Allergy antigens and their epitopes

Abstract

This invention provides novel allergy antigens that are proteins. It also provides polypeptide antigens containing epitopes. [Solution] A novel antigen of a protein to which IgE antibodies in the serum of patients with wheat allergy specifically bind. Furthermore, since the epitope has a relatively short amino acid sequence, if the same amino acid sequence exists in different allergen components, the IgE antibody can bind to multiple allergen components. As a result of the existence of a common epitope in different allergen components, the IgE antibodies of allergic patients bind to both, and the antigen exhibits cross-reactivity.

Description

Technical Field

[0001] The present invention relates to a novel antigen for allergies to wheat and the like. The present invention also relates to a diagnostic kit, a diagnostic composition, and a diagnostic method for allergies to wheat and the like. The present invention also relates to a composition containing the antigen, and a raw material or processed product in which the antigen is removed or reduced. The present invention further relates to a tester composition for determining the presence or absence of wheat antigen in a target object.

[0002] The present invention also relates to an antigen of a polypeptide containing an epitope. The present invention also relates to a diagnostic kit, a diagnostic composition, and a diagnostic method for allergies containing the polypeptide. The present invention also relates to a method for providing an index for diagnosing an allergy of a subject. The present invention also relates to a composition containing the polypeptide, and a raw material or processed product in which the polypeptide is removed or reduced. The present invention further relates to a method for producing a raw material or processed product in which the polypeptide is removed or reduced. The present invention further also relates to a tester composition for determining the presence or absence of an antigen containing the polypeptide in a target object.

Background Art

[0003] In the sera and tissues of allergic patients, IgE antibodies specific to a specific antigen (hereinafter also referred to as an allergen) are produced. An allergic reaction is induced by the physiological result caused by the interaction between this IgE antibody and the specific antigen. An antigen generally refers to foods, foodstuffs, etc. that cause allergic symptoms, and specifically refers to a protein (hereinafter also referred to as an allergen component) contained in foods, foodstuffs, etc. to which specific IgE antibodies bind.

[0004] Conventional allergy test reagents often simply involve grinding up candidate allergen foods and ingredients to create antigen reagents (Patent Document 1). As a result, a positive reaction in allergy testing can only be detected if the amount of a particular allergen component in the antigen reagent exceeds a threshold that allows for a positive reaction to be determined by binding to IgE antibodies. Consequently, the diagnostic efficiency is not sufficiently high.

[0005] Several allergen components have been suggested in candidate foods and ingredients, and these have been commercialized as test kits. To improve the reliability of allergy testing, it is necessary to comprehensively identify allergen components, but the patient detection rate based on the measurement of these allergen components is still insufficient. Identifying novel wheat allergens is crucial not only for improving the accuracy of diagnostic agents but also for targeting low-allergen foods, ingredients, and therapeutic drugs.

[0006] On the other hand, regarding the separation and purification of proteins, in recent years, two-dimensional electrophoresis has been used as a method for separating and purifying diverse proteins from small amounts of sample. This method involves isoelectric focusing in the first dimension and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) in the second dimension. The applicants have developed two-dimensional electrophoresis methods with high separation capabilities (Patent Documents 2-5).

[0007] Allergen-specific IgE antibodies recognize and bind to epitopes, which are specific amino acid sequences within allergen components. However, while there are a few examples of allergen components being analyzed down to the epitope level (Non-Patent Literature 1), such cases are extremely rare. Furthermore, there are currently no allergy diagnostic kits on the market that utilize polypeptides containing epitopes. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2002-286716 [Patent Document 2] Japanese Patent Publication No. 2011-33544 [Patent Document 3] Japanese Patent Publication No. 2011-33546 [Patent Document 4] Japanese Patent Publication No. 2011-33547 [Patent Document 5] Japanese Patent Publication No. 2011-33548 [Non-patent literature]

[0009] [Non-Patent Document 1] Matsuo, H., et al., J. Biol. Chem., (2004), Vol.279, No.13, pp.12135-12140 [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] This invention provides novel allergy antigens that are proteins. This invention also provides a diagnostic method and a diagnostic kit for allergies containing the antigen. Furthermore, this invention provides compositions containing the antigen, and raw materials or processed products from which the antigen has been removed or reduced. This invention further provides a tester composition for determining the presence or absence of the antigen in a target object.

[0011] The present invention also provides antigens of polypeptides containing epitopes. The present invention also provides allergy diagnostic kits, diagnostic compositions, and diagnostic methods containing said polypeptides. The present invention also provides methods for providing indicators for diagnosing allergies in question. The present invention also provides compositions containing said polypeptides, and raw materials or processed products from which the antigen containing said polypeptides has been removed or reduced. The present invention further relates to a method for producing raw materials or processed products from which the antigen has been removed or reduced. The present invention further provides tester compositions for determining the presence or absence of the antigen containing said polypeptides in a subject. [Means for solving the problem]

[0012] To solve the above problems, the inventors diligently conducted research to identify the causative antigen of wheat allergy. As a result, they succeeded in identifying a novel antigen of a protein to which IgE antibodies in the serum of patients with wheat allergy specifically bind.

[0013] Furthermore, since epitopes have relatively short amino acid sequences, if the same amino acid sequence exists in different allergen components, the IgE antibody can bind to multiple allergen components. As a result of the existence of common epitopes in different allergen components, the IgE antibodies of allergy patients bind to both, and the antigens exhibit cross-reactivity. Therefore, the epitopes identified by this invention enable the diagnosis and treatment of allergies, including cross-reactivity, and the detection of multiple allergen components containing the epitope.

[0014] In this specification, unless otherwise specified, “antigen” is used to mean both a protein-based antigen in the narrow sense and a protein-derived “epitope.” Where specified, “antigen” is used to mean either a protein or a protein-derived epitope.

[0015] Based on the above findings, the present invention was completed. The present invention includes, but is not limited to, the following embodiments: [Embodiment 1] An allergy diagnostic kit comprising at least one of the following polypeptides (E1)-(E50).

[0016] (E1) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 29-130 and 2880; (E2) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 131-137 and 139-190; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 191-280 (excluding 227, 259, 269 and 273) and 2881; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 281-341, 343-345; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 346-398, 400-413 and 2882; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 414-492, 2883 and 2884; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 493-561; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 562-639; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 640-705 and 2885-2888; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 706-775 and 2889-2891; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 776-898 and 2892; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 899-973, 2893 and 2895; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 974-1009; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 1010-1088 and 2896-2899; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 1089-1141; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1142 - 1165 and 2900; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1166 - 1208; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1209 - 1256; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1257 - 1296; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1297 - 1366; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1367 - 1402; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1403 - 1507; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1508 - 1565 and 2901; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1566 - 1597; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1598 - 1607; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1608 - 1684, 2902 and 2903; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1685 - 1752, 2904 and 2905; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1753 - 1788, 2906 and 2907; (E29) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1789-1835, 2908, and 2909; (E30) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1836-1894, 2910, and 2911; (E31) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1895-1976, 2912, and 2913; (E32) Polypeptides containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1977-2003; (E33) Polypeptide containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2004-2026; (E34) Polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2027-2067 and 2914; (E35) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2068-2153, 2915, and 2916; (E36) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2154-2216; (E37) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2217-2264 and 2917-2921; (E38) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2265-2308 and 2922-2925; (E39) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2309-2341; (E40) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2342-2418; (E41) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2419-2490; (E42) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2491-2544, 2926, and 2927; (E43) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2545-2612; (E44) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2613-2630; (E45) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2631-2673; (E46) Polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2674-2700 and 2928; (E47) Polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2701-2773 and 2929; (E48) Polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2774-2808, 2930, and 2931; (E49) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2809-2829; or (E50) Polypeptide containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2830-2879 and 2932 [Aspect 2] A diagnostic composition for allergies, comprising at least one polypeptide specified as (E1) to (E50) in Embodiment 1. [Aspect 3] A method for providing indicators for diagnosing a target allergy, comprising the following steps: (i) The sample obtained from the subject is brought into contact with the antigen, where the sample is a solution containing IgE antibodies; (ii) Detect the binding of IgE antibodies to the antigen in the sample obtained from the subject; (iii) If binding of the target IgE antibody to the antigen is detected, it provides an indicator that the subject is allergic; The method comprising, wherein the antigen is at least one of the polypeptides identified as (E1) to (E50) in Embodiment 1. [Aspect 4] The antigen is at least one of the polypeptides identified as (E1) to (E50) in Embodiment 1, and is the cause of allergies. [Aspect 5] A composition comprising at least one of the antigens described in Embodiment 4. [Aspect 6] A composition according to embodiment 5 for treating allergies. [Aspect 7] A tester composition for determining the presence or absence of an antigen in a target, comprising an antibody that binds to at least one of the polypeptides specified as (E1) to (E50) in Embodiment 1. [Aspect 8] A tester composition for determining the presence or absence of an antigen in a target object, comprising at least one primer containing a portion of the base sequence of a nucleic acid encoding a polypeptide specified as (E1) to (E50) in Embodiment 1 and / or a portion of its complementary strand. [Aspect 9] A tester composition for determining the presence or absence of IgE antibodies in a subject, comprising polypeptides identified as (E1) to (E50) in Embodiment 1. [Aspect 10] A method for determining the presence or absence of polypeptides specified as (E1) to (E50) in a raw material or processed product in Embodiment 1, comprising detecting the polypeptides specified as (E1) to (E50) in Embodiment 1 in the raw material or processed product. [Aspect 11] A raw material or processed product in which an antigen has been removed or reduced, wherein the antigen is at least one of the polypeptides specified as (E1) to (E50) in Embodiment 1. [Aspect 12] A method for producing a raw material or processed product from which an antigen has been removed or reduced, comprising a step of confirming that the antigen has been removed or reduced during the manufacturing process of the processed product, wherein the antigen is at least one of the polypeptides specified as (E1) to (E50) in Embodiment 1. [Effects of the Invention]

[0017] The present invention can provide novel antigens for allergies (e.g., allergies to wheat). Since novel allergen components that cause allergies have been identified in the present invention, it is possible to provide a highly sensitive diagnostic method and kit for allergies, a composition containing the antigen, a raw material or processed product from which the antigen has been removed or reduced, and a tester composition for determining the presence or absence of the antigen in a target object.

[0018] Furthermore, the present invention can provide a novel polypeptide antigen containing a protein antigen epitope. By utilizing the polypeptide of the present invention, it is possible to provide a highly sensitive diagnostic kit for allergies (e.g., allergy to wheat), a diagnostic composition, and a diagnostic method, a composition containing the polypeptide, a tester composition for determining the presence or absence of an antigen containing the polypeptide in a target object, and a raw material or processed product from which the polypeptide has been removed or reduced, and a method for producing the raw material or processed product. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 is a photograph of a gel showing the electrophoretic patterns of proteins in wheat, obtained by two-dimensional electrophoresis. The band on the left side of the photograph represents the molecular weight marker band, and the numbers on the left side of the photograph represent the molecular weight (KDa) of each molecular weight marker. The numbers at the top of the photograph represent the isoelectric point. [Figure 2]Figure 2 shows an immunoblot image of the two-dimensional electrophoresis pattern of proteins contained in wheat, using serum from wheat allergy patients. Spots 1-16, where IgE antibodies in the serum of wheat allergy patients specifically reacted compared to healthy individuals, are indicated by white frames. In wheat allergy patients, WDEIA cases are represented as P2-36, while children with typical immediate-type wheat allergy cases that do not require exercise to trigger the onset are represented as P50-57, and adults as P1-9. [Figure 3] Figure 3 shows the results of cross-reactivity analysis using ELISA on serum from patients with concomitant wheat allergies, for peptides possessing the amino acid sequences of each epitope. [Figure 4] Figure 4 shows the results of cross-reactivity analysis using ELISA on serum from patients with concomitant wheat allergies, for peptides possessing the amino acid sequences of each epitope. [Figure 5] Figure 5 shows the results of cross-reactivity analysis using ELISA on serum from patients with concomitant wheat allergies, for peptides possessing the amino acid sequences of each epitope. [Figure 6] Figure 6 shows the results of cross-reactivity analysis using ELISA on serum from patients with concomitant wheat allergies, for peptides possessing the amino acid sequences of each epitope. [Modes for carrying out the invention]

[0020] The present invention will be described in detail below, but the present invention is not limited to these descriptions.

[0021] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings that are generally understood by those skilled in the art.

[0022] In this specification, allergy refers to a condition in which a body exhibits an adverse hypersensitivity reaction when a body sensitized to a certain antigen is re-introduced to that antigen. Allergic reactions can occur upon contact with an antigen or upon ingestion of that antigen. Here, contact refers to touching an object, and in particular to the human body, it refers to adhesion to the skin, mucous membranes (eyes, lips, etc.), etc. Ingestion refers to taking something into the body, and includes inhalation, oral ingestion, etc. Generally, allergic reactions that occur when food is ingested are specifically called food allergies. In a preferred embodiment, the allergy may be a food allergy. In many food allergic diseases, antigen-specific IgE antibodies are produced in the blood and tissues. IgE antibodies bind to mast cells or basophils. When an antigen specific to the IgE antibodies re-enters the body of a patient with an allergic disease, the antigen combines with the IgE antibodies bound to mast cells or basophils, producing the physiological effect of IgE antibody-antigen interaction. These physiological effects include the release of histamine, serotonin, heparin, eosinophil chemotactic factors, or various leukotrienes. These released substances trigger allergic reactions caused by combinations of IgE antibodies and specific antigens. Specifically, IgE antibodies recognize and bind to epitopes, which are specific amino acid sequences in specific antigens, and the allergic reaction to that antigen manifests through the above pathway.

[0023] The allergies covered by this invention are not particularly limited, as long as they are allergies to allergens (antigens) containing the epitope used. In one embodiment, allergens include grains, seafood, fruits, vegetables, nuts (seeds and seeds), edible grasses, meat, milk, dairy products, etc., which are ingested by living organisms (especially humans), or parasites that infest living organisms (especially humans).

[0024] Grains are a general term for food obtained from plants, consisting mainly of starchy seeds used for food. In a narrow sense, grains refer only to the seeds of grasses (Poaceae), while in a broader sense, they include the seeds of legumes (Fabaceae) and seeds of crops from other families. Among grains in the broader sense, the seeds of dicotyledonous plants that are used as grains because they resemble the seeds of cereals (seeds of monocotyledonous grasses) are collectively called pseudocereals. Pseudocereals include buckwheat (Polygonaceae), amaranth (Amaranthaceae), and quinoa (Chenopodiaceae).

[0025] In the present invention, non-limitingly, grass grains include bread wheat (Triticum aestivum), barley, rye, wild oats, orchard grass, timothy grass, sweet vernal grass, millet, foxtail millet, barnyard millet, corn, and Job's tears. Polygonaceae grains include, for example, buckwheat (Fagopyrum esculentum) of the genus Fagopyrum in the Polygonaceae family. In one embodiment, the antigen (allergen) is derived from a grain. In one embodiment, the allergen is derived from a grass grain. In one embodiment, the allergen is derived from wheat.

