New antigens in milk allergy

Abstract

To provide a milk allergy detection method that is high in reliability, and to provide a medical treatment-purpose composition required for the detection.SOLUTION: The present invention provides: a new antigen of an allergy to milk; a medical treatment and a medical treatment kit of the allergy to the milk; a composition including the antigen; milk or a milk finished product having the antigen removed; and a tester composition for determining presence or absence of a milk antigen in an object. Also, the present invention relates to: polypeptide including epitope of the antigen; an allergy medical treatment kit including the polypeptide; a medical treatment-purpose composition; a medical treatment method; a composition including the polypeptide; and a raw material or the finished product having an antigen including the polypeptide removed or reduced. The present invention further relates to the tester composition for determining the presence or absence of the antigen in the object.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel antigen for cow's milk allergy. The present invention also relates to a diagnostic kit, diagnostic composition, and diagnostic method for cow's milk allergy. The present invention also relates to a composition containing the antigen, and raw materials or processed products in which the antigen has been removed or reduced. The present invention further relates to a tester composition for determining the presence or absence of a cow's milk antigen in a subject.

[0002] The present invention also relates to an antigen of the polypeptide comprising the epitope. The present invention also relates to a diagnostic kit, diagnostic composition, and diagnostic method for allergy comprising the polypeptide. The present invention also relates to a method for providing an indicator for diagnosing allergy in a subject. The present invention also relates to a composition comprising the polypeptide, and a raw material or processed product from which the polypeptide has been removed or reduced. The present invention further relates to a method for producing a raw material or processed product from which the polypeptide has been removed or reduced. The present invention still further relates to a tester composition for determining the presence or absence of an antigen comprising the polypeptide in a subject. [Background technology]

[0003] IgE antibodies specific to specific antigens (hereinafter referred to as allergens) are produced in the serum and tissues of allergic patients. The physiological consequences of the interaction between these IgE antibodies and specific antigens trigger an allergic reaction. In a broad sense, an antigen refers to foods and ingredients that cause allergic symptoms, and in a narrow sense, it refers to proteins contained in foods and ingredients (hereinafter referred to as allergen components) that are bound by specific IgE antibodies.

[0004] In conventional allergy test drugs, antigen reagents are often prepared by simply grinding foods or ingredients that are allergen candidates (Patent Document 1). For this reason, it was only possible to detect a positive reaction in an allergy test if the content of the many allergen components contained in the conventional antigen reagent exceeded the threshold at which a positive reaction could be determined for binding with IgE antibodies, and the diagnostic efficiency was not sufficiently high.

[0005] Several allergen components have been identified in candidate foods and ingredients, and test kits have been commercialized. To increase the reliability of allergy testing, comprehensive identification of allergen components is necessary; however, the patient detection rate by measuring the above allergen components is still insufficient. Identifying novel allergens in milk is extremely important not only to improve the accuracy of diagnostic reagents, but also as targets for hypoallergenic foods, hypoallergenic ingredients, and therapeutic drugs.

[0006] Meanwhile, with regard to the separation and purification of proteins, two-dimensional electrophoresis, in which isoelectric focusing is performed in the first dimension and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is performed in the second dimension, has recently been used as a method for separating and purifying a variety of proteins from a small amount of sample. The applicants have previously developed two-dimensional electrophoresis methods with high resolution (Patent Documents 2 to 5).

[0007] Allergen-specific IgE antibodies recognize and bind to epitopes, which are specific amino acid sequences in allergen components. However, although there are a few examples in which epitopes have been analyzed for allergen components (Non-Patent Document 1), the current situation is extremely limited. 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] Patent Publication No. 2002-286716 [Patent Document 2] Patent Publication No. 2011-33544 [Patent Document 3] Patent Publication No. 2011-33546 [Patent Document 4] Patent Publication No. 2011-33547 [Patent Document 5] 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 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention provides a novel protein antigen for allergies. The present invention also provides a method and kit for diagnosing allergies that includes the antigen. The present invention also provides a composition that includes the antigen, and raw materials or processed products in which the antigen has been removed or reduced. The present invention further provides a tester composition for determining the presence or absence of the antigen in a subject.

[0011] The present invention also provides an antigen of the polypeptide comprising the epitope. The present invention also provides a diagnostic kit, diagnostic composition, and diagnostic method for allergies comprising the polypeptide. The present invention also provides a method for providing an indicator for diagnosing allergies in a subject. The present invention also provides a composition comprising the polypeptide, and a raw material or processed product from which an antigen comprising the polypeptide has been removed or reduced. The present invention further relates to a method for producing a raw material or processed product from which the antigen has been removed or reduced. The present invention still further provides a tester composition for determining the presence or absence of an antigen comprising the polypeptide in a subject. [Means for solving the problem]

[0012] In order to solve the above problems, the present inventors have conducted extensive research into identifying the antigen responsible for cow's milk allergy, and as a result have succeeded in identifying a novel antigen, a protein that specifically binds to IgE antibodies in the serum of patients with cow's milk allergy.

[0013] Furthermore, because epitopes have relatively short amino acid sequences, if the same amino acid sequence is present in different allergen components, the IgE antibody can bind to multiple allergen components. As a result of the presence of a common epitope in different allergen components, IgE antibodies from allergy patients bind to both, making the antigen cross-reactive. Therefore, the epitopes identified by the present 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, the term "antigen" is used to include both an antigen in the narrow sense, which is a protein, and an "epitope" derived from a protein. When specified, the term "antigen" is used to mean either a protein or an epitope derived from a protein.

[0015] Based on the above findings, the present invention has been completed. The present invention includes, but is not limited to, the following aspects. [Aspect 1] A kit for diagnosing allergies, comprising at least one of the following polypeptides (E1)-(E25): (E1) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 27-58, and 1115; (E2) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 59-97; (E3) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 98-144; (E4) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 145-195; (E5) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 196-254 and 1116-1118; (E6) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 255-288; (E7) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 289-333, and 1119-1120; (E8) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 334-352; (E9) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 353-376, and 1121-1122; (E10) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 377-384; (E11) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 385-411; (E12) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 412-450; (E13) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 451-500, and 1123-1124; (E14) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 501-604 and 1125-1127; (E15) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 605-632, and 1128; (E16) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 633-673; (E17) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 674-693; (E18) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 694-757, and 1129; (E19) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 758-791; (E20) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 792-868, and 1130; (E21) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 869-927; (E22) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 928-996; (E23) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 997-1037; (E24) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1038-1075; or (E25) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1076-1114 and 1131. [Aspect 2] A composition for diagnosing allergies, comprising at least one of the polypeptides identified as (E1) to (E25) in embodiment 1. [Aspect 3] 1. A method for providing an indicator for diagnosing allergy in a subject, comprising the steps of: (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) if binding of the subject's IgE antibodies to the antigen is detected, an indication that the subject is allergic is provided; wherein the antigen is at least one of the polypeptides identified as (E1) to (E25) in embodiment 1. [Aspect 4] The antigen is at least one of the polypeptides identified as (E1) to (E25) in Aspect 1, and is the cause of allergy. [Aspect 5] A composition comprising at least one antigen according to embodiment 4. [Aspect 6] 6. The composition according to embodiment 5, for treating allergies. [Aspect 7] A tester composition for determining the presence or absence of an antigen in a subject, comprising an antibody that binds to at least one of the polypeptides identified as (E1) to (E25) in embodiment 1. [Aspect 8] A tester composition for determining the presence or absence of an antigen in a subject, comprising at least one primer comprising a portion of the base sequence of a nucleic acid encoding a polypeptide identified as (E1) to (E25) in Aspect 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 the polypeptides identified as (E1) to (E25) in embodiment 1. [Aspect 10] A method for determining the presence or absence of a polypeptide identified as (E1) to (E25) in aspect 1 in a raw material or processed product, the method comprising detecting a polypeptide identified as (E1) to (E25) in aspect 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 (E25) in Aspect 1. [Aspect 12] A method for producing a raw material or processed product from which an antigen has been removed or reduced, the method comprising a step of confirming that the antigen has been removed or reduced during the production of the raw material or processed product, wherein the antigen is at least one of the polypeptides specified as (E1) to (E25) in Aspect 1. [Effects of the Invention]

[0016] The present invention provides a novel antigen for allergies (e.g., allergy to cow's milk). Because a novel allergen component that causes allergies has been identified in the present invention, it is possible to provide a highly sensitive method and kit for diagnosing allergies, a composition containing the antigen, raw materials or processed products in which the antigen has been removed or reduced, and a tester composition for determining the presence or absence of the antigen in a target substance.

[0017] The present invention also provides a novel polypeptide antigen containing an epitope of a protein antigen. By utilizing the polypeptide of the present invention, it is possible to provide a highly sensitive diagnostic kit, diagnostic composition, and diagnostic method for allergies (e.g., allergy to cow's milk), a composition containing the polypeptide, a tester composition for determining the presence or absence of an antigen containing the polypeptide in a subject, as well as 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 drawings]

[0018] [Figure 1] Figure 1 is a photograph of a gel showing the migration pattern of proteins in milk by two-dimensional electrophoresis. The bands on the left side of the photograph are molecular weight marker bands, and the numbers on the left side of the photograph are 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 a photograph of an immunoblot using serum from a patient with milk allergy against the two-dimensional electrophoresis pattern of proteins contained in milk. Spots 1 to 14, which specifically reacted with IgE antibodies in the serum of patients with milk allergy compared to healthy controls, are each enclosed in a white frame. [Figure 3] FIG. 3 shows the results of examining cross-reactivity by ELISA for peptides having the amino acid sequence of each epitope using serum from a patient (P1) who has an allergy to cow's milk. [Figure 4] FIG. 4 shows the results of examining cross-reactivity by ELISA for peptides having the amino acid sequence of each epitope using serum from a patient (P5) who is allergic to cow's milk. [Figure 5] FIG. 5 shows the results of examining cross-reactivity by ELISA for peptides having the amino acid sequence of each epitope using serum from a patient (P8) who is allergic to cow's milk. [Figure 6] FIG. 6 shows the results of examining cross-reactivity by ELISA for peptides having the amino acid sequence of each epitope using serum from a patient (P20) who has an allergy to cow's milk. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be specifically described below, but the present invention is not limited thereto.

[0020] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those skilled in the art.

[0021] As used herein, allergy refers to a state in which an organism sensitized to a certain antigen exhibits an adverse hypersensitivity reaction when that antigen is reintroduced. An allergic reaction can occur when the organism comes into contact with or ingests the antigen. Here, "contact" refers to touching an object, and in the case of the human body, it particularly refers to adhesion to the skin or mucous membranes (eyes, lips, etc.). Furthermore, "ingestion" refers to taking something into the body, such as by inhalation or oral administration. Generally, an allergic reaction that occurs when food is ingested is specifically referred to as a food allergy. 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. The IgE antibodies bind to mast cells or basophils. When an antigen specific to the IgE antibody reintroduces itself into the body of an allergic patient, the antigen combines with the IgE antibody bound to mast cells or basophils, resulting in the physiological effects of IgE antibody-antigen interaction. These physiological effects include the release of histamine, serotonin, heparin, eosinophil chemotactic factor, and various leukotrienes. These released substances trigger allergic reactions caused by the combination of IgE antibodies and specific antigens. Specifically, IgE antibodies recognize and bind to epitopes, which are specific amino acid sequences in specific antigens, and allergic reactions caused by these antigens manifest through the above pathways.

[0022] The allergy targeted by the present invention is not particularly limited as long as it is an allergy to an allergen (antigen) containing the epitope used. In one embodiment, the allergen includes milk, dairy products, processed milk products, meat, processed meat products, grains, seafood, fruits, vegetables, nuts (seeds), edible plants, seafood, eggs, processed eggs, etc., which are ingested by living organisms (especially humans), as well as parasites that infect living organisms (especially humans).

[0023] The origin of the milk is not particularly limited. In one embodiment, it includes milk from cows, goats, sheep, etc. In one embodiment, it is cow's milk. Milk includes, but is not limited to, raw milk, pasteurized milk, modified milk, low-fat milk, and non-fat milk. Dairy products are products processed from milk, which is the main ingredient. Dairy products include, but are not limited to, cream, butter, butter oil, cheese, whey, concentrated whey, ice cream, concentrated milk, concentrated skim milk, unsweetened evaporated milk, unsweetened evaporated skim milk, sweetened evaporated milk, sweetened evaporated skim milk, whole milk powder, skim milk powder, cream powder whey powder, protein-enriched whey powder, buttermilk powder, sweetened milk powder, modified milk powder, modified liquid milk, fermented milk such as yogurt, lactic acid bacteria drinks, and milk drinks. Milk processed products refer to dairy products processed from milk, which is the main ingredient, and foods containing this as an ingredient. Non-limiting examples of processed milk products include cream, butter, butter oil, cheese, whey, concentrated whey, ice cream, concentrated milk, concentrated skim milk, unsweetened evaporated milk, unsweetened evaporated skim milk, sweetened condensed milk, sweetened evaporated skim milk, whole milk powder, skim milk powder, cream powder whey powder, protein-enriched whey powder, buttermilk powder, sweetened milk powder, modified milk powder, modified liquid milk, fermented milk such as yogurt, lactic acid bacteria drinks, milk drinks, and cakes, pastries, custard pudding, milk agar, biscuits, cookies, snacks, chocolates, bread, white sauce, potage, cream stew, gratin, curry roux, and stew roux, which contain these as ingredients.

