Allergic antigens and their epitopes

Abstract

To provide a polypeptide containing an epitope of a novel antigen for fish allergy, an allergy diagnosis kit containing the polypeptide, a diagnosis composition, and a diagnosis method, and a pharmaceutical composition containing the polypeptide, as well as raw materials or processed products from which the antigen containing the polypeptide has been removed or reduced, and further to provide a tester for determining the presence or absence of an antigen in an object.SOLUTION: The present invention provides a polypeptide that specifically binds to an IgE antibody of an allergy patient, the polypeptide containing a specific amino acid sequence, where the allergy is an allergy to an allergen containing the polypeptide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel antigen for fish allergy. The present invention also relates to a diagnostic kit, diagnostic composition, and diagnostic method for fish allergy. The present invention also relates to a pharmaceutical composition containing the antigen, and to fish, fish eggs, fish or fish egg products, or fish that produce or are hatched from the fish eggs, in which the antigen has been removed or reduced. The present invention further relates to a tester for determining the presence or absence of a fish antigen in a subject.

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

[0003] In the blood and tissues of allergic patients, IgE antibodies specific to specific antigens are produced, and the physiological consequences of the interaction between these IgE antibodies and specific antigens result in the allergic reaction.

[0004] In conventional allergy test reagents, antigen reagents are often prepared by simply grinding foods, ingredients, etc. that are allergen candidates (Patent Document 1). As a result, the reagent contains numerous proteins, with the content of each protein being very low. Therefore, a positive reaction in an allergy test could only be detected if, among the numerous proteins contained in conventional antigen reagents, a protein whose content exceeds the threshold for determining a positive reaction in binding with IgE antibodies is a specific antigen protein (allergen component) that causes the allergic reaction. Conversely, for patients whose IgE antibodies bind to allergen components that are present in small amounts in foods, ingredients, etc., a positive reaction cannot be determined even with conventional allergy test reagents, and diagnostic efficiency has not been sufficiently high.

[0005] Furthermore, the symptoms and severity of allergic reactions do not necessarily correlate with the amount of allergen components contained in the food or ingredient. Even if a patient's IgE antibodies react to trace amounts of allergen components contained in a suspected allergen food, they may cause allergic symptoms or contribute to the severity of the symptoms.

[0006] Attempts are being made to improve diagnostic efficiency by examining IgE antibodies against each protein that makes up foods and ingredients, thereby distinguishing between sensitization that directly leads to diagnosis and sensitization due to cross-reactivity with panallergens, etc. Regarding fish allergens, the following are currently known (Non-Patent Documents 1 to 4).

[0007] [Table 1] However, to improve the reliability of allergy testing, it is necessary to comprehensively identify allergen components in candidate foods and ingredients, but the patient detection rate by measuring the above allergen components is still insufficient. Identifying new fish allergens is extremely important not only to improve the accuracy of diagnostic drugs, but also as targets for hypoallergenic foods, hypoallergenic ingredients, and therapeutic drugs.

[0008] On the other hand, various methods have been investigated for separating and purifying proteins and nucleic acids from cell extracts, etc. Dialysis and centrifugation using salt concentration are examples of such methods.

[0009] In addition, many purification methods that utilize the charge of protein or nucleic acid residues or differences in molecular weight are being investigated. Examples of purification methods that utilize charge include column chromatography using ion exchange resins and isoelectric focusing. Examples of purification methods that utilize differences in molecular weight include centrifugation, column chromatography using molecular weight sieves, and SDS-PAGE (dodecane-dioxide-polymerization gel electrophoresis). Examples include sodium silyl sulfate-polyacrylamide gel electrophoresis.

[0010] In recent years, two-dimensional electrophoresis, in which isoelectric focusing is performed in the first dimension and SDS-PAGE is performed in the second dimension, has been used as a method for separating and purifying various proteins from a small amount of sample. The applicants have previously developed two-dimensional electrophoresis methods with high resolution (Patent Documents 2 to 5).

[0011] Allergen-specific IgE antibodies recognize and bind to epitopes, which are specific amino acid sequences in allergen components. However, although there are a few examples in which epitopes have been analyzed for allergen components (Non-Patent Document 5), the current situation is extremely limited. Furthermore, there are currently no allergy diagnostic kits on the market that utilize polypeptides containing epitopes. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] Patent Publication No. 2002-286716 [Patent Document 2] Patent Publication No. 2011-33544 [Patent Document 3] Patent Publication No. 2011-33546 [Patent Document 4] Patent Publication No. 2011-33547 [Patent Document 5] Patent Publication No. 2011-33548 [Non-patent literature]

[0013] [Non-Patent Document 1] Allergen Nomenclature, WHO / IUIS Allergen Nomenclature Sub-Committee, [Retrieved June 9, 2016], Internet <url: http: www.allergen.org search.php?allergenname="&allergensource=&TaxSource=Animalia+Chordata&TaxOrder=&foodallerg=1&bioname="> [Non-patent document 2] Kuehn, A., et al., Clin. Exp. Allergy, (2013), Vol.43, No.7, pp.811-22 [Non-patent document 3] Gonzalez-Mancebo, E., et al., Ann. Allergy Asthma Immunol., (2014), Vol.113, No.1, pp.114-115 [Non-patent document 4] van der Ventel, ML, et al., Mol. Immunol., (2011), Vol.48, No.4, pp.637-646 [Non-Patent Document 5] Matsuo, H., et al., J. Biol. Chem., (2004), Vol.279, No.13, pp.12135-12140 Summary of the Invention [Problem to be solved by the invention]

[0014] The present invention provides a novel antigen for fish allergy. The present invention also provides a method and a diagnostic kit for fish allergy. The present invention also provides a pharmaceutical composition containing the antigen, and fish, fish eggs, fish or fish egg processed products, or fish that lay the fish eggs or are hatched from the fish eggs, in which the antigen has been removed or reduced. The present invention further provides a tester for determining the presence or absence of a fish antigen in a subject.

[0015] The present invention also provides a polypeptide comprising an epitope of an antigen, and a diagnostic kit, diagnostic composition, and diagnostic method for allergies, each comprising the polypeptide. The present invention also provides a pharmaceutical composition comprising the polypeptide, and a raw material or processed product from which an antigen comprising the polypeptide has been removed or reduced. The present invention further relates to a method for producing a processed product from which the antigen has been removed or reduced. The present invention still further provides a tester for determining the presence or absence of an antigen comprising the polypeptide in a subject. [Means for solving the problem]

[0016] In order to solve the above problems, the present inventors have conducted extensive research into identifying the antigen responsible for fish allergies. As a result, they have succeeded in identifying a novel antigen to which IgE antibodies in the serum of patients with fish allergies specifically bind. Based on this finding, the present invention has been completed.

[0017] That is, in one aspect, the present invention may be as follows.

[0018] [1] A diagnostic kit for fish allergy, comprising at least one of the following proteins (10) to (14): (10) (10A) Myosin heavy chain, fast skeletal muscle-like or a variant thereof, for use in fish Any of the following proteins (10A-a) to (10A-e) which are allergic antigens: (10A-a) One or several amino acids are deleted, substituted, inserted or is a protein containing the added amino acid sequence; (10A-b) Amino acids having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 70 Proteins containing sequences; (10A-c) One or several nucleotides are deleted, substituted, or inserted in SEQ ID NO: 69 or a protein containing an amino acid sequence encoded by the added base sequence; (10A-d) A base sequence having an identity of 70% or more with the base sequence shown in SEQ ID NO: 69 a protein comprising the amino acid sequence encoded by (10A-e) A nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 69 a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to (10B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 70 to 108; (11)(11A) Glycogen phosphorylase, muscle foam-like any one of the following proteins (11A-a) to (11A-e), which is an antigen for fish allergy, and is a protein selected from the group consisting of: (11A-a) One or several amino acids are deleted, substituted, inserted or deleted in SEQ ID NO: 110 or proteins containing added amino acid sequences; (11A-b) Amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 110 Proteins containing acid sequences; (11A-c) One or several nucleotides are deleted, substituted, or inserted in SEQ ID NO: 109 a protein comprising an amino acid sequence encoded by the inserted or added base sequence; (11A-d) A base sequence having an identity of 70% or more with the base sequence shown in SEQ ID NO: 109 a protein comprising an amino acid sequence encoded by (11A-e) A nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 109 a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent acid conditions; or (11B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 110 to 119; (12) (12A) Myosin-binding protein C, fast-type-like or a mutant thereof for fish Any of the following proteins (12A-a) to (12A-e) which are allergic antigens: (12A-a) One or several amino acids are deleted, substituted, inserted or deleted in SEQ ID NO: 121 or proteins containing added amino acid sequences; (12A-b) Amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 121 Proteins containing acid sequences; (12A-c) One or several nucleotides are deleted, substituted, or inserted in SEQ ID NO: 120 a protein comprising an amino acid sequence encoded by the inserted or added base sequence; (12A-d) A base sequence having an identity of 70% or more with the base sequence shown in SEQ ID NO: 120 a protein comprising an amino acid sequence encoded by (12A-e) A nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 120 a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent acid conditions; or (12B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 121 to 136; (13) (13A) ATP synthase subunit β, mitochondrial (ATP synthase any one of the following proteins (13A-a) to (13A-e) which is a fish allergy antigen and is a protein selected from the group consisting of mitochondrial subunit beta (mitochondrial) and a mutant thereof: (13A-a) One or several amino acids are deleted, substituted, inserted or deleted in SEQ ID NO: 138 or proteins containing added amino acid sequences; (13A-b) Amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 138 Proteins containing acid sequences; (13A-c) One or several nucleotides are deleted, substituted, or inserted in SEQ ID NO: 137 a protein comprising an amino acid sequence encoded by the inserted or added base sequence; (13A-d) A base sequence having an identity of 70% or more with the base sequence shown in SEQ ID NO: 137 a protein comprising an amino acid sequence encoded by (13A-e) A nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 137 a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent acid conditions; or (13B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 138 to 142; (14) (14A) L-lactate dehydrogenase A chain-like or a mutant thereof, which is an antigen for fish allergy, is any one of the following proteins (14A-a) to (14A-e): (14A-a) One or several amino acids are deleted, substituted, inserted or deleted in SEQ ID NO: 144 or proteins containing added amino acid sequences; (14A-b) Amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 144 Proteins containing acid sequences; (14A-c) One or several nucleotides are deleted, substituted, or inserted in SEQ ID NO: 143 a protein comprising an amino acid sequence encoded by the inserted or added base sequence; (14A-d) A base sequence having an identity of 70% or more with the base sequence shown in SEQ ID NO: 143 a protein comprising an amino acid sequence encoded by (14A-e) A nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 143 a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent acid conditions; or (14B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 144 to 149; as an antigen.

[0019] [2] A composition for diagnosing fish allergy, comprising (10) to (14) in the above [1] The diagnostic composition as defined above, comprising as an antigen at least one of the proteins identified as:

[0020] [3] A method for providing an indicator for diagnosing fish allergy in a subject, comprising the steps of: (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) if binding of the subject's IgE antibodies to the antigen is detected, an indication that the subject is allergic to fish is provided; wherein the antigen is at least one of the proteins identified as (10) to (14) in [1] above.

[0021] [4] A pharmaceutical composition comprising at least one of the proteins identified as (10) to (14) in [1] above.

[0022] [5] The pharmaceutical composition according to [4] above for treating fish allergy.

[0023] [6] A fish or fish product characterized in that an antigen has been removed or reduced, wherein the antigen is at least one of the proteins identified as (10) to (14) in [1] above.

[0024] [7] A tester for determining the presence or absence of a fish antigen in a target object, characterized by comprising an antibody that binds to at least one of the proteins identified as (10) to (14) in [1] above.

[0025] [8] A tester for determining the presence or absence of an antigen that causes fish allergy in a subject, characterized by including a primer having a base sequence complementary to at least a portion of the base sequence shown in SEQ ID NO: 69, 109, 120, 137 or 143.

[0026] [9] A fish-derived antigen, which is at least one of the proteins specified as (10) to (14) in [1] above, and which causes allergy to fish.

[0027] The present inventors have also succeeded in finding epitopes for antigens derived from fish, including the above antigens.

[0028] Because epitopes have relatively short amino acid sequences, if the same amino acid sequence is present in different allergen components, the IgE antibody can bind to multiple allergen components. As a result of the presence of a common epitope in different allergen components, IgE antibodies from allergy patients bind to both of them, making the antigen cross-reactive. Therefore, the epitopes identified in the present application enable the diagnosis and treatment of allergies, including cross-reactivity, and the detection of multiple allergen components containing the epitope.

[0029] Based on this finding, the present invention has been completed. That is, in another aspect, the present invention may be as follows.

[0030]

[10] A polypeptide that specifically binds to IgE antibodies of an allergic patient, comprising: (1α) at ​​least one selected from the group consisting of SEQ ID NOs: 150 to 154 and 205 to 227 a polypeptide containing one amino acid sequence; (2α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 155, 228 to 230; (3α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 156, 157, and 231 to 237; (4α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 159, and 238 to 247; (5α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 160 to 162 and 248 to 261; (6α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 163 to 167 and 262 to 279; (7α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 168 to 171 and 280 to 300; (8α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 172 to 174 and 301 to 310; (9α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 175 to 178 and 311 to 326; (10α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 179 to 185 and 327 to 365; (11α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 186, 366 to 370; (12α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 187 to 196 and 371 to 413; (13α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 197, 414 to 417; (14α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 198, 418 to 420; (15α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 199, 421 to 425; (16α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 200 to 202 and 426 to 436; (17α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 203, 204, and 437 to 444; The polypeptide, wherein the polypeptide is any one of the following:

[0031]

[11] The polypeptide according to

[10] above, which has 500 or less amino acid residues.

[0032]

[12] A kit for diagnosing allergies, comprising at least one of the polypeptides described in

[10] or

[11] above.

[0033]

[13] A composition for diagnosing allergies, comprising at least one of the polypeptides described in

[10] or

[11] above as an antigen.

[0034]

[14] A method for providing an indicator for diagnosing allergies in a subject, comprising the steps of: (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) if binding of the subject's IgE antibodies to the antigen is detected, an indication that the subject is allergic is provided; wherein the antigen is at least one of the polypeptides described in

[10] or

[11] above.

[0035]

[15] A pharmaceutical composition comprising at least one of the polypeptides described in

[10] or

[11] above.

[0036]

[16] The pharmaceutical composition according to

[15] above for treating allergies.

[0037]

[17] A tester for determining the presence or absence of an antigen in a subject, characterized by comprising an antibody that binds to at least one of the polypeptides described in

[10] or

[11] above.

[0038]

[18] One of the following primers: (a) a primer containing a portion of the nucleotide sequence of a nucleic acid encoding the polypeptide according to

[10] or

[11] above and / or a portion of its complementary strand; or (b) a primer which is a part of at least one of the nucleotide sequences shown in SEQ ID NO: 69, 109, 120, 137 or 143 and / or a primer which is a part of a sequence complementary to at least one of the nucleotide sequences shown in SEQ ID NO: 69, 109, 120, 137 or 143; A tester for determining the presence or absence of an antigen in a subject, comprising:

[0039]

[19] A raw material or processed product characterized in that an antigen has been removed or reduced, wherein the antigen is at least one of the polypeptides described in

[10] or

[11] above.

[0040]

[20] A method for producing a processed product in which antigens have been removed or reduced, comprising a step of confirming that the antigens have been removed or reduced during the production process of the processed product, wherein the antigen is at least one of the polypeptides described in

[10] or

[11] above. [Effects of the Invention]

[0041] The present invention provides a novel antigen for fish allergy. Because the present invention identifies a novel antigen (allergen component) that causes fish allergy, it can provide a highly sensitive method and kit for diagnosing fish allergy, a pharmaceutical composition containing the antigen, fish, fish eggs, processed fish or fish eggs products, or fish that produce or are hatched from the fish eggs, in which the antigen has been removed or reduced, and a tester for determining the presence or absence of the fish antigen in a target object.

[0042] The present invention also provides a novel polypeptide containing an epitope of an antigen. By utilizing the polypeptide of the present invention, it is possible to provide a highly sensitive allergy diagnostic kit, diagnostic composition, and diagnostic method, a pharmaceutical composition containing the polypeptide, a tester for determining the presence or absence of an antigen containing the polypeptide in a target substance, as well as a raw material or processed product from which the polypeptide has been removed or reduced, and a method for producing such a processed product. [Brief explanation of the drawings]

[0043] [Figure 1A] Figure 1A shows a gel photograph (left) showing the migration pattern of proteins contained in salmon meat extract by two-dimensional electrophoresis, and a photograph (right) of an immunoblot using serum from fish allergy patient 1 against the two-dimensional electrophoresis pattern. The bands on the left side of the gel photograph are molecular weight marker bands, and the numbers on the left side of the gel photograph are the molecular weight (KDa) of each molecular weight marker. The numbers at the top of the photograph indicate the isoelectric point. [Figure 1B] Figure 1B shows two-dimensional electrophoresis patterns of proteins contained in salmon meat extract, as well as a photograph of an immunoblot using serum from fish allergy patient 2 (left) and a photograph of an immunoblot using serum from fish allergy patient 3 (right). The numbers at the top of each photograph indicate the isoelectric point. [Figure 2] Figure 2 shows a photograph of an immunoblot using serum from fish allergy patient 1 against two-dimensional electrophoresis patterns of proteins contained in extracts of 10 types of fish flesh. [Figure 3] FIG. 3 is a photograph of an immunoblot performed using serum from a subject without fish allergy symptoms (non-fish allergic subject) against two-dimensional electrophoresis patterns of proteins contained in extracts of 10 types of fish flesh. [Figure 4] FIG. 4 is a photograph of an immunoblot using serum from patient 4 with fish allergy against two-dimensional electrophoresis patterns of proteins contained in extracts of six types of fish flesh. [Figure 5] 5 shows the results of chemiluminescence measurements using serum from fish allergy patients and non-fish allergy subjects for a peptide of epitope No. 11 (SEQ ID NO: 160) having the amino acid sequence "SMVLVKMKEIAEAYL" derived from salmon heat shock cognate 70 kDa protein and a peptide having the amino acid sequence "AMVLVKMKETAEAYL" derived from flounder heat shock cognate 70 kDa protein. The values ​​on the vertical axis indicate absorbance at a wavelength of 450 nm. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention will be specifically explained below, but the present invention is not limited thereto.

[0045] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those skilled in the art.

[0046] As used herein, allergy refers to a state in which an undesirable hypersensitivity reaction occurs when a living organism that has been sensitized to a certain antigen re-enters that antigen. An allergic reaction can occur when the antigen is contacted or ingested. Here, "contact" refers to touching an object, particularly in the case of the human body, and refers to adhesion to the skin, mucous membranes (eyes, lips, etc.). Furthermore, "ingestion" refers to taking into the body, such as by inhalation or oral administration. Generally, an allergic reaction that occurs when food is ingested is specifically referred to as a food allergy. In a preferred embodiment, the allergy may be a food allergy. In many food allergic diseases, antigen-specific IgE antibodies are produced in the blood and tissues. The IgE antibodies bind to mast cells or basophils. When an antigen specific for the IgE antibody re-enters the body of an allergic patient, the antigen combines with the IgE antibodies bound to mast cells or basophils, and the IgE antibodies cross-link on the cell surface, resulting in the physiological effects of IgE antibody-antigen interaction. These physiological effects include the release of histamine, serotonin, heparin, eosinophil chemotactic factor, and various leukotrienes. These released substances trigger allergic reactions caused by the combination of IgE antibodies and specific antigens. Specifically, IgE antibodies recognize and bind to epitopes, which are specific amino acid sequences in specific antigens, and allergic reactions caused by these antigens manifest through the above pathways.