[0026] In a non-limiting sense, seafood includes shrimp, crabs, and other creatures belonging to the order Decapoda (shrimp order). Most creatures generally recognized as "crustaceans" belong to the order Decapoda (shrimp order). In a non-limiting sense, seafood also includes squid and octopuses belonging to the orders Teuthida and Octopoda. Seafood further includes fish belonging to the families Scombridae and Gadidae. Seafood also includes mollusks of the family Veneridae.

[0027] In a non-limiting sense, fruits include, for example, fruits belonging to the Actinidiaceae, Bromeliaceae, Anacardiaceae, Cucurbitaceae, Musaceae, Rutaceae, and Rosaceae families. Vegetables include, for example, fruits belonging to the Solanaceae, Cucurbitaceae, and Lauraceae families. Nuts include, for example, nuts belonging to the Anacardiaceae, Rosaceae, and Juglandaceae families. Edible grasses include, for example, edible grasses belonging to the Asteraceae family. Grains include, for example, grains belonging to the Poaceae and Polygonaceae families.

[0028] The type of meat is not particularly limited. In one embodiment, it includes the meat of birds (chicken, duck, etc.), pork, beef, lamb, etc. In one embodiment, it is the meat of birds. In one embodiment, it is the meat of chickens (Gallus gallus).

[0029] The origin of milk is not particularly limited. In one embodiment, it includes milk from cows, goats, sheep, etc. In one embodiment, it is cow's milk (Bos Taurus). Dairy products are processed products of milk. Non-limited, this includes butter, cream, cheese, yogurt, and ice cream.

[0030] The term "parasite" is not limited to, for example, a parasite of the family Anisakidae. Parasites of the family Anisakidae include Anisakis simplex.

[0031] In this specification, allergy refers to a condition in which an allergic reaction occurs in response to an antigen, such as a protein contained in a raw material or processed product (e.g., wheat or wheat products). Allergies can occur when an allergic reaction occurs upon contact with an antigen contained in a raw material or processed product (e.g., wheat or wheat products) or upon ingestion of such antigen. Generally, an allergic reaction that occurs when food is ingested is specifically called a food allergy. An allergy to wheat may be a food allergy.

[0032] In this specification, an antigen is a substance that triggers an allergic reaction. If it is a protein contained in raw materials such as food ingredients, it is also referred to as an allergen component. Antigens are preferably proteins.

[0033] In this specification, a protein is a molecule having a structure in which natural amino acids are linked by peptide bonds. The number of amino acids contained in a protein is not particularly limited. In this specification, the term "polypeptide" also means a molecule having a structure in which natural amino acids are linked by peptide bonds. The number of amino acids contained in a polypeptide is not particularly limited. "Polypeptide" is a concept that includes "protein". Furthermore, a polypeptide in which approximately 2 to 50 amino acids are linked by peptide bonds is sometimes specifically called a peptide.

[0034] Where optical isomers exist for an amino acid, the L-isomer is indicated unless otherwise specified. The notation for amino acid sequences of proteins, polypeptides, or peptides used herein is based on standard usage and conventions in the industry, and is represented by a single-letter abbreviation of the amino acid, with the left direction being the amino-terminus and the right direction being the carboxyl-terminus. In the single-letter abbreviation of an amino acid, X may be any substance having an amino group and a carboxyl group that can bind to the amino acids at both ends, and may be any of the 20 natural amino acids.

[0035] The alanine scan method (or "alanine-lysine scan" method) is a method for creating mutants in which residues in a protein are mutated one by one to alanine (or glycine if the original amino acid was alanine), and for site-specific identification of residues that are important for the structure and function of the protein. If binding to the patient's IgE antibody remains even after the mutation to alanine (or glycine if the original amino acid was alanine), then that residue is not important for binding to the IgE antibody, and the binding will remain even if it is changed to any other amino acid. IgE antibody binding refers to the detection of binding and reaction between the target epitope and the IgE antibody. In the sequence listing of this application, the residues represented by X are amino acid residues at sites where binding to the IgE antibody of an allergic patient remains even after substitution with alanine (or glycine if the original amino acid was alanine) using the alanine-lysine scan method shown in Example 4. It is well known to those skilled in the art that there is a high probability that binding to the IgE antibody will remain even if such sites are substituted with any other amino acid. In other words, it is a residue that can be substituted not only with alanine and glycine, but also with any amino acid residue other than alanine.

[0036] The binding and maintenance of IgE to antigens (epitopes) is crucial for subsequent allergic reactions, and this binding and maintenance is mediated by the epitope's charge, hydrophobic bonds, hydrogen bonds, and aromatic interactions. The fact that binding and maintenance can still be achieved by replacing these with alanine and glycine suggests that the amino acids themselves are not important.

[0037] Identification of antigens Proteins contained in wheat (bread wheat (Triticum aestivum)) were subjected to two-dimensional electrophoresis under the following conditions to identify allergens related to wheat.

[0038] For the first dimension of electrophoresis, isoelectric focusing was performed using an IPG gel Immobiline Drystrip (pH3-10NL) manufactured by GE Healthcare Biosciences Corporation (hereinafter abbreviated as GE). The gel length was within the range of 5-10 cm, the pH range of the gel was within the range of 3-10, and the pH gradient of the gel relative to the electrophoresis direction was within the range of 0.15-0.3, with the gel length up to pH 5 being defined as a, the gel length from pH 5 to 7 being b, and the gel length above pH 7 being c. Specifically, isoelectric focusing was performed using an IPG gel Immobiline Drystrip (pH3-10NL) manufactured by GE Healthcare Biosciences Corporation (hereinafter abbreviated as GE). The electrophoresis apparatus used was an IPGphor manufactured by GE. The current limit of the electrophoresis equipment was set to 75 μA per gel, and the voltage program was as follows: (1) A constant voltage process was performed at 300 V for up to 750 Vhr (the current change range during the 30 minutes of electrophoresis before the end of this process was 5 μA), (2) the voltage was gradually increased to 1000 V over 300 Vhr, (3) the voltage was further gradually increased to 5000 V over 4500 Vhr, and (4) the first dimension of isoelectric focusing was performed at a constant voltage of 5000 V until the total Vhr reached 12000.

[0039] For the second dimension of electrophoresis, a polyacrylamide gel was used, with the gel concentration at the proximal end of the electrophoresis direction set to 3-6% and the gel concentration at the proximal end of the electrophoresis direction set to a higher concentration. Specifically, SDS-PAGE was performed using a NuPAGE 4-12% Bis-Tris Gels IPG well mini 1mm from Life Technologies. The electrophoresis apparatus used was the XCell SureLock Mini-Cell from Life Technologies. 50 mM MOPS, 50 mM Tris base, 0.1% (w / v) SDS, and 1 mM EDTA were used as electrophoresis buffers, and electrophoresis was performed at a constant voltage of 200 V for approximately 45 minutes.

[0040] As a result, we revealed that in the gel obtained by performing two-dimensional electrophoresis on wheat protein under the above conditions, the antigens at spots 1 to 16 below specifically bind to IgE antibodies of patients with wheat allergies (Figure 2).

[0041] Antigen (protein) Sequence identification was performed for each spot using mass spectrometry. The mass data obtained from the mass spectrometer was compared and analyzed with Uniprot protein data. As a result, it was found that each of the spots from 1 to 16 matched known sequences. Information on each spot is summarized in Table 1 below.

[0042] [Table 1]

[0043] TIFF2026062649000002.tif190169

[0044] TIFF2026062649000003.tif176162

[0045] TIFF2026062649000004.tif121167

[0046] TIFF2026062649000005.tif177161

[0047] TIFF2026062649000006.tif170165

[0048] TIFF2026062649000007.tif172166

[0049] TIFF2026062649000008.tif163160

[0050] TIFF2026062649000009.tif174159

[0051] TIFF2026062649000010.tif187170

[0052] TIFF2026062649000011.tif188167

[0053] TIFF2026062649000012.tif184169

[0054] TIFF2026062649000013.tif174169

[0055] Spots 11 and 12 and spots 15 and 16 originated from the same protein, and a total of 14 proteins (1)-(14) were identified as novel wheat-derived antigens.

[0056] Non-limitingly, in the present invention, the antigen of spot 1 may be any of the following (1-a) to (1-f).

[0057] (1-a) Proteins containing the amino acid sequence of Sequence ID No. 2; (1-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 2; (1-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 2; (1-d) Proteins containing an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 1; (1-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 1; or (1-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 1.

[0058] (2-a) Proteins containing the amino acid sequence of Sequence ID No. 4; (2-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 4; (2-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 4; (2-d) Proteins containing amino acid sequences encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 3; (2-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 3; or (2-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 3.

[0059] (3-a) Proteins containing the amino acid sequence of Sequence ID No. 6; (3-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 6; (3-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 6; (3-d) Proteins containing an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 5; (3-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 5; or (3-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 5.

[0060] (4-a) Proteins containing the amino acid sequence of Sequence ID No. 8; (4-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 8; (4-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 8; (4-d) Proteins containing an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 9; (4-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 9; or (4-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 9.

[0061] (5-a) Proteins containing the amino acid sequence of SEQ ID NO: 10; (5-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 10; (5-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 10; (5-d) Proteins containing amino acid sequences encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 9; (5-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 9; or (5-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 9.

[0062] (6-a) Proteins containing the amino acid sequence of SEQ ID NO: 12; (6-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 12; (6-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 12; (6-d) Proteins containing an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 11; (6-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 11; or (6-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 11.

[0063] (7-a) Proteins containing the amino acid sequence of Sequence ID No. 14; (7-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 14; (7-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in SEQ ID NO: 14; (7-d) Proteins containing an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 13; (7-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 13; or (7-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 13.

[0064] (8-a) Proteins containing the amino acid sequence of SEQ ID NO: 16; (8-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 16; (8-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 16; (8-d) Proteins containing an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 15; (8-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 15; or (8-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 15.

[0065] (9-a) Proteins containing the amino acid sequence of Sequence ID No. 18; (9-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 18; (9-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 18; (9-d) Proteins containing an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 17; (9-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 17; or (9-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 17.

[0066] (10-a) Protein containing the amino acid sequence of SEQ ID NO: 20; (10-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 20; (10-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 20; (10-d) Proteins containing amino acid sequences encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 19; (10-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 19; or (10-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in sequence number 19.

[0067] (11-a) Proteins containing the amino acid sequence of Sequence ID No. 22; (11-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 22; (11-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 22; (11-d) Proteins containing an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 21; (11-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 21; or (11-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 21.

[0068] (12-a) Proteins containing the amino acid sequence of sequence number 24; (12-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 24; (12-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 24; (12-d) Proteins containing amino acid sequences encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 23; (12-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 23; or (12-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 23.

[0069] (13a) Protein containing the amino acid sequence of SEQ ID NO: 26; (13-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 26; (13-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in SEQ ID NO: 26; (13-d) Proteins containing an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 25; (13-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 25; or (13-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 25.

[0070] (14a) Protein containing the amino acid sequence of SEQ ID NO: 28; (14-b) Proteins containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 28; (14-c) Proteins containing an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 28; (14-d) Proteins containing an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 27; (14-e) A protein containing an amino acid sequence encoded by a nucleotide sequence that is 70% or more identical to the nucleotide sequence shown in Sequence ID No. 27; or (14-f) A protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 27.

[0071] The antigen proteins described in (1) to (14) above, and the polypeptides described later in (E1) to (E50), also include embodiments in which the amino acid residues of the protein or polypeptide have been modified by phosphorylation, glycosylation, aminoacylation, ring opening, deamination, etc.

[0072] Preferably, the antigenic proteins described in (1) to (14) above and the polypeptides described later in (E1) to (E50) are allergy antigens.

[0073] In this specification, when we say that an amino acid sequence has "one or several amino acids deleted, substituted, inserted, or added," we mean an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added. "Several amino acids" means, non-limitingly, up to 200, 100, 50, 30, 20, 15, 12, 10, 8, 6, 4, or 3 amino acids. Alternatively, "several amino acids" means 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2%, or 1% of the total length of the amino acid sequence.

[0074] Of the above, substitutions are preferably conservative substitutions. A conservative substitution is the replacement of a specific amino acid residue with a residue having similar physicochemical characteristics, but any substitution is acceptable as long as it does not substantially change the structural characteristics of the original sequence. For example, any substitution is acceptable as long as the substituted amino acid does not disrupt the helical structure present in the original sequence or disrupt other types of secondary structures that characterize the original sequence. Conservative substitutions of amino acid residues are exemplified below, categorized by the residues that can be substituted, but the amino acid residues that can be substituted are not limited to those listed below. Group A: Leucine, Isoleucine, Valine, Alanine, Methionine, Glycine, Cysteine, Proline Group B: Aspartic acid, Glutamic acid Group C: Asparagine, Glutamine Group D: Lysine, Arginine, Group E: Serine, Threonine Group F: Phenylalanine, tyrosine, tryptophan, histidine In the case of non-conservative substitutions, one member of one of the above types can be exchanged for a member of another type. For example, amino acids from groups B, D, and E above may be substituted with amino acids from other groups to eliminate unintended glycosylation. Alternatively, cysteine ​​may be deleted or replaced with another amino acid to prevent it from folding into the protein in the tertiary structure. Or, amino acids may be substituted considering the amino acid hydropathic index (J. Kyte and R. Doolittle, J. Mol. Biol., Vol.157, p.105-132, 1982), which is an index of hydrophobicity / hydrophilicity for amino acids, to maintain a hydrophilic / hydrophobic balance or to increase hydrophilicity to facilitate synthesis.

[0075] In other embodiments, substitutions may be made with amino acids that have less steric hindrance than the original amino acid, for example, from group F to groups A, B, C, D, or E; or from charged amino acids to uncharged amino acids, for example, from group B to group C. Doing so may improve the binding affinity to IgE antibodies.

[0076] In this specification, the percentage of identity between two amino acid sequences can be determined by visual inspection and mathematical calculation. The percentage of identity can also be determined using a computer program. Examples of such computer programs include BLAST and ClustalW. In particular, the various conditions (parameters) for identity searches using the BLAST program are described by Altschul et al. (Nucl. Acids. Res., 25, p.3389-3402, 1997) and are publicly available from the NCBI and DNA Data Bank of Japan (DDBJ) websites (BLAST manual, Altschul et al. NCB / NLM / NIH Bethesda, MD 20894; Altschul et al.). Furthermore, identity can also be determined using genetic information processing software such as GENETYX Ver.7 (Genetics), DNASIS Pro (Hitachi Software), and Vector NTI (Infomax).

[0077] In this specification, when a base sequence is described as having "one or several nucleotides deleted, substituted, inserted, or added," it refers to a base sequence in which one or several nucleotides are deleted, substituted, inserted, and / or added. "Several nucleotides" means, non-limitingly, up to 600, 300, 150, 100, 50, 30, 20, 15, 12, 10, 8, 6, 4, or 3 nucleotide acids. Alternatively, "several nucleotides" means 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2%, or 1% of the total length of the base sequence. It is preferable that the deletion, substitution, insertion, or addition of the above nucleotides does not cause a frameshift in the amino acid encoding sequence.