[0024] The type of meat is not particularly limited. In one embodiment, it includes meat from birds (chicken, duck, etc.) and mammals (pork, beef, lamb, etc.). In one embodiment, it is mammalian meat. In one embodiment, it is bovine (Bos taurus) meat. Processed meat products refer to products processed from meat as the main ingredient, and foods containing it as an ingredient. Processed meat products include, but are not limited to, ham, sausage, bacon, bouillon, consommé, steak, yakiniku, roast beef, steak tartare, hamburger steak, hamburger, meatballs, and nuggets.

[0025] Grains are a general term for food ingredients obtained from plants, and refer to edible seeds, primarily starchy. In a narrow sense, grains refer only to the seeds of grass-family crops (cereals), but in a broader sense, this includes seeds of legume-family crops (pulses) and seeds of crops from other families. Within the broad definition of grains, the seeds of dicotyledonous plants used as grains are collectively called pseudocereals or pseudocereals because they resemble cereal seeds (seeds of monocotyledonous grass-family crops). Pseudocereals include buckwheat (Polygonaceae), amaranth (Amaranthaceae), and quinoa (Chenopodiaceae).

[0026] In the present invention, examples of cereal grains of the Poaceae family include, but are not limited to, bread wheat (Triticum aestivum), barley, rye, oat, timothy grass, orchard grass, sweetgrass, millet, foxtail millet, barnyard millet, corn, and Job's tears. Examples of cereal grains of the Polygonaceae family include buckwheat (Fagopyrum esculentum) of the Polygonaceae family. In one embodiment, the antigen (allergen) is derived from a cereal grain. In one embodiment, the allergen is derived from a cereal grain of the Poaceae family. In one embodiment, the allergen is derived from wheat.

[0027] Seafood includes, but is not limited to, shrimp, crabs, and the like, which belong to the order Decapoda (Shrimp). Most of what is generally recognized as "crustaceans" are included in the order Decapoda (Shrimp). Seafood also includes, but is not limited to, squid, which belong to the orders Scombridae and Octopus, and octopus. Seafood further includes fish, which belong to the families Scombridae and Gadidae. Seafood also includes shellfish, which belong to the family Veneridae.

[0028] Non-limiting examples of fruits include fruits belonging to the Actinidiaceae, Bromeliaceae, Anacardiaceae, Cucurbitaceae, Musaceae, Rutaceae, and Rosaceae families. Vegetables include fruits belonging to the Solanaceae, Cucurbitaceae, and Lauraceae families. Nuts (seeds) include nuts (seeds) belonging to the Anacardiaceae, Rosaceae, and Juglandaceae families, and nuts belonging to the Fabaceae family such as peanuts. Edible herbs include edible herbs belonging to the Asteraceae family. Grains include grains belonging to the Poaceae and Polygonaceae families.

[0029] In the present invention, eggs include not only the above-mentioned seafood eggs but also avian eggs. Non-limiting examples of avian eggs include quail eggs and chicken eggs. Processed egg products refer to products made from eggs, which are the main ingredient, and foods containing eggs as ingredients. Non-limiting examples of processed egg products include processed fish eggs such as salted cod roe, spicy mentaiko, salted salmon roe, salmon roe pickled in soy sauce, herring roe, and dried mullet roe, as well as processed chicken eggs such as mayonnaise, hollandaise sauce, boiled quail eggs, boiled eggs, tamagoyaki (rolled omelet), atsuyaki (thick omelet), dashi-maki (rolled omelet), chawanmushi (steamed egg custard), omelets, omurice (omelette), baked eggs, poached eggs, quiche, Scotch eggs, French toast, cakes, pastries, custard pudding, biscuits, cookies, snacks, and bread.

[0030] Parasites include, but are not limited to, parasites of the Anisakidae family, including Anisakis simplex.

[0031] As used herein, allergy refers to a state in which an allergic reaction occurs due to an antigen, such as a protein contained in a raw material or processed product (e.g., milk or a processed milk product). An allergic reaction can occur when a person comes into contact with an antigen contained in a raw material or processed product (e.g., milk or a processed milk product) or when the person ingests the antigen. Generally, an allergic reaction that occurs when a food is ingested is specifically referred to as a food allergy. An allergy to milk may be a food allergy.

[0032] As used herein, an antigen is a substance that induces an allergic reaction. When the antigen is a protein contained in a raw material such as a food ingredient, it is also called an allergen component. The antigen is preferably a protein.

[0033] As used herein, 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. As used herein, the term "polypeptide" also refers to 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] When an amino acid has optical isomers, the L-isomer is indicated unless otherwise specified. The notation method for the amino acid sequence of a protein, polypeptide, or peptide used herein is based on standard usage and common practice in the art, and is represented by a single-letter notation for the amino acid, with the left-hand direction being the amino-terminal direction and the right-hand direction being the carboxy-terminal direction. In the single-letter notation for an amino acid, X represents any substance having an amino group and a carboxyl group that can bond to the amino acids at both ends, and in particular any of the 20 naturally occurring amino acids.

[0035] The alanine scanning method (or "alanine glycine scanning" method) is a method for site-specifically identifying residues important for the structure and function of a protein by mutating each residue in the protein to alanine (or glycine if the original amino acid is alanine) to create mutants. If binding to a patient's IgE antibody remains even after mutation to alanine (or glycine if the original amino acid is alanine), that residue is not important for IgE antibody binding, and binding will remain even if the residue is changed to another 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 the present application, the residue represented by X is an amino acid residue at a site that remains binding to an allergic patient's IgE antibody even when substituted with alanine (glycine if the original amino acid is alanine) by alanine glycine scanning as shown in Example 4. It is well known to those skilled in the art that such sites are highly likely to retain binding to the IgE antibody even when substituted with any other amino acid. That is, 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 important for subsequent allergic reactions, and this binding and maintenance is mediated by the charge, hydrophobic bonds, hydrogen bonds, and aromatic interactions of the epitope. The fact that binding and maintenance are possible even when these are lost by changing to alanine or glycine means that the amino acid is not important.

[0037] Identification of antigens Proteins contained in milk were subjected to two-dimensional electrophoresis under the conditions below to identify antigens causing milk allergy.

[0038] For the first-dimension electrophoresis, the gel length was 5-10 cm, the pH range was 3-10, and the pH gradient of the gel in the direction of migration was 0.15-0.3, 0.4-0.7, and 0.15-0.3, where the total length of the gel is 1, the gel length up to pH 5 is a, the gel length from pH 5 to 7 is b, and the gel length at pH 7 or higher is c. Specifically, isoelectric focusing was performed using IPG Gel Immobiline Drystrip (pH 3-10NL) manufactured by GE Healthcare Biosciences, Inc. (hereinafter abbreviated as GE). The electrophoresis apparatus used was an IPGphor manufactured by GE. The upper limit of the current value of the electrophoresis equipment was set to 75 μA per gel, and the voltage program was as follows: (1) a constant voltage step of 300 V was performed up to 750 Vhr (the current change during the 30 minutes of electrophoresis before the end of this step 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) first-dimension isoelectric focusing was then performed at a constant voltage of 5000 V until the total Vhr reached 12000.

[0039] Second-dimensional electrophoresis was performed using polyacrylamide gels with a gel concentration of 3-6% at the base of the electrophoresis direction and a higher gel concentration at the leading edge of the electrophoresis direction. Specifically, SDS-PAGE was performed using a NuPAGE 4-12% Bis-Tris Gels IPG Well Mini 1mm (Life Technologies). The electrophoresis apparatus used was an XCell SureLock Mini-Cell (Life Technologies). The electrophoresis buffer used was 50 mM MOPS, 50 mM Tris base, 0.1% (w / v) SDS, and 1 mM EDTA, and electrophoresis was performed at a constant voltage of 200 V for approximately 45 minutes.

[0040] As a result, when two-dimensional electrophoresis of milk proteins was performed under the above conditions, it was revealed that the antigens in spots 1 to 14 on the gel below specifically bind to the IgE antibodies of patients with milk allergies (Figure 2).

[0041] antigen (protein) The sequence of each spot was identified by mass spectrometry. The mass data obtained from the mass spectrometer was analyzed by collating it with protein data from Uniprot and NCBI. As a result, it was found that each of spots 1 to 14 matched with a known sequence. Information on each spot is summarized in Table 1 below.

[0042] [Table 1-1]

[0043] [Table 1-2]

[0044] [Table 1-3]

[0045] [Table 1-4]

[0046] [Table 1-5]

[0047] [Table 1-6]

[0048] [Table 1-7]

[0049] [Table 1-8]

[0050] [Table 1-9]

[0051] [Table 1-10]

[0052] [Table 1-11]

[0053] [Table 1-12]

[0054] [Table 1-13]

[0055] [Table 1-14] Spots 2 and 3 are derived from the same protein, and a total of 14 proteins (1)-(13) were identified as novel milk-derived antigens: (1) Immunoglobulin M heavy chain secretory form (spot 1); (2) Beta-1 metal-binding globulin (spots 2 and 3); (3) Lactoperoxidase (spot 4); (4) Ig heavy chain precursor (B / MT.4A.17.H5.A5) (spot 5); (5) Perilipin (spot 6); (6) Polymeric immunoglobulin receptor (spot 7); (7) C3-beta-c (C3-beta-c) (spot 8); (8) Immunoglobulin light chain, lambda gene cluster (spot 9); (9) Glycoprotein 2 (spot 10); (10) Lactadherin (spot 11); (11) Zinc-alpha-2-glycoprotein (spot 12); (12) Intracellular cholesterol transporter 2 (spot 13); (13) Apolipoprotein A-IV (spot 14).

[0056] As shown in Table 2, proteins (1)-(13) are classified into four groups with common functions: Group 1 (signal transduction function): proteins (1), (2), (3), (4), (5); Group 2 (immune system function): proteins (1), (4), (6), (7), (8); Group 3 (glycoprotein function): proteins (9), (10), (11); Group 4 (cholesterol binding function): proteins (12), (13).

[0057] In the present invention, the antigen of spot 1 may be, but is not limited to, any one of the following (1-a) to (1-f).

[0058] (1-a) a protein comprising the amino acid sequence of SEQ ID NO: 2; (1-b) a protein comprising the amino acid sequence of SEQ ID NO: 2 in which one or several amino acids are deleted, substituted, inserted, or added; (1-c) a protein containing an amino acid sequence having an identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 2; (1-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 1 have been deleted, substituted, inserted or added; (1-e) a protein containing an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 1; or (1-f) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1.

[0059] In the present invention, the antigens of spots 2 and 3 may be, but are not limited to, any of the following (2-a) to (2-f).

[0060] (2-a) a protein comprising the amino acid sequence of SEQ ID NO: 4; (2-b) a protein comprising the amino acid sequence of SEQ ID NO: 4 in which one or several amino acids are deleted, substituted, inserted, or added; (2-c) a protein containing an amino acid sequence having an identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 4; (2-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 3 have been deleted, substituted, inserted, or added; (2-e) a protein comprising an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 3; or (2-f) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 3.

[0061] In the present invention, the antigen of spot 4 may be, but is not limited to, any one of the following (3-a) to (3-f).

[0062] (3-a) a protein comprising the amino acid sequence of SEQ ID NO: 6; (3-b) a protein comprising the amino acid sequence of SEQ ID NO: 6 in which one or several amino acids are deleted, substituted, inserted, or added; (3-c) a protein containing an amino acid sequence having an identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 6; (3-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 5 have been deleted, substituted, inserted, or added; (3-e) a protein comprising an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 5; or (3-f) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 5.

[0063] In the present invention, the antigen of spot 5 may be any one of the following (4-a) to (4-f), but is not limited thereto.

[0064] (4-a) a protein comprising the amino acid sequence of SEQ ID NO: 8; (4-b) a protein comprising the amino acid sequence of SEQ ID NO: 8 in which one or several amino acids are deleted, substituted, inserted, or added; (4-c) a protein containing an amino acid sequence having an identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 8; (4-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 7 have been deleted, substituted, inserted, or added; (4-e) a protein comprising an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 7; or (4-f) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 7.

[0065] In the present invention, the antigen of spot 6 may be any one of the following (5-a) to (5-f), but is not limited thereto.

[0066] (5-a) a protein comprising the amino acid sequence of SEQ ID NO: 10; (5-b) a protein comprising the amino acid sequence of SEQ ID NO: 10 in which one or several amino acids are deleted, substituted, inserted, or added; (5-c) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 10; (5-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 9 have been deleted, substituted, inserted, or added; (5-e) a protein comprising an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 9; or (5-f) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 9.

[0067] In the present invention, the antigen of spot 7 may be any one of the following (6-a) to (6-f), but is not limited thereto.

[0068] (6-a) a protein comprising the amino acid sequence of SEQ ID NO: 12; (6-b) a protein comprising the amino acid sequence of SEQ ID NO: 12 in which one or several amino acids are deleted, substituted, inserted, or added; (6-c) a protein containing an amino acid sequence having an identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 12; (6-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 11 have been deleted, substituted, inserted, or added; (6-e) a protein comprising an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 11; or (6-f) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 11.

[0069] In the present invention, the antigen of spot 8 may be any one of the following (7-a) to (7-f), but is not limited thereto.

[0070] (7-a) a protein comprising the amino acid sequence of SEQ ID NO: 14; (7-b) a protein comprising the amino acid sequence of SEQ ID NO: 14 in which one or several amino acids are deleted, substituted, inserted, or added; (7-c) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 14; (7-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 13 have been deleted, substituted, inserted, or added; (7-e) a protein comprising an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 13; or (7-f) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 13.

[0071] In the present invention, the antigen of spot 9 may be any one of the following (8-a) to (8-f), but is not limited thereto.

