New antigen for milk allergy

Abstract

To provide new antigens for milk allergy, a diagnosis method and diagnosis kit for milk allergy, a composition containing the antigens, milk or milk products from which the antigens have been removed, and a tester composition for determining the presence or absence of milk antigens in a subject.SOLUTION: The present invention relates to a polypeptide containing an epitope of an antigen, a diagnostic kit for allergy containing the polypeptide, a diagnostic composition, a diagnostic method, a composition containing the polypeptide, and a raw material or a processed product in which the antigen containing the polypeptide has been removed or reduced. The present invention also relates to a tester composition containing an antibody against at least one polypeptide containing an amino acid sequence.SELECTED DRAWING: None

Description

Technical Field

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

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

Background Art

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

[0004] In conventional allergy test drugs, it is often the case that an antigen reagent is prepared simply by grinding food, food ingredients, etc. that are allergy candidate substances (Patent Document 1). For this reason, among the numerous allergen components contained in conventional antigen reagents, a positive reaction can be detected only when the content exceeds the threshold value at which a positive reaction can be determined for binding to IgE antibodies, and the diagnostic efficiency was not sufficiently high.

[0005] In food and food ingredients that are allergy candidates, several allergen components have been suggested and commercialized as test kits. In order to improve the reliability of allergy tests, it is necessary to comprehensively identify allergen components. However, the patient detection rate based on the measurement of the above allergen components is still insufficient. Identifying novel allergens in milk is very important not only for improving the accuracy of diagnostic drugs, but also as targets for low-allergen foods, low-allergen food ingredients, and therapeutic drugs.

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

[0007] Allergen-specific IgE antibodies recognize and bind to an epitope, which is a specific amino acid sequence in an allergen component. However, although there are a few examples in which the allergen component has been analyzed up to the epitope (Non-Patent Document 1), the current situation is that there are extremely few. In addition, at present, there is no allergy diagnostic kit on the market that uses a polypeptide containing an epitope.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Document

[0009]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] The present invention provides a novel antigen of allergy which is a protein. The present invention also provides a diagnostic method and a diagnostic kit for allergy containing the antigen. The present invention also provides a composition containing the antigen, and a raw material or processed product in which the antigen is removed or reduced. The present invention further provides a tester composition for determining the presence or absence of the antigen in an object.

[0011] The present invention also provides an antigen of a polypeptide containing an epitope. The present invention also provides a diagnostic kit, a diagnostic composition, and a diagnostic method for allergy containing the polypeptide. The present invention also provides a method for providing an index for diagnosing the allergy of a subject. The present invention also provides a composition containing the polypeptide, and a raw material or processed product in which the antigen containing the polypeptide is removed or reduced. The present invention further relates to a method for producing a raw material or processed product in which the antigen is removed or reduced. The present invention further provides a tester composition for determining the presence or absence of the antigen containing the polypeptide in an object.

Means for Solving the Problem

[0012] In order to solve the above problems, the present inventors intensively studied the identification of causative antigens for milk allergy. As a result, they succeeded in identifying a novel antigen of a protein to which IgE antibodies in the sera of patients with milk allergy specifically bind.

[0013] In addition, since the epitope has a relatively short amino acid sequence, if the same amino acid sequence exists in different allergen components, the IgE antibody can bind to a plurality of allergen components. As a result of the existence of an epitope common to different allergen components, the IgE antibody of an allergic patient binds to both, and thus the antigen has cross-reactivity. Therefore, the epitope identified by the present invention enables the diagnosis and treatment of allergies including cross-reactivity, and the detection of a plurality of allergen components including the epitope.

[0014] In the present specification, "antigen" is used in the meaning including both a narrow-sense antigen which is a protein and an "epitope" derived from a protein, unless otherwise specified. When specified, "antigen" is used in the meaning of either a protein or an epitope derived from a protein.

[0015] Based on the above findings, the present invention has been completed. Non-limitingly, the present invention includes the following aspects. [Aspect 1] An allergy diagnosis kit containing at least one of the following polypeptides (E1)-(E25): (E1) A polypeptide containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 27-58 and 1115; (E2) A polypeptide containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 59-97; (E3) A polypeptide containing at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 98-144; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 145-195; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 196-254 and 1116-1118; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 255-288; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 289-333 and 1119-1120; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 334-352; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 353-376 and 1121-1122; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 377-384; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 385-411; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 412-450; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 451-500 and 1123-1124; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 501-604 and 1125-1127; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 605-632 and 1128; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 633-673; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 674-693; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 694-757 and 1129; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 758-791; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 792-868 and 1130; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 869-927; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 928-996; 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 diagnostic composition for allergy, comprising at least one of the polypeptides specified as (E1)-(E25) in Aspect 1. [Aspect 3] A method for providing an indicator for diagnosing an allergy in a subject, comprising the following steps: (i) contacting a sample obtained from the subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting the binding of IgE antibodies in the sample obtained from the subject to the antigen; (iii) when the binding of the subject's IgE antibodies to the antigen is detected, providing an indicator that the subject has an allergy; wherein the antigen is at least one of the polypeptides specified as (E1)-(E25) in Aspect 1. [Aspect 4] At least one of the polypeptides specified as (E1) to (E25) in Aspect 1, and the antigen that causes allergy. [Aspect 5] A composition comprising at least one of the antigens described in Aspect 4. [Aspect 6] The composition according to Aspect 5 for treating allergy. [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 specified as (E1) to (E25) in Aspect 1. [Aspect 8] A tester composition for determining the presence or absence of an antigen in a subject, comprising at least one primer that includes a part of the base sequence of a nucleic acid encoding the polypeptide specified as (E1) to (E25) in Aspect 1 and / or a part of its complementary strand. [Aspect 9] A tester composition for determining the presence or absence of IgE antibodies in a subject, comprising the polypeptide specified as (E1) to (E25) in Aspect 1. [Aspect 10] A method for determining the presence or absence of the polypeptide specified as (E1) to (E25) in Aspect 1 in a raw material or processed product, the method including detecting the polypeptide specified as (E1) to (E25) in Aspect 1 in the raw material or processed product. [Aspect 11] A raw material or processed product in which the antigen has been removed or reduced, the antigen being at least one of the polypeptides specified as (E1) to (E25) in Aspect 1. [Aspect 12] A method for manufacturing a raw material or processed product in which the antigen has been removed or reduced, the method having a step of confirming that the antigen has been removed or reduced during the manufacturing process of the raw material or processed product, where the antigen is at least one of the polypeptides specified as (E1) to (E25) in Aspect 1. [Advantages of the Invention]

[0016] According to the present invention, a novel antigen for allergy (for example, allergy to milk) can be provided. Since a novel allergen component that causes allergy has been identified in the present invention, a highly sensitive diagnostic method and diagnostic kit for allergy, a composition containing the antigen, a raw material or processed product from which the antigen has been removed or reduced, and a tester composition for determining the presence or absence of the antigen in a target object can be provided.

[0017] Further, according to the present invention, an antigen of a novel polypeptide containing an epitope of a protein antigen can be provided. By using the polypeptide of the present invention, a highly sensitive diagnostic kit, diagnostic composition, and diagnostic method for allergy (for example, allergy to milk), a composition containing the polypeptide, a tester composition for determining the presence or absence of an antigen containing the polypeptide in a target object, and a raw material or processed product from which the polypeptide has been removed or reduced, and a method for producing the raw material or processed product can be provided.

Brief Description of the Drawings

[0018]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

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

[0020] Unless otherwise defined specifically in this specification, scientific terms and technical terms used in connection with the present invention shall have the meanings generally understood by those skilled in the art.

[0021] As used herein, allergy refers to a state in which a living body sensitized to a certain antigen exhibits an allergic reaction, which is unfavorable to the living body, when the antigen enters again. An allergic reaction can occur when contacting an antigen or ingesting the antigen. Here, "contact" means touching an object, and in particular, for the human body, it means adhering to the skin, mucous membranes (such as eyes, lips, etc.). Also, "ingestion" means taking in into the body, and taking in by means such as inhalation or oral ingestion. An allergic reaction that generally occurs when ingesting food is particularly referred to as food allergy. In a preferred embodiment, the allergy may be a food allergy. In many allergic diseases in food, antigen-specific IgE antibodies are produced in the blood and tissues. The IgE antibody binds to mast cells or basophils. When the antigen specific to the IgE antibody enters the body of a patient with an allergic disease again, 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, or various leukotrienes, etc. These released substances cause an allergic reaction caused by the combination of an IgE antibody and a specific antigen. Specifically, the IgE antibody recognizes and binds to an epitope, which is a specific amino acid sequence in a specific antigen, and the allergic reaction caused by the antigen appears through the above-mentioned pathway.

[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 to be used. As one aspect, the allergen includes milk, dairy products, milk processed products, livestock meat, livestock meat processed products, grains, seafood, fruits, vegetables, nuts (seeds), edible herbs, seafood, eggs, egg processed products, etc. that are ingested by a living body (especially a human), or parasites that parasitize a living body (especially a human), etc.

[0023] The origin of milk is not particularly limited. In one aspect, it includes milk derived from cows, goats, sheep, etc. In one aspect, it is cow's milk. Without limitation, milk includes raw milk, pasteurized milk, adjusted-component milk, low-fat milk, and fat-free milk. Dairy products are products processed from milk as the main raw material. Without limitation, dairy products include cream, butter, butter oil, cheese, whey, concentrated whey, ice creams, condensed milk, skimmed condensed milk, unsweetened condensed milk, unsweetened skimmed condensed milk, sweetened condensed milk, sweetened skimmed condensed milk, whole milk powder, skimmed milk powder, cream powder, whey powder, protein-concentrated whey powder, butter milk powder, sweetened milk powder, prepared milk powder, prepared liquid milk, fermented milk such as yogurt, lactic acid bacteria beverages, and milk beverages. Processed cow's milk products refer to dairy products processed from cow's milk as the main raw material and foods containing this as a raw material. Without limitation, processed cow's milk products include cream, butter, butter oil, cheese, whey, concentrated whey, ice creams, condensed milk, skimmed condensed milk, unsweetened condensed milk, unsweetened skimmed condensed milk, sweetened condensed milk, sweetened skimmed condensed milk, whole milk powder, skimmed milk powder, cream powder, whey powder, protein-concentrated whey powder, butter milk powder, sweetened milk powder, prepared milk powder, prepared liquid milk, fermented milk such as yogurt, lactic acid bacteria beverages, milk beverages, and cakes, pastries, custard puddings, milk jelly, biscuits, cookies, snacks, chocolates, bread, white sauce, puree, cream stew, gratin, curry roux, and stew roux containing these as raw materials.

[0024] The type of livestock meat is not particularly limited. In one aspect, it includes the meat of birds (such as chicken and duck) and the meat of mammals (such as pork, beef, and mutton). In one aspect, it is the meat of mammals. In one aspect, it is the meat of Bos taurus. Processed livestock meat products refer to products processed from livestock meat as the main raw material and foods containing this as a raw material. Without limitation, processed livestock meat products include ham, sausage, bacon, bouillon, consommé, steak, grilled meat, roast beef, tartare steak, hamburger, hamburger patty, meatball, and nugget.

[0025] Cereals are one of the general terms for food ingredients obtained from plants, and are seeds mainly composed of starch for consumption. Narrowly defined, cereals refer only to the seeds of Gramineae crops (cereals), and broadly defined, they include the seeds of leguminous crops (pulses) and the seeds of other crops. Among the broadly defined cereals, the seeds of dicotyledonous plants that are similar to the seeds of Gramineae (seeds of monocotyledonous Gramineae crops) and are used as cereals are collectively called pseudo-cereals or pseudocereals. Pseudocereals include buckwheat (Polygonaceae), amaranth (Amaranthaceae), quinoa (Chenopodiaceae), etc.

[0026] In the present invention, non-limitingly, Gramineae cereals include common wheat (Triticum aestivum), barley, rye, spelt, wild rice, Japanese millet, foxtail millet, proso millet, corn, pearl barley, etc. Polygonaceae cereals include, for example, buckwheat (Fagopyrum esculentum) of the genus Fagopyrum in the Polygonaceae family. In one aspect, the antigen (allergen) is derived from cereals. In one aspect, the allergen is derived from Gramineae cereals. In one aspect, the allergen is derived from wheat.