[0078] In this specification, the percentage of identity between two nucleotide sequences can be determined by visual inspection and mathematical calculation. The percentage of identity can also be determined using a computer program. Examples of such sequence comparison computer programs include the BLASTN program (Altschul et al. (1990) J. Mol. Biol. 215: 403-10): version 2.2.7, available from the U.S. National Library of Medicine website: https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, or the WU-BLAST2.0 algorithm. Standard default parameter settings for WU-BLAST2.0 can be found on the following website: http: / / blast.wustl.edu.

[0079] In this specification, "under stringent conditions" means hybridizing under moderate or highly stringent conditions. Specifically, moderate stringent conditions can be easily determined by those skilled in the art based on, for example, the length of the DNA. Basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Chapters 6-7, Cold Spring Harbor Laboratory Press, 2001. Preferably, moderate stringent conditions for hybridization include 1×SSC to 6×SSC at 42°C to 55°C, more preferably 1×SSC to 3×SSC at 45°C to 50°C, and most preferably 2×SSC at 50°C. If the hybridization solution contains, for example, about 50% formamide, a temperature 5 to 15°C lower than the above temperatures is used. Washing conditions include 0.5×SSC to 6×SSC and 40°C to 60°C. During hybridization and washing, 0.05% to 0.2%, preferably about 0.1% SDS, may be added. Highly stringent conditions can also be easily determined by those skilled in the art, for example, based on the length of the DNA. Generally, highly stringent conditions include hybridization and / or washing at higher temperatures and / or lower salt concentrations than moderately stringent conditions. For example, hybridization conditions include 0.1×SSC to 2×SSC and 55°C to 65°C, more preferably 0.1×SSC to 1×SSC and 60°C to 65°C, and most preferably 0.2×SSC and 63°C. Washing conditions include 0.2×SSC to 2×SSC, 50°C to 68°C, more preferably 0.2×SSC, 60°C to 65°C.

[0080] Non-limitingly, in the variants corresponding to items (b)-(f) of (1)-(14) above, it is preferable to include the amino acid sequence of the epitope (variant) described later. The included amino acid sequence of the epitope is not limited to one, and preferably includes all the epitope sequences derived from each protein. For example, the epitopes derived from protein (1) are (E9), (E31), (E11), (E36), and (E43). (1-b)-(1-f) preferably include one or more amino acid sequences (including variants) of the (E9), (E31), (E11), (E36), or (E43) epitopes.

[0081] The antigen may be obtained by separating and purifying it from wheat using a combination of protein purification methods well known to those skilled in the art. Alternatively, the antigen may be obtained by expressing it as a recombinant protein using genetic engineering techniques well known to those skilled in the art, and then separating and purifying it using protein purification methods well known to those skilled in the art.

[0082] Methods for purifying proteins include, for example, methods that utilize solubility such as salting out and solvent precipitation; methods that utilize differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE; methods that utilize charge such as ion exchange chromatography and hydroxyl apatite chromatography; methods that utilize specific affinity such as affinity chromatography; methods that utilize differences in hydrophobicity such as reverse-phase high-performance liquid chromatography; and methods that utilize differences in isoelectric point such as isoelectric focusing.

[0083] The preparation of proteins using genetic engineering technology involves preparing an expression vector containing nucleic acids encoding an antigen, introducing the expression vector into suitable host cells by gene transfer or transformation, culturing the host cells under conditions suitable for recombinant protein expression, and then recovering the recombinant protein expressed in the host cells.

[0084] A "vector" is a nucleic acid that can be used to introduce a nucleic acid linked to it into a host cell, while an "expression vector" is a vector capable of inducing the expression of a protein encoded by the nucleic acid introduced by the vector. Vectors include plasmid vectors, viral vectors, and the like. Those skilled in the art can select an expression vector appropriate for the expression of recombinant proteins depending on the type of host cell used.

[0085] A "host cell" is a cell that undergoes gene transfer or transformation by a vector. The host cell can be appropriately selected by those skilled in the art depending on the vector used. The host cell can be derived from a prokaryote, such as Escherichia coli (E. coli). When a prokaryotic cell such as E. coli is used as the host, the antigen of the present invention may include an N-terminal methionine residue to facilitate the expression of the recombinant protein within the prokaryotic cell. This N-terminal methionine can also be cleaved from the recombinant protein after expression. Alternatively, the host cell can be derived from a eukaryote such as a single-celled eukaryote like yeast, a plant cell, an animal cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell, or insect cell), or a silkworm.

[0086] Gene transfer or transformation of host cells using an expression vector can be carried out as appropriate by methods known to those skilled in the art. Furthermore, those skilled in the art can express recombinant proteins by selecting appropriate conditions suitable for recombinant protein expression according to the type of host cell and culturing the host cells. Then, the antigen expressed as a recombinant protein can be isolated and purified by homogenizing the host cells expressing the recombinant protein and combining the above-described protein purification methods with the obtained homogenate as appropriate. Antigens can also be prepared by introducing the above-mentioned expression vector or synthesized double-stranded DNA, or mRNA transcribed from them, into a cell-free protein synthesis system for expression, and then separating and purifying the expressed protein.

[0087] The antigen of the present invention preferably specifically binds to IgE antibodies in allergy patients.

[0088] Diagnostic kits and diagnostic methods (1) The present invention provides a method for providing an indicator for diagnosing a target allergy, comprising the following steps: (i) The sample obtained from the subject is brought into contact with the antigen, where the sample is a solution containing IgE antibodies; (ii) Detect the binding of IgE antibodies to the antigen in the sample obtained from the subject; (iii) If binding of the target IgE antibody to the antigen is detected, it provides an indicator that the subject is allergic; The present invention provides the method comprising, wherein the antigen is at least one of the proteins identified as antigens in (1) to (14) above.

[0089] "Allergy" is, in a non-limiting manner, in one embodiment, an allergy to grains, preferably an allergy to grass grains, and an allergy to wheat.

[0090] In this specification, “diagnosis” generally includes not only a (definitive) diagnosis by a physician, but also mere “detection” including possibility. In this specification, “diagnosis” and “detection” are, in one embodiment, “in vivo,” “in vitro,” or “ex vivo” “diagnosis” and “detection.” Preferably, they are “in vitro” “diagnosis” and “detection.”

[0091] The sample obtained from the subject is a solution containing IgE antibodies collected from the subject. Such solutions include, for example, blood, saliva, sputum, nasal mucus, urine, sweat, and tears. The sample obtained from the subject may be pretreated to increase the concentration of IgE antibodies in the sample before contact with the antigen. Sample pretreatment may include, for example, obtaining serum or plasma from blood. Furthermore, the Fab portion, which is the antigen-binding portion, may be purified. In a particularly preferred embodiment, step (i) above is carried out by contacting the antigen with the IgE antibodies in the serum obtained from the subject.

[0092] The IgE antibody may be the IgE antibody itself, or it may be a mast cell to which the IgE antibody is bound.

[0093] The contact between a sample obtained from a target and an antigen, and the detection of its binding, can be performed by known methods. Such methods include, for example, ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, and immunochromatography. All of these methods involve contacting the antigen with the target IgE antibody to allow it to bind, reacting the IgE antibody specifically bound to the antigen with an enzyme-labeled secondary antibody, and then adding an enzyme substrate (usually a colorimetric or luminescent reagent) to detect the product of the enzymatic reaction, thereby detecting the binding between the antigen and the target IgE antibody. Alternatively, a fluorescently labeled secondary antibody can be detected. Furthermore, detection using measurement methods capable of evaluating the binding of antigens to IgE antibodies, such as surface plasmon resonance (SPR), can also be employed. Multiple types of antigen-specific IgE antibodies may be mixed.

[0094] The antigen may be in a state where the isolated antigen is immobilized on a carrier. In this case, ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance, etc., can be used in steps (i) and (ii) above, and step (i) above is performed by bringing the sample obtained from the target into contact with the surface on which the antigen is immobilized. The isolated antigen may be obtained by separating and purifying it from the target (raw material, processed product, etc.) using a combination of protein purification methods well known to those skilled in the art, or by preparing it using genetic recombination technology. It may also be an antibody that has been attached to it.

[0095] The antigen may not be immobilized on a carrier. In this case, in the above steps (i) and (ii), flow cytometry or the like can be used to confirm the presence of the antigen to which the antibody has bound by laser light. For example, a basophil activation test (BAT) or the like can be mentioned. Also, a histamine release test (HRT) can be mentioned, in which the antigen is further brought into contact with blood cells in the sample to examine whether histamine is released.

[0096] Alternatively, the antigen may be transferred from the state separated by two-dimensional electrophoresis and detected by immunoblotting. Two-dimensional electrophoresis is a technique for separating protein samples by performing isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. In this case, the conditions for two-dimensional electrophoresis are not particularly limited as long as the antigen of the present invention can be separated. For example, the conditions for two-dimensional electrophoresis described in the section "Identification of Antigen" above can be used. Alternatively, the electrophoresis conditions can be determined referring to the descriptions in Patent Documents 1 to 4 above. For example, as follows: (A) As the isoelectric focusing gel in the first dimension, the gel length is within the range of 5 to 10 cm, the pH range of the gel is 3 to 10, and when the gel length up to pH 5 is a, the gel length from pH 5 to 7 is b, and the gel length of pH 7 or more is c, the relationships of "a < b" and "b > c" are satisfied; (B) In the case of (A), when the total length of the gel is 1, a is within the range of 0.15 to 0.3, b is within the range of 0.4 to 0.7, and c is within the range of 0.15 to 0.3; (C) In the isoelectric focusing in the first dimension, a constant voltage step is performed by applying a constant voltage within the range of 100 V to 600 V per gel containing the sample, and after the migration change width per 30 minutes of migration is within the range of 5 μA, the voltage increase step of increasing the voltage from the constant voltage is started; (D) In the case of (C), the final voltage of the voltage increase step is within the range of 3000 V to 6000 V; (E) The gel length in the longitudinal direction of the isoelectric focusing gel in the first dimension is 5 to 10 cm, and the gel concentration at the gel concentration base end in the migration direction of the two-dimensional electrophoresis gel is 3 to 6%; and In cases (F) and (E), the gel concentration at the leading end of the electrophoresis direction of the second-dimensional electrophoresis gel is set higher than the gel concentration at the proximal end of the electrophoresis direction; Two-dimensional electrophoresis can be performed under conditions that satisfy at least one selected from the group consisting of the following.

[0097] The antigens listed in (1) to (14) above are antigens that specifically bind to the IgE antibodies of allergic patients (for example, patients with wheat allergies). Therefore, if the binding of the target IgE antibody to the antigen is detected, it provides an indicator that the subject has an allergy.

[0098] The present invention also provides an allergy diagnostic kit comprising at least one of the antigens described in (1) to (14) above. The diagnostic kit of the present invention may be used in a method for providing an indicator for diagnosing the above allergies, or in the diagnostic method described below. In addition to comprising at least one of the antigens described in (1) to (14) above, the diagnostic kit of the present invention may also comprise an enzyme-labeled anti-IgE antibody and a chromogenic or luminescent substrate that serves as a substrate for the enzyme. Alternatively, a fluorescently labeled anti-IgE antibody may be used. In the diagnostic kit of the present invention, the antigen may be provided immobilized on a carrier. The diagnostic kit of the present invention may also be provided with instructions for a diagnostic procedure and a package containing such instructions.

[0099] In another embodiment, the diagnostic kit described above includes a companion diagnostic for allergies. A companion diagnostic is used to identify patients who are expected to benefit from a drug, or patients who are at risk of serious side effects from a drug, or to assess the responsiveness of a drug in order to optimize treatment with that drug. Treatment optimization here includes, for example, determining the dosage and administration, deciding when to discontinue administration, and confirming which allergen component will induce immune tolerance.

[0100] The present invention also provides an allergy diagnostic composition comprising at least one of the antigens (1) to (14) described above. The diagnostic composition of the present invention can be used in the following diagnostic methods. The diagnostic composition of the present invention may optionally contain pharmaceutically acceptable carriers and additives that are commonly used with the antigens of the present invention.

[0101] In one embodiment, the present invention is a method for diagnosing a target allergy, comprising the following steps: (i) Contact the sample obtained from the subject with the antigen; (ii) Detect the binding of IgE antibodies to the antigen in the sample obtained from the subject; (iii) If binding of the target IgE antibody to the antigen is detected, the subject is determined to be allergic; The present invention provides a method which includes, where the antigen is at least one of the proteins identified as antigens in (1) to (14) above. Herein, steps (i) and (ii) are carried out as described in the steps of the method for providing an indicator for diagnosing allergies.

[0102] In another embodiment, the present invention provides a method for diagnosing an allergy to a subject, comprising administering at least one of the antigens (1) to (14) above to the subject. The method may be carried out in the form of a skin test, characterized by applying the antigen to the skin. Skin tests include forms such as a prick test, in which a diagnostic composition is applied to the skin and then a small wound is made without causing bleeding to allow the antigen to penetrate the skin and observe the skin reaction; a scratch test, in which the skin is lightly scratched over the applied diagnostic composition and the reaction is observed; a patch test, in which a diagnostic composition in the form of a cream or ointment is applied to the skin and the reaction is observed; and an intradermal test, in which the antigen is administered intradermally and the reaction is observed. If a skin reaction such as swelling occurs in the area where the antigen was applied, the subject is diagnosed with an allergy. Here, the amount of antigen applied to the skin may be, for example, 100 μg or less per application.

[0103] In diagnosing allergies, oral food challenge tests are often performed to identify antigens. At least one of the antigens listed in (1) to (14) above can be used as an active ingredient in an oral food challenge test to diagnose allergies. The antigen protein used in the oral food challenge test may be an expressed and purified protein, or it may be an antigen expressed in a raw material or processed product, such as pollen rice, which is produced by transforming rice with the gene for cedar pollen antigen and expressing the antigen protein in the rice.

[0104] In one embodiment, the above-mentioned diagnostic composition and diagnostic kit can be used for prick tests, scratch tests, patch tests, intradermal tests, and the like.

[0105] In another embodiment, the present invention provides at least one of the antigens (1) to (14) described above for use in the diagnosis of allergies, which also includes providing at least one of the antigens (1) to (14) described above in mixture with a known antigen.

[0106] In yet another embodiment, the present invention provides the use of at least one of the antigens (1) to (14) above in the manufacture of a diagnostic composition for allergies.

[0107] Composition / Treatment method (1) The present invention provides a composition comprising at least one of the antigens described in (1) to (14) above.

[0108] In one embodiment, the composition of the present invention is a pharmaceutical composition. In another embodiment, the composition of the present invention is a quasi-drug composition, a non-pharmaceutical composition (for example, a cosmetic composition, a food composition).