[0047] The allergies targeted by the present invention are not particularly limited, as long as they are allergies to allergens containing the epitope used. Such allergies may include allergies to plants such as Oleaceae, Asteraceae, Poaceae, Bromeliaceae, Juglandaceae, Cucurbitaceae, and Fabaceae, allergies to animals such as Phasianidae and Bovidae, allergies to seafood such as Carangidae, Sparidae, Salmonidae, Penaeidae, Lithopodidae, Octopusidae, and Veneridae, and allergies to parasites such as Anisakididae. Examples of plants in the Oleaceae family include olive (scientific name: Olea europaea), and examples of plants in the Asteraceae family include artichoke ( Examples of plants in the Poaceae family include bread wheat (scientific name: Triticum aestivum) and talc wheat (scientific name: Aegilops tauschii). Examples of plants include pineapple (scientific name: Ananas comosus), etc.; examples of plants in the Juglandaceae family include walnut (scientific name: Juglans regia), etc.; examples of plants in the Cucurbitaceae family include pumpkin (scientific name: Cucurbita moschata), etc.; and examples of plants in the Fabaceae family include soybean (scientific name: Glycine max). Examples of animals in the Phasianidae family include quail (scientific name: Conturnix japonica) and turkey (scientific name: Meleagris gallopavo), etc.; and examples of animals in the Bovidae family include cows (scientific name: Bos taurus). Examples of fish and shellfish in the Carangidae family include amberjack (scientific name: Seriola dumerili), etc. Examples of Sparidae fish include red sea bream (Pagrus major), and examples of Salmonidae fish include rainbow trout (Oncorhynchus mykiss). Examples of seafood include the white shrimp (scientific name Litopenaeus vannamei), and seafood from the king crab family include the red king crab (scientific name Paralithodes camtschaticus). Examples of seafood from the Octopus family include the Pacific octopus (scientific name Enteroctopus dofleini). Examples of seafood in the family Ruditapes include the clam (scientific name Ruditapes philippinarum). Examples of parasites in the family Sillago include Anisakis (scientific name: Anisakis simplex).

[0048] In this specification, fish refers to bony fish and cartilaginous fish, preferably bony fish, more preferably those belonging to the Salmoniformes, Perciformes, Anguilliformes, Gadiformes and Pleuronectiformes, even more preferably those belonging to the Salmonidae, Carangidae, Conger Eelidae, Sparidae, Scombridae, Gadidae, Anguilliformes and Pleuronectidae families, and even more preferably salmon, horse mackerel, conger eel, black porgy, mackerel, sea bream, cod, yellowtail, eel and flounder. Fish may be edible.

[0049] In this specification, the term "fish eggs" refers to fish eggs, and is distinguished from "eggs," which primarily refers to avian eggs. Fish eggs may be edible.

[0050] As used herein, allergy to fish refers to a state in which an allergic reaction occurs due to an antigen, such as a protein contained in fish. An allergy to fish can occur when a person comes into contact with an antigen contained in fish or when the antigen is ingested. Generally, an allergic reaction that occurs when a food is ingested is specifically referred to as a food allergy. An allergy to fish may be a food allergy.

[0051] As used herein, an antigen is a substance that induces an allergic reaction and is also referred to as an allergen component. Antigens are preferably proteins.

[0052] As used herein, a protein is a molecule having a structure in which natural amino acids are linked by peptide bonds. The number of amino acids contained in a protein is not particularly limited. As used herein, the term "polypeptide" also refers to a molecule having a structure in which natural amino acids are linked by peptide bonds. The number of amino acids contained in a polypeptide is not particularly limited. "Polypeptide" encompasses the concept of "protein." Furthermore, polypeptides in which approximately 2 to 50 amino acids are linked by peptide bonds are sometimes referred to as peptides. Furthermore, when optical isomers of amino acids are possible, the L-isomer is indicated unless otherwise specified. The notation method for the amino acid sequence of a protein, polypeptide, or peptide used herein is based on standard usage and the notation method commonly used in the art, and is represented by the single-letter notation of amino acids, with the left direction being the amino-terminal direction and the right direction being the carboxy-terminal direction. In the single-letter notation of an amino acid, X represents any substance having an amino group and a carboxyl group that can bond to the amino acids at both ends, and specifically represents any of the 20 natural amino acids. The residue represented by X is an amino acid residue at a site where the binding to IgE antibodies of allergy patients is maintained even when substituted with alanine, as determined by the alanine scan shown in Example 10. It is well known to those skilled in the art that such a site is highly likely to maintain the binding to the IgE antibody even when substituted with any other amino acid.

[0053] Identification of antigens The above-mentioned method was used to identify the antigens responsible for fish allergies. Specifically, proteins were extracted from fish flesh and subjected to two-dimensional electrophoresis under the following conditions.

[0054] The first-dimension electrophoresis gel used was an isoelectric focusing gel with a gel length of 5-10 cm, a gel pH range of 3-10, and a pH gradient of the gel in the direction of migration, where the total length of the gel is 1, the gel length up to pH 5 is a, the gel length from pH 5 to 7 is b, and the gel length above pH 7 is c. Specifically, the gel used was an IPG gel Immobiline Drystrip (pH 3-10NL) manufactured by GE Healthcare Biosciences, Inc. (hereafter abbreviated as GE). The electrophoresis equipment used was an IPGphor manufactured by GE. The upper limit of the current value of the electrophoresis device was set to 75 μA per gel, and the voltage program was as follows: (1) a constant voltage step of 300 V up to 750 Vhr (the current change during the 30 minutes of electrophoresis before the end of this step was 5 μA), (2) the voltage was gradually increased to 1000 V over 300 Vhr, (3) the voltage was further increased gradually to 5000 V over 4500 Vhr, and (4) the first-dimension isoelectric focusing was then performed at a constant voltage of 5000 V until a total Vhr of 12000 was reached.

[0055] For the second-dimensional electrophoresis, polyacrylamide gel was used, with the gel concentration at the base end of the migration direction set to 3-6% and the gel concentration at the leading end of the migration direction set to be higher than the gel concentration at the base end of the migration direction. Specifically, SDS-PAGE was performed using NuPAGE 4-12% Bris-Tris Gels IPG well mini 1mm manufactured by Life Technologies. The electrophoresis equipment was manufactured by Life Technologies. The electrophoresis buffer used was 50 mM MOPS, 50 mM Tris base, 0.1% (w / v) SDS, and 1 mM EDTA were used, and electrophoresis was carried out at a constant voltage of 200 V for approximately 45 minutes.

[0056] As a result, when fish proteins were subjected to two-dimensional electrophoresis under the above conditions, the following spots on the gel were found to specifically bind to IgE antibodies from patients with fish allergies who had been diagnosed with immediate-type allergies. Spot 1: Molecular weight 80-160 kDa, pI 3.0-7.0 Spot 2: Molecular weight 110-260 kDa, pI 4.0-8.0 Spot 3: Molecular weight 80-160 kDa, pI 4.0-10.0 Spot 4: Molecular weight 80-160 kDa, pI 5.0-11.0 Spot 5: Molecular weight 50-110 kDa, pI 3.0-7.0 Spot 6: Molecular weight 60-110 kDa, pI 4.0-8.0 Spot 7: Molecular weight 40-80 kDa, pI 4.0-8.0 Spot 8: Molecular weight 40-80 kDa, pI 3.0-7.0 Spot 9: Molecular weight 20-50 kDa, pI 3.0-7.0 Spot 10: molecular weight 160-300, pI 3.0-7.0 Spot 11: molecular weight 80-160, pI 4.0-8.0 Spot 12: molecular weight 100-160, pI 4.0-7.0 Spot 13: molecular weight 30-70, pI 3.0-7.0 Spot 14: molecular weight 20-50, pI 5.0-9.0 antigen (10) Spot 10 antigen Spot 10 was subjected to sequence identification by mass spectrometry, and the amino acid sequences of SEQ ID NOs: 71 to 108 were detected.

[0057] In addition, for spot 10, the mass data (SEQ ID NOs: 71 to 74) obtained from the mass spectrometer were 108) was analyzed against the NCBI protein data and identified as myosin heavy chain, fast skeletal muscle-like (amino acid sequence: SEQ ID NO: 70, coding nucleotide sequence: SEQ ID NO: 69). SEQ ID NO:71 corresponds to amino acids 13 to 19 of SEQ ID NO:70, SEQ ID NO:72 corresponds to amino acids 262 to 271 of SEQ ID NO:70, SEQ ID NO:73 corresponds to amino acids 996 to 1014 of SEQ ID NO:70, SEQ ID NO:74 corresponds to amino acids 951 to 961 of SEQ ID NO:70, SEQ ID NO:75 corresponds to amino acids 1293 to 1303 of SEQ ID NO:70, SEQ ID NO:76 corresponds to amino acids 1082 to 1091 of SEQ ID NO:70, SEQ ID NO:77 corresponds to amino acids 1847 to 1856 of SEQ ID NO:70, SEQ ID NO:78 corresponds to amino acids 172 to 186 of SEQ ID NO:70, SEQ ID NO:79 corresponds to amino acids 919 to 937 of SEQ ID NO:70, SEQ ID NO:8 0 is amino acids 249 to 258 of SEQ ID NO:70, SEQ ID NO:81 is amino acids 706 to 717 of SEQ ID NO:70, SEQ ID NO:82 is amino acids 50 to 59 of SEQ ID NO:70, SEQ ID NO:83 is amino acids 415 to 430 of SEQ ID NO:70, SEQ ID NO:84 is amino acids 834 to 845 of SEQ ID NO:70, SEQ ID NO:85 is amino acids 617 to 630 of SEQ ID NO:70, SEQ ID NO:86 is amino acids 1556 to 1567 of SEQ ID NO:70, SEQ ID NO:87 is amino acids 1391 to 1408 of SEQ ID NO:70, SEQ ID NO:88 is amino acids 1025 to 1040 of SEQ ID NO:70, SEQ ID NO:89 is amino acids 715 to 730 of SEQ ID NO:70 SEQ ID NO: 90 represents amino acids 743 to 755 of SEQ ID NO: 70, SEQ ID NO: 91 represents amino acids 1172 to 1192 of SEQ ID NO: 70, SEQ ID NO: 92 represents amino acids 353 to 364 of SEQ ID NO: 70, SEQ ID NO: 93 represents amino acids 1261 to 1292 of SEQ ID NO: 70, SEQ ID NO: 94 represents amino acids 1783 to 1789 of SEQ ID NO: 70, SEQ ID NO: 95 represents amino acids 1502 to 1519 of SEQ ID NO: 70, SEQ ID NO: 96 represents amino acids 1484 to 1497 of SEQ ID NO: 70, SEQ ID NO: 97 represents amino acids 1194 to 1212 of SEQ ID NO: 70, SEQ ID NO: 98 represents amino acids 1261 to 1292 of SEQ ID NO: 70, SEQ ID NO: 99 represents amino acids 1302 to 1312 of SEQ ID NO: 70, SEQ ID NO: 1002 to 1014 of SEQ ID NO: 1015 SEQ ID NO: 70 represents amino acids 1304 to 1314, SEQ ID NO: 99 represents amino acids 369 to 384 of SEQ ID NO: 70, SEQ ID NO: 100 represents amino acids 1315 to 1322 of SEQ ID NO: 70, SEQ ID NO: 101 represents amino acids 1536 to 1555 of SEQ ID NO: 70, SEQ ID NO: 102 represents amino acids 237 to 248 of SEQ ID NO: 70, SEQ ID NO: 103 represents amino acids 1699 to 1725 of SEQ ID NO: 70, SEQ ID NO: 104 represents amino acids 1092 to 1104 of SEQ ID NO: 70, SEQ ID NO: 105 represents amino acids 407 to 414 of SEQ ID NO: 70, SEQ ID NO: 106 represents amino acids 1897 to 1917 of SEQ ID NO: 70,SEQ ID NO: 107 corresponds to amino acids 1458 to 1470 of SEQ ID NO: 70, and SEQ ID NO: 108 corresponds to amino acids 1373 to 1388 of SEQ ID NO: 70.

[0058] Therefore, in the present application, the antigen of spot 10 may be any of the following (10A-a) to (10A-e) and (10B). (10A-a) A protein comprising the amino acid sequence of SEQ ID NO: 70 in which one or several amino acids have been deleted, substituted, inserted or added. (10A-b) A protein comprising an amino acid sequence that has an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence set forth in SEQ ID NO: 70. (10A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 69 have been deleted, substituted, inserted or added. (10A-d) A protein comprising an amino acid sequence encoded by a nucleotide sequence that has an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the nucleotide sequence shown in SEQ ID NO: 69. (10A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 69. (10B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 70 to 108, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, A protein comprising any one of 35, 36, 37, 38 or all of the sequences, wherein the amino acid sequence shown in any of SEQ ID NOs: 70 to 108 may have one or several amino acids deleted, substituted, inserted or added.

[0059] The proteins (10A-a) to (10A-e) and (10B) above also include proteins whose amino acid residues have been modified by phosphorylation, glycosylation, aminoacylation, ring-opening, deamination, etc.

[0060] The proteins (10A-a) to (10A-e) and (10B) above may be proteins that appear as spots with a molecular weight of 160 kDa to 300 kDa, preferably a molecular weight of 160 kDa to 260 kDa, and more preferably a molecular weight of about 200 kDa to 230 kDa, and an isoelectric point of 3.0 to 7.0, preferably an isoelectric point of 4.0 to 6.0, and more preferably an isoelectric point of 4.5 to 5.5, in a gel when two-dimensional electrophoresis is performed under the conditions described in the above section "Identification of antigen."

[0061] Preferably, the protein that is the antigen of spot 10 is an antigen for fish allergies.

[0062] Furthermore, the epitopes of the antigens (10A-a) to (10A-e) and (10B) above are present at amino acids 61 to 75 (SEQ ID NO: 179), 471 to 485 (SEQ ID NO: 180), 621 to 635 (SEQ ID NO: 181), 981 to 995 (SEQ ID NO: 182), 1011 to 1025 (SEQ ID NO: 183), 1041 to 1055 (SEQ ID NO: 184), and 1741 to 1755 (SEQ ID NO: 185) of SEQ ID NO: 70. When the antigen of spot 10 contains a mutation in the amino acid sequence of SEQ ID NO: 70, the sequence of SEQ ID NO: 70 may be retained for at least one amino acid corresponding to the epitope.

[0063] Furthermore, from the viewpoint that the binding properties with IgE antibodies, such as sensitivity (the degree to which a patient can be judged as positive) and specificity (the degree to which a healthy person is not judged as positive), remain even if the amino acid sequence is mutated, the antigen in spot 10 may be the following mutant.

[0064] Of the epitope having the sequence of SEQ ID NO: 179, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 327 or 328, which corresponds to the 1st to 11th amino acid positions of SEQ ID NO: 179, or in SEQ ID NO: 330 or 331, which corresponds to the 1st to 9th amino acid positions of SEQ ID NO: 179. Therefore, in another preferred embodiment, when the antigen in spot 10 contains a mutation in the amino acid sequence of SEQ ID NO: 70, the antigen may be one in which the 1st to 11th amino acids of amino acids 61 to 75 in SEQ ID NO: 70 are the amino acid sequence of SEQ ID NO: 327 or 328, or the 1st to 9th amino acids are the amino acid sequence of SEQ ID NO: 330 or 331.

[0065] Of the epitope having the sequence of SEQ ID NO: 180, the site important for binding to IgE antibodies of allergy patients is the site represented by amino acids other than X in SEQ ID NO: 333 or 334, which corresponds to the 7th to 15th amino acids of SEQ ID NO: 180. Therefore, in another preferred embodiment, when the antigen in spot 10 contains a mutation in the amino acid sequence of SEQ ID NO: 70, the antigen may have the amino acid sequence of SEQ ID NO: 333 or 334 at the 7th to 15th amino acids of amino acids 471 to 485 in SEQ ID NO: 70.

[0066] Of the epitopes having the sequence of SEQ ID NO: 181, the epitope having the sequence of SEQ ID NO: 3, which corresponds to the 1st to 10th amino acid positions of SEQ ID NO: 181, is important for binding to IgE antibodies of allergy patients. 336 or 337. Therefore, in another preferred embodiment, when the antigen of spot 10 contains a mutation in the amino acid sequence of SEQ ID NO: 70, the 1st to 10th amino acids of amino acids 621 to 635 in SEQ ID NO: 70 may be the amino acid sequence of SEQ ID NO: 336 or 337.

[0067] Of the epitope having the sequence of SEQ ID NO: 182, those important for binding to IgE antibodies of allergy patients are sites represented by amino acids other than X in SEQ ID NO: 339 or 340, which correspond to the 2nd to 9th amino acid positions of SEQ ID NO: 342 or 343, which correspond to the 2nd to 11th amino acid positions of SEQ ID NO: 345 or 346, which correspond to the 7th to 15th amino acid positions, or SEQ ID NO: 348 or 349, which correspond to the 9th to 14th amino acid positions of SEQ ID NO: 182. Therefore, in another preferred embodiment, when the antigen in spot 10 contains a mutation in the amino acid sequence of SEQ ID NO: 70, the antigen may be such that the 2nd to 9th amino acids of amino acids 981 to 995 in SEQ ID NO: 70 correspond to the amino acid sequence of SEQ ID NO: 339 or 340, the 2nd to 11th amino acids correspond to SEQ ID NO: 342 or 343, the 7th to 15th amino acids correspond to SEQ ID NO: 345 or 346, or the 9th to 14th amino acids correspond to the amino acid sequence of SEQ ID NO: 348 or 349.

[0068] Of the epitope having the sequence of SEQ ID NO: 183, the site important for binding to IgE antibodies of allergy patients is the site represented by amino acids other than X in SEQ ID NO: 351 or 352, which corresponds to the 5th to 14th amino acids of SEQ ID NO: 183. Therefore, in another preferred embodiment, when the antigen in spot 10 contains a mutation in the amino acid sequence of SEQ ID NO: 70, the antigen may have the amino acid sequence of SEQ ID NO: 351 or 352 at the 5th to 14th amino acids of amino acids 1011 to 1025 in SEQ ID NO: 70.

[0069] Of the epitope having the sequence of SEQ ID NO: 184, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 354 or 355, which corresponds to the 5th to 12th amino acid positions of SEQ ID NO: 184, or in SEQ ID NO: 357 or 358, which corresponds to the 9th to 15th amino acid positions of SEQ ID NO: 184. Therefore, in another preferred embodiment, when the antigen in spot 10 contains a mutation in the amino acid sequence of SEQ ID NO: 70, the antigen may be one in which the 5th to 12th amino acids of amino acids 1041 to 1055 in SEQ ID NO: 70 are the amino acid sequence of SEQ ID NO: 354 or 355, or the 9th to 15th amino acids are the amino acid sequence of SEQ ID NO: 357 or 358.

[0070] Of the epitope having the sequence of SEQ ID NO: 185, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 360 or 361, which corresponds to the 5th to 12th amino acid positions of SEQ ID NO: 185, or in SEQ ID NO: 363 or 364, which corresponds to the 7th to 15th amino acid positions of SEQ ID NO: 185. Therefore, in another preferred embodiment, when the antigen in spot 10 contains a mutation in the amino acid sequence of SEQ ID NO: 70, the antigen may be one in which the 5th to 12th amino acids of amino acids 1741 to 1755 in SEQ ID NO: 70 are the amino acid sequence of SEQ ID NO: 360 or 361, or the 7th to 15th amino acids are the amino acid sequence of SEQ ID NO: 363 or 364.

[0071] (11) Spot 11 antigen Spot 11 was subjected to sequence identification by mass spectrometry, and the amino acid sequences of SEQ ID NOs: 111 to 119 were detected.

[0072] In addition, for spot 11, the mass data (SEQ ID NOs: 111 to 119) obtained from the mass spectrometer were analyzed by collating them with the protein data of NCBI. This protein was identified as glycogen phosphorylase, muscle form-like (amino acid sequence: SEQ ID NO:110, nucleotide sequence encoding it: SEQ ID NO:109). SEQ ID NO:111 corresponds to amino acids 471 to 479 of SEQ ID NO:110, SEQ ID NO:112 corresponds to amino acids 547 to 555 of SEQ ID NO:110, SEQ ID NO:113 corresponds to amino acids 203 to 215 of SEQ ID NO:110, SEQ ID NO:114 corresponds to amino acids 727 to 741 of SEQ ID NO:110, SEQ ID NO:115 corresponds to amino acids 508 to 520 of SEQ ID NO:110, SEQ ID NO:116 corresponds to amino acids 742 to 755 of SEQ ID NO:110, SEQ ID NO:117 corresponds to amino acids 13 to 29 of SEQ ID NO:110, SEQ ID NO:118 corresponds to amino acids 775 to 784 of SEQ ID NO:110, and SEQ ID NO:119 corresponds to amino acids 643 to 656 of SEQ ID NO:110.