[0027] Non-limitingly, fishery products include shrimp, crabs, etc. belonging to the order Decapoda (shrimp order). Generally, what is recognized as "crustaceans" is mostly included in the order Decapoda (shrimp order). Non-limitingly, fishery products also include squid and octopus belonging to the order Teuthida and the order Octopoda. Fishery products further include fish belonging to the families Scombridae and Gadidae. Fishery products also further include shellfish of the family Mytilidae.

[0028] Non-limitingly, fruits include, for example, fruits belonging to the families Actinidiaceae, Bromeliaceae, Urticaceae, Cucurbitaceae, Musaceae, Rutaceae, and Rosaceae. Vegetables include, for example, fruits belonging to the families Solanaceae, Cucurbitaceae, and Lauraceae. Nuts (seed fruits) include, for example, nuts (seed fruits) belonging to the families Urticaceae, Rosaceae, Juglandaceae, and nuts belonging to the family Fabaceae such as peanuts. Edible herbs include, for example, edible herbs belonging to the family Asteraceae. Cereals include, for example, cereals belonging to the families Gramineae and Polygonaceae.

[0029] In the present invention, the eggs include, in addition to the eggs of the above-mentioned seafood, the eggs of birds. Non-limiting examples of the eggs of birds include quail eggs and chicken eggs. An egg processed product refers to a product obtained by processing eggs as the main raw material and foods containing this as a raw material. Non-limiting examples of the egg processed products include processed fish roe products such as salted cod roe, spicy pollock roe, salted salmon roe, soy sauce-pickled salmon roe, flying fish roe, and squid roe, and processed chicken egg products such as mayonnaise, Hollandaise sauce, boiled quail eggs, boiled eggs, hard-boiled eggs, fried eggs, thick omelets, steamed eggs with dashi, steamed egg custard, omelets, omurice, baked eggs, poached eggs, quiches, Scotch eggs, French toast, cakes, pastries, custard puddings, biscuits, cookies, snacks, and bread.

[0030] The parasite is, non-limitingly, for example, a parasite of the family Anisakidae. The parasites of the family Anisakidae include Anisakis simplex.

[0031] As used herein, allergy refers to a state having an allergic reaction caused by a protein or the like contained in a raw material or a processed product (for example, milk or a milk processed product) as an antigen. An allergy can cause an allergic reaction when contacting or ingesting an antigen contained in a raw material or a processed product (for example, milk or a milk processed product). An allergic reaction that generally occurs when ingesting food is particularly referred to as food allergy. An allergy to milk may be a food allergy.

[0032] As used herein, an antigen is a substance that causes an allergic reaction. When it is a protein contained in a raw material such as a food ingredient, it is also referred to as an allergen component. An 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 means a molecule having a structure in which natural amino acids are linked by peptide bonds. The number of amino acids contained in a polypeptide is not particularly limited. "Polypeptide" is a concept that includes "protein". In addition, a polypeptide in which about 2 to 50 amino acids are linked by peptide bonds may be particularly referred to as a peptide.

[0034] When there may be optical isomers with respect to an amino acid, the L-form is indicated unless otherwise specified. The notation of the amino acid sequence of a protein, polypeptide or peptide used herein is represented by the one-letter notation of amino acids based on standard usage and the notation commonly used in the art, with the left direction being the amino-terminal direction and the right direction being the carboxy-terminal direction. In the one-letter notation of amino acids, X may be any substance having an amino group and a carboxyl group that can bind to the amino acids at both ends, and particularly represents that it may be any of the 20 natural amino acids.

[0035] The alanine scan method (or "alanine-glycine scan" method) is a method for site-specifically identifying residues that are important for the structure and function of a protein by creating mutants in which each residue in the protein is mutated to alanine (or glycine if the original amino acid is alanine). When the binding to the patient's IgE antibody remains even after mutation to alanine (or glycine if the original amino acid is alanine), it means that the residue is not important for the binding to the IgE antibody and the binding will remain even if it is changed to other amino acids. The binding to the IgE antibody means that the binding and reaction of the target epitope and the IgE antibody are detected. In the sequence listing of the present application, the residue represented by X is an amino acid residue at a site where the binding to the IgE antibody of an allergic patient remains even after substitution with alanine (or glycine if the original amino acid is alanine) by the alanine-glycine scan shown in Example 4. It is well known to those skilled in the art that for such sites, the probability that the binding to the IgE antibody will remain even if it is substituted with any other amino acid is high. 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 and the antigen (epitope) are important for subsequent allergic reactions, and this binding and maintenance are carried out by the charge, hydrophobic bond, hydrogen bond, and aromatic interaction of the epitope. The fact that these can be lost by changing to alanine or glycine but the binding and maintenance can still occur means that the amino acid is not important.

[0037] Identification of antigen The protein contained in milk was subjected to two-dimensional electrophoresis under the following conditions to identify the antigen for milk allergy.

[0038] For the first-dimensional electrophoresis, as the isoelectric focusing gel, a gel with a gel length in the range of 5 to 10 cm, a gel pH range of 3 to 10, and a pH gradient of the gel with respect to the electrophoresis direction, with the total length of the gel being 1, the gel length up to pH 5 being a, the gel length from pH 5 to 7 being b, and the gel length of pH 7 or higher being c, is used. Specifically, isoelectric focusing is performed using an IPG gel Immobiline Drystrip (pH3-10NL) manufactured by GE Healthcare Biosciences Corporation (hereinafter abbreviated as GE). The electrophoresis equipment 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. The voltage program was as follows: (1) A constant voltage step was performed at 300 V constant voltage until 750 Vhr (the current change range during the last 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. (4) Subsequently, the first-dimensional isoelectric focusing was performed at 5000 V constant voltage until the total Vhr reached 12000.

[0039] For the second-dimensional electrophoresis, a polyacrylamide gel with a gel concentration of 3 to 6% set at the base end in the electrophoresis direction and a gel concentration at the tip side in the electrophoresis direction set higher than the gel concentration at the base end in the electrophoresis direction was used. Specifically, SDS-PAGE was performed using NuPAGE 4-12% Bis-Tris Gels IPG well mini 1mm manufactured by life technologies. The electrophoresis equipment used was an XCell SureLock Mini-Cell manufactured by life technologies. As the electrophoresis buffer, 50 mM MOPS, 50 mM Tris base, 0.1% (w / v) SDS, and 1 mM EDTA were used, and electrophoresis was performed at 200 V constant voltage for approximately 45 minutes.

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

[0041] Antigen (protein) For each spot, sequence identification by mass spectrometry was performed. The mass data obtained from the mass spectrometer was compared and analyzed with the protein data of Uniprot and NCBI. As a result, it was found that each of the spots 1 to 14 matched the known sequences. The information of each spot was 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 types of proteins from (1) to (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 (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, the proteins (1)-(13) are classified into four groups each having a common function: 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] Non-limitingly, the antigen at Spot 1 in the present invention may be any of the following (1-a) to (1-f).

[0058] A protein comprising the amino acid sequence of SEQ ID NO: 2; A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 2; A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 2; A protein comprising an amino acid sequence encoded by a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 1; A protein comprising an amino acid sequence encoded by a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1; or 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 represented by SEQ ID NO: 1.

[0059] Non-limitingly, the antigens of spots 2 and 3 in the present invention may be any of the following (2-a) to (2-f).

[0060] A protein comprising the amino acid sequence of SEQ ID NO: 4; A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 4; A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 4; A protein comprising an amino acid sequence encoded by a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 3; A protein comprising an amino acid sequence encoded by a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 3; or 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 represented by SEQ ID NO: 3.

[0061] Non-limitingly, the antigen of spot 4 in the present invention may be 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 an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 6; (3-c) A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 6; (3-d) A protein comprising an amino acid sequence encoded by a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 5; (3-e) A protein comprising an amino acid sequence encoded by a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by 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 nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 5.

[0063] Non-limitingly, the antigen of spot 5 in the present invention may be any one of the following (4-a) to (4-f).

[0064] (4-a) A protein comprising the amino acid sequence of SEQ ID NO: 8; (4-b) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 8; (4-c) A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 8; (4-d) A protein comprising an amino acid sequence encoded by a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 7; (4-e) A protein comprising an amino acid sequence encoded by a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 7; or 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 represented by SEQ ID NO: 7.

[0065] Non-limitingly, the antigen of spot 6 in the present invention may be any one of the following (5-a) to (5-f).

[0066] (5-a) A protein comprising the amino acid sequence of SEQ ID NO: 10; (5-b) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 10; (5-c) A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 10; (5-d) A protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 9; (5-e) A protein comprising an amino acid sequence encoded by a base sequence having 70% or more identity with the base sequence represented by 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 represented by SEQ ID NO: 9.

[0067] Non-limitingly, the antigen of spot 7 in the present invention may be any one of the following (6-a) to (6-f).

[0068] (6-a) A protein comprising the amino acid sequence of SEQ ID NO: 12; (6-b) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 12; (6-c) A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 12; (6-d) A protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 11; A protein comprising an amino acid sequence encoded by a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by 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 nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 11.

[0069] Non-limitingly, the antigen at spot 8 in the present invention may be any one of the following (7-a) to (7-f).

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

[0071] Non-limitingly, the antigen at spot 9 in the present invention may be any one of the following (8-a) to (8-f).

[0072] (8-a) A protein comprising the amino acid sequence of SEQ ID NO: 16; (8-b) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 16; (8-c) A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 16; A protein comprising an amino acid sequence encoded by a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted or added at SEQ ID NO: 15; (8-e) A protein comprising an amino acid sequence encoded by a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by 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 nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 15.

[0073] Non-limitingly, the antigen of spot 10 in the present invention may be any of the following (9-a) to (9-f).

[0074] (9-a) A protein comprising the amino acid sequence of SEQ ID NO: 18; (9-b) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added at SEQ ID NO: 18; (9-c) A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 18; (9-d) A protein comprising an amino acid sequence encoded by a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted or added at SEQ ID NO: 17; (9-e) A protein comprising an amino acid sequence encoded by a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by 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 nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 17.

[0075] Non-limitingly, the antigen of spot 11 in the present invention may be any of the following (10-a) to (10-f).

[0076] (10-a) A protein comprising the amino acid sequence of SEQ ID NO: 20; A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added at SEQ ID NO: 20; A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 20; A protein comprising an amino acid sequence encoded by a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted or added at SEQ ID NO: 19; A protein comprising an amino acid sequence encoded by a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 19; or 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 represented by SEQ ID NO: 19.

[0077] Non-limitingly, the antigen at spot 12 in the present invention may be any of the following (11-a) to (11-f).

[0078] A protein comprising the amino acid sequence of SEQ ID NO: 22; A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added at SEQ ID NO: 22; A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 22; A protein comprising an amino acid sequence encoded by a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted or added at SEQ ID NO: 21; A protein comprising an amino acid sequence encoded by a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 21; or 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 represented by SEQ ID NO: 21.

[0079] Non-limitingly, the antigen of spot 13 in the present invention may be any one of the following (12-a) to (12-f).

[0080] (12-a) A protein comprising the amino acid sequence of SEQ ID NO: 24; (12-b) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 24; (12-c) A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 24; (12-d) A protein comprising an amino acid sequence encoded by a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 23; (12-e) A protein comprising an amino acid sequence encoded by a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by 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 nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 23.

[0081] Non-limitingly, the antigen of spot 14 in the present invention may be any one of the following (13-a) to (13-f).

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

[0083] The proteins that are the antigens of (1) to (13) above and the polypeptides of (E1) to (E25) described below also include modes in which the amino acid residues of the protein or polypeptide are modified by phosphorylation, sugar chain modification, aminoacylation, ring opening, deamination, etc.

[0084] Preferably, the proteins that are the antigens of (1) to (13) above and the polypeptides of (E1) to (E25) described below are antigens for allergy.

[0085] In this specification, when it is said that "one or several amino acids are deleted, substituted, inserted or added" with respect to an amino acid sequence, it means an amino acid sequence in which one or several amino acids are deleted, substituted with other amino acids, inserted with other amino acids, and / or added with other amino acids in the target amino acid sequence. "Several amino acids" means, without limitation, within 200, within 100, within 50, within 30, within 20, within 15, within 12, within 10, within 8, within 6, within 4, within 3 amino acids. Alternatively, several amino acids means 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1% of the total length of the amino acid sequence.