[0072] (8-a) a protein comprising the amino acid sequence of SEQ ID NO: 16; (8-b) a protein comprising the amino acid sequence of SEQ ID NO: 16 in which one or several amino acids are deleted, substituted, inserted, or added; (8-c) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 16; (8-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 15 have been deleted, substituted, inserted, or added; (8-e) a protein comprising an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 15; or (8-f) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 15.

[0073] In the present invention, the antigen of spot 10 may be any one of the following (9-a) to (9-f), but is not limited thereto.

[0074] (9-a) a protein comprising the amino acid sequence of SEQ ID NO: 18; (9-b) a protein comprising the amino acid sequence of SEQ ID NO: 18 in which one or several amino acids are deleted, substituted, inserted, or added; (9-c) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 18; (9-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 17 have been deleted, substituted, inserted, or added; (9-e) a protein comprising an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 17; or (9-f) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 17.

[0075] In the present invention, the antigen of spot 11 may be any one of the following (10-a) to (10-f), but is not limited thereto.

[0076] (10-a) a protein comprising the amino acid sequence of SEQ ID NO: 20; (10-b) a protein comprising the amino acid sequence of SEQ ID NO: 20 in which one or several amino acids are deleted, substituted, inserted, or added; (10-c) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 20; (10-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 19 have been deleted, substituted, inserted, or added; (10-e) a protein comprising an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 19; or (10-f) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 19.

[0077] In the present invention, the antigen of spot 12 may be any one of the following (11-a) to (11-f), but is not limited thereto.

[0078] (11-a) a protein comprising the amino acid sequence of SEQ ID NO: 22; (11-b) a protein comprising the amino acid sequence of SEQ ID NO: 22 in which one or several amino acids are deleted, substituted, inserted, or added; (11-c) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 22; (11-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 21 are deleted, substituted, inserted, or added; (11-e) a protein comprising an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 21; or (11-f) A protein comprising 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 SEQ ID NO: 21.

[0079] In the present invention, the antigen of spot 13 may be any one of the following (12-a) to (12-f), but is not limited thereto.

[0080] (12-a) a protein comprising the amino acid sequence of SEQ ID NO: 24; (12-b) a protein comprising the amino acid sequence of SEQ ID NO: 24 in which one or several amino acids are deleted, substituted, inserted, or added; (12-c) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence set forth in SEQ ID NO: 24; (12-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 23 have been deleted, substituted, inserted, or added; (12-e) a protein comprising an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 23; or (12-f) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 23.

[0081] In the present invention, the antigen of spot 14 may be any one of the following (13-a) to (13-f), but is not limited thereto.

[0082] (13a) a protein comprising the amino acid sequence of SEQ ID NO: 26; (13-b) a protein comprising the amino acid sequence of SEQ ID NO: 26 in which one or several amino acids are deleted, substituted, inserted, or added; (13-c) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence set forth in SEQ ID NO: 26; (13-d) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 25 have been deleted, substituted, inserted, or added; (13-e) a protein comprising an amino acid sequence encoded by a nucleotide sequence having an identity of 70% or more to the nucleotide sequence shown in SEQ ID NO: 25; or (13-f) A protein comprising 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 SEQ ID NO: 25.

[0083] The antigenic proteins (1) to (13) above and the polypeptides (E1) to (E25) described below also include those in which amino acid residues of the proteins or polypeptides have been modified by phosphorylation, glycosylation, aminoacylation, ring-opening, deamination, or the like.

[0084] Preferably, the antigenic proteins (1) to (13) above and the polypeptides (E1) to (E25) described below are allergens.

[0085] As used herein, the phrase "one or several amino acids have been deleted, substituted, inserted, or added" in reference to an amino acid sequence refers to an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added. The term "several amino acids" refers to, but is not limited to, 200 or fewer, 100 or fewer, 50 or fewer, 30 or fewer, 20 or fewer, 15 or fewer, 12 or fewer, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, or 3 or fewer amino acids. Alternatively, the term "several amino acids" refers to 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2%, or 1% of the total amino acid sequence.

[0086] Among the above, the substitution is preferably a conservative substitution. A conservative substitution is a replacement of a specific amino acid residue with a residue having similar physicochemical characteristics, but any substitution may be made as long as it does not substantially change the structural characteristics of the original sequence, for example, any substitution may be made as long as the substituted amino acid does not disrupt the helix present in the original sequence or other types of secondary structure that characterize the original sequence. Below, conservative substitutions of amino acid residues are categorized by substitutable residues and exemplified, but the substitutable amino acid residues 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 D group: lysine, arginine, Group E: serine, threonine Group F: phenylalanine, tyrosine, tryptophan, histidine In the case of non-conservative substitutions, one member of the above-mentioned classes can be exchanged for another member of the other classes. For example, amino acids in the above-mentioned groups B, D, and E may be substituted with amino acids from other groups to eliminate inadvertent glycosylation. Alternatively, cysteines may be deleted or substituted with other amino acids to prevent folding into a protein in a tertiary structure. Alternatively, amino acids may be substituted taking into account the hydropathic index of amino acids, which is an index of hydrophobicity / hydrophilicity for amino acids (J. Kyte and R. Doolittle, J. Mol. Biol., Vol. 157, pp. 105-132, 1982), to maintain a balance of hydrophilicity / hydrophobicity or to increase hydrophilicity for easier synthesis.

[0087] In another embodiment, substitution with an amino acid that is less sterically hindering than the original amino acid, for example, substitution of group F with group A, B, C, D, or E, or substitution of a charged amino acid with an uncharged amino acid, for example, substitution of group B with group C, may be performed. This may improve the binding affinity to IgE antibodies.

[0088] As used herein, the percent identity between two amino acid sequences can be determined by visual inspection and mathematical calculation. Percent 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 in Altschul et al. (Nucl. Acids. Res., 25, pp. 3389-3402, 1997) and are publicly available from the websites of NCBI and the DNA Data Bank of Japan (DDBJ) (BLAST Manual, Altschul et al., NCB / NLM / NIH, Bethesda, MD 20894; Altschul et al.). Percent identity can also be determined using genetic information processing software programs such as GENETYX Ver. 7 (Genetyx), DNASIS Pro (Hitachi Software), and Vector NTI (Infomax).

[0089] As used herein, the phrase "one or several nucleotides deleted, substituted, inserted, or added" in reference to a base sequence refers to a base sequence in which one or several nucleotides have been deleted, substituted, inserted, and / or added. The term "several nucleotides" refers, but is not limited to, to 600 or less, 300 or less, 150 or less, 100 or less, 50 or less, 30 or less, 20 or less, 15 or less, 12 or less, 10 or less, 8 or less, 6 or less, 4 or less, or 3 or less nucleotides. Alternatively, "several nucleotides" refers to 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2%, or 1% of the total length of the base sequence. It is preferred that the deletion, substitution, insertion, or addition of the nucleotides does not result in a frameshift in the amino acid-encoding sequence.

[0090] As used herein, the percent identity between two nucleotide sequences can be determined by visual inspection and mathematical calculation. Percent 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 at the following internet site: http: / / blast.wustl.edu.

[0091] As used herein, "under stringent conditions" refers to hybridization under moderately or highly stringent conditions. Specifically, moderately stringent conditions can be easily determined by those skilled in the art, for example, based on the length of the DNA. Basic conditions are set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Chapters 6-7, Cold Spring Harbor Laboratory Press, 2001. Moderately stringent conditions are preferably hybridization conditions of 1xSSC to 6xSSC at 42°C to 55°C, more preferably 1xSSC to 3xSSC at 45°C to 50°C, and most preferably 2xSSC at 50°C. When 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, generally, 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 (highly stringent) conditions include hybridization and / or washing at a higher temperature and / or lower salt concentration 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, and more preferably 0.2×SSC, 60°C to 65°C.

[0092] In the variants corresponding to the above items (b) to (f) of (1) to (13), it is preferred that the variants contain the amino acid sequences of the epitopes (variants) described below. The amino acid sequences of the epitopes contained are not limited to one, and preferably contain all of the sequences of the epitopes derived from each of the proteins. For example, epitopes derived from protein (1) are (E1) and (E2). (1-b) to (1-f) preferably contain one or more amino acid sequences (including variants) of the (E1) or (E2) epitopes.

[0093] The antigen may be obtained by isolating and purifying it from milk using a combination of protein purification methods well known to those skilled in the art, or by expressing the antigen as a recombinant protein using gene recombination techniques well known to those skilled in the art, and then isolating and purifying it using protein purification methods well known to those skilled in the art.

[0094] Examples of protein purification methods include 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 hydroxylapatite chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reversed-phase high-performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing.

[0095] Proteins are prepared by recombinant DNA technology by preparing an expression vector containing a nucleic acid encoding an antigen, introducing the expression vector into suitable host cells by gene transfer or transformation, culturing the host cells under conditions suitable for expression of the recombinant protein, and recovering the recombinant protein expressed in the host cells.

[0096] A "vector" is a nucleic acid that can be used to introduce a nucleic acid linked thereto into a host cell, and an "expression vector" is a vector that can induce the expression of a protein encoded by the nucleic acid introduced by the vector. Vectors include plasmid vectors, viral vectors, etc. Those skilled in the art can select an appropriate expression vector for expressing a recombinant protein depending on the type of host cell to be used.

[0097] A "host cell" is a cell that is transfected or transformed with a vector. Those skilled in the art can appropriately select a host cell depending on the vector used. The host cell can be derived from a prokaryote, such as E. coli. When a prokaryote such as E. coli is used as a host, the antigen of the present invention may contain an N-terminal methionine residue to facilitate recombinant protein expression in the prokaryote. This N-terminal methionine can be cleaved from the recombinant protein after expression. Alternatively, the host cell can be a cell derived from a eukaryote, such as a unicellular eukaryote such as yeast, a plant cell, an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a silkworm.

[0098] Gene transfer or transformation of an expression vector into a host cell can be carried out appropriately by methods known to those skilled in the art. Furthermore, those skilled in the art can express a recombinant protein by culturing the host cells under appropriate conditions for recombinant protein expression, depending on the type of host cell. The host cells expressing the recombinant protein can then be homogenized, and the antigen expressed as the recombinant protein can be isolated and purified from the resulting homogenate by appropriately combining the above-mentioned protein purification methods. Antigens can also be prepared by introducing the above-mentioned expression vector, synthesized double-stranded DNA, or mRNA transcribed therefrom into a cell-free protein synthesis system, expressing the vector, and then isolating and purifying the expressed protein.

[0099] The antigen of the present invention specifically binds to IgE antibodies in allergic patients.

[0100] Diagnostic kits and methods (1) The present invention provides a method for providing an indicator for diagnosing allergies in a subject, comprising the steps of: (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) if binding of the subject's IgE antibodies to the antigen is detected, an indication that the subject is allergic is provided; wherein the antigen is at least one of the proteins identified as antigens in (1) to (13) above.

[0101] "Allergy" is, but is not limited to, in one embodiment, an allergy to milk, preferably an allergy to cow's milk.

[0102] As used herein, "diagnosis" generally includes not only a (definitive) diagnosis by a physician but also simple "detection," including possibility. In one aspect, "diagnosis" and "detection" as used herein refer to in vivo, in vitro, or ex vivo "diagnosis" and "detection." Preferably, "diagnosis" and "detection" are in vitro.

[0103] A sample obtained from a subject is a solution containing IgE antibodies collected from a subject. Examples of such solutions include blood, saliva, sputum, nasal discharge, urine, sweat, and tears. The sample obtained from a subject may be pretreated to increase the IgE antibody concentration in the sample before contacting it with an antigen. Pretreatment of the sample may include, for example, obtaining serum or plasma from the blood. Furthermore, the Fab portion, which is the binding portion to the antigen, may be purified. In a particularly preferred embodiment, the above step (i) is carried out by contacting the antigen with IgE antibodies in serum obtained from the subject.

[0104] The IgE antibody may be the IgE antibody itself, or may be a mast cell to which the IgE antibody is bound.

[0105] Contact between a sample obtained from a subject and an antigen and detection of its binding can be performed by known methods. Examples of such methods include ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, and immunochromatography. All of these methods involve contacting the subject's IgE antibody with the antigen to allow it to bind, allowing an enzyme-labeled secondary antibody to act on the IgE antibody specifically bound to the antigen, and then adding an enzyme substrate (usually a colorimetric or luminescent reagent) to detect the product of the enzyme reaction, thereby detecting the binding between the antigen and the subject's IgE antibody. Alternatively, fluorescently labeled secondary antibodies can be detected. Alternatively, surface plasmon resonance (SPR), or other measurement methods capable of evaluating antigen-IgE antibody binding, can also be used. Multiple antigen-specific IgE antibodies may be mixed.

[0106] The antigen may be isolated and immobilized on or within 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 contacting a sample obtained from a subject with a surface on which the antigen is immobilized. The isolated antigen may be obtained by separating and purifying from a subject (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. Alternatively, the antigen may have an antibody attached thereto.

[0107] 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. Further, a histamine release test (HRT) can also be mentioned, in which the antigen is further brought into contact with blood cells in the sample to examine whether histamine is released.

[0108] 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 of "Identification of Antigen" above can be used. Alternatively, the electrophoresis conditions can be determined referring to the descriptions of 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 pH 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 to one gel containing the specimen, 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 in 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 end in the migration direction of the two-dimensional electrophoresis gel is 3 to 6%; and (F) In the case of (E), the gel concentration at the leading end of the second-dimensional electrophoresis gel in the migration direction is set higher than the gel concentration at the base end in the migration direction; Two-dimensional electrophoresis can be performed under conditions that satisfy at least one selected from the group consisting of:

[0109] The antigens (1) to (13) above are antigens that specifically bind to IgE antibodies in allergic patients (e.g., patients allergic to cow's milk). Therefore, when binding between the subject's IgE antibodies and the antigens is detected, it provides an indication that the subject has an allergy.