[0073] Therefore, in the present application, the antigen of spot 11 may be any of the following (11A-a) to (11A-e) and (11B). (11A-a) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 110. (11A-b) A protein comprising an amino acid sequence that has an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence shown in SEQ ID NO: 110. (11A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 109 have been deleted, substituted, inserted or added. (11A-d) A protein comprising an amino acid sequence encoded by a nucleotide sequence that has an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the nucleotide sequence shown in SEQ ID NO: 109. (11A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 109. (11B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 110 to 119, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, or all of the amino acid sequences, wherein one or several amino acids may be deleted, substituted, inserted, or added in the amino acid sequence represented by any of SEQ ID NOs: 110 to 119.

[0074] The proteins (11A-a) to (11A-e) and (11B) above also include proteins whose amino acid residues have been modified by phosphorylation, glycosylation, aminoacylation, ring-opening, deamination, etc.

[0075] The above proteins (11A-a) to (11A-e) and (11B) may be proteins that appear as spots with a molecular weight of 80 kDa to 160 kDa, preferably a molecular weight of 80 kDa to 110 kDa, and more preferably a molecular weight of about 90 kDa to 110 kDa, and an isoelectric point of 4.0 to 8.0, preferably an isoelectric point of 5.0 to 7.5, and more preferably an isoelectric point of 6.5 to 7.0, in a gel when two-dimensional electrophoresis is performed under the conditions described in the above section "Identification of antigen."

[0076] Preferably, the protein that is the antigen of spot 11 is an antigen for fish allergies.

[0077] Furthermore, the epitopes of the antigens (11A-a) to (11A-e) and (11B) above are located at amino acids 261 to 275 (SEQ ID NO: 186) of SEQ ID NO: 110. When the antigen of spot 11 contains a mutation in the amino acid sequence of SEQ ID NO: 110, the amino acids corresponding to the epitope may retain the sequence of SEQ ID NO: 110.

[0078] Furthermore, from the viewpoint of maintaining the binding properties with IgE antibodies, such as sensitivity and specificity, even if the amino acid sequence is mutated, the antigen of spot 11 may be the following mutant.

[0079] Of the epitope having the sequence of SEQ ID NO: 186, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 366, which corresponds to the 1st to 10th amino acid positions of SEQ ID NO: 186, or in SEQ ID NO: 368 or 369, which corresponds to the 11th to 15th amino acid positions of SEQ ID NO: 186. Therefore, in another preferred embodiment, when the antigen of spot 11 contains a mutation in the amino acid sequence of SEQ ID NO: 110, the antigen may be one in which the 1st to 10th amino acids of amino acids 261 to 275 in SEQ ID NO: 110 are the amino acid sequence of SEQ ID NO: 366, or the 11th to 15th amino acids of amino acids 261 to 275 in SEQ ID NO: 110 are the amino acid sequence of SEQ ID NO: 368 or 369.

[0080] (12) Spot 12 antigen Spot 12 was subjected to sequence identification by mass spectrometry, and the amino acid sequences of SEQ ID NOs: 122 to 136 were detected.

[0081] Furthermore, mass data (SEQ ID NOS: 122 to 136) obtained from a mass spectrometer for spot 12 was analyzed by collating it with NCBI protein data, and it was identified as myosin-binding protein C, fast-type-like (amino acid sequence: SEQ ID NOS: 121, encoding nucleotide sequence: SEQ ID NOS: 120). SEQ ID NOS: 122 corresponds to amino acids 197 to 204 of SEQ ID NOS: 121, SEQ ID NOS: 123 corresponds to amino acids 421 to 434 of SEQ ID NOS: 121, SEQ ID NOS: 124 corresponds to amino acids 329 to 336 of SEQ ID NOS: 121, SEQ ID NOS: 125 corresponds to amino acids 413 to 420 of SEQ ID NOS: 121, SEQ ID NOS: 126 corresponds to amino acids 240 to 246 of SEQ ID NOS: 121, SEQ ID NOS: 127 corresponds to amino acids 214 to 228 of SEQ ID NOS: 121, SEQ ID NOS: 128 corresponds to amino acids 1014 to 1024 of SEQ ID NOS: 121, and SEQ ID NOS: 129 corresponds to amino acids 1014 to 1024 of SEQ ID NOS: 121. SEQ ID NO: 130 corresponds to amino acids 260 to 274 of SEQ ID NO: 121, SEQ ID NO: 131 corresponds to amino acids 672 to 685 of SEQ ID NO: 121, SEQ ID NO: 132 corresponds to amino acids 506 to 512 of SEQ ID NO: 121, SEQ ID NO: 133 corresponds to amino acids 205 to 213 of SEQ ID NO: 121, SEQ ID NO: 134 corresponds to amino acids 165 to 182 of SEQ ID NO: 121, SEQ ID NO: 135 corresponds to amino acids 321 to 328 of SEQ ID NO: 121, and SEQ ID NO: 136 corresponds to amino acids 70 to 82 of SEQ ID NO: 121.

[0082] Therefore, in the present application, the antigen of spot 12 may be any of the following (12A-a) to (12A-e) and (12B). (12A-a) A protein comprising the amino acid sequence of SEQ ID NO: 121 in which one or several amino acids have been deleted, substituted, inserted or added. (12A-b) A protein comprising an amino acid sequence that has an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence shown in SEQ ID NO: 121. (12A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 119 have been deleted, substituted, inserted or added. (12A-d) A protein comprising an amino acid sequence encoded by a nucleotide sequence that has an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the nucleotide sequence shown in SEQ ID NO: 119. (12A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 119. (12B) At least one amino acid sequence selected from the group consisting of SEQ ID NOs: 121 to 136 A protein containing one of the amino acid sequences, preferably a protein containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all of the amino acid sequences, wherein the amino acid sequence shown in any of SEQ ID NOs: 121 to 136 may have one or several amino acids deleted, substituted, inserted, or added.

[0083] The proteins (12A-a) to (12A-e) and (12B) above also include proteins whose amino acid residues have been modified by phosphorylation, glycosylation, aminoacylation, ring-opening, deamination, etc.

[0084] The above proteins (12A-a) to (12A-e) and (12B) may be proteins that appear as spots with a molecular weight of 100 kDa to 160 kDa, preferably a molecular weight of 110 kDa to 150 kDa, and more preferably a molecular weight of about 120 kDa to 140 kDa, and an isoelectric point of 4.0 to 7.0, preferably an isoelectric point of 4.0 to 6.0, and more preferably an isoelectric point of 5.0 to 6.0, in a gel when two-dimensional electrophoresis is performed under the conditions described in the above section "Identification of antigen."

[0085] Preferably, the protein that is the antigen of spot 12 is an antigen for fish allergies.

[0086] Furthermore, the epitopes of the antigens (12A-a) to (12A-e) and (12B) are located at amino acids 181 to 195 (SEQ ID NO: 187), 211 to 225 (SEQ ID NO: 188), 221 to 235 (SEQ ID NO: 189), 231 to 245 (SEQ ID NO: 190), 251 to 265 (SEQ ID NO: 191), 371 to 385 (SEQ ID NO: 192), 491 to 505 (SEQ ID NO: 193), 651 to 665 (SEQ ID NO: 194), 831 to 845 (SEQ ID NO: 195), and 951 to 965 (SEQ ID NO: 196) of SEQ ID NO: 121. When the antigen of spot 12 contains a mutation in the amino acid sequence of SEQ ID NO: 121, the sequence of SEQ ID NO: 121 may be retained for at least one amino acid corresponding to the epitope.

[0087] Furthermore, from the viewpoint of maintaining the binding properties with IgE antibodies, such as sensitivity and specificity, even if the amino acid sequence is mutated, the antigen in spot 12 may be the following mutant.

[0088] Of the epitope having the sequence of SEQ ID NO: 187, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 371 or 372, which corresponds to the 2nd to 11th amino acid positions of SEQ ID NO: 187, or in SEQ ID NO: 374 or 375, which corresponds to the 7th to 14th amino acid positions of SEQ ID NO: 187. Therefore, in another preferred embodiment, when the antigen of spot 12 contains a mutation in the amino acid sequence of SEQ ID NO: 121, the antigen may be one in which the 2nd to 11th amino acids of amino acids 181 to 195 in SEQ ID NO: 121 are the amino acid sequence of SEQ ID NO: 371 or 372, or the 7th to 14th amino acids are the amino acid sequence of SEQ ID NO: 374 or 375.

[0089] Of the epitope having the sequence of SEQ ID NO: 188, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 377 or 378, which corresponds to the 1st to 10th amino acid positions of SEQ ID NO: 188, or in SEQ ID NO: 380 or 381, which corresponds to the 8th to 15th amino acid positions of SEQ ID NO: 188. Therefore, in another preferred embodiment, when the antigen in spot 12 contains a mutation in the amino acid sequence of SEQ ID NO: 121, the antigen may be one in which the 1st to 10th amino acids of amino acids 211 to 225 in SEQ ID NO: 121 are the amino acid sequence of SEQ ID NO: 377 or 378, or the 8th to 15th amino acids are the amino acid sequence of SEQ ID NO: 380 or 381.

[0090] Of the epitope having the sequence of SEQ ID NO: 189, the site important for binding to IgE antibodies of allergy patients is the site represented by amino acids other than X in SEQ ID NO: 383, which corresponds to the 2nd to 8th amino acid positions of SEQ ID NO: 189. Therefore, in another preferred embodiment, when the antigen in spot 12 contains a mutation in the amino acid sequence of SEQ ID NO: 121, the antigen may have the amino acid sequence of SEQ ID NO: 383 at the 2nd to 8th amino acids of amino acids 221 to 235 in SEQ ID NO: 121.

[0091] Of the epitope having the sequence of SEQ ID NO: 190, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 385, which corresponds to the 2nd to 9th amino acid positions of SEQ ID NO: 387, which corresponds to the 1st to 10th amino acid positions of SEQ ID NO: 190, or in SEQ ID NO: 389, which corresponds to the 9th to 15th amino acid positions of SEQ ID NO: 190. Therefore, in another preferred embodiment, when the antigen in spot 12 contains a mutation in the amino acid sequence of SEQ ID NO: 121, the antigen may be such that the 2nd to 9th amino acids of amino acid 231 to 245 in SEQ ID NO: 121 are represented by the amino acid sequence of SEQ ID NO: 385, the 1st to 10th amino acids are represented by the amino acid sequence of SEQ ID NO: 387, or the 9th to 15th amino acids are represented by the amino acid sequence of SEQ ID NO: 389.

[0092] Of the epitope having the sequence of SEQ ID NO: 191, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 391, which corresponds to the 6th to 11th amino acid positions of SEQ ID NO: 191, or in SEQ ID NO: 393 or 394, which corresponds to the 6th to 12th amino acid positions of SEQ ID NO: 191. Therefore, in another preferred embodiment, when the antigen of spot 12 contains a mutation in the amino acid sequence of SEQ ID NO: 121, the antigen may be one in which the 6th to 11th amino acids of amino acids 251 to 265 in SEQ ID NO: 121 are the amino acid sequence of SEQ ID NO: 391, or the 6th to 12th amino acids are the amino acid sequence of SEQ ID NO: 393 or 394.

[0093] Of the epitope having the sequence of SEQ ID NO: 192, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 396, which corresponds to the 5th to 8th amino acid positions of SEQ ID NO: 192, or in SEQ ID NO: 398 or 399, which corresponds to the 7th to 15th amino acid positions of SEQ ID NO: 192. Therefore, in another preferred embodiment, when the antigen in spot 12 contains a mutation in the amino acid sequence of SEQ ID NO: 121, the antigen may be one in which the 5th to 8th amino acids of amino acids 371 to 385 in SEQ ID NO: 121 are the amino acid sequence of SEQ ID NO: 396, or the 7th to 15th amino acids of the amino acid sequence of SEQ ID NO: 398 or 399.

[0094] Of the epitope having the sequence of SEQ ID NO: 193, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 401, which corresponds to the 1st to 7th amino acid positions of SEQ ID NO: 193, or in SEQ ID NO: 403 or 404, which corresponds to the 6th to 15th amino acid positions of SEQ ID NO: 193. Therefore, in another preferred embodiment, when the antigen of spot 12 contains a mutation in the amino acid sequence of SEQ ID NO: 121, the antigen may be one in which the 1st to 7th amino acids of amino acid 491 to 505 in SEQ ID NO: 121 are the amino acid sequence of SEQ ID NO: 401, or the 6th to 15th amino acids of amino acid 491 to 505 in SEQ ID NO: 121 are the amino acid sequence of SEQ ID NO: 403 or 404.

[0095] Of the epitopes having the sequence of SEQ ID NO: 194, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 406, which corresponds to the 3rd to 7th amino acid positions of SEQ ID NO: 194. Therefore, in another preferred embodiment, when the antigen in spot 12 contains a mutation in the amino acid sequence of SEQ ID NO: 121, the antigen is a site represented by an amino acid other than X in the 3rd to 7th amino acid positions of amino acids 651 to 665 in SEQ ID NO: 121. The amino acid may be the amino acid sequence of SEQ ID NO:406.

[0096] Of the epitope having the sequence of SEQ ID NO: 195, the site important for binding to IgE antibodies of allergy patients is the site represented by amino acids other than X in SEQ ID NO: 408, which corresponds to the 2nd to 13th amino acids of SEQ ID NO: 195. Therefore, in another preferred embodiment, when the antigen of spot 12 contains a mutation in the amino acid sequence of SEQ ID NO: 121, the antigen may have the amino acid sequence of SEQ ID NO: 408 at the 2nd to 13th amino acids of amino acids 831 to 845 in SEQ ID NO: 121.

[0097] Of the epitope having the sequence of SEQ ID NO: 196, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 410, which corresponds to the 1st to 10th amino acid positions of SEQ ID NO: 196, or in SEQ ID NO: 412, which corresponds to the 1st to 8th amino acid positions of SEQ ID NO: 196. Therefore, in another preferred embodiment, when the antigen of spot 12 contains a mutation in the amino acid sequence of SEQ ID NO: 121, the antigen may be one in which the 1st to 10th amino acids of amino acids 951 to 965 in SEQ ID NO: 121 are the amino acid sequence of SEQ ID NO: 410, or the 1st to 8th amino acids of amino acids 951 to 965 in SEQ ID NO: 121 are the amino acid sequence of SEQ ID NO: 412.

[0098] (13) Spot 13 antigen Spot 13 was subjected to sequence identification by mass spectrometry, and the amino acid sequences of SEQ ID NOs: 139 to 142 were detected.

[0099] Furthermore, analysis of mass data (SEQ ID NOS: 139-142) obtained from a mass spectrometer against NCBI protein data identified spot 13 as ATP synthase subunit beta, mitochondrial (amino acid sequence: SEQ ID NOS: 138, encoding nucleotide sequence: SEQ ID NOS: 137). SEQ ID NOS: 139 corresponds to amino acids 191-201 of SEQ ID NOS: 138, 140 corresponds to amino acids 449-469 of SEQ ID NOS: 138, 141 corresponds to amino acids 202-214 of SEQ ID NOS: 138, and 142 corresponds to amino acids 178-187 of SEQ ID NOS: 138.

[0100] Therefore, in the present application, the antigen of spot 13 may be any of the following (13A-a) to (13A-e) and (13B). (13A-a) A protein comprising the amino acid sequence of SEQ ID NO: 138 in which one or more amino acids have been deleted, substituted, inserted or added. (13A-b) A protein containing an amino acid sequence that has an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence shown in SEQ ID NO: 138. (13A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 137 have been deleted, substituted, inserted or added. (13A-d) A protein containing an amino acid sequence encoded by a nucleotide sequence that has an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the nucleotide sequence shown in SEQ ID NO: 137. (13A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 137. (13B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 138 to 142, preferably a protein comprising at least two, three, four, or all of the amino acid sequences, wherein the amino acid sequence represented by any of SEQ ID NOs: 138 to 142 has one or several amino acids deleted, substituted, inserted, or added. It's fine.

[0101] The proteins (13A-a) to (13A-e) and (13B) above also include proteins whose amino acid residues have been modified by phosphorylation, glycosylation, aminoacylation, ring-opening, deamination, etc.

[0102] The above proteins (13A-a) to (13A-e) and (13B) may be proteins that appear as spots with a molecular weight of 30 kDa to 70 kDa, preferably a molecular weight of 40 kDa to 60 kDa, and more preferably a molecular weight of about 45 kDa to 55 kDa, and an isoelectric point of 3.0 to 7.0, preferably an isoelectric point of 3.0 to 6.0, and more preferably an isoelectric point of 4.0 to 5.5, in a gel when two-dimensional electrophoresis is performed under the conditions described in the above section "Identification of antigen."

[0103] Preferably, the protein that is the antigen of spot 13 is an antigen for fish allergies.

[0104] Furthermore, the epitopes of the antigens (13A-a) to (13A-e) and (13B) above are located at amino acids 211 to 225 (SEQ ID NO: 197) of SEQ ID NO: 138. When the antigen of spot 13 contains a mutation in the amino acid sequence of SEQ ID NO: 138, the amino acids corresponding to the epitope may retain the sequence of SEQ ID NO: 138.

[0105] Furthermore, from the viewpoint of maintaining the binding properties with IgE antibodies, such as sensitivity and specificity, even if the amino acid sequence is mutated, the antigen of spot 13 may be the following mutant.

[0106] Of the epitope having the sequence of SEQ ID NO: 197, the site important for binding to IgE antibodies of allergy patients is the site represented by an amino acid other than X in SEQ ID NO: 414, which corresponds to the 7th to 10th amino acid positions of SEQ ID NO: 197, or in SEQ ID NO: 416, which corresponds to the 9th to 14th amino acid positions of SEQ ID NO: 197. Therefore, in another preferred embodiment, when the antigen of spot 13 contains a mutation in the amino acid sequence of SEQ ID NO: 138, the antigen may be one in which the 7th to 10th amino acids of amino acid 211 to 225 in SEQ ID NO: 138 are the amino acid sequence of SEQ ID NO: 414, or the 9th to 14th amino acids of amino acid 211 to 225 in SEQ ID NO: 138 are the amino acid sequence of SEQ ID NO: 416.

[0107] (14) Spot 14 antigen Spot 14 was subjected to sequence identification by mass spectrometry, and the amino acid sequences of SEQ ID NOs: 145 to 149 were detected.

[0108] Furthermore, analysis of mass data (SEQ ID NOS: 145 to 149) obtained from a mass spectrometer against NCBI protein data identified spot 14 as L-lactate dehydrogenase A chain-like (amino acid sequence: SEQ ID NOS: 144, encoding nucleotide sequence: SEQ ID NOS: 143). SEQ ID NOS: 145 corresponds to amino acids 119 to 126 of SEQ ID NOS: 144, 146 corresponds to amino acids 7 to 22 of SEQ ID NOS: 144, 147 corresponds to amino acids 9 to 22 of SEQ ID NOS: 144, 148 corresponds to amino acids 270 to 278 of SEQ ID NOS: 144, and 149 corresponds to amino acids 91 to 118 of SEQ ID NOS: 144.

[0109] Therefore, in the present application, the antigen of spot 14 may be any of the following (14A-a) to (14A-e) and (14B). (14A-a) One or several amino acids are deleted, substituted, inserted or is a protein containing an added amino acid sequence. (14A-b) A protein having an amino acid sequence that is 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identical to the amino acid sequence shown in SEQ ID NO: 144. (14A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides in SEQ ID NO: 143 have been deleted, substituted, inserted or added. (14A-d) A protein containing an amino acid sequence encoded by a nucleotide sequence that has an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the nucleotide sequence shown in SEQ ID NO: 143. (14A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 143. (14B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 144 to 149, preferably a protein comprising at least two, three, four, five, or all of the amino acid sequences, wherein the amino acid sequence represented by any of SEQ ID NOs: 144 to 149 may have one or several amino acids deleted, substituted, inserted, or added.