[0086] Among the above, the substitution is preferably a conservative substitution. A conservative substitution is to replace a specific amino acid residue with a residue having similar physicochemical characteristics, but any substitution may be used as long as it does not substantially change the characteristics related to the structure of the original sequence. For example, any substitution may be used as long as the substituted amino acid does not disrupt the helix present in the original sequence or other types of secondary structures that characterize the original sequence. Hereinafter, conservative substitutions of amino acid residues will be classified and exemplified for each substitutable residue, but the substitutable amino acid residues are not limited to those described below. Group A: Leucine, Isoleucine, Valine, Alanine, Methionine, Glycine, Cysteine, Proline Group B: Aspartic acid, Glutamic acid Group C: Asparagine, Glutamine Group D: Lysine, Arginine Group E: Serine, Threonine Group F: Phenylalanine, Tyrosine, Tryptophan, Histidine In the case of non-conservative substitution, one member of the above types can be exchanged with a member of another type. For example, in order to eliminate inadvertent sugar chain modification, the amino acids in groups B, D, and E above may be substituted with amino acids in other groups. Alternatively, cysteine may be deleted or substituted with another amino acid in order to prevent folding in the protein in the tertiary structure. Or, the amino acids may be substituted in consideration of the hydrophobicity / hydrophilicity index of amino acids (J. Kyte and R. Doolittle, J. Mol. Biol., Vol.157, p.105-132, 1982), which is an index of hydrophobicity / hydrophilicity regarding amino acids, so that the balance of hydrophilicity / hydrophobicity is maintained or the hydrophilicity is increased to facilitate synthesis.

[0087] As another aspect, substitution with an amino acid having less steric hindrance than the original amino acid, for example, substitution from group F to groups A, B, C, D, and E; substitution from a charged amino acid to an uncharged amino acid, for example, substitution from group B to group C, may be performed. By doing so, the binding property with the IgE antibody may be improved.

[0088] In this specification, the identity percentage of two amino acid sequences can be determined by visual inspection and mathematical calculation. It 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 search by the BLAST program are those described by Altschul et al. (Nucl. Acids. Res., 25, p.3389-3402, 1997) and can be publicly obtained from the websites of NCBI and DNA Data Bank of Japan (DDBJ) (BLAST Manual, Altschul et al., NCB / NLM / NIH Bethesda, MD 20894; Altschul et al.). It can also be determined using programs such as the genetic information processing software GENETYX Ver.7 (Genetics), DNASIS Pro (Hitachi Software), and Vector NTI (Infomax).

[0089] In this specification, when it is stated that "one or several nucleotides are deleted, substituted, inserted, or added" for a nucleotide sequence, it refers to a nucleotide sequence in which one or several nucleotides are deleted, substituted with other nucleotides, inserted with other nucleotides, and / or added with other nucleotides in the target nucleotide sequence. "Several nucleotides" means, without limitation, within 600, within 300, within 150, within 100, within 50, within 30, within 20, within 15, within 12, within 10, within 8, within 6, within 4, or within 3 nucleotide acids. Alternatively, several nucleotides mean 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2%, or 1% of the nucleotides with respect to the full length of the nucleotide sequence. It is preferable that the deletion, substitution, insertion, or addition of the above nucleotides does not cause a frameshift in the sequence encoding the amino acid.

[0090] In this specification, the identity percentage of two nucleotide sequences can be determined by visual inspection and mathematical calculation. It can also be determined using a computer program. Examples of such sequence comparison computer programs include, for example, the BLASTN program available from the website of the National Library of Medicine, USA: https: / / blast.ncbi.nlm.nih.gov / Blast.cgi (Altschul et al. (1990) J. Mol. Biol. 215: 403-10), version 2.2.7, or the WU-BLAST2.0 algorithm. For the standard default parameter settings for WU-BLAST2.0, those described on the following Internet site: http: / / blast.wustl.edu can be used.

[0091] As used herein, "stringent conditions" means hybridization under moderately or highly stringent conditions. Specifically, moderately stringent conditions can be easily determined by those of ordinary skill in the art based on, for example, the length of the DNA. The basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Chapters 6-7, Cold Spring Harbor Laboratory Press, 2001. Preferably, moderately stringent conditions include, as hybridization conditions, 1×SSC to 6×SSC, 42°C to 55°C, more preferably 1×SSC to 3×SSC, 45°C to 50°C, and most preferably 2×SSC, 50°C. When, for example, about 50% formamide is included in the hybridization solution, a temperature 5 to 15°C lower than the above temperature is employed. As washing conditions, 0.5×SSC to 6×SSC, 40°C to 60°C are included. Generally, 0.05% to 0.2%, preferably about 0.1% SDS may be added during hybridization and washing. Highly stringent conditions can also be easily determined by those of ordinary skill in the art based on, for example, the length of the DNA. Generally, highly stringent (high-stringent) conditions include hybridization and / or washing at a higher temperature and / or a lower salt concentration than moderately stringent conditions. For example, as hybridization conditions, 0.1×SSC to 2×SSC, 55°C to 65°C, more preferably 0.1×SSC to 1×SSC, 60°C to 65°C, and most preferably 0.2×SSC, 63°C are included. As washing conditions, 0.2×SSC to 2×SSC, 50°C to 68°C, more preferably 0.2×SSC, 60 to 65°C are included.

[0092] Optionally, in the variants corresponding to items (b)-(f) of (1)-(13) above, it is preferable to include the amino acid sequences of the epitopes (variants) described below. The amino acid sequences of the epitopes included are not limited to one, and preferably all the sequences of the epitopes respectively derived from each protein are included. For example, the epitopes derived from protein (1) are (E1) and (E2). (1-b)-(1-f) preferably include one or more of the amino acid sequences (including variants) of the (E1) or (E2) epitope.

[0093] The antigen may be obtained by separating and purifying it from milk by combining protein purification methods well-known to those skilled in the art. Alternatively, the antigen may be obtained by expressing the antigen as a recombinant protein by gene recombination techniques well-known to those skilled in the art and then separating and purifying it by protein purification methods well-known to those skilled in the art.

[0094] Examples of protein purification methods include methods using solubility such as salting out and solvent precipitation, methods using differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE, methods using charge such as ion exchange chromatography and hydroxylapatite chromatography, methods using specific affinity such as affinity chromatography, methods using differences in hydrophobicity such as reverse phase high performance liquid chromatography, and methods using differences in isoelectric point such as isoelectric focusing electrophoresis.

[0095] The preparation of a protein by gene recombination techniques involves preparing an expression vector containing a nucleic acid encoding the antigen, introducing the expression vector into an appropriate host cell by gene transfer or transformation, culturing the host cell under conditions suitable for the expression of the recombinant protein, and recovering the recombinant protein expressed in the host cell.

[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 direct the expression of a protein encoded by the nucleic acid introduced by the vector. Vectors include plasmid vectors, viral vectors, and the like. Those skilled in the art can select an appropriate expression vector for the expression of a recombinant protein according to the type of host cell to be used.

[0097] A "host cell" is a cell that is transfected or transformed by a vector. The host cell can be appropriately selected by those skilled in the art according to the vector to be used. The host cell can be, for example, derived from a prokaryote such as Escherichia coli (E. coli). When using a prokaryotic cell such as E. coli as a host, the antigen of the present invention may contain an N-terminal methionine residue to facilitate the expression of the recombinant protein in the prokaryotic cell. This N-terminal methionine can also be cleaved from the recombinant protein after expression. Alternatively, it can be a cell derived from a eukaryote such as a single-celled eukaryote such as yeast, a plant cell, an animal cell (for example, a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell or an insect cell), or a silkworm.

[0098] The introduction of the expression vector into the host cell or transformation can be appropriately carried out by methods known to those skilled in the art. Also, those skilled in the art can appropriately select conditions suitable for the expression of the recombinant protein according to the type of host cell, and culture the host cell to express the recombinant protein. Then, the host cell expressing the recombinant protein is homogenized, and the antigen expressed as a recombinant protein can be separated and purified by appropriately combining the above-described protein purification methods from the obtained homogenate. The antigen can also be prepared by introducing the above expression vector or synthesized double-stranded DNA, or mRNA transcribed therefrom into a cell-free protein synthesis system for expression, and separating and purifying the expressed protein.

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

[0100] Diagnostic kit · Diagnostic method (1) The present invention is a method for providing an index for diagnosing an allergy of a subject, comprising the following steps: (i) contacting a sample obtained from the subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting the binding of the IgE antibodies in the sample obtained from the subject to the antigen; (iii) when the binding of the subject's IgE antibodies to the antigen is detected, providing an index indicating that the subject has an allergy; wherein the antigen is at least one of the proteins specified as the antigens in (1) to (13) above, and the method is provided.

[0101] "allergy" is, in one aspect, without limitation, 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 doctor but also mere "detection" including the possibility. As used herein, "diagnosis" and "detection" are, in one aspect, "diagnosis" and "detection" in vivo, in vitro or ex vivo. Preferably, they are "diagnosis" and "detection" in vitro.

[0103] The sample obtained from the subject is a solution containing IgE antibodies collected from the subject. Such solutions include, for example, blood, saliva, sputum, nasal discharge, urine, sweat, and tears. The sample obtained from the subject may be subjected to a pretreatment for increasing the IgE antibody concentration in the sample before contacting it with the antigen. The pretreatment of the sample may include, for example, obtaining serum or plasma from blood. Furthermore, the Fab portion, which is the binding portion with the antigen, may be purified. In a particularly preferred aspect, step (i) is performed by contacting the IgE antibodies in the serum obtained from the subject with the antigen.

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

[0105] Contact between a sample obtained from a subject and an antigen and detection of their binding can be carried out by known methods. Such methods can use, for example, detection by ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, immunochromatography. All of these are methods for detecting the binding of an antigen and an IgE antibody of a subject by bringing the IgE antibody of the subject into contact with and binding it to the antigen, allowing a secondary antibody labeled with an enzyme to act on the IgE antibody specifically bound to the antigen, adding a substrate of the enzyme (usually a chromogenic or luminescent reagent), and detecting the product of the enzyme reaction. Alternatively, it is a method for detecting a fluorescently labeled secondary antibody. Alternatively, detection by a measurement method capable of evaluating the binding of an antigen and an IgE antibody, such as surface plasmon resonance (SPR), can also be used. A plurality of antigen-specific IgE antibodies may be mixed.

[0106] The antigen may be in a state where the isolated antigen is immobilized on or inside a carrier. In this case, ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance, etc. can be used in the above steps (i) and (ii), and in the above step (i), it is carried out by bringing the sample obtained from the subject into contact with the surface on which the antigen is immobilized. The isolated antigen may be obtained by separating and purifying it by combining protein purification methods well known to those skilled in the art from a subject (raw material, processed product, etc.), or by preparing it by genetic recombination technology. Further, it may be one to which an antibody is attached.

[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 means of 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 "Identification of Antigen" above can be used. Alternatively, the electrophoresis conditions can be determined with reference to the descriptions in Patent Documents 1 to 4 mentioned above. For example, as follows: (A) As the isoelectric focusing gel in the first dimension, the gel length is in the range of 5 to 10 cm, the pH range of the gel is 3 to 10, and when the gel length up to pH 5 is a, the gel length from pH 5 to 7 is b, and the gel length of pH 7 or more is c, the relationships of "a < b" and "b > c" are satisfied; (B) In the case of (A), when the total length of the gel is taken as 1, a is in the range of 0.15 to 0.3, b is in the range of 0.4 to 0.7, and c is in 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 each gel containing the sample, and after the change width of the electrophoresis per 30 minutes of electrophoresis is within the range of 5 μA, the voltage increase step of increasing the voltage from the constant voltage is started; (D) In the case of (C), the final voltage of the voltage increase step is within the range of 3000 V to 6000 V; (E) The gel length in the longitudinal direction of the isoelectric focusing gel in the first dimension is 5 to 10 cm, and the gel concentration at the base end in the electrophoresis direction of the two-dimensional electrophoresis gel is 3 to 6%; and In the case of (F)(E), the gel concentration of the portion on the leading end side in the electrophoresis direction of the two-dimensional electrophoresis gel is set higher than the gel concentration of the base end portion in the electrophoresis direction; Two-dimensional electrophoresis can be performed under conditions satisfying at least one selected from the group consisting of.

[0109] The antigens of (1) to (13) above are antigens that specifically bind to IgE antibodies of allergic patients (for example, patients allergic to milk). Therefore, when the binding of the IgE antibody of the subject to the antigen is detected, an indicator that the subject has an allergy is provided.