[0110] The present invention also provides a diagnostic kit for allergies, comprising at least one of the antigens (1) to (13) above. The diagnostic kit of the present invention may be used in the above-mentioned method for providing an indicator for diagnosing allergies, or in the diagnostic method described below. In addition to comprising at least one of the antigens (1) to (13) 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. A fluorescently labeled anti-IgE antibody may also be used. In the diagnostic kit of the present invention, the antigen may be provided in a state immobilized on or within a carrier. The diagnostic kit of the present invention may also be provided together with instructions on the diagnostic procedure or a package containing such instructions.

[0111] In another embodiment, the diagnostic kit includes a companion diagnostic for allergies. The companion diagnostic is used to identify patients who are expected to benefit from a drug or who are at risk of serious side effects from the drug, or to examine the responsiveness of the drug to optimize treatment with the drug. Here, optimization of treatment includes, for example, determining the dosage and administration, deciding whether to discontinue administration, and identifying which allergen component is used to induce immune tolerance.

[0112] The present invention also provides a composition for diagnosing allergies, which contains at least one of the antigens (1) to (13) above. The diagnostic composition of the present invention can be used in the following diagnostic methods. The diagnostic composition of the present invention may contain, as needed, pharmaceutically acceptable carriers and additives that are commonly used together with the antigen of the present invention.

[0113] In one aspect, the present invention provides a method for diagnosing allergy in a subject, comprising the steps of: (i) contacting a sample obtained from a subject with an antigen; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) determining that the subject is allergic if binding of the subject's IgE antibody to the antigen is detected; wherein the antigen is at least one of the proteins identified as antigens in (1) to (13) above, wherein steps (i) and (ii) are carried out as described for each step of the method for providing an indicator for diagnosing allergies.

[0114] In another aspect, the present invention provides a method for diagnosing allergies in a subject, the method comprising administering to the subject at least one of the antigens (1) to (13) above. The method may be performed in the form of a skin test, characterized by applying the antigen to the skin. Skin tests include a prick test, in which a diagnostic composition is applied to the skin, followed by a small scratch that does not cause bleeding, allowing the antigen to penetrate the skin and observe a skin reaction; a scratch test, in which a diagnostic composition is applied and then the skin is slightly scratched to observe a reaction; a patch test, in which a diagnostic composition in the form of a cream or ointment is applied to the skin and a reaction is observed; and an intradermal test, in which an antigen is administered intradermally and a reaction is observed. If a skin reaction, such as swelling, occurs in the area where the antigen was applied, the subject is diagnosed as having an allergy. The amount of antigen applied to the skin may be, for example, 100 μg or less per administration.

[0115] In the diagnosis of allergies, a challenge test is often performed to identify the antigen. At least one of the antigens (1) to (13) above can be used as an active ingredient in the challenge test to diagnose allergies. Here, the antigen protein used in the challenge test may be an expressed and purified protein, or may be expressed in a raw material or processed product, such as pollen rice, in which a cedar pollen antigen gene is transformed into rice and the antigen protein is expressed in the rice.

[0116] 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.

[0117] In another aspect, the present invention provides at least one of the antigens (1) to (13) above for use in diagnosing allergies, including providing at least one of the antigens (1) to (13) above in combination with a known antigen.

[0118] In yet another aspect, the present invention provides use of at least one of the antigens (1) to (13) above in the production of a composition for diagnosing allergies.

[0119] Composition / Treatment method (1) The present invention provides a composition containing at least one of the antigens (1) to (13) above.

[0120] In one embodiment, the composition of the present invention is a pharmaceutical composition. In one embodiment, the composition of the present invention is a quasi-medicinal composition, a non-medicinal composition (e.g., a cosmetic composition, a food composition).

[0121] In one embodiment, the composition is used to treat allergies (e.g., allergies to cow's milk). As used herein, "treating allergies" refers to increasing the limit of the amount of antigen that can be ingested without causing symptoms, ultimately aiming to achieve a state (remission) in which symptoms do not appear with normal intake of the antigen.

[0122] The present invention also provides a method for treating allergies, which comprises administering at least one of the antigens (1) to (13) above to a patient in need of treatment for allergies.

[0123] In another aspect, the present invention provides at least one of the antigens (1) to (13) above for use in treating allergies. In yet another aspect, the present invention provides use of at least one of the antigens (1) to (13) above for the manufacture of a medicament for treating allergies.

[0124] In the treatment of allergies, hyposensitization therapy is often performed, with the goal of inducing immune tolerance by administering an antigen to a patient. At least one of the antigens (1) to (13) above can be used as an active ingredient for hyposensitization therapy for allergies. Here, the antigen protein used in hyposensitization therapy may be an expressed and purified protein, or may be expressed in a raw material or processed product, such as pollen rice, in which a cedar pollen antigen gene is transformed into rice and the antigen protein is expressed in the rice.

[0125] The compositions of the present invention can be administered by any conventional route of administration, including, for example, oral, sublingual, transdermal, intradermal, subcutaneous, intravascular, intranasal, intramuscular, intraperitoneal, and rectal administration.

[0126] The compositions of the present invention can be prepared by adding commonly used pharmaceutically acceptable adjuvants, excipients, or various additives (e.g., stabilizers, solubilizers, emulsifiers, buffers, preservatives, colorants, etc.) to the antigens of the present invention in a conventional manner, as needed. 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, troches, sublingual tablets, injections, nasal sprays, poultices, liquids, creams, lotions, suppositories, etc. The dosage, frequency of administration, and / or administration period of the compositions of the present invention can be appropriately selected by a physician depending on the route of administration, symptoms, and patient characteristics such as age and body weight. For example, in the case of adults, the composition may be administered at a dose of 100 μg or less per administration. The administration interval may be, for example, daily, once a week, twice a month, or once every three months. The administration period may be, for example, from several weeks to several years. The administration method may involve gradually increasing the dosage during the administration period.

[0127] Tester Composition (1) The present invention provides a tester composition containing an antibody against at least one of the antigens (1) to (13) above.

[0128] The antibody can be produced by a conventional method. For example, it may be produced by immunizing a mammal such as a rabbit with the antigens (1) to (13) 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').

[0129] In the tester composition, the antibody may be provided in a form immobilized or bound to a carrier or inside the carrier. The carrier is not particularly limited as long as it can be used to detect antibody-antigen binding. Any carrier known to those skilled in the art can be used.

[0130] The following methods can be used to check whether or not an antigen is contained. A tester composition containing the prepared Ig antibody is brought into contact with a sample obtained from a raw material, processed product, etc., and the binding between the Ig antibody and the antigen in the sample is detected using, for example, ELISA, and if binding between the Ig antibody and the antigen is detected, it is determined that the target raw material, processed product, etc. contains the antigen. A method in which raw materials or processed products are soaked in filter paper or the like, and an antibody solution is reacted to detect the antigens contained therein.

[0131] Another aspect of the present invention provides a tester composition for determining the presence or absence of an allergic antigen in a subject, comprising a primer having a nucleotide sequence complementary to at least a portion of the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25. For example, but not limited to, the primer may have a nucleotide sequence complementary to the 3'-terminal or central portion, preferably 12, 15, 20, or 25 bases, of at least a portion of the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25. In particular, when targeting mRNA, the primer may have a complementary primer to a polyA tail. In a preferred embodiment, the tester composition containing the above-mentioned primers may further contain a primer containing a base sequence of the 5'-terminal portion of at least one of the base sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, preferably a base sequence consisting of 12 bases, 15 bases, 20 bases, or 25 bases.

[0132] For example, the presence or absence of the antigen is determined by using DNA or mRNA obtained from milk as a template and the complementary primers to amplify cDNA by PCR (Polymerase Chain Reaction) including RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and comparing the sequence of the amplified cDNA with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25. Examples of PCR amplification methods include the RACE (Rapid Amplification of cDNA End) method. In this case, when the amplified cDNA is compared with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, if there is a point mutation encoding the same amino acid, or even if there is an insertion, deletion, substitution, or addition of bases in the base sequence of the amplified cDNA relative to the base sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, the antigen is determined to be present if the amino acid sequence encoded by the cDNA has an identity of 70% or more, preferably 80, 90, 95, 98, or 99% or more, to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26.

[0133] In one embodiment, the tester composition is used to determine whether or not an antigen is present in a target substance, such as food ingredients (milk) or a food production line. The tester composition may be used by manufacturers for quality inspection of production lines and pre-shipment products, or by consumers themselves to check whether or not the target raw materials or processed products contain the antigen. It may also be used to check for the presence of an antigen by measuring the increase or decrease in the peak of the corresponding protein using a mass spectrometer.

[0134] Method for determining the presence or absence of antigen (1) The present invention includes a method for determining the presence or absence of any of the above antigens (1) to (13) in a target substance, which comprises contacting a raw material or processed product (including a liquid) with an antibody against at least one of the above antigens (1) to (13).

[0135] The raw material may be a food ingredient, a cosmetic raw material, a pharmaceutical raw material, etc. The processed product may be an edible processed product, a cosmetic, a pharmaceutical, etc.

[0136] The antibody, the method for producing the antibody, the method for contacting the antibody with raw materials or processed products, the binding of the antibody with the antigen, etc. are as described above in "Tester composition (1)."

[0137] Antigen-removed foods, etc. (1) The present invention provides a raw material or processed product characterized by having at least one of the antigens (1) to (13) removed or reduced. In one embodiment, the "raw material or processed product" is "milk or a processed milk product" and may also be "beef or a processed beef product."

[0138] The method for removing or reducing the antigens of the present invention in raw materials or processed products is not limited, and the removal or reduction of the antigens may be carried out by any method as long as the antigens of the present invention are removed or reduced.

[0139] For example, a raw material (eg, milk or beef) in which the antigens of the present invention have been removed or reduced may be obtained from cattle in which the expression of the antigens of the present invention has been modified using genetic modification techniques.

[0140] Genetic modification techniques can be any technique known to those skilled in the art. For example, Oishi et al. (Scientific Reports, Vol. 6, Article number: 23980, 2016, doi:10.1038 / srep23980) describes the application of the genome editing technology CRISPER / Cas9 to chicken primordial germ cells to obtain an ovomucoid gene-deficient individual. A similar technique can be used to obtain cattle in which the expression of the antigen of the present invention has been modified, and then beef or milk from the cattle in which the antigen of the present invention has been removed or reduced can be obtained.

[0141] Alternatively, a cow in which expression of the antigen of the present invention has been eliminated or reduced may be obtained by mating, by artificial insemination or the like, with a cow that does not express or expresses the antigen in a low amount, and beef or milk in which the antigen of the present invention has been eliminated or reduced may be obtained from that cow. Artificial mating of cows can be carried out by conventional methods.

[0142] The processed product of the present invention from which the antigens of the present invention have been removed or reduced may be a processed product made from raw materials from which the antigens of the present invention have been removed or reduced. When using ordinary raw materials, treatment to remove or reduce the antigens of the present invention is carried out before or after preparation of the processed product. Methods for removing or reducing the antigens of the present invention in processed products made from ordinary raw materials include methods for removing protein components from raw materials and processed products, such as high-pressure treatment and elution with a neutral salt solution or high-temperature steam, and methods for hydrolysis, denaturation, and amino acid changes (chemical modification and elimination of side chains, etc.) by heat treatment and acid treatment.

[0143] Methods for producing raw materials or processed products in which antigens have been removed or reduced (1) The present invention provides a method for producing raw materials or processed products from which antigens have been removed or reduced, the method comprising a step of confirming that the antigens have been removed or reduced during the production process of the raw materials or processed products, wherein the antigen is at least one of the antigens (1) to (13) above.

[0144] The step of confirming that antigens have been removed or reduced during the manufacturing process of raw materials or processed products from which antigens have been removed or reduced may be carried out by confirming whether or not the antigens are contained using the method described above in the section "Tester composition (1)."

[0145] In addition, milk or processed milk products, or beef or processed beef products from which antigens have been removed or reduced may be produced by the method described above in the section "Antigen-removed foods, etc. (1)."

[0146] epitope For the antigens identified as shown in Examples 1-3, epitopes and amino acids within the epitopes that are important for binding to IgE antibodies from allergic patients were identified as shown in Example 4.

[0147] The results are summarized in Table 2. In Table 2, P1 to P25 are patient numbers assigned to each of the 25 allergy patients.

[0148] [Table 2-1]

[0149] [Table 2-2]

[0150] [Table 2-3]

[0151] [Table 2-4]

[0152] [Table 2-5]

[0153] [Table 2-6]

[0154] [Table 2-7]

[0155] [Table 2-8]

[0156] Table 2-9

[0157] Table 2-10

[0158] Table 2-11

[0159] Table 2-12

[0160] Table 2-13

[0161] Table 2-14

[0162] Table 2-15

[0163] Table 2-16

[0164] Table 2-17

[0165] Table 2-18

[0166] Table 2-19

[0167] [Table 2-20]

[0168] [Table 2-21]

[0169] [Table 2-22]

[0170] [Table 2-23]

[0171] [Table 2-24]

[0172] [Table 2-25]

[0173] [Table 2-26] Tables 2-1 to 2-25 show specific sequences identified as epitopes that bind to IgE antibodies in Example 4, and sequences for which epitope cross-reactivity was confirmed in Example 5. Table 2-26 shows examples of foodstuffs to which 25 patients were allergic, and summarizes the analysis cases of epitope cross-reactivity in Example 5.