[0109] In one embodiment, the above composition is used to treat allergies (for example, allergies to wheat). In this specification, "treatment of allergies" means increasing the limit of antigens that can be taken into the body without causing a reaction, ultimately aiming for a state in which no reaction occurs with normal levels of antigen intake (remission).

[0110] The present invention also provides a method for treating allergies, comprising administering at least one of the antigens (1) to (14) above to a patient who requires treatment for allergies.

[0111] In another embodiment, the present invention provides at least one of the antigens (1) to (14) above for use in the treatment of allergies. In yet another embodiment, the present invention provides the use of at least one of the antigens (1) to (14) above for the manufacture of therapeutic drugs for allergies.

[0112] In the treatment of allergies, desensitization therapy is often performed, which aims to induce immune tolerance by administering antigens to patients. At least one of the antigens listed in (1) to (14) above can be used as an active ingredient for desensitization therapy for allergies. Here, the antigen protein used in desensitization therapy may be an expressed and purified protein, or it may be an expression in a raw material or processed product, such as pollen rice, in which rice is transformed with the gene for cedar pollen antigen and the antigen protein is expressed in the rice.

[0113] The compositions of the present invention can be administered by conventional routes of administration. Conventional routes of administration include, for example, oral, sublingual, transdermal, intradermal, subcutaneous, intravascular, intranasal, intramuscular, intraperitoneal, and rectal administration.

[0114] The composition of the present invention can be used as a composition prepared by conventional methods, with the antigen of the present invention optionally accompanied by commonly used pharmaceutically acceptable adjuvants, excipients, or various additives (e.g., stabilizers, solubilizers, emulsifiers, buffers, preservatives, colorants, etc.). The dosage form of the composition can be appropriately selected by those skilled in the art depending on the route of administration. For example, it may be in the form of tablets, capsules, lozenges, sublingual tablets, injections, intranasal sprays, poultices, liquids, creams, lotions, suppositories, etc. The dosage, frequency and / or duration of administration of the composition of the present invention can be appropriately selected by a physician depending on the route of administration, symptoms, patient characteristics such as age and weight, etc. For example, in adults, a dose of 100 μg or less per dose may be administered. The administration interval may be, for example, daily, once a week, twice a month, or once every three months. The duration of administration may be, for example, several weeks to several years. The dosage may be increased gradually during the duration of administration.

[0115] Tester composition (1) The present invention provides a tester composition comprising an antibody against at least one of the antigens (1) to (14) described above.

[0116] The antibody can be produced by conventional methods. For example, it may be produced by immunizing a mammal such as a rabbit with the antigens described in (1) to (14) above. The antibody may be an Ig antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (e.g., Fab, F(ab')2, Fab').

[0117] Furthermore, in the above-described tester composition, the antibody may be provided bound to a carrier. The carrier is not particularly limited as long as it is a carrier that can be used to detect the binding of the antibody to the antigen. Any carrier known to those skilled in the art can be used.

[0118] Methods for determining the presence or absence of antigens include the following: A method for determining whether the target raw material or processed product contains an antigen if the binding of the Ig antibody to the antigen is detected when the binding of the Ig antibody to the antigen is detected, by contacting a sample obtained from a raw material or processed product with a tester composition containing the prepared Ig antibody, for example, using ELISA. A method in which raw materials or processed products are impregnated into filter paper or similar material, and an antibody solution is reacted to detect the antigens contained therein.

[0119] In another aspect of the present invention, a tester composition for determining the presence or absence of an allergen in a target object is provided, characterized by comprising a primer having a nucleotide sequence complementary to at least one portion of the nucleotide sequences represented by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. Non-limitingly, the primer has a nucleotide sequence complementary to, for example, a 3' terminal portion or a central portion of the sequence of at least one portion of the nucleotide sequences represented by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, preferably 12 residues, 15 bases, 20 bases, or 25 bases. In particular when targeting mRNA, a poly-A tail complementary primer is provided. In a preferred embodiment, the tester composition comprising the above-mentioned primers may further include a primer comprising a nucleotide sequence of the 5' end portion of at least one of the nucleotide sequences represented by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, preferably a nucleotide sequence consisting of 12, 15, 20, or 25 nucleotides.

[0120] For example, using DNA or mRNA obtained from wheat as a template, and using the complementary primers, cDNA is amplified by PCR (Polymerase Chain Reaction), including RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The presence or absence of the antigen is determined by comparing the amplified cDNA sequence with sequence numbers 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. Examples of PCR amplification methods include the RACE (Rapid Amplification of cDNA End) method. At this time, if, in a comparison between the amplified cDNA and sequence numbers 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, there are point mutations encoding the same amino acids, or if, even if there are insertions, deletions, substitutions, or additions of bases to the sequence numbers 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 in the nucleotide sequence of the amplified cDNA, the amino acid sequence encoded by the cDNA is 70% or more identical to the amino acid sequence of sequence numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 28, preferably 80%, 90%, 95%, 98%, or 99% or more, then it is determined that the antigen is present.

[0121] In one embodiment, the above-described tester composition is used to determine, for example, the presence or absence of an antigen in a target object such as a food ingredient (wheat) or a food manufacturing line. The above-described tester composition may also be used by manufacturers for quality inspection of manufacturing lines and pre-shipment products, or by consumers themselves to check for the presence or absence of an antigen in target raw materials or processed products. It may also be used to check for antigen content by increasing or decreasing the peak of the relevant protein using a mass spectrometer.

[0122] Method for determining the presence or absence of an antigen (1) The present invention includes a method for determining the presence or absence of the antigens (1) to (14) in a target substance, which involves contacting an antibody against at least one of the antigens (1) to (14) above with a raw material or processed product (including a liquid).

[0123] The raw materials may be food ingredients, cosmetic ingredients, pharmaceutical ingredients, etc. The processed products may be processed food products, cosmetics, pharmaceuticals, etc.

[0124] The antibody, the method for producing the antibody, the method for bringing the antibody into contact with the raw materials / processed products, and the binding of the antibody to the antigen are as described above in "Tester Composition (1)".

[0125] Antigen-removed foods, etc. (1) The present invention provides a raw material or processed product characterized in that at least one of the antigens (1) to (14) above is removed or reduced. In one embodiment, the "raw material or processed product" is "wheat or a wheat processed product".

[0126] The method for removing or reducing the antigen of the present invention in raw materials or processed products is not limited. The removal or reduction of the antigen may be carried out by any method, as long as the antigen of the present invention is removed or reduced.

[0127] For example, raw materials (e.g., wheat) from which the antigen of the present invention has been removed or reduced may be used to prepare raw materials in which the expression of the antigen of the present invention has been modified using genetic modification technology.

[0128] Genetic modification techniques can be any of the methods known to those skilled in the art. For example, Oishi, et al. (Scientific Reports, Vol.6, Article number: 23980, 2016, doi:10.1038 / srep23980) describe applying the genome editing technology CRISPER / Cas9 to chicken primordial germ cells to obtain individuals with ovomucoid gene deletion. A similar method may be used to obtain raw materials from which the antigen of the present invention has been removed.

[0129] Furthermore, wheat with reduced antigen content according to the present invention may be obtained by artificial insemination with a raw material that does not contain or contains little antigen (for example, wheat). Artificial crossbreeding of raw materials can be carried out by conventional methods, such as the pollination method between selected varieties for the purpose of improving food varieties. For example, as a selected variety, low-allergen wheat 1BS-18 Hokushin can be selected as antigen-deficient wheat.

[0130] A processed product from which the antigen of the present invention has been removed or reduced may be a processed product made from raw materials from which the antigen of the present invention has been removed or reduced. When using ordinary raw materials, the process to remove or reduce the antigen of the present invention is performed before or after the preparation of the processed product. Methods for removing or reducing the antigen of the present invention in a processed product made from ordinary raw materials include methods to remove protein components in the raw materials and processed products, such as high-pressure treatment and elution with a neutral salt solution, or high-temperature steam, as well as methods of hydrolysis, denaturation, and amino acid changes (chemical modification and elimination of side chains, etc.) by heat treatment and acid treatment.

[0131] Method for producing raw materials or processed products from which antigens have been removed or reduced (1) The present invention provides a method for producing a raw material or processed product from which an antigen has been removed or reduced, the method comprising the step of confirming that the antigen has been removed or reduced during the manufacturing process of the processed product, wherein the antigen is at least one of the antigens (1) to (14) above.

[0132] The step of confirming that the antigen has been removed or reduced in the manufacturing process of the raw material or processed product from which the antigen has been removed or reduced may be performed by confirming the presence or absence of the antigen by the method described in the "Tester Composition (1)" section above.

[0133] Furthermore, the manufacture of wheat or wheat products from which antigens have been removed or reduced may be carried out by the methods described in the "Antigen-Removed Foods, etc." section above.

[0134] Epitope As shown in Examples 1-3, for the identified antigens, the epitopes and the amino acids within those epitopes that are important for binding to IgE antibodies in allergy patients were identified, as shown in Example 4.

[0135] The results are summarized in Table 2.

[0136] [Table 2]

[0137] TIFF2026062649000016.tif98152

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[0139] TIFF2026062649000018.tif200155

[0140] TIFF2026062649000019.tif71151

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[0144] TIFF2026062649000023.tif54153

[0145] TIFF2026062649000024.tif212160

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[0150] TIFF2026062649000029.tif187155

[0151] TIFF2026062649000030.tif188152

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[0190] TIFF2026062649000069.tif206160

[0191] TIFF2026062649000070.tif66152

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[0196] TIFF2026062649000075.tif74161

[0197] In Table 2 of TIFF2026062649000076.tif157158, in patients with wheat allergy, WDEIA cases were represented as P2-36, while children with typical immediate-type wheat allergy cases that do not require exercise to trigger the onset were represented as P50-57, and adults as G1-9.

[0198] The present invention provides a polypeptide containing amino acid sequences (E1) to (E50) that include, or consist of, each of the amino acid sequences listed in Table 2 below, SEQ ID NOs. 29-2932 (excluding SEQ ID NOs. 138, 227, 259, 269, 273, 342, 399, and 2894), as a polypeptide containing an amino acid sequence that specifically binds to IgE antibodies in allergy patients. (E1) to (E50) are amino acid sequences that bind to IgE antibodies derived from the proteins listed in "Origin" in Table 2 (hereinafter sometimes referred to as "epitopes").

[0199] (1) Alpha / beta-gliadin MM1 protein: (E9), (E31), (E11), (E36), (E43); (2) Derived from LMW-m glutenin subunit 8 protein: (E2), (E13), (E21); (3) Derived from gamma-gliadin P08453 protein: (E14), (E24), (E5), (E25); (4) Derived from fructose-bisphosphate aldolase protein: (E33), (E20), (E34), (E10), (E15), (E6), (E16), (E40), (E30), (E41); (5) Alpha-gliadin Gli2-LM2-12 protein: (E9), (E31), (E11), (E17), (E7), (E22), (E42); (6) Alpha-gliadin (Fragment) A0A0E3Z522 protein: (E9), (E31), (E11), (E36), (E35); (7) Derived from gamma-gliadin A1 protein: (E4), (E19); (8) Alpha / beta-gliadin I0IT53 protein: (E23), (E12) (9) Derived from LMW-D8 (LMW-GS) protein: (E8), (E1), (E18), (E3), (E39); (10) Derived from globulin 1-S allele 1 protein: (E49), (E50); (11) Derived from globulin 3 protein: (E49), (E50); (12) Derived from elongation factor 1-alpha protein: (E38), (E45), (E28), (E26), (E46), (E47), (E27), (E29), (E48), (E32); (13) Alpha-gliadin (Fragment) A0A0E3UR64 protein: (E23), (E11), (E36) (14) Gamma-gliadin I7KM78 protein (E9) and (E31) are identical sequences. (E9) and (E31) are present in three proteins: (1), (5), and (6). (E11) is present in four proteins: (1), (5), (6), and (13). (E23) is present in two proteins: (8) and (13). (E36) is present in three proteins: (1), (6), and (13).

[0200] Non-limitingly, the epitope antigen of the present invention preferably comprises at least one of the following polypeptides.

[0201] (E1) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 29-130 and 2880; (E2) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 131-137 and 139-190; (E3) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 191-280 (excluding 227, 259, 269 and 273) and 2881; (E4) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 281-341 and 343-345; (E5) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 346-398, 400-413 and 2882; (E6) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 414-492, 2883, and 2884; (E7) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 493-561; (E8) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 562-639; (E9) Polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 640-705 and 2885-2888; (E10) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 706-775 and 2889-2891; (E11) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 776-898 and 2892; (E12) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 899-973, 2893, and 2895; (E13) Polypeptides containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 974-1009; (E14) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1010-1088 and 2896-2899; (E15) Polypeptides containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1089-1141; (E16) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1142-1165 and 2900; (E17) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1166-1208; (E18) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1209-1256; (E19) Polypeptides containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1257-1296; (E20) Polypeptides containing at least one amino acid sequence selected from the amino acid sequences of sequence numbers 1297-1366; (E21) Polypeptides containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1367-1402; (E22) Polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1403-1507; (E23) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1508-1565 and 2901; (E24) Polypeptides containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1566-1597; (E25) Polypeptides containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1598-1607; (E26) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1608-1684, 2902, and 2903; (E27) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1685-1752, 2904, and 2905; (E28) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of sequence numbers 1753-1788, 2906, and 2907; (E29) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1789-1835, 2908, and 2909; (E30) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1836-1894, 2910, and 2911; (E31) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 1895-1976, 2912, and 2913; (E32) Polypeptides containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1977-2003; (E33) Polypeptide containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2004-2026; (E34) Polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2027-2067 and 2914; (E35) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2068-2153, 2915, and 2916; (E36) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2154-2216; (E37) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2217-2264 and 2917-2921; (E38) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2265-2308 and 2922-2925; (E39) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2309-2341; (E40) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2342-2418; (E41) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2419-2490; (E42) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2491-2544, 2926, and 2927; (E43) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2545-2612; (E44) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2613-2630; (E45) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2631-2673; (E46) Polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2674-2700 and 2928; (E47) Polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2701-2773 and 2929; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 2774 - 2808, 2930, and 2931; (E49) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 2809 - 2829; or (E50) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 2830 - 2879 and 2932 Preferably, the polypeptides of (E1) to (E50) described above are specific sequences identified as epitopes that bind to IgE antibodies in the examples of this specification, as shown in Table 2. The epitope antigens of the present invention may include, in addition to the polypeptides of (E1) to (E50) in the above-described preferred embodiments, variants and the like described below. Hereinafter, the embodiments (variants) that can be included in the epitope antigens of the present invention will be described.