[0110] The present invention also provides a diagnostic kit for allergy containing at least one of the antigens of (1) to (13) above. The diagnostic kit of the present invention may be used in a method for providing an indicator for diagnosing the above allergy or the following diagnostic method. The diagnostic kit of the present invention may contain at least one of the antigens of (1) to (13) above, and may also contain an enzyme-labeled anti-IgE antibody and a chromogenic substrate or a luminescent substrate that serves as a substrate for the enzyme. Further, a fluorescently labeled anti-IgE antibody may be used. In the diagnostic kit of the present invention, the antigen may be provided in a state fixed on or inside a carrier. The diagnostic kit of the present invention may also be provided together with an instruction manual for the procedure for diagnosis and a package containing the instruction.

[0111] In another aspect, the above diagnostic kit contains a companion diagnostic agent for allergy. A companion diagnostic agent is used for identifying patients expected to benefit from a pharmaceutical, identifying patients at risk of serious side effects of a pharmaceutical, or examining the reactivity of a pharmaceutical for optimizing treatment with the pharmaceutical. Here, optimization of treatment includes, for example, determination of dosage volume, judgment of discontinuation of administration, confirmation of which allergen component to use for immunotolerance, and the like.

[0112] The present invention also provides a composition for diagnosing allergy, which contains at least one of the antigens described in (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 pharmaceutically acceptable carriers and additives that are generally used together with the antigen of the present invention as needed.

[0113] In one aspect, the present invention provides a method for diagnosing an allergy in a subject, comprising the following steps: (i) contacting a sample obtained from the subject with an antigen; (ii) detecting the binding of the IgE antibody in the sample obtained from the subject to the antigen; (iii) when the binding of the IgE antibody of the subject to the antigen is detected, determining that the subject has an allergy; wherein the antigen is at least one of the proteins specified as the antigens in (1) to (13) above. Here, each of the steps (i) and (ii) is performed as described for each step of the method for providing an index for diagnosing an allergy.

[0114] In another aspect, the present invention provides a method for diagnosing an allergy in a subject, comprising administering at least one of the antigens in (1) to (13) above to the subject. The method may be performed in the form of a skin test, which is characterized by applying the antigen to the skin. Skin tests include a prick test in which a minute wound is made to the skin to the extent that it does not bleed after applying the diagnostic composition on the skin to allow the antigen to penetrate the skin and observing the skin reaction, a scratch test in which the skin is slightly scratched over the applied diagnostic composition and the reaction is observed, a patch test in which the diagnostic composition in the form of a cream or ointment is applied to the skin and the reaction is observed, an intradermal test in which the antigen is administered intradermally and the reaction is observed, and the like. When a skin reaction such as swelling occurs in the skin where the antigen is applied, the subject is diagnosed as having an allergy. Here, the amount of the antigen applied to the skin may be, for example, a dose of 100 μg or less per application.

[0115] In the diagnosis of allergies, a provocation test aimed at identifying an antigen is often performed. At least one of the antigens (1) to (13) above can be used as an active ingredient of a provocation test for diagnosing allergies. Here, the antigen protein used in the provocation test may be a protein expressed and purified, or may be one expressed in a raw material or processed product, such as pollen rice obtained by transforming rice with the gene of Japanese cedar pollen antigen and expressing the antigen protein in the rice.

[0116] In one aspect, the above-described diagnostic composition and diagnostic kit can be used for prick test, scratch test, patch test, intradermal test, etc.

[0117] In another aspect, the present invention provides at least one of the antigens (1) to (13) for use in the diagnosis of allergies. Here, providing at least one of the antigens (1) to (13) by mixing with a known antigen is also included.

[0118] In yet another aspect, the present invention provides the use of at least one of the antigens (1) to (13) in the production of a diagnostic composition for 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 aspect, the composition of the present invention is a pharmaceutical composition. In one aspect, the composition of the present invention is a composition outside the pharmaceutical department, a composition not for pharmaceutical use (for example, a cosmetic composition, a food composition).

[0121] In one aspect, the above composition is used for treating allergies (for example, allergy to milk). In this specification, "treatment of allergy" means increasing the limit amount of an antigen that does not cause a disease even when taken into the body, and ultimately aiming at a state (remission) in which a disease does not occur with the normal intake amount of an antigen.

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

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

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

[0125] The composition of the present invention can be administered by a normal administration route. Normal administration routes include, for example, oral, sublingual, transdermal, intradermal, subcutaneous, intravenous, intranasal, intramuscular, intraperitoneal, and rectal administrations.

[0126] The composition of the present invention can be used as a composition in which, if necessary, together with the antigen of the present invention, a generally used pharmaceutically acceptable adjuvant, excipient, or various additives (for example, stabilizers, solubilizing agents, emulsifying agents, buffers, preservatives, coloring agents, etc.) are added by a conventional method. The dosage form of the composition can be appropriately selected by those skilled in the art according to the administration route. For example, it may be in the form of tablets, capsules, troches, sublingual tablets, injections, intranasal sprays, patches, solutions, creams, lotions, suppositories, etc. The dosage, frequency of administration and / or administration period of the composition of the present invention can be appropriately selected by a doctor according to the administration route, symptoms, characteristics of the patient such as age and weight, etc. For example, in the case of adults, it may be administered at a dose of 100 μg or less per administration. The administration interval may be, for example, once a day, once a week, twice a month, or once every three months. The administration period may be, for example, from several weeks to several years. During the administration period, an administration method in which the dosage is gradually increased may also be used.

[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 (for example, Fab, F(ab’)2, Fab’).

[0129] Further, in the above tester composition, the antibody may be provided in a form fixed or bound on or inside a carrier. The carrier is not particularly limited as long as it can be used for detecting the binding of the antibody and the antigen. Any carrier known to those skilled in the art can be used.

[0130] Examples of the method for examining the presence or absence of antigen inclusion include the following methods. A method of contacting a tester composition containing the produced Ig antibody with a sample obtained from a raw material, processed product, etc., detecting the binding between the Ig antibody and an antigen in the sample using, for example, ELISA, etc., and determining that the antigen is contained in the target raw material, processed product, etc. when the binding between the Ig antibody and the antigen is detected. ·A method of impregnating a raw material or processed product into filter paper or the like and reacting an antibody solution so as to detect an antigen contained therein.

[0131] In another aspect of the present invention, it includes a tester composition for determining the presence or absence of an allergenic antigen in an object, characterized by containing a primer having a base sequence complementary to at least a part of the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25. Non-limitingly, the above primer has, for example, a base sequence complementary to a part of the 3'-terminal part or the central part of the sequence of at least one sequence of the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, preferably 12 residues, 15 bases, 20 bases, 25 bases. Particularly when targeting mRNA, it has a complementary primer for the polyA tail. In a preferred embodiment, the tester composition containing the above primer may further contain a primer containing a base sequence of a part of the 5'-terminal part of the sequence of at least one sequence of the base sequence represented by 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, 25 bases.

[0132] For example, using DNA or mRNA obtained from milk as a template, amplifying cDNA by PCR (Polymerase Chain Reaction) including RT-PCR (Reverse Transcription-Polymerase Chain Reaction) using the complementary primer, 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 to determine the presence or absence of the antigen. The method of amplifying by PCR is RACE (Rapid Examples include the method of (Amplification of cDNA End). At this time, in the comparison between the amplified cDNA and SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, if there are point mutations encoding the same amino acid, or in the nucleotide sequence of the amplified cDNA, there are insertions, deletions, substitutions, or additions of nucleotides with respect to the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25, when the amino acid sequence encoded by the cDNA has an identity of 70% or more, preferably 80%, 90%, 95%, 98%, 99% or more with respect to the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, it is determined that the antigen is present.

[0133] In one aspect, the above-described tester composition is used, for example, to examine the presence or absence of an antigen in an object such as a food material (milk) or in a food production line. The above-described tester composition may be used for quality inspection of a production line by a manufacturer and pre-shipment products, or may be used by a consumer himself / herself to check for the presence or absence of an antigen in a target raw material or processed product. It may also be used to check for the presence or absence of an antigen by the increase or decrease in the peak of the corresponding protein by 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 an antigen in a target substance of the above antigens (1) to (13), which includes contacting an antibody against at least one of the above antigens (1) to (13) with a raw material or processed product (including a liquid).

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

[0136] Regarding the antibody, the method for preparing the antibody, the method for contacting the antibody with the raw material or processed product, the binding between the antibody and the antigen, etc., they are as described above in "Tester Composition (1)".

[0137] Food etc. with antigen removed (1) The present invention provides a raw material or processed product characterized in that at least one of the antigens (1) to (13) above is removed or reduced. In one aspect, the "raw material or processed product" may be "milk or milk processed product", or may be "beef or beef processed product".

[0138] In the raw material or processed product, the method for removing or reducing the antigen of the present invention is not limited. The removal or reduction of the antigen may be carried out by any method as long as the antigen of the present invention is removed or reduced.

[0139] For example, the raw material (e.g., milk or beef) in which the antigen of the present invention is removed or reduced may be obtained from cattle in which the expression of the antigen of the present invention is modified using genetic modification techniques.

[0140] Any of the techniques known to those skilled in the art can be used as the genetic modification technique. For example, Oishi, et al. (Scientific Reports, Vol. 6, Article number: 23980, 2016, doi:10.1038 / srep23980) described the application of the genome editing technique CRISPER / Cas9 to chicken primordial germ cells to obtain ovalbumin gene-deleted individuals. Using a similar technique, cattle in which the expression of the antigen of the present invention is modified can be obtained, and beef or milk in which the antigen of the present invention is removed or reduced can be obtained from the cattle.

[0141] Alternatively, by mating with cattle that do not express the antigen or express a small amount of the antigen, such as artificial insemination, cattle in which the expression of the antigen of the present invention is removed or reduced can be obtained, and beef or milk in which the antigen of the present invention is removed or reduced can be obtained from the cattle. Artificial mating of cattle can be carried out by conventional methods.

[0142] The processed product with the antigen of the present invention removed or reduced may be a processed product made from the raw material with the antigen of the present invention removed or reduced. When using ordinary raw materials, a treatment for removing or reducing the antigen of the present invention is performed before or after the preparation of the processed product. As a method for removing or reducing the antigen of the present invention in a processed product made from ordinary raw materials, there are high-pressure treatment and elution with a neutral salt solution, methods for removing protein components in raw materials and processed products such as 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] Method for producing raw material or processed product with antigen removed or reduced (1) The present invention provides a method for producing a raw material or a processed product with the antigen removed or reduced, which includes a step of confirming that the antigen is removed or reduced during the production process of the raw material or the processed product, where the antigen is at least one of the antigens described in (1) to (13) above.

[0144] The step of confirming that the antigen is removed or reduced during the production process of the raw material or the processed product with the antigen removed or reduced may be performed by confirming the presence or absence of the antigen by the method described in the item of the above "Tester Composition (1)".

[0145] Also, the production of milk or milk processed products, or beef or beef processed products with the antigen removed or reduced may be performed by the method described in the item of the above "Antigen-Removed Food, etc. (1)".

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

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

[0174] The present invention provides polypeptides comprising, or consisting of, each amino acid sequence of SEQ ID NOs: 27-1131 described in Table 2, which polypeptides comprise amino acid sequences that specifically bind to IgE antibodies of allergic patients, and which polypeptides comprise the amino acid sequences of (E1) to (E25). (E1) to (E25) are amino acid sequences that bind to IgE antibodies derived from the proteins described in the "Protein Name" column of Table 2 (hereinafter sometimes referred to as "epitopes").

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

[0176] Non-limitingly, 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 sequence of SEQ ID NO: 59-97; (E3) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 98-144; (E4) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 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 sequence of SEQ ID NO: 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 sequence of SEQ ID NO: 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 sequence of SEQ ID NO: 377-384; (E11) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 385-411; (E12) A polypeptide comprising at least one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 412-450; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 451-500 and 1123-1124; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 501-604 and 1125-1127; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 605-632 and 1128; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 633-673; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 674-693; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 694-757 and 1129; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 758-791; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 792-868 and 1130; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 869-927; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 928-996; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 997-1037; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 1038-1075; A polypeptide comprising at least one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1076-1114 and 1131.

[0178] As a preferred embodiment, the polypeptides (E1) to (E25) described above are specific sequences identified as epitopes that bind to IgE antibodies in the examples of this specification, as shown in Table 2. The epitope antigens of the present invention may include, in addition to the polypeptides (E1) to (E25) of the preferred embodiments described above, variants and the like described below. Hereinafter, embodiments (variants) that can be included in the epitope antigens of the present invention will be described.