[0174] The present invention provides polypeptides comprising amino acid sequences (E1) to (E25) that specifically bind to IgE antibodies of allergy patients, and that comprise or consist of any of the amino acid sequences of SEQ ID NOS: 27-1131 listed in Table 2. (E1) to (E25) are amino acid sequences that bind to IgE antibodies derived from the proteins listed under "Protein Name" in Table 2 (hereinafter, these may be referred to as "epitopes").

[0175] (1) Immunoglobulin M heavy chain secretory form protein: (E1), (E2); (2) Beta-1 metal-binding globulin proteins: (E3), (E4), (E5), (E6), (E7), (E8); (3) Lactoperoxidase protein derived: (E9), (E10); (4) Ig heavy chain precursor (B / MT.4A.17.H5.A5) protein: (E14), (E15); (5) Perilipin protein: (E16), (E17); (6) Polymeric immunoglobulin receptor protein: (E11), (E12); (7) C3-beta-c (C3-beta-c) protein derived: (E13); (8) Immunoglobulin light chain, lambda gene cluster protein (E23); (9) Glycoprotein 2 protein: (E20), (E21); (10) Lactadherin protein: (E22); (11) Zinc-alpha-2-glycoprotein protein: (E18), (E19); (12) Intracellular cholesterol transporter 2 protein (E24); (13) Apolipoprotein A-IV protein (E25).

[0176] Although not limited thereto, the epitope antigen of the present invention preferably comprises at least one of the following polypeptides:

[0177] (E1) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 27-58, and 1115; (E2) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 59-97; (E3) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 98-144; (E4) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 145-195; (E5) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 196-254 and 1116-1118; (E6) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 255-288; (E7) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 289-333, and 1119-1120; (E8) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 334-352; (E9) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 353-376, and 1121-1122; (E10) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 377-384; (E11) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 385-411; (E12) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 412-450; (E13) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 451-500, and 1123-1124; (E14) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 501-604 and 1125-1127; (E15) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 605-632, and 1128; (E16) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 633-673; (E17) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 674-693; (E18) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 694-757, and 1129; (E19) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 758-791; (E20) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 792-868, and 1130; (E21) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 869-927; (E22) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 928-996; (E23) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 997-1037; (E24) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1038-1075; (E25) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1076-1114 and 1131.

[0178] The polypeptides (E1) to (E25) described above as preferred embodiments 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 not only the polypeptides (E1) to (E25) of the preferred embodiments described above, but also the variants described below. The following describes embodiments (variants) that may be included in the epitope antigens of the present invention.

[0179] The 25 sequences listed under "15-residue sequences" in Table 2, SEQ ID NOs: 27, 59, 98, 145, 196, 255, 289, 334, 353, 377, 385, 412, 451, 501, 605, 633, 674, 694, 758, 792, 869, 928, 997, 1038, and 1076, are common 15-amino acid residue sequences identified by overlapping epitope mapping as epitopes that bind to IgE antibodies in each of the epitopes (E1) to (E25). In one aspect, the present invention relates to polypeptides comprising or consisting of these amino acid sequences. The epitope sequences contained in the polypeptides of the present invention may be the entire 15 amino acid residues of this common epitope, or a portion thereof. The epitope sequence is 4 or more amino acid residues, 5 or more amino acid residues, 6 or more amino acid residues, 7 or more amino acid residues, 8 or more amino acid residues, 9 or more amino acid residues, 10 or more amino acid residues, 11 or more amino acid residues, 12 or more amino acid residues, 13 or more amino acid residues, or 14 or more amino acid residues.

[0180] In the Examples herein, for example, many polypeptides consisting of four amino acid residues (e.g., SEQ ID NOs: 34, 67, 116, 195, 229, 275, etc.) were identified as epitopes that bind to IgE antibodies. Furthermore, many polypeptides consisting of five or more amino acid residues were also confirmed to bind to IgE antibodies. Therefore, any sequence containing at least four amino acid residues is useful as an epitope sequence.

[0181] One embodiment of the polypeptide variants of the present invention includes polypeptides containing four or more amino acid residues of the amino acid sequences specifically described above in (E1) to (E25). For example, variants of (E1) may include "polypeptides containing four or more amino acid residues from at least one of the amino acid sequences of SEQ ID NOS: 27-58 and 1115." Preferably, variants of (E1) contain four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or fourteen or more amino acid residues from at least one of the amino acid sequences of SEQ ID NOS: 27-58 and 1115. The same applies to (E2) to (E25).

[0182] In Table 2, "preferred" sequences are shorter partial sequences of the "15-residue sequence" that can function as an epitope. "More preferred" sequences are sequences that are more preferable than the above-mentioned shorter partial fragments in order to improve binding to IgE antibodies. "Key" sequences indicate sequences that are considered particularly important among the "15-residue sequences." Among the "key" sequences, amino acid sequences marked with "X" are amino acid residues that have been confirmed by alanine-glycine scanning to retain IgE antibody binding even when changed to any alanine (or glycine if the original amino acid residue is alanine). Therefore, X is any amino acid residue, preferably alanine (or glycine). Note that "key" sequences that do not contain a sequence marked with "X" are those in which no amino acid residues confirmed to retain IgE antibody binding were found by alanine-glycine scanning.

[0183] For each epitope listed in Table 2, the amino acid residues designated X are amino acid residues that have been confirmed to retain IgE antibody binding even when altered. In the present invention, one or more of the amino acid residues designated X in the "key" sequence corresponding to each "preferred" sequence may preferably be substituted with any amino acid residue. For example, in one embodiment, the preferred sequence SEQ ID NO: 27 has multiple corresponding key sequences, such as SEQ ID NOs: 28 and 34. The same applies hereinafter to other "preferred" sequences, "key" sequences, and (E2)-(E25) of (E1).

[0184] The number of amino acid residues that may be substituted is not limited, but is preferably 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less. The same applies hereinafter to (E2) to (E25).

[0185] The sequences listed in the "Synthetic Sequence" and / or "SEQ ID NO:" columns to the right of the graph No. in Table 2 are sequences for which epitope cross-reactivity was confirmed in Example 5. "Epitope cross-reactivity was confirmed" means that the IgE antibodies in the serum of each allergic patient showed higher binding affinity than the IgE antibodies in the serum of non-allergic subjects (specifically, greater than "1" in Figures 3-6). Thus, in one aspect, the polypeptides of the present invention include polypeptides comprising or consisting of these amino acid sequences. In one aspect, the IgE antibodies in the serum of each allergic patient show binding affinity to the polypeptides of the present invention that is 1.05-fold or more, 1.10-fold or more, 1.15-fold or more, 1.20-fold or more, or 1.25-fold or more higher than the IgE antibodies in the serum of non-allergic subjects.

[0186] As used herein, "a polypeptide comprising an amino acid sequence of (E1) to (E25)" includes, as preferred embodiments, polypeptides comprising or consisting of the amino acid sequences of the polypeptides (E1) to (E25) described above, as well as variants (mutants) in which amino acid residues have been substituted as described above. "Comprising an amino acid sequence of SEQ ID NOS: 1 to 1" means that any other amino acid sequence may be included as long as it does not affect the binding of the amino acid sequences of SEQ ID NOS: 1 to 1 (including the substituted variants) to IgE antibodies (i.e., their epitope function). A "polypeptide comprising an amino acid sequence of (E1) to (E25)" may also be a polypeptide in which two or more polypeptides comprising the amino acid sequences of (E1) to (E25) described above are linked together with or without a spacer. The type of spacer is not particularly limited, and any spacer commonly used by those skilled in the art for linking multiple peptides can be used. The spacer may be, for example, a hydrocarbon chain such as Acp(6)-OH, an amino acid chain, or other polypeptide.

[0187] Amino acid residues in the polypeptide other than those specifically specified in the amino acid sequence can be selected arbitrarily as long as they do not affect the binding to IgE antibodies (i.e., their function as epitopes). Without limitation, it is preferable that they be appropriately selected from the sequence of the corresponding base epitope or the sequence of the base protein. For example, SEQ ID NO: 34 specifies only "HNKE," but if other amino acid residues are added, it is preferable that they be appropriately selected from the base SEQ ID NO: 27. Furthermore, for the sequence listed as (E1) in Table 2-1, it is preferable that amino acid residues from the sequence derived from the base protein (1) (corresponding to spot (1)) are added.

[0188] Polypeptides containing the amino acid sequences (E1)-(E25) above may be prepared by chemical synthesis techniques 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, followed by isolation and purification using protein production methods well known to those skilled in the art. Two or more types of polypeptides may be linked in combination, or one type of epitope may be linked repeatedly. In this case, binding to Ig antibodies is generally improved.

[0189] The length of a polypeptide containing the amino acid sequence of (E1)-(E25) is not particularly limited. In preferred embodiments, the length of a polypeptide containing the amino acid sequence of (E1)-(E25) 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. When a polypeptide is formed by linking one or more of the amino acid sequences of (E1)-(E25) once or twice or more times, in preferred embodiments, the length of the amino acid sequence portion may be 1,000 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 in the preferred embodiments of the length of the polypeptide refers to the sum of the lengths of the sequences before and after the spacer (excluding the spacer).

[0190] The antigen of the present invention specifically binds to IgE antibodies in allergic patients.

[0191] Diagnostic kits and methods (2) The present invention provides a method for providing an indicator for diagnosing allergies in a subject, comprising the steps of: (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) if binding of the subject's IgE antibodies to the antigen is detected, an indication that the subject is allergic is provided; wherein the antigen is at least one polypeptide comprising the amino acid sequence of (E1)-(E25), or a polypeptide in which two or more polypeptides comprising the amino acid sequences of (E1)-(E25) are linked together with or without a spacer.

[0192] A polypeptide that is at least one of the polypeptides containing the amino acid sequence of (E1)-(E25) above, or a polypeptide in which two or more polypeptides containing the amino acid sequence of (E1)-(E25) above are linked together with or without a spacer, may be referred to herein as an "antigen containing (E1)-(E25) above." The type of spacer is not particularly limited, and any spacer commonly used by those skilled in the art for linking multiple peptides can be used. The spacer may be, for example, a hydrocarbon chain such as Acp(6)-OH, an amino acid chain, or other polypeptide.

[0193] When polypeptides containing the amino acid sequences (E1) to (E25) are linked with or without a spacer, the number of linked polypeptides is not particularly limited. In one embodiment, the number is 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 another embodiment, the number is 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.

[0194] The polypeptides containing the amino acid sequences (E1)-(E25) may be repeated or may be multiple linked polypeptides containing different amino acid sequences. Even when multiple linked polypeptides containing the amino acid sequences (E1)-(E25) are linked, they can be used as the polypeptide of the present invention in the methods, kits, and compositions of the present invention.

[0195] The sample obtained from the subject is as described above in the section "Diagnostic kit and diagnostic method (1)."

[0196] Contact between a sample obtained from a subject and the polypeptide and detection of its binding can be performed by known methods described in the above section "Diagnostic Kit / Diagnostic Method (1)," such as ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblot, immunoprecipitation, immunochromatography, etc.

[0197] The polypeptide comprising the amino acid sequence (E1)-(E25) may be immobilized on or within a carrier. In this case, ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance, or the like can be used in steps (i) and (ii). Step (i) is performed by contacting a sample obtained from a subject with a surface onto which the polypeptide comprising the amino acid sequence (E1)-(E25) is immobilized. Alternatively, the subject's IgE antibody may be immobilized on or within a carrier, and the binding of the subject's IgE antibody to the polypeptide comprising the amino acid sequence (E1)-(E25) may be detected by the above-described method. A spacer or a tag such as biotin may be attached to the N-terminus or C-terminus of the polypeptide to facilitate binding to the carrier, to provide a space between the carrier and the polypeptide, or to facilitate antibody contact with the polypeptide. In the case of binding with biotin, the carrier preferably contains avidin.

[0198] The polypeptide comprising the amino acid sequence (E1)-(E25) may not be immobilized on or within a carrier. In this case, flow cytometry or the like can be used in steps (i) and (ii) to confirm the presence of the polypeptide comprising the amino acid sequence (E1)-(E25) to which IgE antibodies have bound using laser light. Examples of this method include the basophil activation test (BAT), which detects the surface antigen CD203c that appears when basophils are activated by contact with a polypeptide comprising the amino acid sequence (E1)-(E25). Another example is the histamine release test (HRT), which examines whether a blood cell in a sample releases histamine by contacting the polypeptide comprising the amino acid sequence (E1)-(E25) with the blood cell.

[0199] A polypeptide comprising the amino acid sequence (E1)-(E25) above is an antigen that specifically binds to the IgE antibody of an allergic patient. Therefore, detection of binding between a subject's IgE antibody and the antigen provides an indication that the subject is allergic, including cross-reactivity. When synthesizing a polypeptide comprising the amino acid sequence (E1)-(E25) above, sequences may be added before and after the epitope to facilitate synthesis using Escherichia coli, for example, to increase the sequence length. Even in such cases, detection of binding between a subject's IgE antibody and the amino acid sequence (E1)-(E25) above provides an indication that the subject is allergic, including cross-reactivity. Therefore, any sequence may be added before or after the amino acid sequence (E1)-(E25) above, which is the epitope.

[0200] The present invention also provides a diagnostic kit for allergies, comprising at least one polypeptide comprising the amino acid sequence of (E1)-(E25). The diagnostic kit of the present invention may be used in the above-mentioned method for providing an indicator for diagnosing allergies, or in the diagnostic method described below. In addition to comprising at least one polypeptide comprising the amino acid sequence of (E1)-(E25), 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. A fluorescently labeled anti-IgE antibody may also be used. In the diagnostic kit of the present invention, the polypeptide comprising the amino acid sequence of (E1)-(E25) may be provided in a state immobilized on or within a carrier. The diagnostic kit of the present invention may also be provided together with instructions on the diagnostic procedure or a package containing such instructions.