[0110] The proteins (14A-a) to (14A-e) and (14B) above also include proteins whose amino acid residues have been modified by phosphorylation, glycosylation, aminoacylation, ring-opening, deamination, etc.

[0111] The above proteins (14A-a) to (14A-e) and (14B) may be proteins that appear as spots with a molecular weight of 20 kDa to 50 kDa, preferably a molecular weight of 30 kDa to 50 kDa, and more preferably a molecular weight of about 30 kDa to 40 kDa, and an isoelectric point of 5.0 to 9.0, preferably an isoelectric point of 6.0 to 8.0, and more preferably an isoelectric point of 6.5 to 7.5, in a gel when two-dimensional electrophoresis is performed under the conditions described in the above section "Identification of antigen."

[0112] Preferably, the protein that is the antigen of spot 14 is an antigen for fish allergies.

[0113] Furthermore, the epitopes of the antigens (14A-a) to (14A-e) and (14B) above are located at amino acids 11 to 25 (SEQ ID NO: 198) of SEQ ID NO: 144. When the antigen of spot 14 contains a mutation in the amino acid sequence of SEQ ID NO: 144, the amino acids corresponding to the epitope may retain the sequence of SEQ ID NO: 144.

[0114] Furthermore, from the viewpoint of maintaining the binding properties with IgE antibodies, such as sensitivity and specificity, even if the amino acid sequence is mutated, the antigen in spot 14 may be the following mutant.

[0115] Of the epitope having the sequence of SEQ ID NO: 198, the site important for binding to IgE antibodies of allergy patients is the site represented by amino acids other than X in SEQ ID NO: 418 or 419, which corresponds to the 5th to 13th amino acids of SEQ ID NO: 198. Therefore, in another preferred embodiment, when the antigen of spot 14 contains a mutation in the amino acid sequence of SEQ ID NO: 144, the antigen may have the amino acid sequence of SEQ ID NO: 418 or 419 at the 5th to 13th amino acids of amino acids 11 to 25 in SEQ ID NO: 144.

[0116] As used herein, when referring to an amino acid sequence, "one or several amino acids have been deleted, substituted, inserted or added" refers to a deletion, substitution, insertion or addition of one or several amino acids (for example, 30%, preferably 25%, 20% or more of the total length of the amino acid sequence) in the target amino acid sequence. "A" refers to an amino acid sequence in which at least one amino acid (0%, 15%, 10%, 5%, 3%, 2% or 1%) has been deleted, substituted, inserted and / or added.

[0117] Among the above, the substitution is preferably a conservative substitution. A conservative substitution is a replacement of a specific amino acid residue with a residue having similar physicochemical characteristics, but any substitution may be made as long as it does not substantially change the structural characteristics of the original sequence. For example, any substitution may be made as long as the substituted amino acid does not disrupt the helix present in the original sequence or other types of secondary structure that characterize the original sequence. Below, conservative substitutions of amino acid residues are categorized by substitutable residues and exemplified, but the substitutable amino acid residues are not limited to those listed below. Group A: leucine, isoleucine, valine, alanine, methionine Group B: aspartic acid, glutamic acid Group C: asparagine, glutamine D group: lysine, arginine, Group E: serine, threonine Group F: phenylalanine, tyrosine In the case of non-conservative substitutions, a member of one of the above classes can be exchanged for a member of another class. For example, amino acids in the above groups B, D, and E may be substituted with amino acids from other groups to prevent inadvertent glycosylation. Alternatively, cysteines may be deleted or substituted with other amino acids to prevent the protein from folding into a tertiary structure. Alternatively, amino acids may be substituted taking into account the hydropathic index of amino acids, which is an index of hydrophobicity / hydrophilicity for amino acids (J. Kyte and R. Doolittle, J. Mol. Biol., Vol. 157, pp. 105-132, 1982), to maintain a balance of hydrophilicity / hydrophobicity or to increase hydrophilicity for easier synthesis.

[0118] In another embodiment, substitution with an amino acid that is less sterically hindering than the original amino acid, for example, substitution of group F with group A, B, C, D, or E, or substitution of a charged amino acid with an uncharged amino acid, for example, substitution of group B with group C, may be performed. This may improve the binding affinity to IgE antibodies.

[0119] As used herein, the percent identity between two amino acid sequences can be determined by visual inspection and mathematical calculation. Percent identity can also be determined using a computer program. Examples of such computer programs include BLAST and ClustalW. In particular, the various conditions (parameters) for identity searches using the BLAST program are described in Altschul et al. (Nucl. Acids. Res., 25, pp. 3389-3402, 1997) and are publicly available from the websites of the National Center for Biotechnology Information (NCBI) and the DNA Data Bank of Japan (DDBJ) (BLAST Manual, Altschul et al., NCB / NLM / NIH, Bethesda, MD 20894; Altschul et al.). Furthermore, the percent identity can also be determined using a computer program. Examples of such computer programs include BLAST and ClustalW. In particular, the various conditions (parameters) for identity searches using the BLAST program are described in Altschul et al. (Nucl. Acids. Res., 25, pp. 3389-3402, 1997) and are publicly available from the websites of the National Center for Biotechnology Information (NCBI) and the 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 information processing software GENETYX Ver. 7 (Genetyx), DINASIS Pro (Hitachi Software), and Vector NTI (Infomax).

[0120] As used herein, when a base sequence is referred to as "having one or several nucleotides deleted, substituted, inserted, or added," it refers to a base sequence in which one or several nucleotides (e.g., 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2%, or 1% of the amino acids relative to the entire length of the base sequence) have been deleted, substituted, inserted, and / or added. It is preferable that the deletion, substitution, insertion, or addition of the nucleotides does not cause a frameshift in the sequence encoding the amino acids.

[0121] As used herein, the percent identity between two nucleotide sequences can be determined by visual inspection and mathematical calculation. Percent identity can also be determined using a computer program. Examples of such sequence comparison computer programs include the BLASTN program (Altschul et al. (1990) J. Mol. Biol. 215: 403-10): version 2.2.7, available from the U.S. National Library of Medicine website: http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / bls.html, or the WU-BLAST2.0 algorithm. Standard default parameter settings for WU-BLAST2.0 can be found at the following internet site: http: / / blast.wustl.edu.

[0122] As used herein, "under stringent conditions" means hybridization under moderately or highly stringent conditions. Specifically, moderately stringent conditions can be easily determined by a person skilled in the art based on, for example, the length of 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 are those under which hybridization is performed under moderately or highly stringent conditions. Hybridization conditions include 1xSSC to 6xSSC at 42°C to 55°C, more preferably 1xSSC to 3xSSC at 45°C to 50°C, and most preferably 2xSSC at 50°C. When the hybridization solution contains, for example, approximately 50% formamide, a temperature 5 to 15°C lower than the above temperatures is used. Washing conditions include 0.5xSSC to 6xSSC at 40°C to 60°C. During hybridization and washing, 0.05% to 0.2%, preferably approximately 0.1%, of SDS may generally be added. Highly stringent conditions can also be easily determined by those skilled in the art, for example, based on the length of the DNA. Generally, highly stringent (highly stringent) conditions involve hybridization and / or washing at a higher temperature and / or a lower salt concentration than moderately stringent conditions. For example, hybridization conditions include 0.1×SSC to 2×SSC at 55° C. to 65° C., more preferably 0.1×SSC to 1×SSC at 60° C. to 65° C., and most preferably 0.2×SSC at 63° C. Washing conditions include 0.2×SSC to 2×SSC at 50° C. to 68° C., more preferably 0.2×SSC at 60° C. to 65° C.

[0123] The antigen may be obtained from fish by isolating and purifying it using a combination of protein purification methods well known to those skilled in the art, or by expressing the antigen as a recombinant protein using gene recombination techniques well known to those skilled in the art, and then isolating and purifying it using protein purification methods well known to those skilled in the art.

[0124] Examples of protein purification methods include methods that utilize solubility, such as salting out and solvent precipitation; methods that utilize differences in molecular weight, such as dialysis, ultrafiltration, gel filtration and SDS-PAGE; methods that utilize charge, such as ion exchange chromatography and hydroxylapatite chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reversed-phase high-performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing.

[0125] Proteins are prepared by recombinant DNA technology by preparing an expression vector containing a nucleic acid encoding an antigen, introducing the expression vector into suitable host cells by gene transfer or transformation, culturing the host cells under conditions suitable for expression of the recombinant protein, and recovering the recombinant protein expressed in the host cells.

[0126] A "vector" can be used to introduce a nucleic acid linked to it into a host cell. An "expression vector" is a vector capable of inducing the expression of a protein encoded by a nucleic acid introduced by the vector. Vectors include plasmid vectors, viral vectors, etc. Those skilled in the art can select an appropriate expression vector for expressing a recombinant protein depending on the type of host cell used. To facilitate purification, the vector may contain an affinity tag such as His × 6 residues. It may also be synthesized to contain a signal sequence so that it is exported outside the cell.

[0127] A "host cell" is a cell that is transfected or transformed with a vector. A host cell can be appropriately selected by those skilled in the art depending on the vector used. The host cell can be derived from a prokaryote, such as E. coli. When prokaryotic cells are used as hosts, the antigens of the present invention may contain an N-terminal methionine residue to facilitate expression of the recombinant protein in the prokaryotic cells. This N-terminal methionine can be cleaved from the recombinant protein after expression. Alternatively, the host may be a cell of eukaryotic origin, such as a unicellular eukaryote such as yeast, a plant cell, an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a silkworm.

[0128] Gene transfer or transformation of an expression vector into a host cell can be carried out appropriately by methods known to those skilled in the art. Furthermore, those skilled in the art can express a recombinant protein by culturing the host cells under appropriate conditions suitable for the expression of the recombinant protein depending on the type of host cell. The host cells expressing the recombinant protein can then be homogenized, and the antigen expressed as the recombinant protein can be isolated and purified from the resulting homogenate by an appropriate combination of the above-mentioned protein purification methods.

[0129] Diagnostic kits and methods (1) The present invention provides a method for providing an indicator for diagnosing fish allergy in a subject, comprising the steps of: (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) if binding of the subject's IgE antibodies to the antigen is detected, an indication that the subject is allergic to fish is provided; wherein the antigen is at least one of the proteins identified as antigens in (10) to (14) above.

[0130] A sample obtained from a subject is a solution containing IgE antibodies collected from a subject. Examples of such solutions include blood, saliva, sputum, nasal discharge, urine, sweat, and tears. The sample obtained from a subject may be pretreated to increase the IgE antibody concentration in the sample before contacting it with an antigen. Pretreatment of the sample may include, for example, obtaining serum or plasma from the blood. Furthermore, the Fab portion, which is the binding portion to the antigen, may be purified. In a particularly preferred embodiment, the above step (i) is carried out by contacting the antigen with IgE antibodies in serum obtained from the subject.

[0131] The IgE antibody may be the IgE antibody itself, or may be a mast cell to which the IgE antibody is bound.

[0132] Contact between a sample obtained from a subject and an antigen and detection of the binding can be performed by known methods, such as ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, immunocloning, etc. Detection by the chromatographic method can be used. This is a method for detecting IgE antibodies that specifically bind to antigens by contacting them and binding them. For example, there are methods that involve the action of an enzyme-labeled secondary antibody and adding an enzyme substrate (usually a color- or luminescent reagent) to detect the product of the enzyme reaction, methods that involve the action of a biotin-labeled secondary antibody and adding an avidin-conjugated dye to detect the dye, and methods that detect fluorescently labeled secondary antibodies. Alternatively, there are methods that can evaluate the binding of antigens and IgE antibodies, such as surface plasmon resonance (SPR). Detection by a measurement method can also be used. Multiple types of antigen-specific IgE antibodies may be mixed.

[0133] The antigen may be isolated and immobilized on a carrier. In this case, ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance, etc. can be used in steps (i) and (ii), and step (i) is performed by contacting a sample obtained from a subject with a surface on which the antigen is immobilized. The isolated antigen may be obtained from fish by separating and purifying it using a combination of protein purification methods well known to those skilled in the art, or by preparing it using genetic recombination technology. Alternatively, an antibody that binds to the antigen may be immobilized.

[0134] The antigen may not be immobilized on a carrier. In this case, flow cytometry or the like can be used in steps (i) and (ii) above, and the presence of the antibody-bound antigen can be confirmed using laser light. For example, a basophil activation test (BAT) can be used, in which the amount of the surface antigen CD203c expressed when the antigen binds to the antibody and activates basophils in the sample is measured. Another example is the histamine release test (HRT), in which the antigen is brought into contact with blood cells via binding to the antibody in the sample to determine whether histamine is released.

[0135] Alternatively, antigens may be transferred from a state separated by two-dimensional electrophoresis and then detected by immunoblotting. Two-dimensional electrophoresis is a technique for separating protein samples by performing isoelectric focusing in the first dimension and SDS-PAGE (SDS-polyacrylamide gel electrophoresis) in the second dimension. In this case, the conditions for two-dimensional electrophoresis are not particularly limited as long as they allow separation of the antigens of the present invention. For example, the two-dimensional electrophoresis conditions described in the above section "Identification of Antigens" can be used. Alternatively, electrophoresis conditions can be determined with reference to the descriptions in the above Patent Documents 1 to 4, for example, as follows: (A) As a first-dimensional isoelectric focusing gel, the gel length is within the range of 5 to 10 cm, the pH range of the gel is 3 to 10, and the pH gradient of the gel in the direction of electrophoresis is defined as a for the gel length up to pH 5, b for the gel length from pH 5 to 7, and c for the gel length at pH 7 or higher.<b]および「b> c" relationship is satisfied; (B) In the case of (A), when the total length of the gel is 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 first-dimensional isoelectric focusing, a constant voltage step is performed by applying a constant voltage within the range of 100 V to 600 V to each gel containing a sample, and after the electrophoretic change amplitude per 30 minutes of electrophoresis reaches a range of 5 μA, a voltage increase step is started in which the voltage is increased from the constant voltage; (D) In ​​the case of (C), the final voltage in the voltage increase step is set to a range of 3000 V to 6000 V; (E) the length of the first-dimension isoelectric focusing gel in the longitudinal direction is 5 to 10 cm, and the gel concentration at the base end of the second-dimension electrophoresis gel in the migration direction is 3 to 6%; and (F) In the case of (E), the gel concentration at the leading end of the second-dimensional electrophoresis gel in the migration direction is set higher than the gel concentration at the base end in the migration direction; Two-dimensional electrophoresis can be performed under conditions that satisfy at least one selected from the group consisting of:

[0136] The antigens (10) to (14) above are antigens that specifically bind to IgE antibodies of patients allergic to fish. Therefore, the binding of the target IgE antibody to the antigen was detected. If so, an indication is provided that the subject is allergic to fish.

[0137] The present invention also provides a diagnostic kit for fish allergy, which contains at least one of the antigens (10) to (14) above. The diagnostic kit of the present invention may be used in the method for providing an indicator for diagnosing fish allergy described above, or in the diagnostic method described below. In addition to containing at least one of the antigens (10) to (14) above, the diagnostic kit of the present invention may also contain an enzyme-labeled anti-IgE antibody and a chromogenic or luminescent substrate that serves as a substrate for the enzyme, or a biotin-labeled anti-IgE antibody and an avidin-binding dye that binds to the biotin. A fluorescently labeled anti-IgE antibody may also be used. In the diagnostic kit of the present invention, the antigen may be provided in a state immobilized on a carrier. The diagnostic kit of the present invention may also be provided together with instructions on the diagnostic procedure or a package containing such instructions.

[0138] In another embodiment, the diagnostic kit includes a companion diagnostic for fish allergy. The companion diagnostic is used to identify patients who are expected to benefit from a drug or who are at risk of serious side effects from the drug, or to examine the responsiveness of the drug to optimize treatment with the drug. Here, optimization of treatment includes, for example, determining the dosage and administration, deciding when to discontinue administration, and identifying which allergen component is used to induce immune tolerance.

[0139] The present invention also provides a diagnostic composition for fish allergy, which contains at least one of the antigens (10) to (14) above. The diagnostic composition of the present invention can be used in the diagnostic methods described below. The diagnostic composition of the present invention may contain, as needed, pharmaceutically acceptable carriers and additives that are commonly used together with the antigen of the present invention.

[0140] In one aspect, the present invention provides a method for diagnosing allergy to fish in a subject, comprising the steps of: (i) contacting a sample obtained from a subject with an antigen; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) if binding of the subject's IgE antibody to the antigen is detected, determining that the subject is allergic to fish; wherein the antigen is at least one of the proteins identified as antigens in (10) to (14) above, wherein steps (i) and (ii) are carried out as described for the steps of the method for providing an indicator for diagnosing fish allergy.

[0141] In another aspect, the present invention provides a method for diagnosing a fish allergy in a subject, the method comprising administering to the subject at least one of the antigens (10) to (14) above. The method may be performed in the form of a skin test, characterized by applying the antigen to the skin. Skin tests include a prick test, in which a diagnostic composition is applied to the skin and then a small scratch is made to penetrate the antigen into the skin, but not to the point of bleeding, to observe a skin reaction; a scratch test, in which a diagnostic composition is applied and then a small scratch is made to observe a reaction; a patch test, in which a diagnostic composition in the form of a cream or ointment is applied to the skin and a reaction is observed; and an intradermal test, in which an antigen is administered intradermally and a reaction is observed. In a prick test or scratch test, the diagnostic composition may be penetrated into the skin by contacting the tip of a lancet with the diagnostic composition and then pricking the skin at the contact site to cause a wound, thereby penetrating the diagnostic composition. If a skin reaction, such as swelling, occurs in the area where the antigen was applied, the subject is diagnosed as having a fish allergy. Here, the amount of antigen applied to the skin may be, for example, 100 μg or less per application.

[0142] In the diagnosis of allergies, a challenge test is often performed to identify the antigen. At least one of the antigens (10) to (14) above can be used as an active ingredient in a challenge test to diagnose fish allergy. The antigen protein used in the challenge test may be an expressed and purified protein, or may be expressed in a food or ingredient, such as pollen rice, in which rice is transformed with a cedar pollen antigen gene and the antigen protein is expressed in the rice.

[0143] In another aspect, the present invention provides at least one of the antigens (10) to (14) above for use in diagnosing fish allergies, including providing at least one of the antigens (10) to (14) above in a mixture with a known antigen.

[0144] In yet another aspect, the present invention provides use of at least one of the antigens (10) to (14) above for the manufacture of a diagnostic agent for fish allergy.

[0145] Pharmaceutical Compositions and Treatment Methods (1) The present invention provides a pharmaceutical composition comprising at least one of the antigens (10) to (14) above.

[0146] In one embodiment, the pharmaceutical composition is used to treat fish allergies. Treating allergies involves increasing the limit of the antigen that can be ingested without causing symptoms, ultimately aiming to achieve a state (remission) in which symptoms do not appear with normal intake of the antigen.

[0147] The present invention also provides a method for treating fish allergy, which comprises administering at least one of the antigens (10) to (14) to a patient in need of treatment for fish allergy.

[0148] In another aspect, the present invention provides at least one of the antigens (10) to (14) above for use in treating fish allergy. In yet another aspect, the present invention provides use of at least one of the antigens (10) to (14) above for the manufacture of a medicament for treating fish allergy.

[0149] In the treatment of allergies, hyposensitization therapy is often performed, with the goal of inducing immune tolerance by administering an antigen to the patient. At least one of the antigens (10) to (14) above can be used as an active ingredient in hyposensitization therapy for fish allergies. Here, the antigen protein used in hyposensitization therapy may be an expressed and purified protein, or may be expressed in a food or ingredient, such as pollen rice, in which rice is transformed with a cedar pollen antigen gene and the antigen protein is expressed in the rice.

[0150] The pharmaceutical compositions of the present invention can be administered by any conventional route of administration, including, for example, oral, sublingual, transdermal, intradermal, subcutaneous, intravascular, intranasal, intramuscular, intraperitoneal, and rectal administration.