[0202] Among the 50 sequences of SEQ ID NO: 29, 131, 191, 281, 346, 414, 493, 562, 640, 706, 776, 899, 974, 1010, 1089, 1142, 1166, 1209, 1257, 1297, 1367, 1403, 1508, 1566, 1598, 1608, 1685, 1753, 1789, 1836, 1895, 1977, 2004, 2027, 2068, 2154, 2217, 2265, 2309, 2342, 2419, 2491, 2545, 2613, 2631, 2674, 2701, 2774, 2809, 2830 described in the "Common 15-residue sequence" of Table 2, each is a common 15-amino acid residue sequence identified as an epitope that binds to IgE antibody in each of the epitopes (E1)-(E50) by epitope mapping based on overlapping. In one aspect, the present invention is a polypeptide containing or consisting of these amino acid sequences. The epitope sequence contained in the polypeptide of the present invention may be the entire common epitope 15 amino acid residues or a part thereof. The epitope sequence is 4 amino acid residues or more, 5 amino acid residues or more, 6 amino acid residues or more, 7 amino acid residues or more, 8 amino acid residues or more, 9 amino acid residues or more, 10 amino acid residues or more, 11 amino acid residues or more, 12 amino acid residues or more, 13 amino acid residues or more, 14 amino acid residues or more.

[0203] In the examples of this specification, for example, polypeptides consisting of 4 amino acid residues (such as SEQ ID NO: 30, 58, 61, 222, 238, 345, etc.) were identified as epitopes that bind to IgE antibody in many cases. Furthermore, in many cases of 5 amino acid residues or more, binding to IgE antibody was confirmed. Therefore, if at least 4 amino acid residues are present, it is useful as an epitope sequence.

[0204] One embodiment of the polypeptide variant of the present invention includes a polypeptide containing four or more amino acid residues from the amino acid sequences specifically described in (E1) to (E50) above. For example, a variant of (E1) may include "a polypeptide containing four or more amino acid residues from at least one amino acid sequence of SEQ ID NOs. 29-130 and 2880". Preferably, it contains four or more amino acid residues, five or more amino acid residues, six or more amino acid residues, seven or more amino acid residues, eight or more amino acid residues, nine or more amino acid residues, ten or more amino acid residues, eleven or more amino acid residues, twelve or more amino acid residues, thirteen or more amino acid residues, and fourteen or more amino acid residues from at least one amino acid sequence of SEQ ID NOs. 29-130 and 2880. The same applies to (E2) to (E50).

[0205] Non-limitingly, one embodiment of each polypeptide of the present invention includes the following amino acid residues.

[0206] E1: At least four amino acid residues from ILWYH; E2: At least four amino acid residues from FPQQHQQ; E3: At least four amino acid residues from QQPFP; E5: At least four amino acid residues from QSFPQ and / or at least four amino acid residues from RPFIQ; E6:At least four amino acid residues from WRAVL, at least four amino acid residues from KIGAT, and / or at least four amino acid residues from ATEPS; E9: At least four amino acid residues from VPLVQ; E10: At least four amino acid residues from ILFEE and / or at least four amino acid residues from QSTKG; E13: At least four amino acid residues from IIILQ; E14: At least four amino acid residues from VPQLQ; E15: At least four amino acid residues from TKGGK and / or at least four amino acid residues from KPFVDI; E16: At least four amino acid residues from TEPSQL and / or at least four amino acid residues from SIDQN; E17: At least four amino acid residues from PGQQQQ, and / or; E18: At least four amino acid residues from PIQQQP; E19: At least four amino acid residues from SMILPRSDCKV; E20: At least four amino acid residues from INVEN, and / or at least four amino acid residues from VEDNRR; E21: At least four amino acid residues from KPWQQ; E22: At least four amino acid residues from YLQPQQP and / or at least four amino residues from SQQQA; E23: At least four amino acid residues from VQQQ and / or at least four amino acid residues from GQQQ; E24: At least four amino acid residues of PFPQT; E25: At least four amino acid residues from AQLEAIRS; E26: At least four amino acid residues from YNPDKV and / or at least four amino acid residues from FVPISG; E27: at least four amino acid residues from KEAANF and / or at least four amino acid residues from SQVIIM; E28: At least four amino acid residues from SYLKK and / or at least four amino acid residues from KVGYNP; E29: At least four amino acid residues from SGKELE and / or at least four amino acid residues from LPKFLKN; E30: At least four amino acid residues from YTVRTLQRT; E31: At least four amino acid residues from QVPLV; E32: At least four amino acid residues from PTGAK, at least four amino acid residues from AKVTK, and / or at least four amino acid residues from AAIK; E33: At least four amino acid residues from TGTIG and / or at least four amino acid residues from KRFAS; E34: At least four amino acid residues from PGALQ and / or at least four amino acid residues from YLSGVIL; E35:At least four amino acid residues from CTIAP, and / or at least four amino acid residues from PFGIFG; E36: at least four amino acid residues from YSQPQQ, at least four amino acid residues from PISQ, and / or at least four amino acid residues from SQQQ; E37: At least four amino acid residues from SRLLR and / or at least four amino acid residues from IRNYRV; E38: At least four amino acid residues from GIDKR and / or at least four amino acid residues from ERFEK; E39: At least four amino acid residues from RAIIY; E40: At least four amino acid residues from HDIDR and / or at least four amino acid residues from VTEIV; E41: At least four amino acid residues from KAWSGKT; E42: At least four amino acid residues from SQVSF, and / or at least four amino acid residues from FQPSQL; E43: at least four amino acid residues from QLVQQ and / or at least four amino acid residues from QQLCC; E44: At least four amino acid residues from LASL and / or at least four amino acid residues from SKRV; E45: At least four amino acid residues from KEAAE and / or at least four amino acid residues from NKRS; E46: At least four amino acid residues of KGPTL; E47: at least four amino acid residues from YKIGG and / or at least four amino acid residues from PVGRV; E48: At least four amino acid residues from KNGD and / or at least four amino acid residues from KMIPT; E49: At least four amino acid residues from SANTH, and / or at least four amino acid residues from RSKW; E50: At least four amino acid residues from IVVEG and / or at least four amino acid residues from GRDGY.

[0207] As shown in Table 2, the above sequences were found to be common sequences among multiple sequences that bind to IgE antibodies for each epitope in the examples.

[0208] In Table 2, the "preferred" sequence is a shorter subsequence of the "common 15-residue sequence" that can function as an epitope. The "more preferred" sequence is a sequence that is preferable to the aforementioned short subsequence in order to improve binding affinity to the IgE antibody. The "key" sequence indicates a sequence from the "common 15-residue sequence" or the "key" sequence that is considered particularly important. Among the "key" sequences, the amino acid sequence indicated by "X" is an amino acid residue that has been confirmed to retain binding affinity to the IgE antibody even when changed to any alanine (or glycine if the original amino acid residue was alanine) by an Alaning lysine scan. Therefore, X is any amino acid residue, preferably alanine (or glycine). Note that if the sequence indicated by "X" is not included in the "key" sequence, it means that no amino acid residue that retains binding affinity to the IgE antibody was found by an Alaning lysine scan.

[0209] Regarding each epitope described in Table 2, the amino acid residues indicated as X are amino acid residues for which it has been confirmed that even if they are changed, the binding property to IgE antibodies remains. The present invention preferably allows one or more amino acid residues indicated as X in the "key sequence" corresponding to each "preferred sequence" to be substituted with any amino acid residue. For example, in one aspect, there are a plurality of key sequences corresponding to SEQ ID NO: 29, which is a preferred sequence, such as SEQ ID NO: 30, SEQ ID NO: 33, etc. The same applies to the other "preferred sequences" and "key sequences" of (E1) and (E2)-(E50) below.

[0210] The number of amino acid residues that may be substituted is not limited, and preferably is 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less. The same applies to (E2)-(E50) below.

[0211] Each sequence described in the column of "synthetic sequence" and / or "SEQ ID NO" on the right side of the graph No. in Table 2 is a sequence for which epitope cross-reaction was confirmed in Example 5. "Epitope cross-reactivity was confirmed" means that the IgE antibodies in the sera of each allergic patient showed higher binding properties than the IgE antibodies in the sera of non-allergic subjects (specifically, greater than "1" in FIGS. 7-12). Therefore, in one aspect, the polypeptide of the present invention includes these amino acid sequences or includes a polypeptide consisting of these amino acid sequences. In one aspect, for the polypeptide of the present invention, the IgE antibodies in the sera of each allergic patient show binding properties 1.05 times or more, 1.10 times or more, 1.15 times or more, 1.20 times or more, 1.25 times or more higher than the IgE antibodies in the sera of non-allergic subjects.

[0212] In this specification, "polypeptide containing the amino acid sequences (E1) to (E50)" includes, in preferred embodiments, a polypeptide containing each of the amino acid sequences of the (E1) to (E50) polypeptides described above, a polypeptide consisting of each of these amino acid sequences, and embodiments (mutants) in which amino acid residues are substituted as described above. "Containing each of the amino acid sequences of SEQ ID NO: ~~" means that any other amino acid sequences may be included, as long as they do not affect the binding of each of the amino acid sequences of SEQ ID NO: ~~ (including their substituted embodiments) to the IgE antibody (i.e., their function as epitopes).

[0213] In the polypeptide, amino acid residues other than those specifically identified can be arbitrarily selected as long as they do not affect binding to the IgE antibody (i.e., their function as epitopes). Non-limitingly, preferably, they should be appropriately selected from the sequences of the corresponding base epitopes and base proteins. For example, while Sequence ID No. 30 identifies only "QPIQ", if other amino acid residues are added, it is desirable to appropriately select them from the base Sequence ID No. 29. Furthermore, for the sequences listed in Table 2-1 as (E1), it is desirable that amino acid residues from the sequence derived from the base protein (9) (corresponding to spot (9)) are added.

[0214] Polypeptides containing the amino acid sequences (E1)-(E50) above may be prepared by chemical synthesis methods such as solid-phase peptide synthesis. Alternatively, polypeptides containing epitopes may be expressed as recombinant polypeptides using genetic recombination techniques well known to those skilled in the art, and then separated and synthesized by protein synthesis methods well known to those skilled in the art. Polypeptides may be formed by linking two or more types together, or by repeatedly linking a single epitope. In the latter case, binding affinity to Ig antibodies is generally improved.

[0215] The length of the polypeptide containing the above amino acid sequence (E1)-(E50) is not particularly limited. In a preferred embodiment, the length of the polypeptide containing the above amino acid sequence (E1)-(E50) may be 500 amino acids or less, 300 amino acids or less, 200 amino acids or less, 100 amino acids or less, 50 amino acids or less, 30 amino acids or less, 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, or 5 amino acids or less. If the polypeptide is formed by repeatedly linking one or more of the above amino acid sequences (E1)-(E50) once or twice or more, in a preferred embodiment, the length of the amino acid sequence portion may be 1000 amino acids or less, 750 amino acids or less, 500 amino acids or less, 250 amino acids or less, 100 amino acids or less, 75 amino acids or less, 50 amino acids or less, 30 amino acids or less, 15 amino acids or less, 10 amino acids or less, or 5 amino acids or less. The number of amino acid residues described in the preferred embodiment as the length of the polypeptide is the sum of the lengths of the sequences before and after the spacer (excluding the spacer).

[0216] The antigen of the present invention preferably specifically binds to IgE antibodies in allergy patients.

[0217] Diagnostic kits and diagnostic methods (2) The present invention provides a method for providing an indicator for diagnosing a target allergy, comprising the following steps: (i) The sample obtained from the subject is brought into contact with the antigen, where the sample is a solution containing IgE antibodies; (ii) Detect the binding of IgE antibodies to the antigen in the sample obtained from the subject; (iii) If binding of the target IgE antibody to the antigen is detected, it provides an indicator that the subject is allergic; The present invention provides a method comprising, wherein the antigen is a polypeptide which is at least one polypeptide containing the above (E1)-(E50) amino acid sequence, or a polypeptide in which two or more polypeptides containing the above (E1)-(E50) amino acid sequence are linked together with or without a spacer.

[0218] Hereinafter, a polypeptide that is at least one of the polypeptides containing the amino acid sequences (E1)-(E50) above, or a polypeptide in which two or more polypeptides containing the amino acid sequences (E1)-(E50) above are linked with or without a spacer, may be referred to as an "antigen containing (E1)-(E50) above" in this specification. The type of spacer is not particularly limited, and any spacer commonly used by those skilled in the art to link multiple peptides can be used. The spacer may be a polypeptide such as a hydrocarbon chain such as Acp(6)-OH or an amino acid chain.

[0219] When polypeptides containing the amino acid sequences (E1)-(E50) above are linked together with or without spacers, the number of linked polypeptides is not particularly limited. In one embodiment, it may be 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, or 15 or more. In one embodiment, it may be 30 or less, 20 or less, 15 or less, 10 or less, 8 or less, 6 or less, 5 or less, 3 or less, or 2 or less.

[0220] Among the polypeptides containing the above amino acid sequences (E1)-(E50), the same sequence may be repeated, or multiple different sequences may be linked together. Even when multiple polypeptides containing the above amino acid sequences (E1)-(E50) are linked together in this way, they can be used as polypeptides of the present invention and applied to the methods, kits, and compositions of the present invention.

[0221] The samples obtained from the subjects are as described in the "Diagnostic Kit and Diagnostic Method (1)" section above.

[0222] Contact between the sample obtained from the target and the polypeptide, and detection of its binding, can be performed by known methods described in the "Diagnostic Kits and Diagnostic Methods (1)" section above, such as ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, and immunochromatography.

[0223] The polypeptide containing the amino acid sequence (E1)-(E50) above may be immobilized on a carrier. In this case, ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance, etc., can be used in steps (i) and (ii) above, and step (i) is performed by bringing the sample obtained from the target into contact with the surface on which the polypeptide containing the amino acid sequence (E1)-(E50) above is immobilized. Alternatively, the IgE antibody of the target may be immobilized on a carrier, and its binding to the polypeptide containing the amino acid sequence (E1)-(E50) above may be detected by the above method. To facilitate binding to the carrier, to create space between the carrier and the polypeptide, or to facilitate contact between the antibody and the polypeptide, a spacer or a tag such as biotin may be attached to the N-terminus or C-terminus of the polypeptide. In the case of binding with biotin, it is preferable that the carrier contains avidin.

[0224] The polypeptide containing the amino acid sequence (E1)-(E50) described above may not be immobilized on a carrier. In this case, in steps (i) and (ii) described above, flow cytometry or the like can be used to confirm the presence of the polypeptide containing the amino acid sequence (E1)-(E50) to which IgE antibodies are bound by laser light. One example of this method is the basophil activation test (BAT), which detects the surface antigen CD203c that appears when basophils are activated by contact with the polypeptide containing the amino acid sequence (E1)-(E50). Another example is the histamine release test (HRT), which examines whether histamine is released by further contacting blood cells in a sample with the polypeptide containing the amino acid sequence (E1)-(E50).

[0225] Polypeptides containing the amino acid sequence (E1)-(E50) above are antigens that specifically bind to the IgE antibodies of allergic patients. Therefore, if binding between the target IgE antibody and the antigen is detected, it provides an indicator that the subject is allergic, including cross-reactivity. In the synthesis of polypeptides containing the amino acid sequence (E1)-(E50) above, sequences may be added before and after the epitope to lengthen the sequence, for example, to facilitate synthesis using E. coli. Even in such cases, if binding between the target IgE antibody and the amino acid sequence (E1)-(E50) above is detected, it provides an indicator that the subject is allergic, including cross-reactivity. Therefore, anything may be added before and after the amino acid sequence (E1)-(E50) above, which is the epitope.