[0201] In another embodiment, the diagnostic kit includes a companion diagnostic for allergies. The companion diagnostic is used to identify patients who are expected to benefit from a drug or who are at risk of serious side effects from the drug, or to examine the responsiveness of the drug to optimize treatment with the drug. Here, optimization of treatment includes, for example, determining the dosage and administration, deciding whether to discontinue administration, and identifying which allergen component is used to induce immune tolerance.

[0202] The present invention also provides a composition for diagnosing allergies, comprising at least one polypeptide comprising the amino acid sequence of any of (E1)-(E25) above. The diagnostic composition of the present invention can be used in the diagnostic methods described below. The diagnostic composition of the present invention may, as needed, contain pharmaceutically acceptable carriers or additives that are commonly used together with the polypeptide of the present invention.

[0203] In one aspect, the present invention provides a method for diagnosing allergy in a subject, comprising the steps of: (i) contacting a sample obtained from a subject with an antigen; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) determining that the subject is allergic if binding of the subject's IgE antibody to the antigen is detected; wherein the antigen is at least one of the polypeptides identified as comprising the amino acid sequence (E1)-(E25) above, wherein steps (i) and (ii) are carried out as described for each step of the method for providing an indicator for diagnosing allergies.

[0204] In another aspect, the present invention provides a method for diagnosing allergies in a subject, the method comprising administering to the subject at least one polypeptide comprising the amino acid sequence of (E1)-(E25). The method may be performed in the form of a skin test, which involves applying a polypeptide comprising the amino acid sequence of (E1)-(E25) to the skin. Skin tests include a prick test, in which a diagnostic composition is applied to the skin, followed by a small scratch that does not cause bleeding, allowing the polypeptide comprising the amino acid sequence of (E1)-(E25) to penetrate the skin and observe a skin reaction; a scratch test, in which a diagnostic composition is applied and then the skin is slightly scratched to observe a reaction; a patch test, in which a diagnostic composition in the form of a cream or ointment is applied to the skin and observe a reaction; and an intradermal test, in which a polypeptide comprising the amino acid sequence of (E1)-(E25) is intradermally administered and observe a reaction. If a skin reaction, such as swelling, occurs in the area where the polypeptide comprising the amino acid sequence of (E1)-(E25) was applied, the subject is diagnosed as having an allergy. Here, the amount of the polypeptide to be applied to the skin may be, for example, 100 μg or less per application.

[0205] In the diagnosis of allergies, oral challenge tests are often performed to identify the antigen, verify antigen intake, and verify the severity of symptoms. At least one of the polypeptides containing the amino acid sequence (E1)-(E25) above can be used as an active ingredient in oral challenge tests to diagnose allergies. The polypeptide used in oral challenge tests may be an expressed and purified polypeptide, or it may be expressed in raw materials or processed products, such as pollen rice, in which a cedar pollen antigen gene is transformed into rice and the polypeptide is expressed in the rice.

[0206] 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.

[0207] In another aspect, the present invention provides at least one polypeptide comprising the amino acid sequence of (E1)-(E25) above for use in diagnosing allergies.

[0208] In yet another aspect, the present invention provides the use of at least one polypeptide comprising the amino acid sequence of (E1)-(E25) above in the manufacture of a diagnostic agent for allergies.

[0209] In this section, the allergy to be diagnosed may be an allergy to a polypeptide comprising the amino acid sequence of (E1)-(E25). That is, the diagnosis of allergy, including the detection of allergy and the provision of a diagnostic indicator, may diagnose not only allergy to a single polypeptide comprising the amino acid sequence of (E1)-(E25), but also allergy including cross-reactivity.

[0210] Composition / Treatment method (2) The present invention provides compositions comprising at least one of the polypeptides comprising the amino acid sequences (E1)-(E25) above.

[0211] In one embodiment, the composition of the present invention is a pharmaceutical composition. In one embodiment, the composition of the present invention is a quasi-medicinal composition, a non-medicinal composition (e.g., a cosmetic composition, a food composition).

[0212] In one embodiment, the composition is used to treat allergies. Treating allergies involves increasing the limit of the amount of polypeptide that can be taken into the body without causing symptoms, ultimately aiming to achieve a state (remission) in which symptoms do not appear with normal intake of the polypeptide.

[0213] The present invention also provides a method for treating allergies, which comprises administering to a patient in need of such treatment at least one of the polypeptides comprising the amino acid sequence of (E1)-(E25) above.

[0214] In another aspect, the present invention provides at least one polypeptide comprising the amino acid sequence of (E1)-(E25) above for use in treating allergies. In yet another aspect, the present invention provides use of at least one polypeptide comprising the amino acid sequence of (E1)-(E25) above for the manufacture of a medicament for treating allergies.

[0215] In the treatment of allergies, hyposensitization therapy is often performed, with the goal of inducing immune tolerance by administering an antigen to the patient. At least one of the polypeptides containing the amino acid sequence (E1)-(E25) above can be used as an active ingredient in hyposensitization therapy for allergies. Here, the antigen used in hyposensitization therapy may be an expressed and purified polypeptide, or it may be expressed in a raw material or processed product, such as pollen rice.

[0216] The administration route, dosage, frequency and / or duration of administration, other components contained in the composition, and dosage form of the composition of the present invention can be those described above in the section "Composition and Treatment Method (1)." When using a polypeptide containing the amino acid sequence of (E1)-(E25) above, the dosage may be, for example, 100 μg or less per administration for an adult.

[0217] In this section, the allergy to be treated may be an allergy to a polypeptide comprising any one of the amino acid sequences (E1)-(E25). That is, the allergy treatment may be not only an allergy to a single polypeptide comprising any one of the amino acid sequences (E1)-(E25), but also an allergy including cross-reactive allergies.

[0218] Tester Composition (2) The present invention provides a tester composition comprising an antibody against at least one of the polypeptides comprising the amino acid sequences (E1)-(E25) above.

[0219] 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 sequence of any one of (E1)-(E25). 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').

[0220] Furthermore, in the above-mentioned tester composition, the antibody may be provided in a solid or bound form on or within a carrier. The carrier is not particularly limited as long as it can be used to detect the binding between the antibody and the polypeptide comprising the amino acid sequence of (E1)-(E25). Any carrier known to those skilled in the art can be used. Furthermore, it is preferable that the antibody against the polypeptide comprising the amino acid sequence of (E1)-(E25) is an antibody against an epitope described above in the "Epitope" section or a polypeptide having an amino acid sequence identical to the epitope in key amino acids. This allows for the preparation of a tester composition that can detect cross-reactivity.

[0221] The following method can be used to examine whether or not a polypeptide containing any of the amino acid sequences (E1) to (E25) is contained.

[0222] A method for determining whether a polypeptide containing the amino acid sequence (E1)-(E25) is contained in a target raw material, processed product, etc. by contacting a tester composition containing the antibody thus prepared with a sample obtained from the raw material, processed product, etc., and detecting the binding between the antibody and the polypeptide containing the amino acid sequence (E1)-(E25) in the sample using, for example, ELISA, and determining that the polypeptide containing the amino acid sequence is contained in the target raw material, processed product, etc. if the binding between the antibody and the polypeptide containing the amino acid sequence (E1)-(E25) is detected. (The "method for determining whether a polypeptide is contained" includes determining that the polypeptide has been removed or reduced if the binding between the antibody and the polypeptide containing the amino acid sequence (E1)-(E25) is reduced.) A method in which raw materials, processed products, etc. are soaked in filter paper, etc., and an antibody solution is reacted to detect polypeptides containing the amino acid sequences (E1)-(E25) contained therein.

[0223] Another aspect of the present invention provides a tester composition for determining the presence or absence of a polypeptide comprising any of the amino acid sequences (E1)-(E25) of an allergen in a subject, the composition comprising primers corresponding to a polypeptide having an amino acid sequence identical to an epitope or key amino acids. The primers may be designed, for example and without limitation, to include a portion of the nucleotide sequence of a nucleic acid encoding the amino acid sequence identified in (E1)-(E25) above, or its complementary strand. Alternatively, the primers may be designed to correspond to the nucleotide sequence of a region upstream of the portion encoding the polypeptide having the epitope or key amino acid sequence identical to any of the amino acid sequences identified in (E1)-(E25) above, or the complementary strand of a region downstream of the portion encoding the polypeptide having the epitope or key amino acid sequence identical to the amino acid sequence identified in (E1)-(E25) above. 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, and 25, 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, and 25. The position of the epitope in the full-length sequence of the antigen is as specified in Table 2 based on the results of the Examples. Furthermore, particularly when targeting mRNA, a primer complementary to a poly(A) tail may be used.

[0224] For example, DNA or mRNA obtained from a sample is used as a template, and the primers are used to amplify DNA by PCR (Polymerase Chain Reaction), including RT-PCR. The presence or absence of an antigen containing (E1)-(E25) is determined by determining whether the amplified DNA sequence contains a nucleic acid encoding the amino acid sequence specified by (E1)-(E25) above. Examples of methods for PCR amplification of mRNA include the RACE method. If one of the amino acid sequences encoded by the three possible open reading frames in the amplified DNA contains the amino acid sequence specified by (E1)-(E25) above, the antigen is determined to be present. If DNA is not amplified, the antigen is determined to be absent.

[0225] In one embodiment, the tester composition is used to determine whether or not a target substance, such as a raw material or a processed product production line, contains a polypeptide comprising the amino acid sequence (E1)-(E25). The raw material may be a food ingredient, a cosmetic raw material, a pharmaceutical raw material, etc. The processed product may be an edible processed product, a cosmetic product, a pharmaceutical product, etc. The tester composition may be used to search for biological species contained in raw materials, for quality inspection by manufacturers on production lines and pre-shipment products, or for consumers or users themselves to check whether or not a target raw material or processed product contains an antigen. It may also be used to check for the presence of a polypeptide by measuring an increase or decrease in the peak of the corresponding polypeptide using a mass spectrometer.

[0226] Method for determining the presence or absence of polypeptide (2) The present invention includes a method for determining the presence or absence of a polypeptide comprising the amino acid sequence of (E1)-(E25) in a raw material or processed product, which method comprises detecting a polypeptide comprising the whole or part of the amino acid sequence of a polypeptide comprising the amino acid sequence of (E1)-(E25) in the raw material or processed product.

[0227] In one aspect, the method of the present invention comprises a step of determining the presence or absence of a polypeptide comprising the amino acid sequence of (E1)-(E25) in a target substance, which step comprises contacting a raw material or processed product (including a liquid) with an antibody against at least one of the polypeptides comprising the amino acid sequence of (E1)-(E25).

[0228] The antibody, raw material / processed product definition, method for producing the antibody, method for contacting the antibody with the raw material / processed product, and binding of the antibody with the antigen are as described above in "Tester composition (2)."

[0229] Alternatively, the method for determining the presence or absence of the antigen also includes detecting an epitope portion of a polypeptide comprising the amino acid sequence (E1)-(E25) contained in the antigen. The "epitope portion" preferably comprises at least four, at least six, or at least eight amino acid residues. Detection of the epitope portion can be performed using known methods for detecting a specific amino acid sequence in a portion of a polypeptide. For example, a method may be used in which proteins from a target raw material or processed product (e.g., foodstuff) are cleaved with a digestive enzyme for antigen removal treatment, followed by separation by HPLC or the like, and the peak of a given epitope peptide is measured to determine whether the antigen removal treatment has reduced the peak. Alternatively, the presence or absence of an antigen comprising a polypeptide comprising the amino acid sequence (E1)-(E25) in a target substance may be determined using an antibody that recognizes a portion of the polypeptide comprising the amino acid sequence (E1)-(E25).

[0230] Antigen removal raw materials, etc. (2) The present invention provides raw materials or processed products characterized in that at least one of the polypeptides comprising the amino acid sequences (E1) to (E25) above has been removed or reduced.

[0231] The method for removing or reducing the antigens of the present invention in raw materials or processed products is not limited. The antigens may be removed or reduced by any method as long as the polypeptides containing the amino acid sequences (E1) to (E25) are removed or reduced. For example, the methods described in the above section "Antigen-removed foods, etc. (1)" may be used.

[0232] "At least one of the polypeptides comprising the amino acid sequences (E1)-(E25) has been removed or reduced" may be achieved by removing or reducing the entire amino acid sequence, or by truncating or removing the amino acid sequence portions (E1)-(E25) from the antigen protein. "Removed" includes deletion and modification of all or part of the sequence portions specified in (E1)-(E25) above.

[0233] For example, a raw material from which the polypeptide comprising the amino acid sequence of (E1)-(E25) has been removed or reduced may be prepared using genetic modification techniques to eliminate expression of the polypeptide comprising the amino acid sequence of (E1)-(E25). Any genetic modification knockout technique known to those skilled in the art can be used.