[0179] The 25 sequences of 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 described in the "15-residue sequence" of Table 2 are common 15-amino acid residue sequences identified as epitopes that bind to IgE antibodies in each of the epitopes of (E1)-(E25) by epitope mapping based on overlapping. In one aspect, the present invention is a polypeptide comprising or consisting of these amino acid sequences. The epitope sequence contained in the polypeptide of the present invention can be the entire common epitope 15 amino acid residues or a part 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, 14 or more amino acid residues.

[0180] In the examples of this specification, for example, many polypeptides consisting of 4 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, in many cases of 5 amino acid residues and more, binding to IgE antibodies was confirmed. Therefore, the presence of at least 4 amino acid residues is useful as an epitope sequence.

[0181] One aspect of the variant of the polypeptide of the present invention includes a polypeptide containing four or more amino acid residues of the amino acid sequences specifically described in (E1) to (E25) above. For example, as a variant of (E1), it may include "a polypeptide containing four or more amino acid residues in at least one amino acid sequence of the amino acid sequences of SEQ ID NOs: 27-58 and 1115". Preferably, in at least one amino acid sequence of SEQ ID NOs: 27-58 and 1115, it includes 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, 14 or more amino acid residues. The same applies to (E2) to (E25).

[0182] In Table 2, the "preferred" sequence is a shorter partial sequence that can function as an epitope among the "15-residue sequences". The "more preferred" sequence is a sequence more preferred than the above short partial fragment in order to improve the binding property to the IgE antibody. The "key" sequence indicates a sequence that is considered particularly important among the "15-residue sequences". Among the "key" sequences, the amino acid sequence indicated by "X" is an amino acid residue that has been confirmed to retain the binding property to the IgE antibody even when changed to any alanine (glycine if the original amino acid residue is alanine) by alanine-glycine scanning. Therefore, X is any amino acid residue, preferably alanine (or glycine). In addition, those that do not contain the sequence indicated by "X" in the "key" sequence are cases where no amino acid residue has been found that retains the binding property to the IgE antibody by alanine-glycine scanning.

[0183] Regarding each epitope described in Table 2, the amino acid residues indicated as X are amino acid residues that have been confirmed to retain binding to IgE antibodies even when changed. The present invention preferably allows one or more of the amino acid residues indicated as X in the "key" sequences corresponding to each "preferred" sequence to be substituted with any amino acid residue. For example, in one embodiment, there are multiple "key" sequences such as SEQ ID NO: 28, 34, etc. corresponding to SEQ ID NO: 27 which is a preferred sequence. The same applies to the other "preferred" sequences, "key" sequences of (E1), and (E2)-(E25) below.

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

[0185] Each sequence described in the column of "synthetic sequence" and / or "SEQ ID NO:" on the right of the graph No. in Table 2 is a sequence for which epitope cross-reactivity was confirmed in Example 5. "Confirmed epitope cross-reactivity" means that the IgE antibodies in the sera of each allergic patient showed higher binding than the IgE antibodies in the sera of non-allergic subjects (specifically, greater than "1" in Figures 3-6). Thus, in one embodiment, the polypeptide of the present invention includes these amino acid sequences or includes a polypeptide consisting of these amino acid sequences. In one embodiment, for the polypeptide of the present invention, the IgE antibodies in the sera of each allergic patient show a binding that is 1.05 times or more, 1.10 times or more, 1.15 times or more, 1.20 times or more, 1.25 times or more higher than the IgE antibodies in the sera of non-allergic subjects.

[0186] In this specification, the "polypeptide containing the amino acid sequences of (E1) to (E25)" preferably includes or consists of each amino acid sequence of the polypeptides of (E1) to (E25) described above, and also includes any of the above-described modes (variants) in which amino acid residues are substituted. "Containing each amino acid sequence of SEQ ID NO: ~~" means that other arbitrary amino acid sequences may be included as long as they do not affect the binding of each amino acid sequence of SEQ ID NO: ~~ (including these substituted modes) to the IgE antibody (i.e., their function as these epitopes). The "polypeptide containing the amino acid sequences of (E1) to (E25)" may be a polypeptide in which two or more of the above amino acid sequences of (E1) to (E25) are linked with or without a spacer. The type of the spacer is not particularly limited, and those commonly used by those skilled in the art for linking multiple peptides can be used. The spacer may be, for example, a polypeptide such as a hydrocarbon chain like Acp(6)-OH or an amino acid chain.

[0187] Amino acid residues other than the specifically specified amino acid sequences in the polypeptide can be arbitrarily selected as long as they do not affect the binding to the IgE antibody (i.e., their function as these epitopes). Non-limitingly, preferably, they are appropriately selected from the sequences of the epitopes that are the corresponding groups and the sequences of the proteins that are the groups. For example, although SEQ ID NO: 34 specifies only "HNKE", when other amino acid residues are added, it is preferably selected from SEQ ID NO: 27 which is the sequence serving as the group as appropriate. And for the sequence described in Table 2-1 as (E1), it is desirable that amino acid residues of the sequence derived from the protein (1) (corresponding to spot (1)) serving as the group are added.

[0188] The polypeptide containing the amino acid sequences of (E1)-(E25) above may be prepared by chemical synthesis methods such as solid-phase synthesis of peptides. Alternatively, the polypeptide containing the epitope may be expressed as a recombinant polypeptide by gene recombination techniques well known to those skilled in the art, and obtained by separating and producing it by protein production methods well known to those skilled in the art. Two or more polypeptides may be combined and linked, or a polypeptide may be obtained by repeatedly linking one type of epitope. In that case, generally, the binding property with Ig antibodies is improved.

[0189] The length of the polypeptide containing the amino acid sequences of (E1)-(E25) above is not particularly limited. In a preferred embodiment, the length of the polypeptide containing the amino acid sequences of (E1)-(E25) above 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 the polypeptide is one obtained by repeatedly linking one or more of the amino acid sequences of (E1)-(E25) above once or more, in a preferred embodiment, the length of the amino acid sequence portion may be 1000 amino acids or less, 750 amino acids or less, 500 amino acids or less, 250 amino acids or less, 100 amino acids or less, 75 amino acids or less, 50 amino acids or less, 30 amino acids or less, 15 amino acids or less, 10 amino acids or less, or 5 amino acids or less. The number of amino acid residues described as a preferred embodiment for the length of the above polypeptide is the total length of the sequences (excluding the spacer) before and after the spacer.

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

[0191] Diagnostic kit · Diagnostic method (2) The present invention is a method for providing an index for diagnosing the allergy of a subject, comprising the following steps: (i) contacting a sample obtained from the subject with the antigen, where the sample is a solution containing IgE antibody; (ii) Detect the binding of the IgE antibody in the sample obtained from the subject to the antigen; (iii) When the binding of the IgE antibody of the subject to the antigen is detected, an indicator that the subject has an allergy is provided; comprising, wherein the antigen is a polypeptide comprising at least one of the polypeptides having the amino acid sequences of (E1)-(E25) above, or a polypeptide in which two or more polypeptides having the amino acid sequences of (E1)-(E25) above are linked with or without a spacer, provide the above method.

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

[0193] When the polypeptides having the amino acid sequences of (E1)-(E25) above are linked with or without a spacer, the number of polypeptides to be linked is not particularly limited. In one aspect, it is 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more. In one aspect, it 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, 2 or less.

[0194] Among the polypeptides having the amino acid sequences of (E1)-(E25) above, the same ones may be repeated, or different ones may be linked in multiple. Even when multiple polypeptides having the amino acid sequences of (E1)-(E25) above are linked in this way, it can be applied to the polypeptides, methods, kits, and compositions of the present invention.

[0195] The sample obtained from the subject is as described in the item of the above "Diagnostic Kit · Diagnostic Method (1)".

[0196] The detection of the contact and binding between the sample obtained from the subject and the polypeptide can be carried out by known methods described in the item of the above "Diagnostic Kit · Diagnostic Method (1)", for example, ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, immunochromatography, etc.

[0197] The polypeptide containing the amino acid sequences of (E1)-(E25) above may be in a state fixed on or inside the carrier. In this case, ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance, etc. can be used in the above steps (i) and (ii). The above step (i) is carried out by contacting the sample obtained from the subject with the surface on which the polypeptide containing the amino acid sequences of (E1)-(E25) is fixed. Also, a sample in which the IgE antibody of the subject is fixed on or inside the carrier may be used, and the binding with the polypeptide containing the amino acid sequences of (E1)-(E25) may be detected by the above method. For binding to the carrier or providing a space with the carrier, or making it easier for the antibody to contact the polypeptide, tags such as spacers or biotin may be attached to the N-terminus or C-terminus of the polypeptide. In the case of binding with biotin, it is preferable for the carrier to have avidin.

[0198] The polypeptide containing the amino acid sequences of (E1)-(E25) above may not be fixed on or inside the carrier. In this case, in the above steps (i) and (ii), flow cytometry or the like can be used, and the presence of the polypeptide containing the amino acid sequences of (E1)-(E25) to which the IgE antibody is bound can be confirmed by laser light. This method includes, for example, a basophil activation test (BAT), which is a method for detecting surface antigen CD203c that appears when basophils are activated by contact with the polypeptide containing the amino acid sequences of (E1)-(E25). Also included is a histamine release test (HRT) for examining whether histamine is released by further contacting the polypeptide containing the amino acid sequences of (E1)-(E25) with blood cells in a sample.

[0199] The polypeptide containing the amino acid sequences of (E1)-(E25) above is an antigen that specifically binds to the IgE antibody of an allergic patient. Therefore, when the binding between the IgE antibody of the subject and the antigen is detected, an indicator that the subject is allergic, including cross-reactivity, is provided. In the synthesis of the polypeptide containing the amino acid sequences of (E1)-(E25) above, for example, in order to facilitate synthesis using Escherichia coli, sequences may be added before and after the epitope to increase the sequence length. Even in such a case, if the binding between the IgE antibody of the subject and the amino acid sequences of (E1)-(E25) above is detected, an indicator that the subject is allergic, including cross-reactivity, is provided. Therefore, anything may be added before and after the amino acid sequences of (E1)-(E25) that are the epitope.

[0200] The present invention also provides a diagnostic kit for allergy comprising at least one polypeptide containing the amino acid sequences of (E1)-(E25) above. The diagnostic kit of the present invention may be used in a method for providing an index for diagnosing the above allergy or in the following diagnostic method. The diagnostic kit of the present invention, in addition to containing at least one polypeptide containing the amino acid sequences of (E1)-(E25) above, may also contain an enzyme-labeled anti-IgE antibody and a chromogenic substrate or a luminescent substrate that serves as a substrate for the enzyme. Further, a fluorescent-labeled anti-IgE antibody may be used. In the diagnostic kit of the present invention, the polypeptide containing the amino acid sequences of (E1)-(E25) above may be provided in a state immobilized on or inside a carrier. The diagnostic kit of the present invention may also be provided together with an instruction manual for the procedure for diagnosis and a package containing the instruction.

[0201] In another aspect, the above diagnostic kit contains a companion diagnostic agent for allergy. A companion diagnostic agent is used for identifying patients for whom the effect of a pharmaceutical is expected, or patients having a high risk of severe side effects of a pharmaceutical, or for examining the reactivity of a pharmaceutical for optimizing the treatment with the pharmaceutical. Here, optimization of treatment includes, for example, determination of dosage volume, judgment of discontinuation of administration, confirmation of which allergen component to use for immunotolerance, and the like.

[0202] The present invention also provides a diagnostic composition for allergy comprising at least one polypeptide containing the amino acid sequences of (E1)-(E25) above. The diagnostic composition of the present invention can be used in the following diagnostic method. The diagnostic composition of the present invention may contain pharmaceutically acceptable carriers and additives that are generally used together with the polypeptide of the present invention as necessary.

[0203] In one aspect, the present invention is a method for diagnosing an allergy in a subject, comprising the following steps: (i) contacting a sample obtained from the subject with an antigen; (ii) detecting the binding between the IgE antibody in the sample obtained from the subject and the antigen; (iii) If the binding of the target IgE antibody to the antigen is detected, it is determined that the subject has an allergy; including, wherein the antigen is at least one of the polypeptides identified as a polypeptide comprising the amino acid sequences of (E1)-(E25) above, and providing the above method. Here, each step of (i) and (ii) is performed as described for each step of the method for providing an index for diagnosing allergies.

