensity (counts) of the substrate (AMPPD). The results are shown in Table 4 below.

[0100]

[0101] As shown in Table 4, by using GFAP-Ab1 (Ab1, Example), even with each antibody combination in the same table, the count value was sufficiently high, and the count value increased correctly depending on the GFAP concentration, confirming that highly accurate detection was possible. Furthermore, regardless of which antibody was combined with GFAP-Ab1, the count value was sufficiently high, equivalent to that of a sample frozen at -80°C (control), for samples stored under conditions such as two cycles of freezing and thawing 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 (rt, 1 day). However, when GFAP-Ab1 (Ab1) was combined with GFAP-Ab2 (Ab2), the decrease in count value relative to the control was smaller than when GFAP-Ab3 (Ab3) was combined with GFAP-Ab1, confirming that the combination tends to be less affected by the storage conditions of the sample.

[0102] As described above, the present invention makes it possible to provide a detection method that can detect at least the amount of GFAP in a sample with high accuracy by immunoassay, a method that thereby assists in the diagnosis of Alzheimer's disease, and a kit for use therein.

Claims

1. A method for detecting GFAP in a sample by immunoassay, comprising a first step of forming a complex between a first antibody and GFAP, and a second step of forming a complex between a second antibody and GFAP, wherein the first antibody is an antibody that binds to a region consisting of 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 a region consisting of amino acids 191 to 200 in the amino acid sequence of GFAP.

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

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 a step of capturing GFAP with a capture body comprising a first antibody and an insoluble carrier, and the second step is a step of labeling GFAP with a label comprising a second antibody and a labeling substance, or the first step is a step of labeling GFAP with a label comprising a first antibody and a labeling substance, and the second step is a step of capturing GFAP with a capture body comprising a second antibody and an insoluble carrier.

6. A method for assisting in the diagnosis of Alzheimer's disease, comprising a step of detecting GFAP in a sample collected from a subject using the detection method described in any one of claims 1 to 5.

7. A kit for use in a method for detecting GFAP in a sample by immunoassay, comprising: a first antibody that binds to a region consisting of amino acids 111 to 115 in the amino acid sequence of GFAP; and a second antibody that binds to a region of 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 a region consisting of amino acids 191 to 200 in the amino acid sequence of GFAP.

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