[0226] The present invention also provides an allergy diagnostic kit comprising at least one polypeptide containing the amino acid sequence (E1)-(E50) described above. The diagnostic kit of the present invention may be used in a method for providing an indicator for diagnosing the above allergy, or in the diagnostic method described below. In addition to comprising at least one polypeptide containing the amino acid sequence (E1)-(E50) described above, the diagnostic kit of the present invention may also comprise an enzyme-labeled anti-IgE antibody and a chromogenic or luminescent substrate that is a substrate for the enzyme. Alternatively, a fluorescently labeled anti-IgE antibody may be used. In the diagnostic kit of the present invention, the polypeptide containing the amino acid sequence (E1)-(E50) described above may be provided immobilized on a carrier. The diagnostic kit of the present invention may also be provided with instructions for a diagnostic procedure and a package containing such instructions.

[0227] In another embodiment, the diagnostic kit described above includes a companion diagnostic for allergies. A companion diagnostic is used to identify patients who are expected to benefit from a drug, or patients who are at risk of serious side effects from a drug, or to assess the responsiveness of a drug in order to optimize treatment with that drug. Treatment optimization here includes, for example, determining the dosage and administration, deciding when to discontinue administration, and confirming which allergen component will induce immune tolerance.

[0228] The present invention also provides an allergy diagnostic composition comprising at least one polypeptide containing the amino acid sequences (E1)-(E50) described above. The diagnostic composition of the present invention can be used in the following diagnostic methods. The diagnostic composition of the present invention may optionally contain pharmaceutically acceptable carriers and additives that are commonly used with the polypeptide of the present invention.

[0229] In one embodiment, the present invention is a method for diagnosing a target allergy, comprising the following steps: (i) Contact the sample obtained from the subject with the antigen; (ii) Detect the binding of IgE antibodies to the antigen in the sample obtained from the subject; (iii) If binding of the target IgE antibody to the antigen is detected, the subject is determined to be allergic; The present invention provides a method comprising, wherein the antigen is at least one polypeptide identified as a polypeptide comprising the amino acid sequence (E1)-(E50) above. Herein, steps (i) and (ii) are carried out as described for each step of the method for providing an indicator for diagnosing allergies.

[0230] In another embodiment, the present invention provides a method for diagnosing an allergy to a subject, comprising administering at least one polypeptide containing the amino acid sequence (E1)-(E50) to the subject. The method may be carried out in the form of a skin test characterized by applying the polypeptide containing the amino acid sequence (E1)-(E50) to the skin. Skin tests include forms such as a prick test, in which a diagnostic composition is applied to the skin, and then a small wound is made without causing bleeding to allow the polypeptide containing the amino acid sequence (E1)-(E50) to penetrate the skin and observe the skin reaction; a scratch test, in which the skin is lightly scratched over the applied diagnostic composition and the reaction is observed; a patch test, in which a diagnostic composition in the form of a cream or ointment is applied to the skin and the reaction is observed; and an intradermal test, in which the polypeptide containing the amino acid sequence (E1)-(E50) is administered intradermally and the reaction is observed. If a skin reaction such as swelling occurs in the area of ​​skin to which the polypeptide containing the amino acid sequence (E1)-(E50) has been applied, the subject is diagnosed with an allergy. Here, the amount of the polypeptide applied to the skin may be, for example, 100 μg or less per application.

[0231] In diagnosing allergies, oral food challenge tests are often performed to identify antigens and verify the severity of symptoms after antigen intake. At least one polypeptide containing the amino acid sequences (E1)-(E50) above can be used as an active ingredient in an oral food challenge test to diagnose allergies. The polypeptide used in the oral food challenge test may be an expressed and purified polypeptide, or it may be one expressed in a raw material or processed product, such as pollen rice, which is produced by transforming rice with a cedar pollen antigen gene and expressing the polypeptide in the rice.

[0232] In one embodiment, the above-mentioned diagnostic composition and diagnostic kit can be used for prick tests, scratch tests, patch tests, intradermal tests, and the like.

[0233] In another embodiment, the present invention provides at least one polypeptide comprising the above (E1)-(E50) amino acid sequence for use in the diagnosis of allergies.

[0234] In yet another embodiment, the present invention provides the use of at least one polypeptide comprising the above-mentioned amino acid sequences (E1)-(E50) in the manufacture of an allergy diagnostic agent.

[0235] In this section, the allergy to be diagnosed may be an allergy to a polypeptide containing the amino acid sequences (E1)-(E50) described above. In other words, the diagnosis of an allergy, including the detection of allergies and the provision of diagnostic indicators, may diagnose not only an allergy to a single polypeptide containing the amino acid sequences (E1)-(E50) described above, but also allergies including cross-reactivity.

[0236] Composition / Treatment method (2) The present invention provides a composition comprising at least one polypeptide containing the above amino acid sequences (E1)-(E50).

[0237] In one embodiment, the composition of the present invention is a pharmaceutical composition. In another embodiment, the composition of the present invention is a quasi-drug composition, a non-pharmaceutical composition (for example, a cosmetic composition, a food composition).

[0238] In one embodiment, the above composition is used to treat allergies. The treatment of allergies involves increasing the limit of polypeptide intake that does not cause a reaction in the body, ultimately aiming for a state in which no reaction occurs with normal polypeptide intake (remission).

[0239] The present invention also provides a method for treating allergies, comprising administering at least one polypeptide comprising the above amino acid sequences (E1)-(E50) to a patient who requires treatment for allergies.

[0240] In another embodiment, the present invention provides at least one polypeptide comprising the above (E1)-(E50) amino acid sequence for use in the treatment of allergies. In yet another embodiment, the present invention provides the use of at least one polypeptide comprising the above (E1)-(E50) amino acid sequence for the manufacture of therapeutic drugs for allergies.

[0241] In the treatment of allergies, desensitization therapy is often performed, which aims to induce immune tolerance by administering an antigen to the patient. At least one polypeptide containing the amino acid sequences (E1)-(E50) above can be used as an active ingredient for desensitization therapy for allergies. Here, the antigen used in desensitization therapy may be an expressed and purified polypeptide, or it may be one that has been expressed in a raw material or processed product, such as pollen rice.

[0242] The route of administration, dosage, number of administrations and / or duration of administration of the composition of the present invention, other components contained in the composition, and dosage form may be as described in the "Composition and Treatment Method (1)" section above. When using a polypeptide containing the amino acid sequence (E1)-(E50) above, the dosage may be, for example, 100 μg or less per dose for adults.

[0243] In this section, the allergy to be treated may be an allergy to a polypeptide containing the amino acid sequence (E1)-(E50) described above. In other words, the treatment of an allergy may not only be an allergy to a single polypeptide containing the amino acid sequence (E1)-(E50) described above, but may also include allergies involving cross-reactivity.

[0244] Tester composition (2) The present invention provides a tester composition comprising an antibody against at least one polypeptide containing the above amino acid sequences (E1)-(E50).

[0245] The antibody can be produced by conventional methods. For example, it may be produced by immunizing a mammal such as a rabbit with a polypeptide containing the amino acid sequences (E1)-(E50) above. The antibody may be an Ig antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (e.g., Fab, F(ab')2, Fab').

[0246] Furthermore, in the above tester composition, the antibody may be provided in a form conjugated to a carrier. The carrier is not particularly limited as long as it is a carrier that can be used to detect the binding of the antibody to a polypeptide containing the amino acid sequence (E1)-(E50) above. Any carrier known to those skilled in the art can be used. In addition, it is preferable that the antibody against the polypeptide containing the amino acid sequence (E1)-(E50) above is an antibody against a polypeptide having the same amino acid sequence as the epitope and important amino acids described in the "Antigen Epitope" section above. This makes it possible to create a tester composition that can detect cross-reactivity as well.

[0247] For example, the following methods can be used to determine whether or not a polypeptide containing the above amino acid sequences (E1)-(E50) is present.

[0248] A method for determining whether a target raw material or processed product contains a polypeptide containing the amino acid sequence (E1)-(E50) is present in the target raw material or processed product by contacting a sample obtained from a raw material or processed product with a tester composition containing the prepared antibody, and detecting the binding of the antibody to a polypeptide containing the amino acid sequence (E1)-(E50) in the sample using, for example, ELISA. (The "method for determining whether or not polypeptide is present" includes determining that the polypeptide has been removed or reduced if the binding of the antibody to the polypeptide containing the amino acid sequence (E1)-(E50) is reduced.) A method of impregnating filter paper or similar material with raw materials or processed products, and reacting it with an antibody solution to detect polypeptides containing the above-mentioned amino acid sequences (E1)-(E50).

[0249] In another embodiment of the present invention, a tester composition for determining the presence or absence of a polypeptide containing the above (E1)-(E50) amino acid sequences of an allergen in a subject is provided, characterized by comprising a primer corresponding to a polypeptide having the same amino acid sequence as the epitope and key amino acid. Not limited thereto, the primer may be designed to include, for example, a portion of the nucleotide sequence or a complementary strand of a nucleic acid encoding the amino acid sequence identified above (E1)-(E50). Alternatively, in a nucleic acid encoding a protein containing a polypeptide having the same amino acid sequence as the epitope and key amino acid, the primer may be designed to be the nucleotide sequence of the upstream region of the portion encoding the polypeptide having the same amino acid sequence as the epitope and key amino acid, or the nucleotide sequence of the complementary strand of the downstream region of the portion encoding the polypeptide having the same amino acid sequence as the epitope and key amino acid. Examples of such primers include primers that are part of at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27, and / or primers that are part of a sequence complementary to at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27. Here, the epitope locations in the full-length antigen sequence are as identified in Table 2 based on the results of the examples. Furthermore, especially when targeting mRNA, complementary primers with a poly-A tail may be used.

[0250] For example, using DNA or mRNA obtained from a sample as a template, the DNA is amplified by PCR (Polymerase Chain Reaction), including RT-PCR, using the primers mentioned above. The presence or absence of an antigen containing (E1)-(E50) is determined by determining whether the amplified DNA sequence contains nucleic acids encoding the amino acid sequences identified in (E1)-(E50) above. Examples of methods for amplifying mRNA by PCR include the RACE method. If one of the three possible open reading frames encoded in the amplified DNA contains the amino acid sequence identified in (E1)-(E50) above, it is determined that the antigen is present. If the DNA is not amplified, it is determined that the antigen is not present.

[0251] In one embodiment, the above tester composition is used to determine whether or not a target object, such as a raw material or processed product manufacturing line, contains a polypeptide containing the above amino acid sequence (E1)-(E50). The raw material may be a food ingredient, a cosmetic ingredient, a pharmaceutical ingredient, etc. The processed product may be a processed food product, a cosmetic, a pharmaceutical, etc. The above tester composition may be used to search for biological species contained in the raw material, to inspect the quality of the manufacturing line and pre-shipment products by the manufacturer, or to check for the presence or absence of antigens in the target raw material or processed product by the consumer or user themselves.

[0252] Method for determining the presence or absence of polypeptides (2) The present invention includes a method for determining the presence or absence of a polypeptide containing the above amino acid sequences (E1)-(E50) in a raw material or processed product. The method includes detecting a polypeptide having all or part of the amino acid sequence of the polypeptide containing the above amino acid sequences (E1)-(E50) in a raw material or processed product.

[0253] In one embodiment, the method of the present invention includes a step of determining the presence or absence of a target substance containing the polypeptides (E1)-(E50) by contacting an antibody against at least one polypeptide containing the above amino acid sequence (E1)-(E50) with a raw material / processed product (including a liquid).

[0254] The definition of the antibody, raw materials / processed products, the method of producing the antibody, the method of contacting the antibody with the raw materials / processed products, and the binding of the antibody to the antigen are as described above in "Tester Composition (2)".

[0255] Alternatively, the method for determining the presence or absence of the above-mentioned antigen also includes detecting the epitope portion of a polypeptide containing the above-mentioned (E1)-(E50) amino acid sequence contained in the antigen. The "eptope portion" is preferably 4 or more amino acid residues, 6 or more amino acid residues, or 8 or more amino acid residues. Detection of the epitope portion can be performed by known methods for detecting a specific amino acid sequence of a part of a polypeptide. For example, one method may involve cleaving the protein of the target raw material or processed product (e.g., food ingredient) with a digestive enzyme for antigen removal, separating it by HPLC, and measuring whether the peak of an arbitrary epitope peptide has decreased due to the antigen removal treatment. Alternatively, the presence or absence of an antigen containing the polypeptide containing the above-mentioned (E1)-(E50) amino acid sequence in the target substance may be determined using an antibody that recognizes the portion of the polypeptide containing the above-mentioned (E1)-(E50) amino acid sequence.

[0256] Antigen removal raw materials, etc. (2) The present invention provides a raw material or processed product characterized in that at least one of the polypeptides containing the above amino acid sequences (E1)-(E50) is removed or reduced.

[0257] The method for removing or reducing the antigen of the present invention in raw materials or processed products is not limited. The removal or reduction of the antigen may be carried out by any method, as long as the polypeptide containing the amino acid sequence (E1)-(E50) above is removed or reduced, for example, by using the methods described in the "Antigen-Removed Foods, etc." section above.

[0258] The removal or reduction of at least one polypeptide containing the amino acid sequences (E1)-(E50) above may be achieved by the removal or reduction of the entire amino acid sequence, or by the cleavage or removal of the amino acid sequence portions (E1)-(E50) above from the antigen protein. "Removed" includes deletion and modification of all or part of the sequence portions specified in (E1)-(E50) above.

[0259] For example, a raw material from which polypeptides containing the above amino acid sequence (E1)-(E50) have been removed or reduced may be prepared using gene modification technology to produce a raw material in which polypeptides containing the above amino acid sequence (E1)-(E50) are no longer expressed. Gene modification knockout technology can be any method known to those skilled in the art.

[0260] Processed products from which polypeptides containing the amino acid sequences (E1)-(E50) above have been removed or reduced may be processed products using raw materials from which polypeptides containing the amino acid sequences (E1)-(E50) above have been removed or reduced, such as powdered milk made from protein digests. When using ordinary raw materials, processing to remove or reduce polypeptides containing the amino acid sequences (E1)-(E50) above is performed before, during, or after the preparation of the processed product. "Preparation of processed products" means, for example, preparing processed food products (e.g., wheat products, such as bread, pasta, udon, cakes, etc.) from food raw materials (e.g., wheat).

[0261] As a method for removing or reducing polypeptides containing the amino acid sequences (E1)-(E50) above in processed products using ordinary raw materials, the methods described in the "Antigen-Removed Foods, etc." section above may be used. As a method for cleaving polypeptides containing the amino acid sequences (E1)-(E50) above, a method of cleaving with a specific digestive enzyme is used.