[0234] The processed product from which the polypeptides containing the amino acid sequences (E1)-(E25) have been removed or reduced may be a processed product made from raw materials from which the polypeptides containing the amino acid sequences (E1)-(E25) have been removed or reduced, such as powdered milk made from protein digests. When using normal raw materials, treatment to remove or reduce the polypeptides containing the amino acid sequences (E1)-(E25) is carried out before, during, or after the preparation of the processed product. "Preparation of a processed product" refers to the preparation of a processed food product from, for example, a food raw material (e.g., milk or beef). For example, "preparation of processed milk products" means the preparation of dairy products made from milk as a main ingredient, and foods containing the same as an ingredient, including, but not limited to, the preparation of dairy products made from milk, such as cream, butter, butter oil, cheese, whey, concentrated whey, ice cream, concentrated milk, concentrated skim milk, unsweetened condensed milk, unsweetened condensed skim milk, sweetened condensed milk, sweetened condensed skim milk, whole milk powder, skim milk powder, cream powder whey powder, protein-enriched whey powder, buttermilk powder, sweetened milk powder, modified milk powder, modified liquid milk, fermented milk such as yogurt, lactic acid bacteria drinks, and dairy drinks, as well as the preparation of cakes, pastries, custard puddings, milk agar, biscuits, cookies, snacks, chocolates, bread, white sauce, potage, cream stew, gratin, curry roux, and stew roux. Furthermore, "preparation of processed beef products" means preparing products made from beef, which is the main ingredient, and foods that contain beef as an ingredient, and includes, but is not limited to, the preparation of processed meat products such as ham, sausage, bacon, bouillon, consommé, steak, yakiniku, roast beef, steak tartare, hamburger, hamburger, meatballs, nuggets, etc.

[0235] The method for removing or reducing the amount of polypeptides containing the amino acid sequence (E1)-(E25) in processed foods made from ordinary raw materials may be the method described in the above section "Antigen-removed foods, etc. (1)." Methods for cleaving polypeptides containing the amino acid sequence (E1)-(E25) include cleavage using a specific digestive enzyme.

[0236] (2) Manufacturing methods for raw materials or processed products in which antigens have been removed or reduced 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)-(E25) has been removed or reduced, the method comprising a step of confirming that the antigen has been removed or reduced during the production of the processed product.

[0237] In the production method, the removal or reduction of polypeptides comprising the amino acid sequence of (E1)-(E25) means that at least one of the polypeptides comprising the amino acid sequence of (E1)-(E25) is removed or reduced, or the sequence portion specified by (E1)-(E25) above is cleaved or removed from the antigen.

[0238] The method for confirming that polypeptides have been removed or reduced during the production of raw materials or processed products is not particularly limited, and any method capable of detecting at least one polypeptide comprising the amino acid sequence (E1)-(E25) above may be used. For example, the presence or absence of a polypeptide in a raw material or processed product may be confirmed by measuring the binding of a sample containing a material resulting from the production of the raw material or processed product to an antibody against at least one of the polypeptides comprising the amino acid sequence (E1)-(E25) above. Details of such a method are as described above in the "Diagnostic Kit / Diagnostic Method (2)" section. That is, in the above production method, the "subject's IgE antibody" in the "Diagnostic Kit / Diagnostic Method (2)" section may be replaced with "an antibody against at least one of the polypeptides comprising the amino acid sequence (E1)-(E25) above," and the "antigen" and "polypeptide" in the "Diagnostic Kit / Diagnostic Method (2)" section may be replaced with "a sample containing a material resulting from the production of the processed product." The method described above in the "Diagnostic Kit / Diagnostic Method (2)" section may be used to confirm that antigens have been removed or reduced during the production of the processed product. In addition, the tester composition described above in the section "Tester composition (2)" can also be used.

[0239] The present invention also relates to the use of a kit in a method for diagnosing allergies, the use of a kit for diagnosing allergies and / or providing an indicator for diagnosis, a composition for use in a method for diagnosing allergies, the use of a composition for diagnosing allergies and / or providing an indicator for diagnosis, a method for diagnosing allergies and / or providing an indicator for diagnosis, the use of an antigen (protein antigen or epitope antigen) in a method for detecting the presence or absence of IgE antibodies in a sample obtained from a subject (a living organism such as a human), an antigen (protein antigen or epitope antigen) for use in treating allergies, a kit or composition comprising an antigen (protein antigen or epitope antigen) for detecting binding between an IgE antibody and an antigen (protein antigen or epitope antigen) in a sample obtained from a subject (a living organism such as a human), and the use of a kit or composition comprising an antigen (protein antigen or epitope antigen) for detecting binding between an IgE antibody and an antigen (protein antigen or epitope antigen) in a sample obtained from a subject (a living organism such as a human). The terms "antigen" and the like are as defined above. [Example]

[0240] Examples of the present invention will be described below, but the technical scope of the present invention is not limited to these examples. Example 1: Protein pattern confirmation The proteins contained in cow's milk (milk from Bos taurus) were examined using the following two-dimensional electrophoresis method.

[0241] Protein extraction Proteins contained in milk were extracted and purified as follows: Urea buffer was added to milk to extract proteins. The composition of the urea buffer is as follows: 30mM Tris 2M thiourea 7M Urea 4% (w / v) CHAPS: 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate A suitable amount of dilute hydrochloric acid Distilled water was added to adjust the total volume to 100 mL. The pH was 8.5. Two precipitation procedures were then performed using the 2D-CleanUP kit (GE). In the first precipitation procedure, TCA (trichloroacetic acid) was added to the recovered protein extract, and the resulting precipitate (TCA precipitate) was collected. In the second precipitation procedure, acetone was added to the recovered TCA precipitate, and the resulting precipitate (sample) was collected.

[0242] Preparation of sample solution A portion of the obtained sample (100 μg protein weight) was dissolved in 150 μl of DeStreak Rehydration Solution (GE), a swelling buffer for the first-dimension isoelectric focusing gel, to prepare the sample solution for first-dimension isoelectric focusing (swelling sample solution). The composition of DeStreak Rehydration Solution is as follows: 7M thiourea 2M urea 4% (w / v) CHAPS 0.5% (v / v) IPG buffer; GE Appropriate amount of BPB (bromophenol blue) Permeation of the sample into the first-dimension isoelectric focusing gel A first-dimension isoelectric focusing gel (GE: IPG Gel Immobiline Drystrip (pH 3-10NL)) was immersed in 140 μl of the specimen solution for first-dimension isoelectric focusing (specimen solution for swelling) and allowed to soak overnight at room temperature.

[0243] In this example, an IPGphor manufactured by GE was used as the electrophoresis device.

[0244] The electrophoresis tray was filled with silicone oil, and water-moistened filter paper was placed on both ends of the gel permeated with the sample. The gel was then set on the electrophoresis tray so that it was covered with silicone oil, and the electrodes were set with the filter paper sandwiched between the gel and the tray.

[0245] The upper limit of the current value of the isoelectric focusing device was set to 75 μA per gel, and the voltage program was as follows: (1) a constant voltage step of 300 V was performed up to 750 Vhr (the current change during the 30 minutes of electrophoresis before the end of this step 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 isoelectric focusing was then performed at a constant voltage of 5000 V until the total Vhr reached 12000.

[0246] SDS equilibration of isoelectric focusing gels After the first-dimension isoelectric focusing, the gel was removed from the isoelectric focusing apparatus and immersed in an equilibration buffer containing a reducing agent and shaken at room temperature for 15 minutes. The composition of the equilibration buffer containing a reducing agent was 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 an alkylating agent and shaken at room temperature for 15 minutes to obtain an SDS-equilibrated gel. The composition of the equilibration buffer containing the alkylating agent was 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 dimension SDS-PAGE In this example, the electrophoresis apparatus used was an XCell SureLock Mini-Cell manufactured by Life Technologies, Inc. The second-dimensional electrophoresis gel used was NuPAGE 4-12% Bis-Tris Gels manufactured by Life Technologies, Inc. The electrophoresis buffer solution with the following composition was prepared and used. 50mM MOPS 50mM Tris base 0.1% (w / v) SDS 1mM EDTA In this example, an adhesive agarose solution was used in which 0.5% (w / v) Agarose S (manufactured by Nippon Gene Co., Ltd.) and an appropriate amount of BPB (bromophenol blue) were dissolved in the electrophoresis buffer.

[0247] The wells of the SDS-PAGE were thoroughly washed with the above-mentioned electrophoresis buffer, and the washing buffer was then removed. A fully dissolved adhesive agarose solution was then added to the wells. The SDS-equilibrated gel was then immersed in the agarose, and the SDS-equilibrated gel and the second-dimensional electrophoresis gel were brought into close contact with each other using tweezers. After confirming that the agarose had solidified sufficiently with both gels in close contact, electrophoresis was performed at a constant voltage of 200 V for approximately 45 minutes.

[0248] Fluorescent staining of gels The gel was fluorescently stained using SYPRO Ruby (Life Technologies).

[0249] First, the sealed container to be used was thoroughly washed with 98% (v / v) ethanol. After electrophoresis, the second-dimensional electrophoresis gel was removed from the SDS-PAGE machine and placed in the washed sealed container. It was then immersed in an aqueous solution containing 50% (v / v) methanol and 7% (v / v) acetic acid for 30 minutes twice. The aqueous solution was then replaced with water and the gel was immersed for 10 minutes. Next, the second-dimensional electrophoresis gel was immersed in 40 ml of SYPRO Ruby and shaken overnight at room temperature. The SYPRO Ruby was then removed, and the second-dimensional electrophoresis gel was washed with water and then shaken for 30 minutes in an aqueous solution containing 10% (v / v) methanol and 7% (v / v) acetic acid. The aqueous solution was then replaced with water and shaken for at least 30 minutes.

[0250] analysis The second-dimensional electrophoresis gels that had undergone the above series of treatments were subjected to fluorescent image scanning using a Typhoon 9500 (GE). The results of two-dimensional electrophoresis of proteins contained in milk are shown in Figure 1. Molecular weight marker bands can be seen on the left side of the gel photographs in each figure, and the position of the bands indicates a specific molecular weight (KDa).

[0251] Example 2: Antigen confirmation by immunoblotting Antigen confirmation by immunoblotting was performed by following the procedure described in Example 1 up to the "second-dimensional SDS-PAGE" step, followed by the following steps of "transfer to membrane," "immunoblot," and "analysis."

[0252] Transfer to membrane The transfer onto the membrane was carried out using the following transfer device and transfer buffer. Transfer device: XCell SureLock Mini-Cell and XCell II Blot Module (Life Technologies) Transfer buffer: NuPAGE transfer buffer (x20) (Life Technologies) was diluted 20 times with milliQ water and used.

[0253] Specifically, proteins in the two-dimensional electrophoresis gel were transferred to a membrane (PVDF membrane) according to the following procedure.

[0254] (1) The PVDF membrane was immersed in 100% methanol, then in milliQ water, and then transferred to a transfer buffer solution to hydrophilize the PVDF membrane.

[0255] (2) The sponge, filter paper, gel after second-dimensional SDS-PAGE, hydrophilic PVDF membrane, filter paper, and sponge were placed in this order, and a constant voltage of 30 V was applied for 1 hour in a transfer device.

[0256] Immunoblot Immunoblots of the membranes were performed using sera from patients with allergies to cow's milk or sera from non-cow's milk allergic subjects as primary antibodies.

[0257] Membrane immunoblotting was performed according to the following procedure. (1) The transferred membrane was shaken in Pierce (registered trademark) Fast Blocking Buffer (Thermo) (hereinafter referred to as "Fast Block") at room temperature for 1 hour. (2) The primary antibody was left to stand in 10% serum / Fast Block solution at room temperature for 1 hour. (3) Washing was performed with PBST solution (PBS buffer containing 0.1% of the nonionic surfactant Tween (registered trademark) 20) (5 minutes x 3 times). (4) Anti-human IgE-HRP (horseradish peroxidase) was used as a secondary antibody, and the sample was shaken at room temperature for 1 hour in a solution diluted 5000 times with Fast Block solution. (5) Wash with PBST solution (5 minutes x 3 times). (6) The sections were left to stand for 5 minutes in Pierce Western Blotting Substrate Plus (Thermo).

[0258] analysis The membrane subjected to the above series of treatments was subjected to fluorescent image scanning using Typhoon 9500 (manufactured by GE).

[0259] Immunoblots using serum from patients with milk allergy were compared with immunoblots using serum from control subjects without milk allergy. In immunoblots using serum from patients with milk allergy for proteins contained in milk, 14 spots were detected that were different from those detected using serum from subjects without milk allergy and that were different from known milk allergen proteins (Figure 2). Analysis of the amino acid sequence in Example 3 revealed that spots 2 and 3 were derived from the same protein. The isoelectric points of each spot are listed in Table 1.

[0260] Example 3: Mass spectrometry and antigen identification The antigens that produced each of the above spots were subjected to identification of the amino acid sequences by mass spectrometry.

[0261] Specifically, protein extraction and mass spectrometry were performed according to the following procedure. (1) For milk, protein extraction, two-dimensional electrophoresis, and membrane transfer were performed according to the procedures in Examples 1 and 2, and the milk was stained with 0.008% Direct blue / 40% ethanol / 10% acetic acid by shaking. (2) The sections were then decolorized by treating them with 40% ethanol and 10% acetic acid for 5 minutes three times, washed with water for 5 minutes, and air-dried. (3) The desired spot was cut out with a clean cutter blade and placed in a centrifuge tube. After hydrophilizing the membrane with 50 μL of methanol, it was washed twice with 100 μL of water and then centrifuged. 20 μL of 20 mM NH4HCO3·50% acetonitrile was added. (4) 1 μL of 1 pmol / μL lysyl endopeptidase (WAKO) was added, and the mixture was left to stand at 37°C for 60 minutes. The solution was then collected in a new centrifuge tube. 20 μL of 20 mM NH₄HCO₃·70% acetonitrile was added to the membrane, which was then left to soak for 10 minutes at room temperature. The membrane was then further collected. The membrane was then dissolved in 10 μL of 0.1% formic acid and 4% acetonitrile, and transferred to a tube. (5) The collected solution was dried under reduced pressure, dissolved in 15 μl of solution A (0.1% formic acid, 4% acetonitrile solution), and subjected to mass spectrometry (ESI-TOF6600, manufactured by AB Sciex). (6) Proteins were identified based on the mass data obtained from the mass spectrometer by searching Uniprot and NCBI.