[0151] The pharmaceutical composition of the present invention can be used as a pharmaceutical composition by adding commonly used pharmaceutically acceptable adjuvants, excipients, or various additives (e.g., stabilizers, solubilizers, emulsifiers, buffers, preservatives, colorants, etc.) together with the antigen of the present invention in a conventional manner, as needed. The dosage form of the pharmaceutical composition can be appropriately selected by those skilled in the art depending on the route of administration. For example, it may be in the form of tablets, capsules, troches, sublingual tablets, injections, nasal sprays, poultices, liquids, creams, lotions, suppositories, etc. The dosage, frequency of administration, and / or duration of administration of the pharmaceutical composition of the present invention can be determined depending on the route of administration, symptoms, and patient characteristics such as age and weight. A physician can appropriately select the dosage depending on the patient's condition, etc. For example, in the case of an adult, the dosage may be 100 μg or less per administration. The administration interval may be, for example, daily, once a week, twice a month, or once every three months. The administration period may be, for example, several weeks to several years. The administration method may be such that the dosage is gradually increased during the administration period.

[0152] Tester (1) The present invention provides a tester containing an antibody against at least one of the antigens (10) to (14) above.

[0153] 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 (10) to (14) above. The antibody may be an IgE antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (e.g., Fab, F(ab')2, Fab').

[0154] In the above tester, the antibody may be provided in a form bound to a carrier. The carrier is not particularly limited as long as it can be used to detect antibody-antigen binding. Any carrier known to those skilled in the art can be used.

[0155] The following methods can be used to check whether or not an antigen is contained. A tester containing the prepared IgE antibody is brought into contact with a sample obtained from food, ingredients, etc., and the binding of the IgE antibody to the antigen in the sample is detected using, for example, ELISA, and if binding of the IgE antibody to the antigen is detected, it is determined that the antigen remains in the target food, ingredients, etc. A method in which proteins are extracted from food and ingredients, electrophoresed, and the presence of antigen spot bands is detected using antibodies. A method in which food or ingredients are soaked in filter paper or the like, and an antibody solution is reacted to detect the antigens contained therein.

[0156] Another embodiment of the present invention includes a tester for determining the presence or absence of a fish allergy antigen in a subject, characterized by comprising a primer having a nucleotide sequence complementary to at least a portion of the nucleotide sequence set forth in SEQ ID NO: 69, 109, 120, 137, or 143. For example, but not limited to, the primer may have a nucleotide sequence complementary to the 3'-terminal or central portion, preferably 12, 15, 20, or 25 bases, of at least one of the nucleotide sequences set forth in SEQ ID NO: 69, 109, 120, 137, or 143. In particular, when targeting mRNA, a complementary primer to the poly(A) tail is provided. In a preferred embodiment, the tester containing the primer may further comprise a primer containing a nucleotide sequence of the 5'-terminal portion, preferably a nucleotide sequence consisting of 12, 15, 20, or 25 bases, of at least one of the nucleotide sequences set forth in SEQ ID NO: 69, 109, 120, 137, or 143.

[0157] For example, DNA or cDNA is amplified by PCR (Polymerase Chain Reaction) including RT-PCR using the above primers as a template for DNA or mRNA obtained from fish. The presence or absence of the antigen is determined by amplifying the mRNA by PCR and comparing the sequence of the amplified DNA or cDNA with that of SEQ ID NO: 69, 109, 120, 137, or 143. Examples of methods for amplifying mRNA by PCR include the RACE method. When comparing the amplified DNA or cDNA with SEQ ID NO: 69, 109, 120, 137, or 143, even if there is a point mutation encoding the same amino acid, or if there is an insertion, deletion, substitution, or addition of a base in the nucleotide sequence of the amplified DNA or cDNA relative to the nucleotide sequence of SEQ ID NO: 69, 109, 120, 137, or 143, the amino acid sequence encoded by the DNA or cDNA is identical to the amino acid sequence of SEQ ID NO: 70, 110, 121, 138, or 144. If the affinity is 70% or more, preferably 80, 90, 95, 98, or 99% or more, the antigen is judged to be present.

[0158] In one embodiment, the tester is used to check for the presence or absence of an antigen in a target object, such as foodstuffs (fish or fish eggs) or in a food production line. The tester may be used by manufacturers to inspect the quality of production lines and products before shipping, or by consumers themselves to check for the presence or absence of an antigen in target foods or ingredients.

[0159] Allergen-free foods, etc. The present invention provides a fish, fish eggs, a processed product of the fish or fish eggs, or a fish that lays the fish eggs or is born from the fish eggs, characterized in that at least one of the antigens (10) to (14) above has been removed or reduced.

[0160] There are no limitations on the method for removing or reducing the antigens of the present invention in fish, fish eggs, the fish or fish egg processed products, or fish that produce the fish eggs or are hatched from the fish eggs. The removal or reduction of the antigens may be carried out by any method as long as the antigens of the present invention are removed or reduced.

[0161] For example, fish or fish eggs in which the expression of the antigen of the present invention has been removed or reduced may be prepared by using gene knockout technology to knock out the expression of the antigen of the present invention.

[0162] Gene knockout techniques can be any technique known to those skilled in the art. For example, Oishi et al. (Scientific Reports, Vol. 6, Article number: 23980, 2016, doi:10.1038 / srep23980) describes the application of the genome editing technology CRISPER / Cas9 to primordial germ cells to obtain individuals lacking an allergen protein gene. Similar techniques can be used to obtain fish or fish eggs from which the antigens of the present invention have been removed. Alternatively, fish or fish eggs from which the antigens of the present invention have been removed or reduced can be obtained by crossbreeding by artificial insemination with fish or fish eggs that do not contain or contain a low amount of the antigen. Artificial crossbreeding of fish or fish eggs can be performed using conventional methods.

[0163] The processed fish product or processed fish roe product of the present invention from which antigens have been removed or reduced may be a processed product made from fish from which antigens of the present invention have been removed or reduced. When using normal fish or fish roe as the raw material, treatment to remove or reduce the antigens of the present invention is carried out before or after preparation of the processed fish product or processed fish roe product. Methods for removing or reducing the antigens of the present invention in processed fish products or processed fish roe products made from normal fish or fish roe include methods for removing protein components from foods or ingredients, such as high-pressure treatment and elution with a neutral salt solution or high-temperature steam, and methods for hydrolysis, denaturation, or amino acid changes (chemical modification or elimination of side chains, etc.) using heat treatment and acid treatment. The fish from which antigens have been removed or reduced may be hatched and grown from the fish roe from which antigens of the present invention have been removed or reduced. The fish roe from which antigens have been removed or reduced may also be obtained from the fish from which antigens of the present invention have been removed or reduced.

[0164] Manufacturing method for allergen-free processed products (1) The present invention provides a method for producing a processed fish product or a processed fish roe product in which antigens have been removed or reduced, the method comprising a step of confirming that the antigens have been removed or reduced during the production process of the processed product, wherein the antigen is at least one of the antigens (10) to (14) above.

[0165] The step of confirming that antigens have been removed or reduced during the manufacturing process of the fish processed product or fish roe processed product is the same as that described in the above item "Tester (1)". This may be done by confirming whether or not the antigen is contained by the method described above.

[0166] Furthermore, processed fish products or processed fish eggs from which antigens have been removed or reduced may be produced by the methods described above in the section "Allergen-removed foods, etc."

[0167] antigen epitope As shown in Example 10, epitopes and amino acids within the epitopes that are important for binding to IgE antibodies from allergic patients were identified for the antigens identified as shown in Examples 2, 3, and 5 to 8, as well as for aldolase and β-enolase, which are known allergy antigens for salmon, etc., and glyceraldehyde-3-phosphate dehydrogenase, which is known allergy antigen for sardines, etc.

[0168] The present invention provides the following polypeptides (1α) to (17α) as polypeptides comprising an amino acid sequence that specifically binds to IgE antibodies of allergy patients.

[0169] (1α) Epitope of α-actinin-3 In the present invention, the polypeptide (1α) may be any polypeptide selected from the group consisting of the following (1α-1) to (1α-6):

[0170] (1α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 150 to 154, 207, 210, 213, 218, 224, and 227.

[0171] A polypeptide comprising the amino acid sequence of (1α-2)XXXXXXKPDX (SEQ ID NO: 205). Preferably, a polypeptide comprising the amino acid sequence of SXXXXXKPDK (SEQ ID NO: 206). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, six, or seven amino acids at positions 1 to 6 and 10 of SEQ ID NO: 207 have been substituted with other amino acids, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, or five amino acids at positions 2 to 6 of SEQ ID NO: 207 have been substituted with other amino acids, preferably alanine.

[0172] In another aspect, a polypeptide comprising the amino acid sequence of DKXXXR (SEQ ID NO: 208), preferably a polypeptide comprising an amino acid sequence in which any one, two or three amino acids at positions 3 to 5 of SEQ ID NO: 210 have been substituted with another amino acid, preferably alanine.

[0173] A polypeptide comprising the amino acid sequence of (1α-3)XXXXXXXDPM (SEQ ID NO: 211). Preferably, a polypeptide comprising the amino acid sequence of YXXXXKDDPM (SEQ ID NO: 212). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, or 7 amino acids at positions 1 to 7 of SEQ ID NO: 213 have been substituted with other amino acids, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, or 4 amino acids at positions 2 to 5 of SEQ ID NO: 213 have been substituted with other amino acids, preferably alanine.

[0174] In another aspect, a polypeptide comprising the amino acid sequence of XXXXXXDXPM (SEQ ID NO: 214). Preferably, a polypeptide comprising the amino acid sequence of YSXXXXDXPM (SEQ ID NO: 215). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, six, or seven amino acids at positions 1 to 6 and 8 of SEQ ID NO: 213 have been substituted with other amino acids, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, or five amino acids at positions 3 to 6 and 8 of SEQ ID NO: 213 have been substituted with other amino acids, preferably alanine.

[0175] In yet another embodiment, a polypeptide comprising the amino acid sequence of PXGXLXX (SEQ ID NO: 216) Preferably, the polypeptide comprises the amino acid sequence of PXGNLNT (SEQ ID NO: 217). Preferably, a polypeptide comprising an amino acid sequence in which any one, two, three or four amino acids at positions 2, 4, 6 or 7 of SEQ ID NO: 218 are substituted with another amino acid, preferably alanine. More preferably, a polypeptide comprising an amino acid sequence in which the second amino acid of SEQ ID NO: 218 is substituted with another amino acid, preferably alanine.

[0176] A polypeptide comprising the amino acid sequence of (1α-4)SXFYHAFAGAEQAET (SEQ ID NO: 219) Preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 152 in which the second amino acid is replaced by another amino acid, preferably alanine.

[0177] A polypeptide comprising the amino acid sequence of (1α-5)TXLXXXNXPXXXXSE (SEQ ID NO: 220) Preferably, the polypeptide comprises the amino acid sequence TXLRLXNRPXXXXSE (SEQ ID NO: 221). More preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, six, seven, eight, or nine amino acids at positions 2, 4 to 6, 8, or 10 to 13 of SEQ ID NO: 153 are substituted with another amino acid, preferably alanine. Still more preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 2, 6, or 10 to 13 of SEQ ID NO: 153 are substituted with another amino acid, preferably alanine.

[0178] A polypeptide comprising the amino acid sequence of (1α-6)SXKTXXXXXEXR (SEQ ID NO: 222). Preferably, a polypeptide comprising the amino acid sequence of SDKTXXXXXELR (SEQ ID NO: 223). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, six, or seven amino acids at positions 2, 5 to 9, and 11 of SEQ ID NO: 224 have been substituted with other amino acids, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, or five amino acids at positions 5 to 9 of SEQ ID NO: 224 have been substituted with other amino acids, preferably alanine.

[0179] In another embodiment, a polypeptide comprising the amino acid sequence of XXRE (SEQ ID NO: 225). Preferably, a polypeptide comprising the amino acid sequence of LXRE (SEQ ID NO: 226). More preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 227 in which either one or two amino acids at positions 1 and 2 have been substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 227 in which the second amino acid has been substituted with another amino acid, preferably alanine.

[0180] In one embodiment, the polypeptide of (1α) may not contain variants or homologs that are identical to the full-length amino acid sequence of α-actinin-3 (SEQ ID NO: 2) or have 90% or more, 80% or more, or 70% or more identity thereto.

[0181] (2α) EEF1A2 binding protein-like epitope In the present invention, the (2α) polypeptide may be any of the polypeptides selected from the group consisting of the following (2α-1) and (2α-2):

[0182] (2α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 155 and 230.

[0183] A polypeptide comprising the amino acid sequence of (2α-2)XXNRXYYXXXE (SEQ ID NO: 228). Preferably, the polypeptide comprises the amino acid sequence LDNRLYYXVAE (SEQ ID NO: 229). Preferably, any one, two, three, four, five or six amino acids at positions 1, 2, 5, 8 to 10 of SEQ ID NO: 230 are substituted with another amino acid, preferably alanine. More preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 230, in which the eighth amino acid is replaced by another amino acid, preferably alanine.

[0184] In one embodiment, the polypeptide of (2α) may be free of variants or homologs identical to, or having 90% or more, 80% or more, or 70% or more identity to, the full-length amino acid sequence of EEF1A2 binding protein-like (SEQ ID NO: 5).

[0185] (3α) Epitope of α-1,4-glucan phosphorylase In the present invention, the polypeptide (3α) may be any polypeptide selected from the group consisting of the following (3α-1) to (3α-3):

[0186] (3α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 156, 157, 232, 235, and 237.

[0187] A polypeptide comprising the amino acid sequence of (3α-2)YXXXXXXR (SEQ ID NO: 231), preferably an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 2 to 7 of SEQ ID NO: 232 have been substituted with another amino acid, preferably alanine.

[0188] In another embodiment, a polypeptide comprising the amino acid sequence of GGYXQXXLXR (SEQ ID NO: 233). Preferably, a polypeptide comprising the amino acid sequence of GGYIQXXLDR (SEQ ID NO: 234). More preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 235 in which any one, two, three, or four amino acids at positions 4, 6, 7, and 9 are substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 235 in which any one or two amino acids at positions 6 and 7 are substituted with another amino acid, preferably alanine.

[0189] A polypeptide comprising the amino acid sequence of (3α-3)RXKXXXDY (SEQ ID NO: 236), preferably an amino acid sequence in which any one, two, three or four amino acids at positions 2 and 4 to 6 of SEQ ID NO: 237 are substituted with another amino acid, preferably alanine.

[0190] In one aspect, the (3α) polypeptide may not contain variants or homologs that are identical to the full-length amino acid sequence of α-1,4-glucan phosphorylase (SEQ ID NO: 10) or have 90% or more, 80% or more, or 70% or more identity thereto.

[0191] (4α) Epitope of elongation factor 2 In the present invention, the (4α) polypeptide may be any of the polypeptides selected from the group consisting of the following (4α-1) to (4α-3):

[0192] (4α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 159, 239, 242, 245, and 247.

[0193] A polypeptide comprising the amino acid sequence of (4α-2)KKXXXR (SEQ ID NO: 238), preferably an amino acid sequence in which any one, two, or three amino acids at positions 3 to 5 of SEQ ID NO: 239 are substituted with another amino acid, preferably alanine.

[0194] In another embodiment, a polypeptide comprising the amino acid sequence of KKXXXRN (SEQ ID NO: 240). Preferably, the polypeptide comprises the amino acid sequence of KKSNXRN (SEQ ID NO: 241). Alternatively, it is a polypeptide comprising an amino acid sequence in which any one, two or three amino acids from the third to fifth positions of SEQ ID NO: 242 are substituted with another amino acid, preferably alanine, and even more preferably, it is a polypeptide comprising an amino acid sequence in which the fifth amino acid of SEQ ID NO: 242 is substituted with another amino acid, preferably alanine.

[0195] A polypeptide comprising the amino acid sequence of (4α-3)LXXXLXXKXXI (SEQ ID NO: 243). Preferably, the polypeptide comprises the amino acid sequence of LXDXLXXKXXI (SEQ ID NO: 244). Preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, six, or seven amino acids at positions 2 to 4, 6, 7, 9, and 10 of SEQ ID NO: 245 are substituted with other amino acids, preferably alanine. Even more preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 2, 4, 6, 7, 9, and 10 of SEQ ID NO: 245 are substituted with other amino acids, preferably alanine.

[0196] In another embodiment, a polypeptide comprising the amino acid sequence of GKXXXXXXXXS (SEQ ID NO: 246) Preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from the 3rd to 10th amino acids of SEQ ID NO: 247 have been substituted with another amino acid, preferably alanine.

[0197] In one aspect, the (4α) polypeptide may be free of variants or homologs identical to or having 90% or more, 80% or more, or 70% or more identity to the full-length amino acid sequence of elongation factor 2 (SEQ ID NO: 19).

[0198] (5α) Heat shock cognate 70kDa protein epitope In the present invention, the (5α) polypeptide may be any of the polypeptides selected from the group consisting of the following (5α-1) to (5α-4):

[0199] (5α-1) SEQ ID NOs: 160 to 162, 250, 252 (amino acid sequence: QYK), 25 A polypeptide comprising at least one amino acid sequence selected from the group consisting of: 5, 258, and 261.

[0200] A polypeptide comprising the amino acid sequence of (5α-2)EXXXXYL (SEQ ID NO: 248). More preferably, a polypeptide comprising the amino acid sequence of EIXXAYL (SEQ ID NO: 249). or a polypeptide comprising an amino acid sequence in which any one, two, three or four amino acids at positions 2 to 5 of SEQ ID NO: 250 are substituted with other amino acids, preferably alanine. More preferably, a polypeptide comprising an amino acid sequence in which any one or two amino acids at positions 3 and 4 of SEQ ID NO: 250 are substituted with other amino acids, preferably alanine.

[0201] A polypeptide comprising the amino acid sequence of (5α-3)QXK (SEQ ID NO: 251). Preferably , a polypeptide comprising an amino acid sequence in which the second amino acid of SEQ ID NO: 252 is replaced with another amino acid, preferably alanine.

[0202] In another embodiment, a polypeptide comprising an amino acid sequence of XDXXXDK (SEQ ID NO: 253). Preferably, the polypeptide comprises the amino acid sequence DDVXXDK (SEQ ID NO: 254). Alternatively, it is a polypeptide comprising an amino acid sequence in which any one, two, three or four amino acids at positions 1, 3 to 5 of SEQ ID NO: 255 are substituted with other amino acids, preferably alanine, and even more preferably, it is a polypeptide comprising an amino acid sequence in which any one or two amino acids at positions 4 and 5 of SEQ ID NO: 255 are substituted with other amino acids, preferably alanine.

[0203] A polypeptide comprising the amino acid sequence of (5α-4)KXSXEXK (SEQ ID NO: 256). More preferably, a polypeptide comprising the amino acid sequence of KLSXEDK (SEQ ID NO: 257). or a polypeptide comprising an amino acid sequence in which any one, two or three amino acids at positions 2, 4 or 6 of SEQ ID NO: 258 are substituted with another amino acid, preferably alanine. More preferably, a polypeptide comprising an amino acid sequence in which the fourth amino acid of SEQ ID NO: 258 is substituted with another amino acid, preferably alanine.

[0204] In another embodiment, a polypeptide comprising the amino acid sequence of QXXXDKC (SEQ ID NO: 259). Preferably, the polypeptide comprises the amino acid sequence of QKIXDKC (SEQ ID NO: 260). Alternatively, it is a polypeptide comprising an amino acid sequence in which any one, two or three amino acids from the second to fourth amino acids of SEQ ID NO: 261 are substituted with another amino acid, preferably alanine, and even more preferably, it is a polypeptide comprising an amino acid sequence in which the fourth amino acid of SEQ ID NO: 261 is substituted with another amino acid, preferably alanine.

[0205] In one embodiment, the (5α) polypeptide may be free of variants or homologs identical to, or having greater than 90%, greater than 80%, or greater than 70% identity to, the full-length amino acid sequence of the heat shock cognate 70 kDa protein (SEQ ID NO: 26).

[0206] (6α) Serotransferrin epitopes In the present invention, the (6α) polypeptide may be any of the polypeptides selected from the group consisting of the following (6α-1) to (6α-5):

[0207] (6α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 163 to 167, 263, 266, 269, 271, 273, 276, and 279.