[0262] Method for producing raw materials or processed products from which antigens have been removed or reduced (2) The present invention provides a method for producing a raw material or processed product in which at least one polypeptide comprising the amino acid sequence (E1)-(E50) is removed or reduced, the method comprising the step of confirming that the antigen is removed or reduced during the manufacturing process of the processed product.

[0263] In the said manufacturing method, the removal or reduction of the polypeptide containing the amino acid sequence (E1)-(E50) means that at least one of the polypeptides containing the above-mentioned amino acid sequence (E1)-(E50)(E1)-(E50) is removed or reduced, or that the sequence portion specified by (E1)-(E50) above is cleaved or removed from the antigen.

[0264] The method for confirming that polypeptides are removed or reduced during the manufacturing process of the raw material or processed product is not particularly limited, and any method capable of detecting at least one polypeptide containing the amino acid sequence (E1)-(E50) above may be used. For example, the presence or absence of the polypeptide in the raw material or processed product may be confirmed by the binding affinity of an antibody against at least one polypeptide containing the amino acid sequence (E1)-(E50) above to a sample containing materials generated during the manufacturing process of the raw material or processed product. Details of such a method are described in the "Diagnostic Kit / Diagnostic Method (2)" section above. That is, in the above manufacturing method, the "target IgE antibody" in the "Diagnostic Kit / Diagnostic Method (2)" section above can be replaced with "antibody against at least one polypeptide containing the amino acid sequence (E1)-(E50) above," and the "antigen" and "polypeptide" in the "Diagnostic Kit / Diagnostic Method (2)" section above can be replaced with "sample containing materials generated during the manufacturing process of the processed product," and the method described in the "Diagnostic Kit / Diagnostic Method (2)" section above can be used to confirm that the antigen is removed or reduced during the manufacturing process of the processed product. Alternatively, the tester compositions described in the "Tester Composition (2)" section above can also be used.

[0265] The present invention also relates to the use of kits in methods for diagnosing allergies, the use of kits for diagnosing allergies and / or providing indicators for diagnosis, compositions for use in methods for diagnosing allergies, the use of compositions for diagnosing allergies and / or providing indicators for diagnosis, methods for diagnosing allergies and / or providing indicators for diagnosis, the use of antigens (protein antigens or epitope antigens) in methods for detecting the presence or absence of IgE antibodies in samples obtained from subjects (living organisms such as humans), antigens (protein antigens or epitope antigens) for use in the treatment of allergies, kits or compositions containing antigens (protein antigens or epitope antigens) for detecting the binding between IgE antibodies and antigens (protein antigens or epitope antigens) in samples obtained from subjects (living organisms such as humans), and the use of kits or compositions containing antigens (protein antigens or epitope antigens) for detecting the binding between IgE antibodies and antigens (protein antigens or epitope antigens) in samples obtained from subjects (living organisms such as humans). The definitions of terms such as "antigen" are as described above. [Examples]

[0266] Examples of the present invention are described below. The technical scope of the present invention is not limited by these examples. Example 1: Confirmation of protein patterns The proteins contained in wheat (Triticum aestivum) were investigated using the following two-dimensional electrophoresis method.

[0267] Protein extraction The extraction and purification of proteins from wheat was performed as follows: Proteins were extracted by adding a mammalian cell lysis kit; MCL1 (manufactured by SIGMA-ALDRICH) to wheat flour. The composition of the mammalian cell lysis kit; MCL1 is as follows: 50 mM Tris-HCl pH 7.5 1 mM EDTA 250 mM NaCl 0.1% (w / v) SDS 0.5%(w / v) Deoxycholic acid sodium salt 1% (v / v) Igepal CA-630 (Surfactant manufactured by SIGMA-ALDRICH: Octylphenoxy polyethoxyethanol) An appropriate amount of protease inhibitor was added, followed by two precipitation steps using a 2D-CleanUP kit (GE). In the first precipitation step, trichloroacetic acid (TCA) was added to the recovered protein extract, and the resulting precipitate (TCA precipitate) was collected. In the second precipitation step, acetone was added to the recovered TCA precipitate, and the resulting precipitate (sample) was collected.

[0268] Preparation of sample solution A portion of the obtained sample (50 μg in protein weight) was dissolved in 150 μl of DeStreak Rehydration Solution (GE), a buffer used for swelling the gel for first-dimensional isoelectric focusing, to prepare the sample solution (swelling sample solution) for first-dimensional isoelectric focusing. The composition of DeStreak Rehydration Solution is as follows: 7M Thiourea 2M urea 4% (w / v) CHAPS 0.5% (v / v) IPG buffer; manufactured by GE. An appropriate amount of BPB (bromophenol blue) Sample infiltration into a gel for first-dimensional isoelectric focusing. A first-dimensional isoelectric focusing gel (GE IPG gel Immobiline Drystrip (pH3-10NL)) was immersed in 140 μl of the aforementioned first-dimensional isoelectric focusing sample solution (expansion sample solution) and allowed to permeate overnight at room temperature.

[0269] In this embodiment, GE's IPGphor was used as the electrophoresis apparatus.

[0270] The electrophoresis tray was filled with silicone oil. Moistened filter paper was placed on both ends of the gel impregnated with the sample, and the gel was placed in the electrophoresis tray so that it was covered with silicone oil. The electrodes were then set with the filter paper sandwiched between the gel and the filter paper.

[0271] The current limit of the isoelectric focusing electrophoresis apparatus was set to 75 μA per gel, and the voltage program was as follows: (1) A constant voltage process was performed at 300 V for up to 750 Vhr (the current change range during the 30 minutes of electrophoresis before the end of this process was 5 μA), (2) the voltage was gradually increased to 1000 V over 300 Vhr, (3) the voltage was further gradually increased to 5000 V over 4500 Vhr, and (4) the first dimension of isoelectric focusing was performed at a constant voltage of 5000 V until the total Vhr reached 12000.

[0272] Equilibration of isoelectric focusing gels with SDS After performing the first-dimensional isoelectric focusing described above, the gel was removed from the isoelectric focusing apparatus and immersed in an equilibration buffer containing a reducing agent, and shaken for 15 minutes at room temperature. The composition of the equilibration buffer containing the reducing agent is as follows. 100 mM Tris-HCl (pH 8.0) 6M urea 30% (v / v) glycerol 2% (w / v) SDS 1% (w / v) DTT Next, the equilibration buffer containing the reducing agent was removed, and the gel was immersed in an equilibration buffer containing the alkylating agent. The gel was shaken for 15 minutes at room temperature to obtain an SDS-equilibrium gel. The composition of the equilibration buffer containing the alkylating agent is as follows. 100 mM Tris-HCl (pH 8.0) 6M urea 30% (v / v) glycerol 2% (w / v) SDS 2.5% (w / v) Iodoacetamide Second-dimensional SDS-PAGE In this embodiment, the XCell SureLock Mini-Cell from Life Technologies was used as the electrophoresis apparatus. NuPAGE 4-12% Bis-Tris Gels from Life Technologies were used as the gel for 2D electrophoresis. Furthermore, an electrophoresis buffer with the following composition was prepared and used. 50mM MOPS 50 mM Tris base 0.1% (w / v) SDS 1 mM EDTA In this example, an agarose solution for adhesion was used, which was prepared by dissolving 0.5% (w / v) agarose S (manufactured by Nippon Gene Co., Ltd.) and an appropriate amount of BPB (bromophenol blue) in the electrophoresis buffer.

[0273] After thoroughly washing the wells of the SDS-PAGE with the electrophoresis buffer described above, the buffer used for washing was removed. Next, a fully dissolved agarose solution for adhesion was added to the wells. Then, the SDS-equalized gel was immersed in the agarose, and the SDS-equalized gel and the gel for the second dimension electrophoresis were brought into close contact using tweezers. After confirming that the agarose had solidified sufficiently with the two gels in close contact, electrophoresis was performed at a constant voltage of 200V for approximately 45 minutes.

[0274] Fluorescence staining of gels Fluorescent staining of the gel was performed using SYPRO Ruby (manufactured by Life Technologies).

[0275] First, the sealed container to be used was thoroughly washed with 98% (v / v) ethanol beforehand. The 2D electrophoresis gel was removed from the SDS-PAGE instrument after electrophoresis and placed in the washed sealed container. This was then immersed twice in an aqueous solution containing 50% (v / v) methanol and 7% (v / v) acetic acid for 30 minutes each. After that, the aqueous solution was replaced with water and the gel was immersed for 10 minutes. Next, the 2D electrophoresis gel was immersed in 40 ml of SYPRO Ruby and shaken overnight at room temperature. Then, the SYPRO Ruby was removed, the 2D electrophoresis gel was washed with water, and then shaken in an aqueous solution containing 10% (v / v) methanol and 7% (v / v) acetic acid for 30 minutes. Finally, the aqueous solution was replaced with water and shaken for 30 minutes or more.

[0276] analysis The gels for 2D electrophoresis, after undergoing the above-described process, were subjected to fluorescence imaging scanning using a Typhoon 9500 (GE). Figure 1 shows the results of 2D electrophoresis for proteins contained in wheat grain. On the left side of each gel photograph, a band of molecular weight markers can be seen, and the position of the band indicates a specific molecular weight (KDa).

[0277] Example 2: Antigen confirmation by immunoblotting Antigen confirmation by immunoblotting was performed by following the procedure described in Example 1 up to "second-dimensional SDS-PAGE," followed by the following operations: "transfer to membrane," "immunoblotting," and "analysis."

[0278] Transfer to membrane Transfer to the membrane was performed using the following transfer apparatus and transfer buffer. Transfer equipment: XCell SureLock Mini-Cell and XCell II blot module (manufactured by Life Technologies) Transfer buffer: NuPAGE Transfer Buffer (×20) (manufactured by Life Technologies) was used, diluted 200-fold with milliQ water.

[0279] Specifically, proteins in the two-dimensional electrophoresis gel were transferred to a membrane (PVDF membrane) following the procedure below.

[0280] (1) The PVDF membrane was immersed in 100% methanol, then in milliQ water, and then transferred to a transfer buffer to perform hydrophilization treatment of the PVDF membrane.

[0281] (2) The sponge, filter paper, gel after the second dimension of SDS-PAGE, hydrophilized PVDF membrane, filter paper, and sponge were set in that order, and the device was powered at a constant voltage of 30V for 1 hour.

[0282] Imbubrot Immunoblotting of the membranes was performed using serum from patients with wheat allergy (WDEIA cases and general immediate-type allergy cases that do not require exercise to trigger) or serum from non-wheat allergy subjects as the primary antibody.

[0283] Immunoblotting of the membrane was performed according to the following procedure. (1) The transferred membrane was shaken at room temperature for 1 hour in a 5% skim milk / PBST solution (PBS buffer containing 0.1% nonionic surfactant Tween 20). (2) As the primary antibody, it was left to stand at room temperature for 1 hour in a 4% serum / 3% skim milk / PBST solution. (3) Washed with PBST solution (5 minutes x 3 times). (4) Anti-human IgE-HRP (horseradish peroxidase) was diluted 1000-fold with 3% skim milk / PBST solution as a secondary antibody and left to stand at room temperature for 1 hour. (5) Washed with PBST solution (5 minutes x 3 times). (6) The sample was left to stand for 5 minutes in Pierce Western Blotting Substrate Plus (Thermo).

[0284] analysis The membranes that underwent the above series of processes were subjected to fluorescence image scanning using a Typhoon 9500 (manufactured by GE).

[0285] Immunoblots using serum from wheat allergy patients were compared with immunoblots using serum from control subjects without wheat allergies. Regarding proteins contained in wheat, the immunoblots using serum from wheat allergy patients detected 16 spots that differed from those using serum from non-wheat allergy subjects and were distinct from known wheat allergen proteins (Figure 2). Analysis of the amino acid sequences in Example 3 revealed that spots 11 and 12, and spots 15 and 16 originated from the same protein. The isoelectric points of each spot are listed in Table 1.

[0286] Example 3: Mass spectrometry and antigen identification The amino acid sequences of the antigens that produce each of the above-mentioned spots were identified by mass spectrometry.

[0287] Specifically, protein extraction and mass spectrometry were performed using the following procedure. (1) For wheat, protein extraction, two-dimensional electrophoresis, and membrane transfer were performed according to the procedures of Examples 1 and 2, and the mixture was stained with 0.008% Direct blue / 40% ethanol / 10% acetic acid by shaking. (2) After that, the material was decolorized by treating it with 40% ethanol and 10% acetic acid for 5 minutes three times, then washed with water for 5 minutes and air-dried. (3) The target spot was cut out with a clean cutter blade and placed in a centrifuge tube. After hydrophilic treatment of the membrane with 50 μL of methanol, it was washed twice with 100 μL of water and removed by centrifugation, and 20 μL of 20 mM NH4HCO3·50% acetonitrile was added. (4) Add 1 μL of 1 pmol / μL lysyl endopeptidase (WAKO), and let stand at 37°C for 60 minutes. Then collect the solution in a new centrifuge tube. Add 20 μL of 20 mM NH4HCO3·70% acetonitrile to the membrane, immerse at room temperature for 10 minutes, and collect again. Dissolve in 10 μL of 0.1% formic acid and 4% acetonitrile, and transfer to a tube. (5) The recovered solution was dried under reduced pressure, then dissolved in 15 μl of solution A (0.1% formic acid, 4% acetonitrile solution), and mass spectrometry (ESI-TOF6600, AB Sciex) was performed. (6) Protein identification based on mass data obtained from mass spectrometers was performed by searching NCBI.

[0288] result The amino acid sequence of each spot was detected, and further analysis of the mass data obtained from the mass spectrometer for each spot using Uniprot revealed that each spot was the protein shown in Table 1.

[0289] Spots 11 and 12, and spots 15 and 16 originate from the same protein. The reason these spots were recognized as separate spots in the immunoblot of Example 2 is that even though the amino acid sequence is the same, post-translational modifications such as glycosylation and phosphate group modification altered the isoelectric point and / or molecular weight. Binding to the IgE antibody was observed despite the differences in post-translational modifications, indicating that post-translational modifications have no effect.

[0290] Example 4: Epitope Identification Epitope of wheat allergen component The epitopes of wheat allergen components were identified using the following procedure.

[0291] (A) Wheat epitope mapping (1) Epitope mapping was performed using a library of overlap peptides (length: 15 amino acids) corresponding to amino acid sequences identified as allergenic components of wheat. Specifically, the overlap peptide library was prepared based on the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28.

[0292] Each peptide synthesized was shifted by 10 amino acids. That is, each peptide has a 5-amino acid overlap with the preceding and succeeding peptides.

[0293] For the preparation of the peptide array, Intavis CelluSpots™ technology was used. Specifically, the following procedure was followed: (1) On an amino-modified cellulose disk, an automated synthesizer (Intavis MultiPep (1) The target peptide was synthesized using RS, (2) the amino-modified cellulose disk was dissolved to obtain a cellulose-bound peptide solution, and (3) the cellulose-bound peptide was spotted onto a coated glass slide to prepare the peptide array. Details of each procedure are as follows.