[0262] result The amino acid sequence of each spot was detected, and the mass data obtained from the mass spectrometer for each spot was analyzed using Uniprot, NCBI. Each spot was identified as the protein shown in Table 1.

[0263] Spots 2 and 3 are derived from the same protein. The reason these spots were recognized as separate spots in the immunoblot in Example 2 is that even though the amino acid sequences are the same, post-translational modifications such as glycosylation and phosphate group modification have changed the isoelectric point and / or molecular weight. Binding to IgE antibodies was observed regardless of the difference in post-translational modifications, indicating that there is no influence of post-translational modifications.

[0264] Example 4: Epitope Identification Epitopes of allergen components of cow's milk Epitopes of the allergen components of milk were identified by the following procedure.

[0265] (A) Milk epitope mapping (1) Epitope mapping was performed using a library of overlapping peptides (15 amino acids in length) corresponding to the amino acid sequences identified as allergenic components of cow's milk. Specifically, a library of overlapping peptides was prepared based on the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.

[0266] Each synthesized peptide was shifted by 10 amino acids, i.e., each peptide overlaps with the previous and subsequent peptides by 5 amino acids each.

[0267] To prepare peptide arrays, we used Intavis CelluSpots™ technology. The peptide arrays were prepared by the following procedure: (1) synthesizing the peptides of interest on amino-modified cellulose discs using an automated synthesizer (Intavis MultiPep RS), (2) dissolving the amino-modified cellulose discs to obtain a cellulose-bound peptide solution, and (3) spotting the cellulose-bound peptides onto coated glass slides. Details of each procedure are as follows:

[0268] (1) Peptide synthesis Peptide synthesis was performed stepwise on amino-modified cellulose discs in a 384-well synthesis plate using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Specifically, amino acids bearing Fmoc groups were activated with a solution of N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in dimethylformamide (DMF) and added dropwise to the cellulose discs to couple the Fmoc-conjugated amino acids to the amino groups on the cellulose discs. The unreacted amino groups were capped with acetic anhydride and washed with DMF. The Fmoc groups were then removed from the amino groups of the amino acids bound to the cellulose discs by treating with piperidine and washing with DMF. The amino termini were then extended by repeating the coupling, capping, and Fmoc removal steps.

[0269] (2) Dissolution of amino-modified cellulose discs The peptide-bound cellulose disks obtained in the above "(1) Peptide Synthesis" section were 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. The deprotected cellulose-bound peptides were then 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 spotting.

[0270] (3) Spotting of cellulose-binding peptide solution The peptide solution for slide spotting obtained in the above "(2) Dissolution of amino-modified cellulose disc" was spotted onto an Intavis CelluSpots™ slide using an Intavis slide spotting robot, and then dried to prepare a peptide array.

[0271] Using the peptide array, whether or not each peptide fragment binds to IgE antibodies in the serum of milk allergy patients through an antigen-antibody reaction was measured 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) The mixture was shaken overnight at 4°C in a refrigerator. (3) PBST (PBS buffer containing 3% nonionic surfactant Tween® 20) Washed with 10 ml of PBS for 5 minutes (×3). (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 (x 3 times). (6) Add Pierce ECL Plus Western Blotting Substrate (Thermo). The mixture was added and shaken at room temperature for 5 minutes. (7) Using an Amersham Imager 600, the above processes (1) to (6) were performed. The chemiluminescence of the peptide was measured.

[0272] The chemiluminescence intensity of the images obtained by the measurement in (7) above was quantified using ImageQuant TL (GE Healthcare). The second highest value among the quantified values ​​from the images obtained using the serum of six subjects without cow's milk allergy was defined as the N2nd value, and peptides with a value of 800,000 or more, calculated by subtracting the N2nd value of each peptide from the quantified values ​​from the images obtained using the serum of 25 patients, were determined to be peptides that bound to patient-specific IgE antibodies.

[0273] As a result, it was confirmed that patient-specific IgE antibodies bound to peptides (sequence numbers 27, 59, 98, 145, 196, 255, 289, 334, 353, 377, 385, 412, 451, 501, 605, 633, 674, 694, 758, 792, 869, 928, 997, 1038, 1076) derived from spots 1 to 14 (spots 2 and 3 are derived from the same protein) that do not contain any known epitopes.

[0274] (B) Milk epitope mapping (2): overlapping Based on the peptide sequences (sequence numbers 27, 59, 98, 145, 196, 255, 289, 334, 353, 377, 385, 412, 451, 501, 605, 633, 674, 694, 758, 792, 869, 928, 997, 1038, 1076) to which patient-specific serum IgE antibodies bound as described in (A) above, a library of overlapping peptide fragments (length: 10 amino acids) was created using the peptide sequences and sequences obtained by adding the sequences before and after the peptide in the amino acid sequence of the allergy component containing the peptide sequence, and epitope mapping was performed.

[0275] Each synthesized peptide was shifted by one amino acid, i.e., each peptide overlaps with the previous and subsequent peptides by 9 amino acids.

[0276] The library was prepared using the same procedure as in (A) above, and each peptide fragment was evaluated for binding to IgE antibodies in patient serum using the same method as above. The values ​​quantified from images obtained using patient serum were compared with the values ​​obtained from the peptides that served as the basis for overlapping, and peptides that lost or significantly reduced binding to patient IgE antibodies when shifted by one amino acid were determined to be peptides that did not bind to IgE antibodies.

[0277] As in (A) above, the amount of chemiluminescence was quantified for the image obtained by measurement. In the values ​​quantified from images obtained using patient serum, the value obtained for the sequences used as the basis for overlapping (SEQ ID NOs: 27, 59, 98, 145, 196, 255, 289, 334, 353, 377, 385, 412, 451, 501, 605, 633, 674, 694, 758, 792, 869, 928, 997, 1038, 1076) was taken as 100%, and a value of less than 30% indicated no binding to IgE antibodies, a value of 30% to less than 50% indicated poor binding to IgE antibodies but still binding to IgE antibodies, a value of 50% to less than 70% indicated some poor binding to IgE antibodies but still binding to IgE antibodies, and a value of 70% or more indicated no difference in binding to IgE antibodies or good binding to IgE antibodies, and the peptide was determined to have residual binding to IgE antibodies.

[0278] This analysis identified regions in the overlapping sequences that were important for binding to the patient's IgE antibodies.

[0279] (C) Milk epitope mapping (3): Alanine glycine scanning For the amino acid sequence identified in (A) above, a library of peptide fragments was prepared by substituting one amino acid at a time from the amino-terminus with alanine (or glycine if the original amino acid was alanine) using a technique called alanine glycine scanning (Non-Patent Document 1), and each peptide fragment was evaluated for binding to IgE antibodies in patient serum using the same technique as above. Amino acids at positions where binding to the patient's IgE antibody was lost or significantly reduced upon alanine glycine substitution were determined to be amino acids important for the expression of the original antigenicity or amino acids that affect the expression of the original antigenicity, while amino acids where binding to the patient's IgE antibody was not lost or significantly reduced were determined to be amino acids that are not important for the expression of the original antigenicity and can be substituted.

[0280] As in (A) above, the amount of chemiluminescence was quantified for the images obtained by measurement. In the values ​​quantified from the images obtained using the serum of 25 patients, the values ​​obtained using the sequences (SEQ ID NOs: 27, 59, 98, 145, 196, 255, 289, 334, 353, 377, 385, 412, 451, 501, 605, 633, 674, 694, 758, 792, 869, 928, 997, 1038, 1076) on which the alanineglycine scan was based were set at 100%. When the above criteria were met, peptides with a binding affinity of less than 30% had no binding affinity to IgE antibodies, peptides with a binding affinity of 30% to 50% had poor binding affinity to IgE antibodies but still had binding affinity, peptides with a binding affinity of 50% to 70% had somewhat poorer binding affinity to IgE antibodies but still had binding affinity, and peptides with a binding affinity of 70% or more had no difference in binding affinity to IgE antibodies or had good binding affinity to IgE antibodies, and were therefore judged to have remaining binding affinity to IgE antibodies.

[0281] Analysis of the results of (A) to (C) revealed that consensus sequences important for the expression of original antigenicity were found in the regions of the sequence that served as the basis for the alanineglycine scan that were important for binding to the patient's IgE antibody. Sequences were identified for all 25 epitopes, and the results are summarized in Table 2.

[0282] Example 5: Confirmation of epitope cross-reactivity For each epitope sequence found for each protein in milk in Table 2 above, amino acids other than those important for maintaining binding with IgE antibodies were used as an arbitrary amino acid (X). Proteins having the same sequence were searched using BLAST for the allergen foods listed in Table 2-26 that each patient also had. As a result, the amino acid sequences included in the sequences of each food listed in the "Cross-validated foods" column in Table 2 were found as hits.

[0283] The peptides containing the amino acid sequences listed in the "Synthetic Sequence" column of Table 2 were tested by ELISA for their binding to IgE antibodies from patients allergic to each of the foods listed in the "Cross-validated Foods" column of Table 2. The peptides were synthesized by the Fmoc method so as to be biotinylated at the N-terminus.

[0284] Specifically, ELISA was performed according to the following procedure.

[0285] (1) The biotinylated peptide was prepared in PBST (0.1% Tween (registered trademark) 20) so that the concentration was 10 μg / mL.

[0286] (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 recovering the solution, the plate was washed five times with PBS.

[0287] (3) 80 μL of Pierce Protein-Free (PBS) Blocking Buffer (Thermo) was added, and the mixture was shaken at room temperature for 1 hour. After removing the solution, the mixture was washed three times with PBST.

[0288] (4) 40 μL of 2% serum / Canget Signal Solution I (TOYOBO) was added, and the mixture was shaken at room temperature for 1 hour. After removing the solution, the mixture was washed five times with PBST.

[0289] (5) 40 μL of secondary antibody dilution solution (1:10000, Canget Signal Solution II (TOYOBO)) was added, and the mixture was shaken at room temperature for 1 hour. After removing the solution, the mixture was washed three times with PBST.

[0290] (6) 40 μL of 1-Step Ultra TMB-ELISA (Thermo) was added and the mixture was shaken at room temperature for 15 minutes.

[0291] (7) 40 μL of 2 M H2SO4 was added, and the absorbance at 450 nm was measured.

[0292] Peptides having these amino acid sequences were prepared in the same manner as in (A) of Example 4, and the binding of IgE antibodies in the sera of allergic patients and non-allergic subjects was measured. For the non-allergic subjects, the sera of two subjects were measured and the value was divided by the average value.

[0293] The results are shown in Figures 3 to 6. P1, P5, P8, and P20 represent patient numbers. The bar graphs in each figure show the absorbance of allergic patients versus that of non-allergic patients (healthy individuals). The names of ingredients listed in each graph are the names of ingredients containing polypeptides that contain the amino acid sequence that forms the basis of each synthetic sequence used. If no ingredient name is listed, the ingredient is "beef."

[0294] As is clear from Figures 3 to 6, all polypeptides having the amino acid sequences shown in the figures showed higher binding affinity (greater than "1") to the IgE antibodies of each allergic patient than to the IgE antibodies in the serum of non-allergic subjects, confirming the cross-reactivity of these polypeptides. This indicates that these epitopes can be used to detect cross-reactivity with antigens other than milk. Furthermore, this supports the idea that the "X" moiety can be any amino acid residue.

Claims

1. A diagnostic kit for milk allergy, comprising at least one of the following polypeptides (E1) and (E2): (E1) a polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 27, 29-31, 33, 34, 38, 41, 44-46, 49, 52, and 54; (E2) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 59, 61, 63-66, 69-73, 75, 80, 82, 85, 87, 92, 93, 95, and 97.

2. A diagnostic composition for cow's milk allergy, comprising at least one of the polypeptides identified as (E1) and (E2) in claim 1.

3. 1. A method for providing an indicator for diagnosing cow's milk allergy in a subject, comprising the steps of: (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) if binding of the subject's IgE antibodies to the antigen is detected, an indication that the subject is allergic to cow's milk is provided; wherein the antigen is at least one of the polypeptides identified as (E1) and (E2) in claim 1.

4. An antigen that causes cow's milk allergy, said antigen being at least one of the polypeptides identified as (E1) and (E2) in claim 1.

5. A composition comprising at least one of the antigens of claim 4.

6. The composition according to claim 5 for treating cow's milk allergy.

7. A tester composition for determining the presence or absence of a cow's milk antigen in a subject, comprising an antibody that binds to at least one of the polypeptides identified as (E1) and (E2) in claim 1.

8. A tester composition for determining the presence or absence of a milk antigen in a subject, comprising at least one primer comprising a portion of the base sequence of a nucleic acid encoding the polypeptides identified as (E1) and (E2) in claim 1 and / or a portion of its complementary strand.

9. A tester composition for cow's milk allergy for determining the presence or absence of IgE antibodies in a subject, comprising the polypeptides identified as (E1) and (E2) in claim 1.

10. A method for determining the presence or absence of polypeptides identified as (E1) and (E2) in claim 1 in raw materials or processed products, wherein the raw materials or processed products are milk or milk processed products, and the method comprises detecting the polypeptides identified as (E1) and (E2) in claim 1 in the raw materials or processed products.

11. A raw material or processed product in which an antigen has been removed or reduced, said raw material or processed product being milk or a milk processed product, and said antigen being at least one of the polypeptides identified as (E1) and (E2) in claim 1.

12. A method for producing raw materials or processed products in which antigens have been removed or reduced, the method comprising a step of confirming that the raw materials or processed products are milk or milk processed products and that antigens have been removed or reduced during the production process of the raw materials or processed products, wherein the antigen is at least one of the polypeptides identified as (E1) and (E2) in claim 1.

Images