[0204] In another aspect, the present invention provides a method for diagnosing a subject's allergy, comprising administering to the subject at least one of the polypeptides comprising the amino acid sequences of (E1)-(E25) above. The method may be performed in the form of a skin test characterized by applying to the skin a polypeptide comprising the amino acid sequences of (E1)-(E25) above. Skin tests include a prick test in which after applying a diagnostic composition on the skin, a minimal wound is made to the extent that it does not bleed to allow the polypeptide comprising the amino acid sequences of (E1)-(E25) above to penetrate the skin and observe the skin reaction, a scratch test in which the skin is slightly scratched over the applied diagnostic composition to observe the reaction, a patch test in which a diagnostic composition in the form of a cream or ointment is applied to the skin to observe the reaction, an intradermal test in which a polypeptide comprising the amino acid sequences of (E1)-(E25) above is administered intradermally to observe the reaction, and the like. If a skin reaction such as swelling occurs in the skin where the polypeptide comprising the amino acid sequences of (E1)-(E25) above is applied, the subject is diagnosed as having an allergy. Here, the amount of the polypeptide applied to the skin may be, for example, a dose of 100 μg or less per application.

[0205] In the diagnosis of allergies, an oral challenge test is often performed for the purpose of identifying antigens, verifying antigen intake, and the degree of symptoms. At least one of the polypeptides containing the amino acid sequences of (E1)-(E25) above can be used as an active ingredient in an oral challenge test for diagnosing allergies. Here, the polypeptide used in the oral challenge test may be an expressed and purified polypeptide. For example, it may be a polypeptide expressed in raw materials or processed products, such as pollen rice obtained by transforming rice with the gene of cedar pollen antigen and expressing the polypeptide in the rice.

[0206] In one aspect, the above-described diagnostic composition and diagnostic kit can be used for prick tests, scratch tests, patch tests, intradermal tests, etc.

[0207] In another aspect, the present invention provides at least one of the polypeptides containing the amino acid sequences of (E1)-(E25) above for use in the diagnosis of allergies.

[0208] In yet another aspect, the present invention provides the use of at least one of the polypeptides containing the amino acid sequences of (E1)-(E25) above in the manufacture of allergy diagnostic agents.

[0209] In this item, the allergy to be diagnosed may be an allergy to the polypeptide containing the amino acid sequences of (E1)-(E25) above. That is, the diagnosis of allergies, including the detection of allergies and the provision of diagnostic indicators, can diagnose not only allergies to a single polypeptide containing the amino acid sequences of (E1)-(E25) above, but also allergies including cross-reactivity.

[0210] Composition · Treatment method (2) The present invention provides a composition containing at least one of the polypeptides containing the amino acid sequences of (E1)-(E25) above.

[0211] In one aspect, the composition of the present invention is a pharmaceutical composition. In one aspect, the composition of the present invention is a composition for external use in medicine, a composition not for medical use (for example, a cosmetic composition, a food composition).

[0212] In one aspect, the above composition is used for treating allergies. Treating allergies means increasing the limit amount of a polypeptide that does not cause an allergic reaction even when taken into the body, and ultimately aiming for a state (remission) where an allergic reaction does not occur even with the normal intake amount of the polypeptide.

[0213] The present invention also provides a method for treating allergies, which includes administering at least one polypeptide containing the amino acid sequences of (E1)-(E25) above to a patient in need of allergy treatment.

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

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

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

[0217] In this item, the allergy to be treated may be an allergy to the polypeptide containing the amino acid sequences of (E1)-(E25) above. That is, the treatment of allergy can treat not only the allergy to a single polypeptide containing the amino acid sequences of (E1)-(E25) above, but also allergies including cross-reactivity.

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

[0219] The antibody can be prepared by a conventional method. For example, it may be prepared by immunizing a mammal such as a rabbit with the polypeptide containing the amino acid sequences of (E1)-(E25) above. The antibody may be an Ig antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (for example, Fab, F(ab’)2, Fab’).

[0220] In the above-described tester composition, the antibody may be provided in a solid or bound form on or inside the carrier. The carrier is not particularly limited as long as it can be used for detecting the binding between the antibody and the polypeptide containing the amino acid sequences of (E1)-(E25) above. Any carrier known to those skilled in the art can be used. Further, it is preferable that the antibody against the polypeptide containing the amino acid sequences of (E1)-(E25) above is an antibody against a polypeptide having the same amino acid sequence as the epitope or important amino acids described in the above item "Epitope". Thereby, a tester composition capable of detecting including cross-reactivity can be obtained.

[0221] Examples of the method for examining the presence or absence of the polypeptide containing the amino acid sequences of (E1)-(E25) above include the following methods.

[0222] · Contacting a tester composition containing the prepared antibody with a sample obtained from a raw material, processed product, etc., and detecting the binding between the antibody and the polypeptide containing the amino acid sequences of (E1)-(E25) in the sample using, for example, ELISA or the like. When the binding between the antibody and the polypeptide containing the amino acid sequences of (E1)-(E25) is detected, it is determined that the target raw material, processed product, etc. contains the polypeptide containing the amino acid sequence. (The "method for examining the presence or absence of polypeptide" includes determining that the polypeptide has been removed or reduced when the binding between the antibody and the polypeptide containing the amino acid sequences of (E1)-(E25) is decreased.) · A method of impregnating a raw material, processed product, etc. into filter paper or the like and reacting an antibody solution so as to detect the polypeptide containing the amino acid sequences of (E1)-(E25) contained therein.

[0223] In another aspect of the present invention, there is provided a tester composition for determining the presence or absence of a polypeptide comprising the amino acid sequences of (E1)-(E25) for allergies in a subject, which comprises primers corresponding to a polypeptide having the same amino acid sequence for an epitope or an important amino acid. Without limitation, the primers may be designed to include, for example, a part of the nucleotide sequence of a nucleic acid encoding the amino acid sequence specified in (E1)-(E25) or its complementary strand. Alternatively, the primer may be a nucleotide sequence in the upstream region of the portion encoding a polypeptide having the same amino acid sequence for an epitope or an important amino acid in a nucleic acid encoding a protein comprising a polypeptide having the same amino acid sequence for an epitope and an important amino acid specified in (E1)-(E25), or a complementary strand of the nucleotide sequence in the downstream region of the portion encoding a polypeptide having the same amino acid sequence for an epitope or an important amino acid. Examples of such primers include primers that are part of at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 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. Here, the positions of the epitopes in the full-length sequence of the antigen are as specified in Table 2 based on the results of the examples. In particular, when targeting mRNA, it may have a poly-A tail complementary primer.

[0224] For example, using the DNA or mRNA obtained from a sample as a template, the DNA is amplified by PCR (Polymerase Chain Reaction) including RT-PCR using the primer, and in the sequence of the amplified DNA, it is determined whether it contains a nucleic acid encoding the amino acid sequence specified in (E1)-(E25) above, thereby determining the presence or absence of the antigen containing (E1)-(E25) above. Examples of methods for amplifying mRNA by PCR include the RACE method. When one of the amino acid sequences encoded by the three possible open reading frames in the amplified DNA contains the amino acid sequence specified in (E1)-(E25) above, it is determined that the antigen is present. When the DNA is not amplified, it is determined that the antigen is absent.

[0225] In one aspect, the above-described tester composition is used to examine the presence or absence of a polypeptide containing the amino acid sequence of (E1)-(E25) above in an object such as in a raw material or a processed product manufacturing line. The raw material may be a food ingredient, or may be a cosmetic raw material, a pharmaceutical raw material, etc. The processed product may be an edible processed product, or may be a cosmetic, a pharmaceutical, etc. The above-described tester composition may be used for searching for the biological species contained as a raw material, may be used for quality inspection of the manufacturing line by the manufacturer and the product before shipment, or may be used for checking the presence or absence of antigen in the target raw material / processed product by the consumer or the user himself / herself. It may also be used for checking the polypeptide content by the increase or decrease of the peak of the corresponding polypeptide by 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 containing the amino acid sequence of (E1)-(E25) above in a raw material / processed product. The method includes detecting a polypeptide having the whole or a part of the amino acid sequence of a polypeptide containing the amino acid sequence of (E1)-(E25) above in the raw material / processed product.

[0227] In one aspect, the method of the present invention includes bringing an antibody against at least one polypeptide containing the amino acid sequences of (E1)-(E25) above into contact with a raw material or processed product (including liquids), and determining the presence or absence of the polypeptide containing the amino acid sequences of (E1)-(E25) above in the target substance.

[0228] The definition of the antibody, the raw material or processed product, the method for producing the antibody, the method for bringing the antibody into contact with the raw material or processed product, the binding between the antibody and the antigen, etc. are as described above in "Tester Composition (2)".

[0229] Alternatively, the method for determining the presence or absence of the antigen also includes a mode of detecting an epitope portion of the polypeptide containing the amino acid sequences of (E1)-(E25) contained in the antigen. The "epitope portion" is preferably 4 or more amino acid residues, 6 or more amino acid residues, 8 or more amino acid residues. Detection of the epitope portion can be performed by a known method for detecting a specific amino acid sequence of a part of the polypeptide. For example, a method of cleaving the protein of the target raw material or processed product (e.g., food) with a digestive enzyme for antigen removal treatment, separating by HPLC, etc., and measuring whether the peak of any epitope peptide has decreased due to the antigen removal treatment, etc. can be considered. Alternatively, an antibody that recognizes a part of the polypeptide containing the amino acid sequences of (E1)-(E25) above may be used to determine the presence or absence of the antigen containing the polypeptide containing the amino acid sequences of (E1)-(E25) above in the target substance.

[0230] Raw material etc. with antigen removed (2) The present invention provides a raw material or processed product characterized in that at least one of the polypeptides containing the amino acid sequences of (E1)-(E25) above is removed or reduced.

[0231] The method for removing or reducing the antigen of the present invention in raw materials or processed products is not limited. The removal or reduction of the antigen may be carried out by any method as long as the polypeptide containing the amino acid sequences of (E1)-(E25) above is removed or reduced. For example, the techniques described in the item "Antigen-removed food, etc. (1)" above may be used.

[0232] The fact that at least one of the polypeptides containing the amino acid sequences of (E1)-(E25) above is removed or reduced may be achieved by removing or reducing the entire amino acid sequence, or may be achieved by cleaving or removing the amino acid sequence portion of (E1)-(E25) from the antigen protein. "Removed" includes deletion and modification of all or part of the sequence portion specified by (E1)-(E25) above.

[0233] For example, for a raw material in which the polypeptide containing the amino acid sequences of (E1)-(E25) above is removed or reduced, a raw material in which the polypeptide containing the amino acid sequences of (E1)-(E25) above is not expressed may be prepared using genetic modification techniques. Any of the techniques known to those skilled in the art for genetic modification knockout techniques can be used.

[0234] Processed products in which the polypeptide containing the amino acid sequences (E1)-(E25) is removed or reduced may be processed products using raw materials in which the polypeptide containing the amino acid sequences (E1)-(E25) is removed or reduced, such as powdered milk using protein digest as a raw material. When using ordinary raw materials, a treatment for removing or reducing the polypeptide containing the amino acid sequences (E1)-(E25) is performed before, during, or after the preparation of the processed product. "Preparation of processed products" means, for example, preparing food processed products from food raw materials (e.g., milk or beef). For example, "preparation of milk processed products" means preparing dairy products obtained by processing the main raw material milk, and foods containing this as a raw material, and includes, without limitation, cream, butter, butter oil, cheese, whey, concentrated whey, ice creams, condensed milk, skimmed condensed milk, unsweetened condensed milk, unsweetened skimmed condensed milk, sweetened condensed milk, sweetened skimmed condensed milk, whole milk powder, skimmed milk powder, cream powder, whey powder, protein - concentrated whey powder, buttermilk powder, sweetened milk powder, prepared milk powder, prepared liquid milk, fermented milk such as yogurt, lactic acid bacteria beverages, milk beverages and other dairy products, and preparing cakes, pastries, custard pudding, milk jelly, biscuits, cookies, snacks, chocolates, bread, white sauce, puree, cream stew, gratin, curry roux, stew roux containing these dairy products as raw materials. Also, "preparation of beef processed products" means preparing products obtained by processing the main raw material beef, and foods containing this as a raw material, and includes, without limitation, processed meat products such as ham, sausage, bacon, bouillon, consommé, steak, grilled meat, roast beef, tartare steak, hamburger, hamburger patty, meatballs, nuggets, etc.