[0208] A polypeptide comprising the amino acid sequence (6α-2)XXCYY (SEQ ID NO: 262). A polypeptide comprising an amino acid sequence in which either one or two amino acids at positions 1 and 2 of SEQ ID NO: 263 are substituted with another amino acid, preferably alanine.

[0209] In another embodiment, a polypeptide comprising the amino acid sequence of VXVXKKXXX (SEQ ID NO: 264). Preferably, the polypeptide comprises the amino acid sequence of VAVAKKGXE (SEQ ID NO: 265). Preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four or five amino acids at positions 2, 4, or 7 to 9 of SEQ ID NO: 266 are substituted with another amino acid, preferably alanine, and more preferably, it is a polypeptide comprising an amino acid sequence in which the eighth amino acid of SEQ ID NO: 266 is substituted with another amino acid, preferably alanine.

[0210] A polypeptide comprising the amino acid sequence of (6α-3)XKXXXXEX (SEQ ID NO: 267). Preferably, a polypeptide comprising the amino acid sequence of XKXGXGEX (SEQ ID NO: 268). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 1, 3 to 6, and 8 of SEQ ID NO: 269 have been substituted with other amino acids, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, or four amino acids at positions 1, 3, 5, and 8 of SEQ ID NO: 269 have been substituted with other amino acids, preferably alanine.

[0211] A polypeptide comprising the amino acid sequence (6α-4)XXKXM (SEQ ID NO: 270). A polypeptide comprising an amino acid sequence in which any one, two or three amino acids at positions 1, 2 or 4 of SEQ ID NO: 271 are substituted with another amino acid, preferably alanine.

[0212] In another embodiment, a polypeptide comprising the amino acid sequence of VTNFXXXS (SEQ ID NO: 272). Preferably, a polypeptide comprising an amino acid sequence in which any one, two or three amino acids at positions 5 to 7 of SEQ ID NO: 273 have been substituted with another amino acid, preferably alanine.

[0213] A polypeptide comprising the amino acid sequence (6α-5)YXYXXXXXC (SEQ ID NO: 274). Or, a polypeptide comprising the amino acid sequence of YXYNXXFXC (SEQ ID NO: 275). or a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 2, 4, 5, or 8 of SEQ ID NO: 276 have been substituted with other amino acids, preferably alanine, or more preferably, any one, two, three, or four amino acids at positions 2, 5, 6, or 8 of SEQ ID NO: 276 have been substituted with other amino acids, preferably alanine.

[0214] In another embodiment, a polypeptide comprising the amino acid sequence of XXXCLV (SEQ ID NO: 277). Preferably, a polypeptide comprising the amino acid sequence of FXLV (SEQ ID NO: 278). is a polypeptide comprising an amino acid sequence in which either one or two amino acids at positions 1 and 2 of SEQ ID NO: 279 are substituted with another amino acid, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which the second amino acid of SEQ ID NO: 279 is substituted with another amino acid, preferably alanine.

[0215] In one embodiment, the polypeptide of (6α) S The amino acid sequence may not include variants or homologs that are identical to the full-length amino acid sequence of ibuprofen (SEQ ID NO: 33) or that have 90% or more, 80% or more, or 70% or more identity thereto.

[0216] (7α) Myosin-binding protein H-like epitope In the present invention, the (7α) polypeptide may be any of the polypeptides selected from the group consisting of the following (7α-1) to (7α-5):

[0217] (7α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 168 to 171, 282, 284, 287, 290, 292, 295, 297 and 300.

[0218] A polypeptide comprising the amino acid sequence of (7α-2)YVKXXXXKI (SEQ ID NO: 280). Or, a polypeptide comprising the amino acid sequence of YVKXVXEKI (SEQ ID NO: 281). or a polypeptide comprising an amino acid sequence in which any one, two, three or four amino acids at positions 4 to 7 of SEQ ID NO: 282 have been substituted with another amino acid, preferably alanine, or more preferably a polypeptide comprising an amino acid sequence in which any one or two amino acids at positions 4 and 6 of SEQ ID NO: 282 have been substituted with another amino acid, preferably alanine.

[0219] In another embodiment, a polypeptide comprising the amino acid sequence of NIXIP (SEQ ID NO: 283). Preferably, a polypeptide comprising an amino acid sequence in which the third amino acid of SEQ ID NO: 284 is substituted with another amino acid, preferably alanine.

[0220] A polypeptide comprising the amino acid sequence of (7α-3)SXEXXXKXXXF (SEQ ID NO: 285). Preferably, the polypeptide comprises the amino acid sequence SXEXCXKXXXF (SEQ ID NO: 286). Preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, six, or seven amino acids at positions 2, 4 to 6, or 8 to 10 of SEQ ID NO: 287 are substituted with other amino acids, preferably alanine. Even more preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 2, 4, 6, or 8 to 10 of SEQ ID NO: 287 are substituted with other amino acids, preferably alanine.

[0221] In another embodiment, a polypeptide comprising the amino acid sequence of KXDXFXXK (SEQ ID NO: 288). Preferably, it is a polypeptide comprising the amino acid sequence of KXDXFXDK (SEQ ID NO: 289). More preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three or four amino acids at positions 2, 4, 6 and 7 of SEQ ID NO: 290 are substituted with another amino acid, preferably alanine. Even more preferably, it is a polypeptide comprising an amino acid sequence in which any one, two or three amino acids at positions 2, 4 and 6 of SEQ ID NO: 290 are substituted with another amino acid, preferably alanine.

[0222] A polypeptide comprising the amino acid sequence of (7α-4)EXXXYXXXXXXXXD (SEQ ID NO: 291), preferably an amino acid sequence in which any one, two, three, four, five, six, seven, eight, nine, ten, or eleven amino acids at positions 2 to 4, or 6 to 13 of SEQ ID NO: 292 are substituted with another amino acid, preferably alanine.

[0223] A polypeptide comprising the amino acid sequence of (7α-5)XNXXYXXIXX (SEQ ID NO: 293). Preferably, a polypeptide comprising the amino acid sequence of DNXXYXXIXT (SEQ ID NO: 294). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, six, or seven amino acids at positions 1, 3, 4, 6, 7, 9, and 10 of SEQ ID NO: 295 have been substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, or five amino acids at positions 3, 4, 6, 7, and 9 of SEQ ID NO: 295 have been substituted with another amino acid, preferably alanine.

[0224] In another embodiment, a polypeptide comprising the amino acid sequence of YXXIXT (SEQ ID NO: 296), preferably a polypeptide comprising the amino acid sequence of SEQ ID NO: 297 in which any one, two or three amino acids at positions 2, 3 or 5 are substituted with another amino acid, preferably alanine.

[0225] In another aspect, a polypeptide comprising the amino acid sequence of XXXISXGG (SEQ ID NO: 298). Preferably, a polypeptide comprising the amino acid sequence of YXMISXGG (SEQ ID NO: 299). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, or four amino acids at positions 1 to 3 and 6 of SEQ ID NO: 300 are substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one or two amino acids at positions 2 and 6 of SEQ ID NO: 300 are substituted with another amino acid, preferably alanine.

[0226] In one embodiment, the polypeptide of (7α) may be identical to the full-length amino acid sequence of myosin binding protein H-like (SEQ ID NO: 43) or may be free of variants or homologs having 90% or more, 80% or more, or 70% or more identity thereto.

[0227] (8α) Desmin (fragment) epitope In the present invention, the (8α) polypeptide may be any of the polypeptides selected from the group consisting of the following (8α-1) to (8α-4):

[0228] (8α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 172, 173, 174, 303, 306, 308, and 310.

[0229] A polypeptide comprising the amino acid sequence of (8α-2)KXXXSX (SEQ ID NO: 301). Preferably, a polypeptide comprising the amino acid sequence of KXXXSD (SEQ ID NO: 302). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three or four amino acids at positions 2 to 4 and 6 of SEQ ID NO: 303 are substituted with other amino acids, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two or three amino acids at positions 2 to 4 of SEQ ID NO: 303 are substituted with other amino acids, preferably alanine. A polypeptide comprising an amino acid sequence in which the amino acid is replaced by another amino acid, preferably alanine.

[0230] In another embodiment, a polypeptide comprising the amino acid sequence of XKXKXXXXN (SEQ ID NO: 304). Preferably, the polypeptide comprises the amino acid sequence of YKSKXSDLN (SEQ ID NO: 305). Preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 1, 3, and 5 to 8 of SEQ ID NO: 306 are substituted with another amino acid, preferably alanine, and more preferably, the fifth amino acid of SEQ ID NO: 306 is substituted with another amino acid, preferably alanine.

[0231] In yet another embodiment, a polypeptide comprising the amino acid sequence of VXKN (SEQ ID NO: 307), preferably a polypeptide comprising the amino acid sequence of SEQ ID NO: 308, with the second amino acid replaced by another amino acid, preferably alanine.

[0232] A polypeptide comprising the amino acid sequence of (8α-4)DXGRXXE (SEQ ID NO: 309). or a polypeptide comprising an amino acid sequence in which any one, two or three amino acids at positions 2, 5 or 6 of SEQ ID NO: 310 have been substituted with another amino acid, preferably alanine.

[0233] In one embodiment, the (8α) polypeptide may be free of variants or homologs identical to, or having 90% or more, 80% or more, or 70% or more identity to, the amino acid sequence of desmin (fragment) (SEQ ID NO: 56).

[0234] (9α) Epitope of muscle Z-line beta capping protein (actin filament) In the present invention, the (9α) polypeptide may be any of the polypeptides selected from the group consisting of the following (9α-1) to (9α-5):

[0235] (9α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 175 to 178, 312, 314, 317, 320, 323, and 326.

[0236] A polypeptide comprising the amino acid sequence of (9α-2)KKXXXG (SEQ ID NO: 311), preferably an amino acid sequence in which any one, two, or three amino acids at positions 3 to 5 of SEQ ID NO: 312 have been substituted with another amino acid, preferably alanine.

[0237] In another embodiment, a polypeptide comprising the amino acid sequence of SKXXK (SEQ ID NO: 313). Preferably, a polypeptide comprising an amino acid sequence in which either one or two of the third and fourth amino acids of SEQ ID NO: 314 are substituted with another amino acid, preferably alanine.

[0238] A polypeptide comprising the amino acid sequence of (9α-3)XXXXXQXKXXG (SEQ ID NO: 315). Preferably, the polypeptide comprises the amino acid sequence HXXXXQXKSSG (SEQ ID NO: 316). Preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, or 8 amino acids at positions 1 to 5, 7, 9, and 10 of SEQ ID NO: 317 are substituted with other amino acids, preferably alanine. Even more preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, or 5 amino acids at positions 2 to 5 and 7 of SEQ ID NO: 317 are substituted with other amino acids, preferably alanine.

[0239] A polypeptide comprising the amino acid sequence of (9α-4)XYXGKXXXX (SEQ ID NO: 318). Or, a polypeptide comprising the amino acid sequence of IYXGKTXDI (SEQ ID NO: 319). or a polypeptide comprising an amino acid sequence in which any one, two, three, four, five or six amino acids at positions 1, 3, and 6 to 9 of SEQ ID NO: 320 are substituted with another amino acid, preferably alanine. More preferably, any one of the amino acids at positions 3 and 7 of SEQ ID NO: 320 is substituted with alanine. or a polypeptide comprising an amino acid sequence in which two amino acids have been replaced by another amino acid, preferably alanine.

[0240] In another embodiment, a polypeptide comprising the amino acid sequence of XDXXNXXRS (SEQ ID NO: 321). Preferably, the polypeptide comprises the amino acid sequence KDXXNXLRS (SEQ ID NO: 322). Preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four or five amino acids at positions 1, 3, 4, 6 or 7 of SEQ ID NO: 323 are substituted with another amino acid, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any one, two or three amino acids at positions 3, 4 or 6 of SEQ ID NO: 323 are substituted with another amino acid, preferably alanine.

[0241] A polypeptide comprising the amino acid sequence of (9α-5)QKYRQXXKXXXX (SEQ ID NO: 324). Preferably, a polypeptide comprising the amino acid sequence of QKYRQXXKXLXQ (SEQ ID NO: 325). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 6, 7, and 9 to 12 of SEQ ID NO: 326 have been substituted with other amino acids, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, or four amino acids at positions 6, 7, 9, and 11 of SEQ ID NO: 326 have been substituted with other amino acids, preferably alanine.

[0242] In one embodiment, the (9α) polypeptide may be free of variants or homologs identical to or having 90% or more, 80% or more, or 70% or more identity to the full-length amino acid sequence of capping protein (actin filament) muscle Z-line beta (SEQ ID NO: 61).

[0243] (10α) Myosin heavy chain, a fast skeletal muscle-like epitope In the present invention, the (10α) polypeptide may be any polypeptide selected from the group consisting of the following (10α-1) to (10α-8):

[0244] (10α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 179 to 185, 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, and 365.

[0245] A polypeptide comprising the amino acid sequence of (10α-2)TXXXXDXXEGK (SEQ ID NO: 327). Preferably, the polypeptide comprises the amino acid sequence of TXXXLDFREGK (SEQ ID NO: 328). Preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 2 to 5, 7, or 8 of SEQ ID NO: 329 are substituted with other amino acids, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, or three amino acids at positions 2 to 4 of SEQ ID NO: 329 are substituted with other amino acids, preferably alanine.

[0246] In another embodiment, a polypeptide comprising the amino acid sequence of XXXEXXD (SEQ ID NO: 330). Preferably, the polypeptide comprises the amino acid sequence of FREXXEXXD (SEQ ID NO: 331). Preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five or six amino acids at positions 1, 2, 4, 5, 7 or 8 of SEQ ID NO: 332 are substituted with another amino acid, preferably alanine. More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three or four amino acids at positions 4, 5, 7 or 8 of SEQ ID NO: 332 are substituted with another amino acid, preferably alanine.

[0247] A polypeptide comprising the amino acid sequence of (10α-3)LXXNFTXXK (SEQ ID NO: 333). Preferably, the polypeptide comprises the amino acid sequence of LXINFTNEK (SEQ ID NO: 334). Alternatively, a polypeptide comprising an amino acid sequence in which any one, two, three or four amino acids at positions 2, 3, 7 and 8 of SEQ ID NO: 335 are substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which the second amino acid of SEQ ID NO: 335 is substituted with another amino acid, preferably alanine.

[0248] A polypeptide comprising the amino acid sequence of (10α-4)XYXPPPXXXK (SEQ ID NO: 336). Preferably, a polypeptide comprising the amino acid sequence of LYXPPPXXXK (SEQ ID NO: 337). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, or five amino acids at positions 1, 3, and 7 to 9 of SEQ ID NO: 338 have been substituted with other amino acids, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, or four amino acids at positions 3, and 7 to 9 of SEQ ID NO: 338 have been substituted with other amino acids, preferably alanine.

[0249] A polypeptide comprising the amino acid sequence of (10α-5)XXDEXVXK (SEQ ID NO: 339). Preferably, a polypeptide comprising the amino acid sequence of SXDEXVXK (SEQ ID NO: 340). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, or four amino acids at positions 1, 2, 5, and 7 of SEQ ID NO: 341 have been substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, or three amino acids at positions 2, 5, and 7 of SEQ ID NO: 341 have been substituted with another amino acid, preferably alanine.

[0250] In another aspect, a polypeptide comprising the amino acid sequence of XXDXXXAKXT (SEQ ID NO: 342). Preferably, a polypeptide comprising the amino acid sequence of SXDEXVAKXT (SEQ ID NO: 343). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 1, 2, 4 to 6, and 9 of SEQ ID NO: 344 have been substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, or three amino acids at positions 2, 5, and 9 of SEQ ID NO: 344 have been substituted with another amino acid, preferably alanine.

[0251] In yet another embodiment, a polypeptide comprising the amino acid sequence of VXXXXKXKK (SEQ ID NO: 345) Preferably, a polypeptide comprising the amino acid sequence VAXXXKXKK (SEQ ID NO: 346). More preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four, or five amino acids at positions 2 to 5 and 7 of SEQ ID NO: 347 are substituted with other amino acids, preferably alanine. Still more preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, or four amino acids at positions 3 to 5 and 7 of SEQ ID NO: 347 are substituted with other amino acids, preferably alanine.

[0252] In yet another embodiment, a polypeptide comprising the amino acid sequence of KXXKXK (SEQ ID NO: 348). Preferably, a polypeptide comprising the amino acid sequence of KXXKEK (SEQ ID NO: 349). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, or three amino acids at positions 2, 3, and 5 of SEQ ID NO: 350 are substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one or two amino acids at positions 2 and 3 of SEQ ID NO: 350 are substituted with another amino acid, preferably alanine.

[0253] A polypeptide comprising the amino acid sequence of (10α-6)XNXXXKXKXK (SEQ ID NO: 351), preferably the amino acid sequence of XNXXXKXKTK (SEQ ID NO: 352), more preferably an amino acid sequence in which any one, two, three, four, five or six amino acids at positions 1, 3 to 5, 7 and 9 of SEQ ID NO: 353 are substituted with another amino acid, preferably alanine. More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, or five amino acids at positions 1, 3 to 5, or 7 of SEQ ID NO: 353 are substituted with another amino acid, preferably alanine.

[0254] A polypeptide comprising the amino acid sequence of (10α-7)DXXXSXXK (SEQ ID NO: 354). Preferably, a polypeptide comprising the amino acid sequence of DXXXSXRK (SEQ ID NO: 355). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, or five amino acids at positions 2 to 4, 6, and 7 of SEQ ID NO: 356 have been substituted with other amino acids, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, or four amino acids at positions 2 to 4 and 6 of SEQ ID NO: 356 have been substituted with other amino acids, preferably alanine.

[0255] In another embodiment, a polypeptide comprising the amino acid sequence of SXXKXEX (SEQ ID NO: 357). Preferably, the polypeptide comprises the amino acid sequence SXRKXEG (SEQ ID NO: 358). Alternatively, a polypeptide comprising an amino acid sequence in which any one, two, three or four amino acids at positions 2, 3, 5 and 7 of SEQ ID NO: 359 are substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one or two amino acids at positions 2 and 5 of SEQ ID NO: 359 are substituted with another amino acid, preferably alanine.

[0256] A polypeptide comprising the amino acid sequence of (10α-8)XXRXXEXK (SEQ ID NO: 360). Preferably, a polypeptide comprising the amino acid sequence of EXRXXEEK (SEQ ID NO: 361). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, or five amino acids at positions 1, 2, 4, 5, or 7 of SEQ ID NO: 362 are substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, or three amino acids at positions 2, 4, or 5 of SEQ ID NO: 362 are substituted with another amino acid, preferably alanine.

[0257] In another embodiment, a polypeptide comprising the amino acid sequence of XXXEXKXKK (SEQ ID NO: 363). Preferably, the polypeptide comprises the amino acid sequence RXXEEKXKK (SEQ ID NO: 364). Preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four or five amino acids at positions 1 to 3, 5 or 7 of SEQ ID NO: 365 are substituted with another amino acid, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any one, two or three amino acids at positions 2, 3 or 7 of SEQ ID NO: 365 are substituted with another amino acid, preferably alanine.

[0258] (11α) Glycogen phosphorylase, a muscle foam-like epitope In the present invention, the (11α) polypeptide may be any of the polypeptides selected from the group consisting of the following (11α-1) and (11α-2):

[0259] (11α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 186, 367, and 370.

[0260] A polypeptide comprising the amino acid sequence of (11α-2)GXYXXXXXXR (SEQ ID NO: 366), preferably an amino acid sequence in which any one, two, three, four, five, six, or seven amino acids at positions 2, and 4 to 9 of SEQ ID NO: 367 are substituted with another amino acid, preferably alanine.

[0261] In another embodiment, a polypeptide comprising the amino acid sequence of NLXXN (SEQ ID NO: 368). Preferably, a polypeptide comprising the amino acid sequence of NLXEN (SEQ ID NO: 369). is a polypeptide comprising an amino acid sequence in which either one or two of the amino acids at positions 3 and 4 of SEQ ID NO: 370 are substituted with another amino acid, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which the amino acid at position 3 of SEQ ID NO: 370 is substituted with another amino acid, preferably alanine.