[0294] (1) Peptide synthesis Peptide synthesis was performed stepwise using the 9-fluorenylmethoxycarbonyl (Fmoc) chemical reaction on an amino-modified cellulose disc in a 384-well synthesis plate. Specifically, an amino acid with an Fmoc group attached to its amino group was activated with a solution of N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in dimethylformamide (DMF), and this solution was added dropwise to the cellulose disc to bond the Fmoc-bound amino acid to the amino group on the cellulose disc (coupling). Unreacted amino groups were capped with acetic anhydride and washed with DMF, then treated with piperidine and washed with DMF again to remove the Fmoc group from the amino group of the amino acid attached to the amino group on the cellulose disc. Peptide synthesis was performed by repeatedly performing the above coupling, capping, and removal of the Fmoc group on the amino acid attached to the amino group on the cellulose disc to extend the amino terminus.

[0295] (2) Dissolution of amino-modified cellulose disc The cellulose disc to which the target peptide obtained in "(1) Peptide Synthesis" above was bound was transferred to a 96-well plate and treated with a side-chain deprotection mixture of trifluoroacetic acid (TFA), dichloromethane, triisopropylsilane (TIPS), and water to deprotect the amino acid side chains. Subsequently, the deprotected cellulose-bound peptide was dissolved in a mixture of TFA, perfluoromethanesulfonic acid (TFMSA), TIPS, and water, precipitated with tetrabutyl methyl ether (TBME), resuspended in dimethyl sulfoxide (DMSO), and mixed with a mixture of NaCl, sodium citrate, and water to obtain a peptide solution for slide spots.

[0296] (3) Spot of cellulose-bound peptide solution The peptide solution for slide spotting obtained in "(2) Dissolution of amino-modified cellulose disc" above was spotted onto an Intavis CelluSpots® slide using an Intavis slide spotting robot, and the resulting slide was dried to prepare a peptide array.

[0297] Using the aforementioned peptide array, we measured whether IgE antibodies in the serum of wheat allergy patients bound to each peptide fragment via antigen-antibody reaction. The measurement was performed according to the following procedure. (1) The peptide was shaken in Pierce Protein-Free (PBS) Blocking Buffer (Thermo) at room temperature for 1 hour. (2) 2% serum / Pierce Protein-Free (PBS) Blocking Buffer (Thermo) Inside, it was shaken overnight at 4°C. (3) PBST (PBS buffer containing 3% nonionic surfactant Tween 20) I washed it for 5 minutes (3 times). (4) Anti-human IgE antibody-HRP (1:20,000, Pierce Protein-Fr ee (PBS) Blocking Buffer (Thermo) was added and the mixture was shaken at room temperature for 1 hour. (5) Washed with PBST for 5 minutes (x3 times). (6) Add Pierce ECL Plus Western Blotting Substrate (manufactured by Thermo). In addition, it was shaken at room temperature for 5 minutes. (7) Using the Amersham Imager 600, perform the processes described in (1) to (6) above. The chemiluminescence of the peptide was measured.

[0298] The chemiluminescence was quantified using ImageQuant TL (GE Healthcare) for the images obtained from the measurements described in (7) above. The second highest value among the quantified values ​​from the images obtained using the serum of 5 non-wheat allergy subjects was defined as the N2nd value. Peptides with a value of 35,000 or more, obtained by taking the difference in N2nd values ​​from the images obtained using the serum of 49 patients (32 WDEIA cases, 8 children with general wheat immediate-type allergy, and 9 adults), were judged to be patient-specific peptides that bound to IgE antibodies.

[0299] As a result, peptides derived from spot 1-spot 16 (spots 11 and 12 and spots 15 and 16 are derived from the same protein) that do not contain known epitopes (SEQ ID NOs: 29, 131, 191, 281, 346, 414, 493, 562, 640, 706, 776, 899, 974, 1010, 1089, 1142, 1166, 1209, 1257, 1297, 1367, Patient-specific IgE antibody binding was confirmed in 1403, 1508, 1566, 1598, 1608, 1685, 1753, 1789, 1836, 1895, 1977, 2004, 2027, 2068, 2154, 2217, 2265, 2309, 2342, 2419, 2491, 2545, 2613, 2631, 2674, 2701, 2774, 2809, and 2830.

[0300] (B) Wheat epitope mapping (2): Overlapping The above (A) shows the sequences of peptides to which patient-specific serum IgE antibodies are bound ((SEQ ID NOs: 29, 131, 191, 281, 346, 414, 493, 562, 640, 706, 776, 899, 974, 1010, 1089, 1142, 1166, 1209, 1257, 1297, 1367, 1403, 1508, 1566, 1598, 1608, 1685, 1753, 1789, 1836, 1895, 1977, 2004, 2027, 2068, Based on (2154, 2217, 2265, 2309, 2342, 2419, 2491, 2545, 2613, 2631, 2674, 2701, 2774, 2809, 2830), a library of overlapping peptide fragments (length: 10 amino acids) was created using the sequence of the peptide in question and the sequences obtained by adding the sequences before and after the peptide in the amino acid sequence of the allergy component containing the peptide, and epitope mapping was performed.

[0301] Each peptide synthesized was shifted by one amino acid. That is, each peptide has a 9-amino acid overlap with the preceding and succeeding peptides.

[0302] The library was prepared using the same procedure as in (A) above, and the binding of IgE antibodies in the patient's serum to each peptide fragment was measured using the same method as above. The numerical values ​​obtained from images obtained from the results when only (1), (4) to (6) described in the (3) Cellulose-bound peptide solution spot section were performed were used as control values, and the difference between the control values ​​of each peptide and the numerical values ​​obtained from images obtained using the patient's serum was compared with the values ​​obtained from the peptide that formed the basis of the overlapping. Peptides in which binding to the patient's IgE antibodies was lost or significantly reduced by the peptide shifted by one amino acid were judged to be peptides that do not bind to IgE antibodies.

[0303] Similar to (A) above, the amount of chemiluminescence was quantified for the images obtained by measurement. The values ​​quantified from the images obtained from the results (secondary antibody measurement values) when only (1), (4)~(6) described in the (3) Cellulose-bound peptide solution spot column were performed were used as control values, and the difference between the control values ​​of each peptide and the values ​​quantified from the images obtained from the results using patient serum was taken, and the sequences that formed the basis of overlapping (SEQ ID NOs: 29, 131, 191, 281, 346, 414, 493, 562, 640, 706, 776, 899, 974, 1010, 1089, 1142, 1166, 1209, 1257, 1297, 1367, 1403, 1508, 1566, 1598, 1608, 1685, 1753, 17 When the values ​​obtained from (89, 1836, 1895, 1977, 2004, 2027, 2068, 2154, 2217, 2265, 2309, 2342, 2419, 2491, 2545, 2613, 2631, 2674, 2701, 2774, 2809, 2830) are set to 100%, values ​​of less than 30% indicate no binding to IgE antibodies, values ​​between 30% and less than 50% indicate poor but present binding to IgE antibodies, values ​​between 50% and less than 70% indicate slightly poor but present binding to IgE antibodies, and values ​​of 70% or more indicate no difference in or good binding to IgE antibodies. Thus, it was determined that the peptide retained binding to IgE antibodies.

[0304] This analysis revealed a region in the sequence used as the basis for overlapping that is crucial for binding to the patient's IgE antibody.

[0305] (C) Wheat epitope mapping (3): Alaning lysine scan For the amino acid sequences identified in (A) above, a library of peptide fragments was prepared by substituting one amino acid at a time with alanine (or glycine if the original amino acid was alanine) from the amino-terminus using a method called Alaning lysine scanning (Non-Patent Document 5). The library was prepared using the same method as above, and the binding of each peptide fragment to IgE antibodies in the patient's serum was measured using the same method as above. Amino acids at positions where binding to the patient's IgE antibodies was lost or significantly reduced due to Alaning lysine substitution were judged to be amino acids important for the expression of the original antigenicity, or amino acids that affect the expression of the original antigenicity. Amino acids where binding to the patient's IgE antibodies was not lost or significantly reduced were judged to be amino acids that are not important for the expression of the original antigenicity and can be substituted.

[0306] Similar to (A) above, the amount of chemiluminescence was quantified for the images obtained by measurement. The values ​​quantified from the images obtained from the secondary antibody measurement were used as control values, and the difference between the control values ​​of each peptide and the values ​​quantified from the images obtained from the results using serum from 49 patients was taken, and the sequences that formed the basis of the Alaning lysine scan (SEQ ID NOs: 29, 131, 191, 281, 346, 414, 493, 562, 640, 706, 776, 899, 974, 1010, 1089, 1142, 1166, 1209, 1257, 1297, 1367, 1403, 1508, 1566, 1598, 1608, 1685, 1753, 1789, 1836, 1895, 1977, 2004) were used. When the values ​​obtained from (2027, 2068, 2154, 2217, 2265, 2309, 2342, 2419, 2491, 2545, 2613, 2631, 2674, 2701, 2774, 2809, 2830) are set to 100%, values ​​of less than 30% indicate no binding to IgE antibodies, values ​​of 30% to less than 50% indicate poor but present binding to IgE antibodies, values ​​of 50% to less than 70% indicate slightly poor but present binding to IgE antibodies, and values ​​of 70% or more indicate no difference in binding to IgE antibodies or good binding to IgE antibodies. Thus, it was determined that the peptide retained binding to IgE antibodies.

[0307] Analysis of the results from (A) to (C) revealed a common sequence crucial for the expression of intrinsic antigenicity in the region of the sequence used as the basis for the Alaning lysine scan that is important for binding to the patient's IgE antibody. Sequences were identified for all 50 epitopes, and the results are summarized in Table 2.

[0308] Example 5: Confirmation of epitope cross-reactivity For each epitope sequence found for each wheat protein in Table 2 above, an arbitrary amino acid (X) was selected, which is an amino acid other than the amino acid important for maintaining binding with IgE antibodies. A search of NCBI for proteins with the same sequence in allergen foods simultaneously present in each patient yielded, for example, amino acid sequences found in the sequences of each food listed in the "Cross-relevance Confirmed Foods" column of Table 2.

[0309] For peptides containing the amino acid sequences listed in the "Synthetic Sequence" column and the "Sequence Number" column (far right) of Table 2, the binding affinity to IgE antibodies from patients allergic to each food listed in the "Cross-reactivity Confirmed Foods" column of Table 2 was confirmed by ELISA. The peptides were synthesized using the Fmoc method so that they were biotinylated at the N-terminus.

[0310] The ELISA was performed according to the following specific procedure.

[0311] (1) The biotinylated peptide was prepared in PBST (0.1% Tween 20) to a concentration of 10 μg / mL.

[0312] (2) 20 μL of each peptide solution was added to each well of a streptavidin-coated 384-well plate and shaken at room temperature for 1 hour. After collecting the solutions, they were washed five times with PBS.

[0313] (3) Add 40 μL of Pierce Protein-Free (PBS) Blocking Buffer (Thermo) and shake at room temperature for 1 hour. After removing the solution, wash five times with PBST.

[0314] (4) Add 20 μL of 2% serum / Canget SignalSolution I (TOYOBO) and shake at room temperature for 1 hour. After removing the solution, wash 5 times with PBST.

[0315] (5) Add 20 μL of secondary antibody diluent (1:10000, Canget SignalSolution II (TOYOBO)) and shake at room temperature for 1 hour. After removing the solution, wash five times with PBST.

[0316] (6) Add 20 μL of 1-Step Ultra TMB-ELISA (Thermo) and shake at room temperature for 15 minutes.

[0317] (7) 20 μL of 2 M H2SO4 was added. The absorbance at 450 nm was measured.

[0318] Peptides having these amino acid sequences were prepared using the same procedure as in Example 4(A), and it was measured whether or not IgE antibodies bound to them in the serum of allergy patients and non-allergy subjects. For non-allergy subjects, serum from two individuals was measured and the average value was used.

[0319] The results are shown in Figure 3-6. The bar graphs in each figure represent "absorbance of allergic patients / absorbance of non-allergic patients (healthy individuals)." The food names listed in each graph are food names containing polypeptides that include the amino acid sequences that form the basis of each synthetic sequence used.

[0320] As is evident from Figure 3-6, all polypeptides with the amino acid sequences shown in the figure exhibited higher binding affinity (greater than "1") to the IgE antibodies of each allergy patient than to the IgE antibodies in the serum of non-allergy subjects, confirming the cross-reactivity of these polypeptides. This indicates that these epitopes can be used to detect cross-reactivity with antigens other than wheat. Furthermore, it supports the idea that the "X" portion can take any amino acid residue.

Claims

1. A polypeptide that specifically binds to IgE antibodies in wheat allergy patients, and is one of the following polypeptides: (E9), (E36), and (E43): (E9) Polypeptides comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 640-705 and 2885-2888; (E36) Polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 2154-2216; (E43) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 2545-2612.

2. A diagnostic kit for wheat allergy comprising at least one polypeptide specified as (E9), (E36), and (E43) in claim 1.

3. A diagnostic composition for wheat allergy, comprising at least one polypeptide specified as (E9), (E36), and (E43) in claim 1.

4. A method for providing indicators for diagnosing a target wheat allergy, comprising the following steps: (i) The sample obtained from the subject is brought into contact with the antigen, where the sample is a solution containing IgE antibodies; (ii) Detect the binding of IgE antibodies to the antigen in the sample obtained from the subject; (iii) If binding of the target IgE antibody to the antigen is detected, it provides an indicator that the subject has a wheat allergy; The method comprising, wherein the antigen is at least one of the polypeptides specified as (E9), (E36), and (E43) in claim 1.

5. The antigen is an antigen that causes wheat allergy, and is at least one of the polypeptides specified as (E9), (E36), and (E43) in claim 1.

6. A composition comprising at least one of the antigens described in claim 5.

7. The composition according to claim 6 for treating wheat allergy.

8. A tester composition for determining the presence or absence of wheat antigen in a subject, comprising an antibody that conjugates to at least one of the polypeptides specified as (E9), (E36), and (E43) in claim 1.

9. A tester composition for determining the presence or absence of wheat antigen in a subject, comprising at least one primer comprising a portion of the base sequence and / or a portion of the complementary strand thereof of a nucleic acid encoding a polypeptide as specified as (E9), (E36), and (E43) in claim 1.

10. A wheat allergy tester composition for determining the presence or absence of IgE antibodies in a subject, comprising the polypeptides specified as (E9), (E36), and (E43) in claim 1.

11. A method for determining the presence or absence of polypeptides specified as (E9), (E36), and (E43) in claim 1 in a raw material or processed product, comprising detecting polypeptides specified as (E9), (E36), and (E43) in claim 1 in the raw material or processed product, wherein the raw material or processed product is wheat or a wheat product.

12. A method for producing a raw material or processed product from which an antigen has been removed or reduced, wherein the raw material or processed product is wheat or a wheat processed product, and the step of confirming that the antigen has been removed or reduced during the manufacturing process of the processed product, wherein the antigen is at least one of the polypeptides specified as (E9), (E36), and (E43) in claim 1.

Images