[0235] As a method for removing or reducing the polypeptide containing the amino acid sequences (E1)-(E25) in processed products using ordinary raw materials, the methods described in the item "Antigen - removed foods, etc. (1)" above may be used. As a method for cleaving the polypeptide containing the amino acid sequences (E1)-(E25), a method of performing a treatment of cleavage with a specific digestive enzyme can be mentioned.

[0236] Method for producing raw material or processed product with antigen removed or reduced (2) The present invention provides a method for producing a raw material or a processed product in which at least one of the polypeptides having the amino acid sequences of (E1)-(E25) is removed or reduced, the method having a step of confirming that an antigen is removed or reduced in the process of producing the processed product.

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

[0238] The method for confirming that a polypeptide is removed or reduced in the process of producing a raw material or a processed product is not particularly limited, and any method capable of detecting at least one of the polypeptides having the amino acid sequences of (E1)-(E25) may be used. For example, the presence or absence of the polypeptide in the raw material or the processed product may be confirmed by the binding property between a sample containing a material generated in the process of producing the raw material or the processed product and an antibody against at least one of the polypeptides having the amino acid sequences of (E1)-(E25). Details of such a method are as described in the item of "Diagnostic Kit · Diagnostic Method (2)". That is, in the production method, the "IgE antibody of the subject" in the item of "Diagnostic Kit · Diagnostic Method (2)" is replaced with "an antibody against at least one of the polypeptides having the amino acid sequences of (E1)-(E25)", and the "antigen" and "polypeptide" in the item of "Diagnostic Kit · Diagnostic Method (2)" are replaced with "a sample containing a material generated in the process of producing the processed product", and the method described in the item of "Diagnostic Kit · Diagnostic Method (2)" can be used to confirm that an antigen is removed or reduced in the process of producing the processed product. Further, the tester composition described in the item of "Tester Composition (2)" can also be used.

[0239] The present invention also relates to the use of a kit in a method for diagnosing allergy, the use of a kit for diagnosing allergy and / or for providing an index for diagnosis, a composition for use in a method for diagnosing allergy, the use of a composition for diagnosing allergy and / or for providing an index for diagnosis, a method for diagnosing allergy and / or for providing an index for diagnosis, the use of an antigen (protein antigen or epitope antigen) in a method for detecting the presence or absence of IgE antibody in a sample obtained from a subject (such as a living body like a human), an antigen (protein antigen or epitope antigen) for use in treating allergy, a kit or composition containing an antigen (protein antigen or epitope antigen) for detecting the binding between an IgE antibody and an antigen (protein antigen or epitope antigen) in a sample obtained from a subject (such as a living body like a human), and the use of a kit or composition containing an antigen (protein antigen or epitope antigen) for detecting the binding between an IgE antibody and an antigen (protein antigen or epitope antigen) in a sample obtained from a subject (such as a living body like a human). Each term such as "antigen" is as described above.

Example

[0240] Examples of the present invention will be described below. The technical scope of the present invention is not limited by these examples. Example 1: Confirmation of protein pattern Using the following two-dimensional electrophoresis method, the proteins contained in milk (milk derived from Bos taurus) were examined.

[0241] Extraction of protein The extraction and purification of the proteins contained in milk were performed as follows. Urea buffer was added to milk to extract the proteins. The composition of the urea buffer is as follows. 30 mM Tris 2 M thiourea 7 M urea 4% (w / v) CHAPS: 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate An appropriate amount of dilute hydrochloric acid Distilled water was added to adjust the total volume to 100 mL. The pH was 8.5. Then, two precipitation operations were performed using a 2D-CleanUP kit (manufactured by GE). In the first precipitation operation, TCA (trichloroacetic acid) was added to the recovered protein extract to cause precipitation, and the resulting precipitate (TCA precipitate) was recovered. In the second precipitation operation, acetone was added to the recovered TCA precipitate to cause precipitation, and the resulting precipitate (specimen) was recovered.

[0242] Preparation of sample solution A part of the obtained specimen (100 μg as protein weight) was dissolved in 150 μl of DeStreak Rehydration Solution (manufactured by GE), which is a buffer for swelling the gel for one-dimensional isoelectric focusing electrophoresis, to obtain a specimen solution for one-dimensional isoelectric focusing electrophoresis (swelling specimen solution). The composition of DeStreak Rehydration Solution is as follows. 7M thiourea 2M urea 4% (w / v) CHAPS 0.5% (v / v) IPG buffer; manufactured by GE An appropriate amount of BPB (bromophenol blue) Penetration of sample into gel for first-dimensional isoelectric focusing A one-dimensional isoelectric focusing electrophoresis gel (manufactured by GE: IPG gel Immobiline Drystrip (pH3-10NL)) was immersed in 140 μl of the above-mentioned specimen solution for one-dimensional isoelectric focusing electrophoresis (swelling specimen solution) and allowed to penetrate overnight at room temperature.

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

[0244] The electrophoresis tray was filled with silicone oil. Filter papers moistened with water were provided at both ends of the gel penetrated with the specimen, and the gel was set in the electrophoresis tray so as to be covered with silicone oil, and the electrodes were set in a state where the filter paper was sandwiched between the gel.

[0245] The upper limit of the current value of the isoelectric focusing apparatus was set to 75 μA per gel, and the voltage program was as follows: (1) a constant voltage step was performed at 300 V up to 750 Vhr (the current change range during the 30-minute 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) thereafter, one-dimensional isoelectric focusing was performed at a constant voltage of 5000 V until the total Vhr reached 12000.

[0246] SDS equilibration of isoelectric focusing gel After performing the above one-dimensional isoelectric focusing, the gel was removed from the isoelectric focusing apparatus, immersed in an equilibration buffer containing a reducing agent, and shaken at room temperature for 15 minutes. The composition of the equilibration buffer containing the reducing agent was as follows. 100 mM Tris-HCl (pH 8.0) 6 M urea 30% (v / v) glycerol 2% (w / v) SDS 1% (w / v) DTT Next, except for the equilibration buffer containing the reducing agent, 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) 6 M urea 30% (v / v) glycerol 2% (w / v) SDS 2.5% (w / v) iodoacetamide Second-dimensional SDS-PAGE In this example, an XCell SureLock Mini-Cell manufactured by life technologies was used as the electrophoresis apparatus. NuPAGE 4-12% Bis-Tris Gels manufactured by life technologies were used as the gels for two-dimensional electrophoresis. Also, an electrophoresis buffer having the following composition was prepared and used. 50 mM MOPS 50 mM Tris base 0.1% (w / v) SDS 1 mM EDTA In addition, in this example, an agarose solution for adhesion in which 0.5% (w / v) of agarose S (manufactured by Nippon Gene Co., Ltd.) and an appropriate amount of BPB (bromophenol blue) were dissolved in the electrophoresis buffer was used.

[0247] After thoroughly washing the wells in the SDS-PAGE with the above electrophoresis buffer, the buffer used for the washing was removed. Next, the agarose solution for adhesion that had been sufficiently dissolved was added to the wells. Next, the SDS-equilibrated gel was immersed in the agarose, and the SDS-equilibrated gel and the second-dimension electrophoresis gel were brought into close contact with each other using forceps. After confirming that the agarose had sufficiently solidified with the two gels in close contact, electrophoresis was performed at a constant voltage of 200 V for about 45 minutes.

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

[0249] First, the sealed container to be used was thoroughly washed in advance with 98% (v / v) ethanol. The second-dimension electrophoresis gel after electrophoresis was removed from the SDS-PAGE apparatus and placed in the washed sealed container, and the treatment of immersing it in an aqueous solution containing 50% (v / v) methanol and 7% (v / v) acetic acid was performed twice for 30 minutes. Then, the aqueous solution was replaced with water and immersed for 10 minutes. Next, the second-dimension electrophoresis gel was immersed in 40 ml of SYPRO Ruby and shaken overnight at room temperature. Next, after removing SYPRO Ruby, the second-dimension electrophoresis gel was washed with water and then shaken in an aqueous solution containing 10% (v / v) methanol and 7% (v / v) acetic acid for 30 minutes. Further, the aqueous solution was replaced with water and shaken for 30 minutes or more.

[0250] Analysis The 2D electrophoresis gel subjected to the above series of processes was subjected to fluorescence image scanning using Typhoon 9500 (manufactured by GE). The results of 2D electrophoresis of the proteins contained in milk are shown in Fig. 1. On the left side of the gel photograph in each figure, bands of molecular weight markers can be seen, and the positions of the bands indicate specific molecular weights (KDa).

[0251] Example 2: Confirmation of antigen by immunoblot For antigen confirmation by immunoblotting, after performing the procedure described in Example 1 up to "2D SDS-PAGE", the following operations of "transfer to membrane", "immunoblotting", and "analysis" were performed.

[0252] Transfer to membrane Transfer to the membrane was performed using the following transfer device and transfer buffer. Transfer device: XCell SureLock Mini-Cell and XCell II blot module (manufactured by life technologies) Transfer buffer: NuPAGE transfer buffer (×20) (manufactured by life technologies) was diluted 20-fold with milliQ water and used.

[0253] Specifically, the proteins in the 2D 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 the transfer buffer for hydrophilic treatment of the PVDF membrane.

[0255] (2) A sponge, filter paper, the gel after 2D SDS-PAGE, the hydrophilic-treated PVDF membrane, filter paper, and sponge were set in this order, and an electric current was passed through at a constant voltage of 30 V for 1 hour in the transfer device.

[0256] Immunoblot The immunoblot of the membrane was performed using the serum of a patient with an allergy to milk or the serum of a non-milk-allergic subject as the primary antibody.

[0257] The immunoblot of the membrane was performed according to the following procedure. (1) The transferred membrane was shaken in Pierce® Fast Blocking Buffer (manufactured by Thermo) (hereinafter referred to as "Fast Block") at room temperature for 1 hour. (2) As the primary antibody, it was left standing at room temperature for 1 hour in a 10% serum / Fast Block solution. (3) It was washed with a PBST solution (PBS buffer containing 0.1% nonionic surfactant Tween® 20) (5 minutes × 3 times). (4) As the secondary antibody, anti-human IgE-HRP (horseradish peroxidase) was shaken in a solution diluted 5000-fold with Fast Block solution at room temperature for 1 hour. (5) It was washed with a PBST solution (5 minutes × 3 times). (6) It was left standing for 5 minutes with Pierce Western Blotting Substrate Plus (manufactured by Thermo).

[0258] Analysis The membrane subjected to the above series of treatments was subjected to fluorescence image scanning using Typhoon9500 (manufactured by GE).

[0259] The immunoblot using the serum of a milk-allergic patient was compared with the immunoblot using the serum of a non-milk-allergic subject as a control. In the immunoblot using the serum of a milk-allergic patient for the proteins contained in milk, 14 spots different from those in the case of using the serum of a non-milk-allergic subject and different from known milk allergen proteins were detected (Figure 2). As a result of the amino acid sequence analysis of Example 3, it was found that Spots 2 and 3 were derived from the same protein. The isoelectric points of each spot are shown in Table 1.

[0260] Example 3: Mass spectrometry and identification of antigen For the antigen that causes each of the above spots, the amino acid sequence was identified 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 of Examples 1 and 2, and stained by shaking with 0.008% Direct blue / 40% ethanol·10% acetic acid. (2) Thereafter, the membrane was decolorized by treating it three times with 40% ethanol·10% acetic acid for 5 minutes each, washed with water for 5 minutes, and then air-dried. (3) The target spot was cut out with a clean cutter blade and placed in a centrifuge tube. After treating the membrane with 50 μL of methanol for hydrophilic treatment, it was washed twice with 100 μL of water and then centrifuged to remove the supernatant, and 20 μL of 20 mM NH4HCO3·50% acetonitrile was added. (4) 1 μL of 1 pmol / μL lysyl endopeptidase (WAKO) was added, and after standing at 37 °C for 60 minutes, the solution was collected in a new centrifuge tube. 20 μL of 20 mM NH4HCO3·70% acetonitrile was added to the membrane, immersed at room temperature for 10 minutes, and further collected. It was dissolved in 10 μL of 0.1% formic acid, 4% acetonitrile and transferred to a tube. (5) After the collected solution was dried under reduced pressure, it was dissolved in 15 μl of Solution A (0.1% formic acid, 4% acetonitrile solution), and mass spectrometry (ESI-TOF6600, manufactured by AB Sciex) was performed. (6) Identification of the protein based on the mass data obtained from the mass spectrometer was performed by searching Uniprot and NCBI.