[0262] (12α) Myosin-binding protein C, fast-type-like epitope In the present invention, the (12α) polypeptide may be any of the polypeptides selected from the group consisting of the following (12α-1) to (12α-11):

[0263] (12α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 187 to 196, 373, 376, 379, 382, ​​384, 386, 388, 390, 392, 395, 397, 400, 402, 405, 407, 409, 411, and 413.

[0264] A polypeptide comprising the amino acid sequence of (12α-2)KXTXXKKKXX (SEQ ID NO: 371). Preferably, a polypeptide comprising the amino acid sequence of KXTXXKKKPV (SEQ ID NO: 372). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, or five amino acids at positions 2, 4, 5, 9, and 10 of SEQ ID NO: 373 have been substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, or three amino acids at positions 2, 4, and 5 of SEQ ID NO: 373 have been substituted with another amino acid, preferably alanine.

[0265] In another embodiment, a polypeptide comprising the amino acid sequence of KXKPVVXX (SEQ ID NO: 374). Preferably, a polypeptide comprising the amino acid sequence of KXKPVVDE (SEQ ID NO: 375). More preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 376 in which any one, two or three amino acids at positions 2, 7 or 8 have been substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 376 in which the second amino acid has been substituted with another amino acid, preferably alanine.

[0266] A polypeptide comprising the amino acid sequence of (12α-3)YXXXXFXXXI (SEQ ID NO: 377). Preferably, a polypeptide comprising the amino acid sequence of YXXIXFEYXI (SEQ ID NO: 378). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, six, or seven amino acids at positions 2 to 5, or 7 to 9 of SEQ ID NO: 379 have been substituted with other amino acids, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, or four amino acids at positions 2, 3, 5, or 9 of SEQ ID NO: 379 have been substituted with other amino acids, preferably alanine.

[0267] In another embodiment, a polypeptide comprising the amino acid sequence of XXIXDXRG (SEQ ID NO: 380). Preferably, a polypeptide comprising the amino acid sequence of YXIXDXRG (SEQ ID NO: 381). More preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 382 in which any one, two, three, or four amino acids at positions 1, 2, 4, and 6 have been substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 382 in which any one, two, or three amino acids at positions 2, 4, and 6 have been substituted with another amino acid, preferably alanine.

[0268] A polypeptide comprising the amino acid sequence of (12α-4)DXRGXXK (SEQ ID NO: 383). Or a polypeptide comprising an amino acid sequence in which any one, two or three amino acids at positions 2, 5 and 6 of SEQ ID NO: 384 are substituted with another amino acid, preferably alanine.

[0269] A polypeptide comprising the amino acid sequence of (12α-5)KMXXXXPK (SEQ ID NO: 385), preferably an amino acid sequence in which any one, two, three, or four amino acids at positions 3 to 6 of SEQ ID NO: 386 are substituted with another amino acid, preferably alanine.

[0270] In another aspect, a polypeptide comprising the amino acid sequence of KXXKXXXPKH (SEQ ID NO: 387), preferably a polypeptide comprising an amino acid sequence in which any one, two, three, four or five amino acids at positions 2, 3, and 5 to 7 of SEQ ID NO: 388 have been substituted with another amino acid, preferably alanine.

[0271] In yet another embodiment, a polypeptide comprising the amino acid sequence of KXXXXFL (SEQ ID NO: 389). Preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, or four amino acids from the second to fifth amino acids of SEQ ID NO: 390 are substituted with another amino acid, preferably alanine.

[0272] A polypeptide comprising the amino acid sequence of (12α-6)KGKKXX (SEQ ID NO: 391), preferably an amino acid sequence in which either one or two amino acids at positions 5 and 6 of SEQ ID NO: 392 are substituted with another amino acid, preferably alanine.

[0273] In another aspect, a polypeptide comprising the amino acid sequence of KXKXXXLXXE (SEQ ID NO: 393). Preferably, a polypeptide comprising the amino acid sequence of KXKXXXLQXE (SEQ ID NO: 394). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 2, 4 to 6, 8, and 9 of SEQ ID NO: 395 have been substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, or five amino acids at positions 2, 4 to 6, and 9 of SEQ ID NO: 395 have been substituted with another amino acid, preferably alanine.

[0274] A polypeptide comprising the amino acid sequence of (12α-7)TXKX (SEQ ID NO: 396), preferably an amino acid sequence in which either one or two of the second and fourth amino acids of SEQ ID NO: 397 are substituted with another amino acid, preferably alanine.

[0275] In another embodiment, a polypeptide comprising the amino acid sequence of KYXXKKXXX (SEQ ID NO: 398). Preferably, the polypeptide comprises the amino acid sequence KYXXKKDGL (SEQ ID NO: 399). Preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four or five amino acids at positions 3, 4, and 7 to 9 of SEQ ID NO: 400 are substituted with another amino acid, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any one or two amino acids at positions 3 and 4 of SEQ ID NO: 400 are substituted with another amino acid, preferably alanine.

[0276] A polypeptide comprising the amino acid sequence (12α-8)YXXXPDG (SEQ ID NO: 401). Alternatively, a polypeptide comprising an amino acid sequence in which any one, two or three amino acids from the second to fourth amino acids of SEQ ID NO: 402 have been substituted with another amino acid, preferably alanine.

[0277] In another embodiment, a polypeptide comprising the amino acid sequence of DGYXXSXSXK (SEQ ID NO: 403). Preferably, a polypeptide comprising the amino acid sequence of DGYXLSLSXK (SEQ ID NO: 404). More preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 405 in which any one, two, three, or four amino acids at positions 4, 5, 7, and 9 have been substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 405 in which any one or two amino acids at positions 4 and 9 have been substituted with another amino acid, preferably alanine.

[0278] A polypeptide comprising the amino acid sequence of (12α-9)PVTXX (SEQ ID NO: 406). or a polypeptide comprising an amino acid sequence in which either one or two amino acids at positions 4 and 5 of SEQ ID NO: 407 are substituted with another amino acid, preferably alanine.

[0279] A polypeptide comprising the amino acid sequence of (12α-10)QXXXXXXXDKXN (SEQ ID NO: 408), preferably an amino acid sequence in which any one, two, three, four, five, six, seven, or eight amino acids at positions 2 to 8 and 11 of SEQ ID NO: 409 are substituted with another amino acid, preferably alanine.

[0280] A polypeptide comprising the amino acid sequence of (12α-11)YXXXXXDXKT (SEQ ID NO: 410), preferably an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 2 to 6 and 8 of SEQ ID NO: 411 are substituted with another amino acid, preferably alanine.

[0281] In another aspect, a polypeptide comprising the amino acid sequence of YXXQXXXK (SEQ ID NO: 412), preferably a polypeptide comprising an amino acid sequence in which any one, two, three, four or five amino acids at positions 2, 3, and 5 to 7 of SEQ ID NO: 413 have been substituted with another amino acid, preferably alanine.

[0282] (13α) Epitopes on ATP synthase subunit β, mitochondrial In the present invention, the (13α) polypeptide may be any of the polypeptides selected from the group consisting of (13α-1) and (13α-2) below.

[0283] (13α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 197, 415, and 417.

[0284] A polypeptide comprising the amino acid sequence of (13α-2)GXYS (SEQ ID NO: 414), preferably comprising the amino acid sequence of SEQ ID NO: 415 in which the second amino acid is replaced by another amino acid, preferably alanine.

[0285] In another aspect, a polypeptide comprising the amino acid sequence of YXXXXG (SEQ ID NO: 416), preferably a polypeptide comprising an amino acid sequence in which any one, two, three or four amino acids at positions 2 to 5 of SEQ ID NO: 417 have been substituted with another amino acid, preferably alanine.

[0286] (14α) L-lactate dehydrogenase A chain-like epitope In the present invention, the (14α) polypeptide may be any of the polypeptides selected from the group consisting of (14α-1) and (14α-2):

[0287] (14α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 198 and 420.

[0288] A polypeptide comprising the amino acid sequence of (14α-2)XPVGSXSKX (SEQ ID NO: 418). Preferably, the polypeptide comprises the amino acid sequence of EPVGSXSKV (SEQ ID NO: 419). Alternatively, a polypeptide comprising an amino acid sequence in which any one, two or three amino acids at positions 1, 6 or 9 of SEQ ID NO: 420 are substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which the amino acid at position 6 of SEQ ID NO: 420 is substituted with another amino acid, preferably alanine.

[0289] (15α) Aldolase epitopes In the present invention, the (15α) polypeptide may be any of the polypeptides selected from the group consisting of the following (15α-1) and (15α-2):

[0290] (15α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 199, 423, and 425.

[0291] A polypeptide comprising the amino acid sequence of (15α-2)XYXRXXXXXXXK (SEQ ID NO: 421). Preferably, a polypeptide comprising the amino acid sequence of SYXRXLXXXAXK (SEQ ID NO: 422). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, six, seven, eight, or nine amino acids at positions 1, 3, and 5 to 11 of SEQ ID NO: 423 have been substituted with other amino acids, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 3, 5, 7 to 9, and 11 of SEQ ID NO: 423 have been substituted with other amino acids, preferably alanine.

[0292] In another embodiment, a polypeptide comprising the amino acid sequence of SXXKXXG (SEQ ID NO: 424). Preferably, a polypeptide comprising an amino acid sequence in which any one, two, three or four amino acids at positions 2, 3, 5 and 6 of SEQ ID NO: 425 are substituted with another amino acid, preferably alanine.

[0293] In one embodiment, the (15α) polypeptide may be free of variants or homologs identical to, or having 90% or more, 80% or more, or 70% or more identity to, the full-length amino acid sequence of aldolase (NCBI protein accession number NP_001133180.1, SEQ ID NO: 445).

[0294] (16α) beta-enolase epitope In the present invention, the (16α) polypeptide may be any of the polypeptides selected from the group consisting of the following (16α-1) to (16α-4):

[0295] (16α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 200 to 202, 428, 431, 434, and 436.

[0296] A polypeptide comprising the amino acid sequence of (16α-2)RYLGKXXX (SEQ ID NO: 426). Preferably, a polypeptide comprising the amino acid sequence of RYLGKGXV (SEQ ID NO: 427). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, or three amino acids at positions 6 to 8 of SEQ ID NO: 428 are substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which the seventh amino acid of SEQ ID NO: 428 is substituted with another amino acid, preferably alanine.

[0297] A polypeptide comprising the amino acid sequence of (16α-3)YXXIXDXXXH (SEQ ID NO: 429). Preferably, a polypeptide comprising the amino acid sequence of YXXIXDLXGH (SEQ ID NO: 430). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, or six amino acids at positions 2, 3, 5, and 7 to 9 of SEQ ID NO: 431 are substituted with other amino acids, preferably alanine. Even more preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, or four amino acids at positions 2, 3, 5, and 8 of SEQ ID NO: 431 are substituted with other amino acids, preferably alanine.

[0298] In another aspect, a polypeptide comprising the amino acid sequence of DXXXHXDVXL (SEQ ID NO: 432), preferably the amino acid sequence of DXXXHKDVIL (SEQ ID NO: 433), more preferably the amino acid sequence in which any one, two, three, four or five amino acids at positions 2 to 4, 6 and 9 of SEQ ID NO: 434 are substituted with another amino acid, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any one, two or three amino acids at positions 2 to 4 of SEQ ID NO: 434 are substituted with another amino acid, preferably alanine.

[0299] A polypeptide comprising the amino acid sequence of (16α-4)IKXXXXKDAT (SEQ ID NO: 435), preferably an amino acid sequence in which any one, two, three, or four amino acids at positions 3 to 6 of SEQ ID NO: 436 are substituted with another amino acid, preferably alanine.

[0300] In one embodiment, the (16α) polypeptide may be free of variants or homologs identical to, or having greater than 90%, greater than 80%, or greater than 70% identity to, the full-length amino acid sequence of beta-enolase (NCBI protein accession number NP_001133193.1, SEQ ID NO: 446).

[0301] (17α) Epitope of glyceraldehyde-3-phosphate dehydrogenase In the present invention, the (17α) polypeptide may be any of the polypeptides selected from the group consisting of the following (17α-1) to (17α-3):

[0302] (17α-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 203, 204, 441, and 444.

[0303] A polypeptide comprising the amino acid sequence of (17α-2)YXXXXXXDXXXGRF (SEQ ID NO: 437) Preferably, the polypeptide comprises the amino acid sequence YXXXXXXXDSTXGRF (SEQ ID NO: 438). More preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, six, seven, eight, nine, or ten amino acids at positions 2 to 8 and 10 to 12 of SEQ ID NO: 203 are substituted with another amino acid, preferably alanine. Still more preferably, it is a polypeptide comprising an amino acid sequence in which any one, two, three, four, five, six, seven, or eight amino acids at positions 2 to 8 and 12 of SEQ ID NO: 203 are substituted with another amino acid, preferably alanine.

[0304] A polypeptide comprising the amino acid sequence of (17α-3)SYXXIXXV (SEQ ID NO: 440), preferably an amino acid sequence in which any one, two, three or four amino acids at positions 3, 4, 6 and 7 of SEQ ID NO: 441 are substituted with another amino acid, preferably alanine.

[0305] In another embodiment, a polypeptide comprising the amino acid sequence of IKKXXK (SEQ ID NO: 442). Preferably, a polypeptide comprising the amino acid sequence of IKKVXK (SEQ ID NO: 443). More preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 444 in which either one or two amino acids at positions 4 and 5 are substituted with another amino acid, preferably alanine. Even more preferably, a polypeptide comprising the amino acid sequence of SEQ ID NO: 444 in which the fifth amino acid is substituted with another amino acid, preferably alanine.

[0306] In one embodiment, the (17α) polypeptide may be free of variants or homologs identical to, or having 90% or more, 80% or more, or 70% or more identity to, the full-length amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase (NCBI protein accession number NP_001117033.1, SEQ ID NO: 447).

[0307] The length of the above polypeptides (1α) to (17α) is not particularly limited. In a preferred embodiment, the length of the above polypeptides (1α) to (17α) is 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, The amino acid sequence may be 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, or 5 amino acids or less.

[0308] The above polypeptides (1α) to (17α) may be prepared by chemical synthesis techniques such as solid-phase peptide synthesis, or the epitope-containing polypeptide may be expressed as a recombinant protein by gene recombination techniques well known to those skilled in the art, and then isolated and purified by protein production methods well known to those skilled in the art.

[0309] Diagnostic kits and methods (2) The present invention provides a method for providing an indicator for diagnosing allergies in a subject, comprising the steps of: (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) if binding of the subject's IgE antibodies to the antigen is detected, an indication that the subject is allergic is provided; wherein the antigen is at least one of the polypeptides (1α) to (17α) above, or a polypeptide in which two or more of the polypeptides (1α) to (17α) above are linked together with or without a spacer.

[0310] Hereinafter, a polypeptide that is at least one of the above polypeptides (1α) to (17α), or a polypeptide in which two or more of the above polypeptides (1α) to (17α) are linked together with or without a spacer, will be referred to as an "antigen containing the above (1α) to (17α)." The type of spacer is not particularly limited, and any spacer commonly used by those skilled in the art for linking multiple peptides can be used. For example, the spacer may be Acp(6)-OH It may also be a hydrocarbon chain such as

[0311] The sample obtained from the subject is as described above in the section "Diagnostic kit and diagnostic method (1)."

[0312] The contact between the sample obtained from the control and the antigen and the binding thereto can be detected by known methods described in the above section "Diagnostic Kit / Diagnostic Method (1)," such as ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblot, and immunoprecipitation. This can be done by immunochromatography or the like.

[0313] The antigens containing (1α) to (17α) may be immobilized on a carrier. In this case, ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance, etc. can be used in the steps (i) and (ii), and the step (i) is carried out by contacting a sample obtained from the subject with a surface on which the antigens containing (1α) to (17α) are immobilized. Alternatively, the binding of the subject's IgE antibodies to the antigens containing (1α) to (17α) may be detected by the above-mentioned method using the IgE antibodies immobilized on a carrier.

[0314] The antigens containing (1α) to (17α) may not be immobilized on a carrier. In this case, flow cytometry or the like can be used in steps (i) and (ii) to confirm the presence of the antigens containing (1α) to (17α) bound to IgE antibodies using laser light. Examples of this method include the basophil activation test (BAT), which detects the surface antigen CD203c that appears when basophils are activated by contact with the antigens containing (1α) to (17α). Other examples include the histamine release test (HRT), which examines whether histamine is released by further contacting blood cells in a sample with the antigens containing (1α) to (17α).

[0315] The antigens containing the above (1α) to (17α) are antigens that specifically bind to the IgE antibodies of allergy patients. Therefore, when the binding between the IgE antibodies of a subject and the antigen is detected, an indicator that the subject has an allergy is provided.

[0316] The present invention also provides a diagnostic kit for allergies, comprising at least one of the antigens comprising (1α) to (17α). The diagnostic kit of the present invention may be used in the above-mentioned method for providing an indicator for diagnosing allergies, or in the diagnostic method described below. In addition to comprising at least one of the antigens comprising (1α) to (17α), the diagnostic kit of the present invention may also comprise an enzyme-labeled anti-IgE antibody and a chromogenic or luminescent substrate that serves as a substrate for the enzyme. A fluorescently labeled anti-IgE antibody may also be used. In the diagnostic kit of the present invention, the antigen comprising (1α) to (17α) may be provided in a state where it is immobilized on a carrier. The diagnostic kit of the present invention may also be provided together with instructions on the diagnostic procedure and a package containing such instructions.

[0317] In another embodiment, the diagnostic kit includes a companion diagnostic for allergies. The companion diagnostic is used to identify patients who are expected to benefit from a drug or who are at risk of serious side effects from the drug, or to examine the responsiveness of the drug to optimize treatment with the drug. Here, optimization of treatment includes, for example, determining the dosage and administration, deciding whether to discontinue administration, and identifying which allergen component is used to induce immune tolerance.

[0318] The present invention also provides a composition for diagnosing allergies, which contains at least one of the antigens (1α) to (17α) described above. The diagnostic composition of the present invention can be used in the following diagnostic methods. The diagnostic composition of the present invention may contain, as needed, pharmaceutically acceptable carriers and additives that are commonly used together with the antigen of the present invention.

[0319] In one aspect, the present invention provides a method for diagnosing an allergy in a subject, comprising the steps of: (i) contacting a sample obtained from the subject with an antigen; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) determining that the subject is allergic if binding of the subject's IgE antibody to the antigen is detected; wherein the antigen is at least one of the proteins identified as antigens including (1α) to (17α), wherein each of steps (i) and (ii) is carried out as described for each step of the method for providing an indicator for diagnosing allergies.

[0320] In another aspect, the present invention provides a method for diagnosing allergy in a subject, the method comprising administering to the subject at least one of the antigens comprising (1α) to (17α). The method may be performed in the form of a skin test, characterized by applying the antigen comprising (1α) to (17α) to the skin. Skin tests include a prick test in which a diagnostic composition is applied to the skin, and then a small scratch is made in the skin to allow the antigen comprising (1α) to (17α) to penetrate the skin without bleeding, and a skin reaction is observed; a scratch test in which a diagnostic composition is applied and then the skin is slightly scratched to observe a reaction; a patch test in which a diagnostic composition in the form of a cream or ointment is applied to the skin to observe a reaction; and an intradermal test in which an antigen comprising (1α) to (17α) is intradermally administered and a reaction is observed. If a skin reaction such as swelling occurs in the area of ​​the skin where the antigen comprising (1α) to (17α) is applied, the subject is diagnosed as having an allergy. Here, the amount of antigens containing the above (1α) to (17α) to be applied to the skin may be, for example, a dose of 100 μg or less per application.

[0321] In the diagnosis of allergies, a challenge test is often performed to identify the antigen. At least one of the antigens (1α) to (17α) can be used as an active ingredient in a challenge test to diagnose allergies. Here, the allergen component used in the challenge test may be an expressed and purified polypeptide, or may be expressed in a food or ingredient, such as pollen rice, in which a cedar pollen antigen gene is transformed into rice and the antigen protein is expressed in the rice.