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

[0263] Spots 2 and 3 are derived from the same protein. In the immunoblot of Example 2, these spots were recognized as separate spots because, even though the amino acid sequences were the same, the isoelectric point and / or molecular weight changed due to post-translational modifications such as sugar chain modification and phosphate group modification. The binding to IgE antibodies was also observed depending on the differences in post-translational modifications, indicating that there is no influence of post-translational modifications.

[0264] Example 4: Identification of epitope Epitopes of milk allergen components Regarding the epitopes of milk allergen components, epitope identification was performed according to the following procedure.

[0265] (A) Milk epitope mapping (1) Epitope mapping was performed using a library of overlapping peptides (length: 15 amino acids) corresponding to the amino acid sequences identified as milk allergen components. 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 peptide to be synthesized was shifted by 10 amino acids. That is, each peptide has an overlap of 5 amino acids with the previous peptide and the next peptide, respectively.

[0267] For the preparation of the peptide array, Intavis CelluSpots (trademark) technology was used. That is, the following procedure: (1) Synthesize the target peptide on an amino-modified cellulose disk using an automated synthesizer (Intavis MultiPep RS), (2) dissolve the amino-modified cellulose disk to obtain a cellulose-bound peptide solution, and (3) spot the cellulose-bound peptide on a coated glass slide to prepare a peptide array. The details of each procedure are as follows.

[0268] (1) Peptide synthesis Peptide synthesis was carried out stepwise on an amino-modified cellulose disk in a 384-well synthesis plate using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. That is, an amino acid with an Fmoc group attached to the amino group was activated with a solution of N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in dimethylformamide (DMF), dropped onto the cellulose disk, and the Fmoc-group-attached amino acid was coupled to the amino group on the cellulose disk. Unreacted amino groups were capped with acetic anhydride, washed with DMF, further treated with piperidine, and washed with DMF to remove the Fmoc group from the amino group of the amino acid attached to the amino group on the cellulose disk. Peptide synthesis was performed by repeating the above coupling, capping, and removal of the Fmoc group to extend the amino terminus.

[0269] (2) Dissolution of the amino-modified cellulose disk The cellulose disk with the target peptide obtained in the above “(1) Peptide synthesis” was transferred to a 96-well plate and treated with a side-chain deprotection mixture of trifluoroacetic acid (TFA), dichloromethane, triisopropylsilane (TIPS), and water for deprotection of the side chains of the amino acids. Thereafter, the deprotected cellulose-bound peptide was dissolved in a mixture of TFA, trifluoromethanesulfonic acid (TFMSA), TIPS, and water, precipitated with tetrabutyl methyl ether (TBME), resuspended in dimethyl sulfoxide (DMSO), mixed with a mixture of NaCl, sodium citrate, and water, and a peptide solution for slide spotting was obtained.

[0270] (3) Spotting of the cellulose-bound peptide solution The peptide solution for slide spots obtained in the above-mentioned “(2) Dissolution of Amino-Modified Cellulose Disk” was spotted onto an Intavis CelluSpots (trademark) slide using an Intavis slide spotting robot, and this was dried to prepare a peptide array.

[0271] Using the peptide array, it was measured for each peptide fragment whether the IgE antibody in the serum of milk allergy patients binds by an antigen-antibody reaction. The measurement was performed according to the following procedure. (1) The peptide was shaken at room temperature for 1 hour in Pierce Protein-Free (PBS) Blocking Buffer (manufactured by Thermo). (2) In 2% serum / Pierce Protein-Free (PBS) Blocking Buffer (manufactured by Thermo) It was shaken overnight at 4°C. (3) It was washed with PBST (PBS buffer containing 3% nonionic surfactant Tween (registered trademark) 20) for 5 minutes (×3 times). (4) Anti-human IgE antibody-HRP (1:20,000, Pierce Protein-Fr ee (PBS) Blocking Buffer (manufactured by Thermo)) was added and shaken at room temperature for 1 hour. (5) It was washed with PBST for 5 minutes (×3 times). (6) Pierce ECL Plus Western Blotting Substrate (manufactured by Thermo) was added and shaken at room temperature for 5 minutes. (7) Using an Amersham Imager 600, the chemiluminescence of the peptide that had been subjected to the above-mentioned treatments (1) to (6) was measured.

[0272] For the image obtained by measurement in (7) above, the chemiluminescence amount was quantified using ImageQuant TL (GE Healthcare). Among the quantified values obtained from the images obtained using the sera of 6 non-cow milk allergy subjects, the second highest value was defined as the N2nd value, and peptides having a value of 800,000 or more in the value obtained by taking the difference from the N2nd value of the peptide from each of the images obtained using the sera of 25 patients were determined to be peptides that specifically bind IgE antibodies in patients.

[0273] As a result, it was confirmed that IgE antibodies specifically bound to peptides (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) derived from Spots 1 - 14 (Spots 2 and 3 are derived from the same protein) that do not contain known epitopes.

[0274] (B) Milk epitope mapping (2): Overlapping Based on the sequences of the peptides (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) to which IgE antibodies in serum specifically bound in patients according to (A) above, in the sequences of the peptides and the sequences before and after the peptides in the amino acid sequences of the allergenic components containing the sequences of the peptides, a library of overlapping peptide fragments (length: 10 amino acids) was created and epitope mapping was performed.

[0275] Each peptide to be synthesized was shifted by 1 amino acid. That is, each peptide has an overlap of 9 amino acids with the previous peptide and the next peptide, respectively.

[0276] The library was prepared in the same procedure as described above in (A), and whether the IgE antibody in the patient's serum binds or not was evaluated for each peptide fragment by the same method as described above. From the values digitized from the images obtained as a result of using the patient's serum, compared with the values obtained from the peptides that were the basis of the overlapping, peptides in which the binding to the patient's IgE antibody was lost or significantly decreased by shifting one amino acid at a time were determined to be peptides without IgE antibody binding.

[0277] Similar to (A) above, the chemiluminescence amount of the measured image was digitized. When the values obtained from 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) that were the basis of the overlapping were taken as 100%, less than 30% was determined to have no binding to the IgE antibody, 30% or more and less than 50% was determined to have poor binding to the IgE antibody but still have binding to the IgE antibody, 50% or more and less than 70% was determined to have slightly poor binding to the IgE antibody but still have binding to the IgE antibody, and 70% or more was determined to have no difference in binding to the IgE antibody or good binding to the IgE antibody, and the peptide was determined to be a peptide with remaining binding to the IgE antibody.

[0278] By this analysis, in the sequences that were the basis of the overlapping, regions important for binding to the patient's IgE antibody were found.

[0279] (C) Milk epitope mapping (3): Alanine glycine scan For the amino acid sequence identified in (A) above, a library of peptide fragments in which each amino acid from the amino-terminal side was substituted with alanine (glycine if the original amino acid was alanine) one by one by a method called alanine-glycine scanning (Non-Patent Document 1) was prepared by the same method as above, and whether the IgE antibody in the patient's serum binds to each peptide fragment was evaluated by the same method as above. Amino acids at positions where the binding ability to the patient's IgE antibody was lost or significantly decreased due to 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, and amino acids for which the binding ability to the patient's IgE antibody was not lost or significantly decreased were determined to be amino acids not important for the expression of the original antigenicity and amino acids that can be substituted.

[0280] Similar to (A) above, the chemiluminescence amount of the obtained image was quantified. Among the values quantified from the images obtained from the results using the sera of 25 patients, when the value obtained by the sequence (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) that was the basis of alanine-glycine scanning was taken as 100%, less than 30% was determined to have no binding ability to the IgE antibody, 30% or more and less than 50% was determined to have poor binding ability to the IgE antibody but have binding ability to the IgE antibody, 50% or more and less than 70% was determined to have somewhat poor binding ability to the IgE antibody but have binding ability to the IgE antibody, and 70% or more was determined to have no difference in binding ability to the IgE antibody or have good binding ability to the IgE antibody, and it was determined that the peptide had residual binding ability to the IgE antibody.

[0281] When the results of (A) to (C) were analyzed, a common sequence important for the expression of the original antigenicity was found in the region important for the binding of the patient's IgE antibody in the sequence that was the basis of alanine-glycine scanning. The sequences of all 25 types of epitopes were identified, and the results were summarized in Table 2.

[0282] Example 5: Confirmation of cross-reactivity of epitope Regarding the key sequences in which the amino acids other than the amino acids important for maintaining the binding to IgE antibodies in each epitope sequence found for each protein in milk in Table 2 above were replaced with an arbitrary amino acid (X), as a result of searching for proteins having the same sequence against the allergenic foods that each patient has simultaneously using BLAST, as an example, the amino acid sequences contained in the sequences of each food described in the column of "Cross-checked foods" in Table 2 were hit.

[0283] Regarding the peptides containing the amino acid sequences described in the column of "Synthetic sequences" in Table 2, the binding properties with the IgE antibodies of patients allergic to each food described in the column of "Cross-checked foods" in Table 2 were confirmed by ELISA. The peptides were synthesized so as to be biotinylated on the N-terminal side by the Fmoc method.

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

[0285] (1) It was prepared with PBST (0.1% Tween (registered trademark) 20) so that the concentration of the biotinylated peptide became 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 collecting the solution, it was washed 5 times with PBS.

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

[0288] (4) 40 μL of 2% serum / Canget Signal Solution I (manufactured by TOYOBO) was added and shaken at room temperature for 1 hour. After removing the solution, it was washed 5 times with PBST.

[0289] (5) 40 μL of secondary antibody dilution solution (1:10,000, Canget SignalSolution II (manufactured by TOYOBO)) was added and shaken at room temperature for 1 hour. After removing the solution, it was washed three times with PBST.

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

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

[0292] Peptides having these amino acid sequences were prepared in the same procedure as in (A) of Example 4, and it was measured whether IgE antibodies in the sera of allergic patients and non-allergic subjects bind. For the sera of non-allergic subjects, the value obtained by dividing by the average value of two measured subjects was used.

[0293] The results are shown in FIGS. 3 to 6. P1, P5, P8, and P20 represent patient numbers. The bar graphs in each figure show the absorbance of allergic patients / the absorbance of non-allergic patients (healthy subjects). The food names described in each graph are the food names including the polypeptide containing the amino acid sequence that is the basis of each synthetic sequence used. Those without a food name description are "beef".

[0294] As is clear from FIGS. 3 to 6, in all polypeptides having the amino acid sequences described in the figures, higher binding (greater than "1") was shown for the IgE antibodies of each allergic patient than for the IgE antibodies in the sera of non-allergic subjects, and the cross-reactivity of these polypeptides was confirmed. This indicates that these epitopes can be used to detect cross-reactivity with antigens other than milk. Furthermore, it supports that the "X" part can take any amino acid residue.

Claims

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.

2. A composition for diagnosing allergies, comprising at least one of the polypeptides identified as (E1) to (E25) in claim 1.

3. A method for providing an indicator for diagnosing an 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 an IgE antibody to the antigen in a sample obtained from a subject; (iii) detecting binding of the subject's IgE antibodies to the antigen provides an indication that the subject is allergic; wherein the antigen is at least one of the polypeptides specified as (E1) to (E25) in claim 1.

4. An antigen which is at least one of the polypeptides specified as (E1) to (E25) in claim 1 and which causes an allergy.

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

6. The composition according to claim 5 for treating allergies.

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 specified as (E1) to (E25) in claim 1.

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 specified as (E1) to (E25) in claim 1 and / or a portion of its complementary strand.

9. A tester composition for determining the presence or absence of IgE antibodies in a subject, comprising a polypeptide identified as (E1) to (E25) in claim 1.

10. A method for determining the presence or absence of a polypeptide identified as (E1) to (E25) in claim 1 in a raw material or processed product, the method comprising detecting a polypeptide identified as (E1) to (E25) in claim 1 in the raw material or processed product.

11. A raw material or processed product in which an antigen has been removed or reduced, said raw material or processed product being at least one of the polypeptides specified as (E1) to (E25) in claim 1.

12. A method for producing a raw material or processed product in which an antigen has been removed or reduced, comprising a step of confirming that the antigen has been removed or reduced during the production process of the raw material or processed product, wherein the antigen is at least one of the polypeptides specified as (E1) to (E25) in claim 1.

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