[0322] In another aspect, the present invention provides at least one antigen comprising the above (1α) to (17α) for use in diagnosing allergies.

[0323] In yet another aspect, the present invention provides use of at least one of the antigens comprising the above (1α) to (17α) for the manufacture of a diagnostic agent for allergies.

[0324] In this item, the allergy to be diagnosed or detected may be an allergy to an antigen including the above (1α) to (17α). That is, the detection of allergy may be not only an allergy to a single antigen including the above (1α) to (17α), but also an allergy including cross-reactivity.

[0325] Pharmaceutical Compositions and Treatment Methods (2) The present invention provides a pharmaceutical composition containing at least one of the antigens (1α) to (17α) described above. In one aspect, the pharmaceutical composition is used to treat allergies. Treating allergies involves increasing the limit amount of antigen that does not cause symptoms even when taken into the body, ultimately aiming to achieve a state (remission) where symptoms do not occur with normal intake of antigen.

[0326] The present invention also provides a method for treating allergies, which comprises administering at least one of the antigens comprising (1α) to (17α) to a patient in need of such treatment.

[0327] In another aspect, the present invention provides at least one antigen comprising the above (1α) to (17α) for use in treating allergies. In yet another aspect, the present invention provides use of at least one antigen comprising the above (1α) to (17α) for the manufacture of a therapeutic agent for allergies.

[0328] In the treatment of allergies, hyposensitization therapy is often performed, aiming to induce immune tolerance by administering an antigen to a patient. At least one of the antigens (1α) to (17α) above can be used as an active ingredient for hyposensitization therapy for allergies. Here, the allergen component used in hyposensitization therapy may be an expressed and purified polypeptide, or may be one expressed in a food or ingredient, such as pollen rice.

[0329] The administration route, dosage, number of doses and / or duration of administration, other ingredients contained in the pharmaceutical composition, and dosage form of the pharmaceutical composition of the present invention can be those described above in the section "Pharmaceutical Compositions and Treatment Methods (1)." When using antigens containing (1α) to (17α), the dose may be, for example, 100 μg or less per administration for adults.

[0330] In this section, the allergy to be treated may be an allergy to an antigen containing the above (1α) to (17α). That is, the treatment of allergy may be not only an allergy to a single antigen containing the above (1α) to (17α), but also a cross-reactive allergy.

[0331] Tester (2) The present invention provides a tester containing an antibody against at least one of the antigens including (1α) to (17α) above.

[0332] The antibody can be prepared by a conventional method. For example, it may be prepared by immunizing a mammal such as a rabbit with an antigen containing any of the above (1α) to (17α). The antibody may be an Ig antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (e.g., Fab, F(ab')2, Fab').

[0333] Furthermore, in the above tester, the antibody may be provided in a form bound to a carrier. The carrier is not particularly limited as long as it can be used to detect the binding of an antibody to an antigen including the above (1α) to (17α). Any carrier known to those skilled in the art can be used. Furthermore, it is preferable that the antibody against the antigen including the above (1α) to (17α) is an antibody against an epitope described in the above section "Epitope of the antigen." This allows for a tester that can detect cross-reactivity as well.

[0334] The following method can be used to examine whether or not the antigens containing (1α) to (17α) are contained. A tester containing the antibody thus prepared is brought into contact with a sample obtained from raw materials, processed products, etc., and the binding of the antibody to the antigen containing the above-mentioned (1α) to (17α) in the sample is detected, for example, using an ELISA method, and if the binding of the antibody to the antigen containing the above-mentioned (1α) to (17α) is detected, it is determined that the antigen remains in the target raw materials, processed products, etc. A method in which raw materials, processed products, etc. are soaked in filter paper, etc., and an antibody solution is reacted to detect the antigens contained therein, including the above (1α) to (17α).

[0335] In another aspect of the present invention, the present invention includes a tester for determining the presence or absence of an allergic antigen comprising any of the above (1α) to (17α) in a subject, characterized by comprising a primer corresponding to the epitope. The primer may be, for example, but not limited to, designed to comprise a portion of the nucleotide sequence of a nucleic acid encoding the amino acid sequence specified in (1α) to (17α) above, or its complementary strand. Alternatively, the primer may be designed to be the nucleotide sequence of a region upstream of the portion encoding the epitope, or the nucleotide sequence of a complementary strand of a region downstream of the portion encoding the epitope, in a nucleic acid encoding a protein comprising the epitope, which is an amino acid sequence specified in (1α) to (17α) above. Examples of such primers include primers that are a portion of at least one of the nucleotide sequences set forth in SEQ ID NOs: 1, 4, 9, 18, 25, 32, 42, 55, 60, 69, 109, 120, 137, 143, 448, 449, or 450, and / or primers that are a portion of a sequence complementary to at least one of the nucleotide sequences set forth in SEQ ID NOs: 1, 4, 9, 18, 25, 32, 42, 55, 60, 69, 109, 120, 137, 143, 448, 449, or 450. The location of the epitope in the full-length antigen sequence is as specified in Table 2 in Example 10 below. Furthermore, particularly when targeting mRNA, a primer complementary to a poly(A) tail may be used.

[0336] For example, DNA or mRNA obtained from a sample is used as a template, and the primers are used to amplify the DNA by PCR (Polymerase Chain Reaction) including RT-PCR. The presence or absence of an antigen containing (1α) to (17α) above is determined by determining whether the DNA sequence obtained contains a nucleic acid encoding the amino acid sequence specified in (1α) to (17α) above. Examples of methods for amplifying mRNA by PCR include the RACE method. If one of the amino acid sequences encoded by the three possible open reading frames in the amplified DNA contains the amino acid sequence specified in (1α) to (17α) above, the antigen is identified. If the DNA is not amplified, it is determined that the antigen is not present.

[0337] In one embodiment, the tester is used to examine whether or not an antigen containing any of the above (1α) to (17α) is contained in a raw material or a target object, such as in a processed product production line. The raw material may be a food ingredient, a cosmetic raw material, a pharmaceutical raw material, etc. The processed product may be an edible processed product, a cosmetic, a pharmaceutical, etc. The tester may be used to search for biological species contained in raw materials, for quality inspection by manufacturers on production lines and pre-shipment products, or for consumers or users themselves to check whether or not the target raw material or processed product contains the antigen.

[0338] Allergen-removing ingredients, etc. The present invention provides a raw material or processed product characterized in that at least one of the antigens including the above (1α) to (17α) has been removed or reduced.

[0339] The method for removing or reducing the antigens of the present invention in raw materials or processed products is not limited. The antigens may be removed or reduced by any method as long as the antigens including the above-mentioned (1α) to (17α) are removed or reduced. For example, the methods described in the above section "Allergen-free foods, etc." may be used.

[0340] The removal or reduction of at least one of the antigens including (1α) to (17α) above may be achieved by removing or reducing the entire antigen, or by cleaving or removing the sequence portion specified by (1α) to (17α) above from the antigen protein. "Removed" includes deletion and modification of all or part of the sequence portion specified by (1α) to (17α) above.

[0341] For example, a material from which the expression of the above antigens including (1α) to (17α) has been removed or reduced may be prepared using gene knockout technology to knock out the expression of the above antigens including (1α) to (17α). The gene knockout technology can be any technique known to those skilled in the art, such as gene modification.

[0342] The processed product from which the antigens containing (1α) to (17α) have been removed or reduced may be a processed product made from raw materials from which the antigens containing (1α) to (17α) have been removed or reduced, such as powdered milk made from purified peptides. When using normal raw materials, treatment to remove or reduce the antigens containing (1α) to (17α) is carried out before or after preparation of the processed product. As a method for removing or reducing the antigens containing (1α) to (17α) in processed products made from normal raw materials, the techniques described in the above section "Allergen-free foods, etc." may be used. As a method for cleaving the antigens containing (1α) to (17α), a cleavage treatment using a specific digestive enzyme may be used.

[0343] Manufacturing method for allergen-free processed products (2) The present invention provides a method for producing a processed product in which antigens have been removed or reduced, the method comprising a step of confirming that the antigens have been removed or reduced during the production process of the processed product, wherein the antigen is at least one of the antigens including (1α) to (17α) above.

[0344] In the production method, the removal or reduction of antigens means that at least one of the antigens including (1α) to (17α) above has been removed or reduced, or the sequence portion specified by (1α) to (17α) above has been cleaved or removed from the antigen.

[0345] There are no particular limitations on the methods for confirming that antigens have been removed or reduced during the manufacturing process of processed foods. Any method capable of detecting at least one of the antigens (1α) to (17α) may be used. For example, the presence or absence of the polypeptide or antigen in a processed product may be confirmed by measuring the binding between a sample containing materials generated during the manufacturing process of the processed product and an antibody against at least one of the antigens (1α) to (17α). Details of such a method are as described above in the section "Diagnostic Kit / Diagnostic Method (2)." That is, in the manufacturing method, the "subject's IgE antibody" in the section "Diagnostic Kit / Diagnostic Method (2)" may be replaced with "an antibody against at least one of the antigens (1α) to (17α)," and the "antigen" in the section "Diagnostic Kit / Diagnostic Method (2)" may be replaced with "a sample containing materials generated during the manufacturing process of the processed product." The method described in the section "Diagnostic Kit / Diagnostic Method (2)" may be used to confirm that the antigen has been removed or reduced during the manufacturing process of the processed product. The tester described in the section "Tester (2)" may also be used. [Example]

[0346] Examples of the present invention will be described below, but the technical scope of the present invention is not limited to these examples.

[0347] Example 1: Protein pattern confirmation The proteins contained in salmon were investigated using the following two-dimensional electrophoresis method.

[0348] Protein extraction Salmon proteins were extracted and purified as follows. A solubilizing agent (Mammalian Lysis Buffer (MCLI), SIGMA) was added to the salmon flesh to extract the proteins, and then a urea buffer was added to obtain a protein extract. The composition of the urea buffer is as follows: 30mM Tris 2M thiourea 7M Urea 4% (w / v) CHAPS: 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate A suitable amount of dilute hydrochloric acid Distilled water was added to bring the total volume to 100 mL, and the pH was 8.5. Then, 25 μg of protein each was mixed to obtain an extract.

[0349] Then, two precipitation procedures were performed using the 2D-CleanUP kit (GE). In the first precipitation procedure, TCA (trichloroacetic acid) was added to the recovered protein extract, and the resulting precipitate (TCA precipitate) was collected. In the second precipitation procedure, acetone was added to the recovered TCA precipitate, and the resulting precipitate (sample) was collected.

[0350] Preparation of sample solution A portion of the obtained sample (40 μg in terms of protein weight) was dissolved in 150 μl of DeStreak Rehydration Solution (GE), which is a swelling buffer for the first-dimensional isoelectric focusing gel. The solution was dissolved and used as the specimen solution for first-dimension isoelectric focusing (specimen solution for swelling). The composition of the DeStreak Rehydration Solution is as follows: 7M thiourea 2M urea 4% (w / v) CHAPS 0.5% (v / v) IPG buffer; GE Appropriate amount of BPB (bromophenol blue) Permeation of the sample into the first-dimension isoelectric focusing gel A first-dimension isoelectric focusing gel (GE: IPG Gel Immobiline Drystrip (pH 3-10NL)) was immersed in 140 μl of the specimen solution for first-dimension isoelectric focusing (specimen solution for swelling) and allowed to soak overnight at room temperature.

[0351] In this example, an IPGphor manufactured by GE was used as the electrophoresis device.

[0352] The electrophoresis tray was filled with silicone oil, and water-moistened filter paper was placed on both ends of the gel permeated with the sample. The gel was then set on the electrophoresis tray so that it was covered with silicone oil, and the electrodes were set with the filter paper sandwiched between the gel and the tray.

[0353] The upper limit of the current value of the isoelectric focusing device was set to 75 μA per gel, and the voltage program was as follows: (1) a constant voltage step of 300 V was performed up to 750 Vhr (the current change during the 30 minutes of electrophoresis before the end of this step was 5 μA), (2) the voltage was gradually increased to 1000 V over 300 Vhr, (3) the voltage was further gradually increased to 5000 V over 4500 Vhr, and (4) the first-dimension isoelectric focusing was then performed at a constant voltage of 5000 V until the total Vhr reached 12000.

[0354] SDS equilibration of isoelectric focusing gels After the first-dimension isoelectric focusing, the gel was removed from the isoelectric focusing apparatus and immersed in an equilibration buffer containing a reducing agent and shaken at room temperature for 15 minutes. The composition of the equilibration buffer containing a reducing agent was as follows: 100 mM Tris-HCl (pH 8.0) 6M urea 30% (v / v) glycerol 2% (w / v) SDS 1% (w / v) DTT Next, the equilibration buffer containing the reducing agent was removed, and the gel was immersed in an equilibration buffer containing an alkylating agent and shaken at room temperature for 15 minutes to obtain an SDS-equilibrated gel. The composition of the equilibration buffer containing the alkylating agent was as follows: 100 mM Tris-HCl (pH 8.0) 6M urea 30% (v / v) glycerol 2% (w / v) SDS 2.5% (w / v) iodoacetamide Second dimension SDS-PAGE In this example, the electrophoresis apparatus used was an XCell SureLock Mini-Cell manufactured by Life Technologies, Inc. The second-dimensional electrophoresis gel used was NuPAGE 4-12% Bis-Tris Gels manufactured by Life Technologies, Inc. The electrophoresis buffer solution with the following composition was prepared and used. 50mM MOPS 50mM Tris base 0.1% (w / v) SDS 1mM EDTA In this example, an adhesive agarose solution was used in which 0.5% (w / v) Agarose S (manufactured by Nippon Gene Co., Ltd.) and an appropriate amount of BPB (bromophenol blue) were dissolved in the electrophoresis buffer.

[0355] After thoroughly washing the inside of the SDS-PAGE wells with the above-mentioned electrophoresis buffer, the buffer used for washing was removed. Next, agarose solution for adhesion, which had been thoroughly dissolved, was added to the wells. Next, the SDS-equilibrated gel was immersed in the agarose, and the SDS-equilibrated gel was removed with tweezers. The equilibrated gel and the second-dimensional electrophoresis gel were brought into close contact with each other. After confirming that the agarose had solidified sufficiently with both gels in close contact, electrophoresis was carried out at a constant voltage of 200 V for approximately 45 minutes.

[0356] Fluorescent staining of gels The gel was fluorescently stained using SYPRO Ruby (Life Technologies).

[0357] First, the sealed container to be used was thoroughly washed with 98% (v / v) ethanol. After electrophoresis, the second-dimensional electrophoresis gel was removed from the SDS-PAGE machine and placed in the washed sealed container. It was then immersed in an aqueous solution containing 50% (v / v) methanol and 7% (v / v) acetic acid for 30 minutes twice. The aqueous solution was then replaced with water and the gel was immersed for 10 minutes. Next, the second-dimensional electrophoresis gel was immersed in 40 ml of SYPRO Ruby and shaken overnight at room temperature. The SYPRO Ruby was then removed, and the second-dimensional electrophoresis gel was washed with water and then shaken for 30 minutes in an aqueous solution containing 10% (v / v) methanol and 7% (v / v) acetic acid. The aqueous solution was then replaced with water and shaken for at least 30 minutes.

[0358] analysis The second-dimensional electrophoresis gel that had undergone the above series of treatments was subjected to fluorescent electrophoresis using a Typhoon 9500 (GE). The light image was scanned. The results of 2D electrophoresis of proteins contained in salmon flesh are shown in the left panel of Figure 1A. Molecular weight marker bands can be seen on the left side of the gel photograph, and the position of the bands indicates a specific molecular weight (KDa).

[0359] Example 2: Antigen confirmation by immunoblotting (1) Antigen confirmation by immunoblotting was performed by following the procedure described in Example 1 up to the "second-dimensional SDS-PAGE" step, followed by the following steps of "transfer to membrane," "immunoblot," and "analysis."

[0360] Transfer to membrane The transfer onto the membrane was carried out using the following transfer device and transfer buffer. Transfer device: XCell SureLock Mini-Cell and XCell II Blot Module (Life Technologies) Transfer buffer: NuPAGE transfer buffer (x20) (Life Technologies) was diluted 20-fold with milliQ water before use.

[0361] Specifically, proteins in the second-dimensional electrophoresis gel were transferred to a membrane (PVDF membrane) according to the following procedure.

[0362] (1) The PVDF membrane was immersed in 100% methanol, then in milliQ water, and then transferred to a transfer buffer solution to hydrophilize the PVDF membrane.

[0363] (2) The sponge, filter paper, second-dimensional electrophoresis gel after second-dimensional SDS-PAGE, hydrophilized PVDF membrane, filter paper, and sponge were placed in this order, and a constant voltage of 30 V was applied for 1 hour in a transfer device.

[0364] Immunoblot Immunoblotting of the membrane was performed using the serum of Patient 1, who had a fish allergy, or the serum of a non-fish allergic subject as the primary antibody. Patient 1 had been diagnosed with immediate-type allergy to fish. This patient developed allergic symptoms to salmon, horse mackerel, conger eel, black porgy, mackerel, sea bream, cod, and yellowtail, and showed positive results in a skin prick test.

[0365] Membrane immunoblotting was performed according to the following procedure. (1) The transferred membrane was shaken in a 5% skim milk / PBST solution (PBS buffer containing 0.1% of the nonionic surfactant Tween 20) at room temperature for 1 hour. (2) The primary antibody was left to stand in a 5% serum / 5% skim milk / PBST solution at room temperature for 1 hour. (3) Wash with PBST solution (5 minutes x 3 times). (4) Anti-human IgE-HRP (horseradish peroxidase) was used as a secondary antibody and the sample was left to stand at room temperature for 1 hour in a solution diluted 5000 times with 5% skim milk / PBST solution. (5) Wash with PBST solution (5 minutes x 3 times). (6) The sections were left to stand for 5 minutes in Pierce Western Blotting Substrate Plus (Thermo).

[0366] analysis The membrane that had undergone the above series of treatments was subjected to fluorescent imaging using Typhoon 9500 (GE). The specimen was then scanned.

[0367] An immunoblot using the serum of fish-allergic patient 1 was compared with an immunoblot using the serum of a control subject without fish allergy. In an immunoblot performed on salmon proteins using the serum of fish-allergic patient 1 (Fig. 1A, right panel), nine spots were detected that were different from those detected when the serum of a subject without fish allergy was used (Fig. 3) and that were distinct from known salmon allergen proteins. The detected spots are shown on the immunoblot (Fig. 1A, right panel).

[0368] The molecular weights and isoelectric points of the above nine spots are as follows: Spot 1: Molecular weight 90-120 kDa, pI 3.0-6.0 Spot 2: Molecular weight 120-160 kDa, pI 4.0-7.0 Spot 3: Molecular weight 90-120 kDa, pI 5.0-8.0 Spot 4: Molecular weight 90-120 kDa, pI 6.0-8.0 Spot 5: Molecular weight 60-90 kDa, pI 3.0-6.0 Spot 6: Molecular weight 70-100 kDa, pI 5.0-7.0 Spot 7: molecular weight 40-70 kDa, pI 4.0-7.0 Spot 8: Molecular weight 40-70 kDa, pI 4.0-7.0 Spot 9: Molecular weight 20-50 kDa, pI 4.0-7.0 Example 3: Mass spectrometry and antigen identification (1) The antigens that produced the above nine spots were subjected to identification of the amino acid sequences by mass spectrometry.

[0369] Specifically, protein extraction and mass spectrometry were performed according to the following procedure. (1) For salmon meat, protein extraction, two-dimensional electrophoresis, and membrane transfer were performed according to the procedures in Examples 1 and 2, and the resulting mixture was analyzed using 0.008% Direct Blue / 40% ethanol / 10% vinegar. Staining was carried out by shaking with acid. (2) The sections were then decolorized by treating them with 40% ethanol and 10% acetic acid for 5 minutes three times, washed with water for 5 minutes, and air-dried. (3) The desired spot was cut out with a clean cutter blade and placed in a centrifuge tube. After hydrophilizing the membrane with 50 μL of methanol, it was washed twice with 100 μL of water and then centrifuged. 20 μL of 20 mM NH4HCO3·50% acetonitrile was added. (4) 1 μL of 1 pmol / μL lysyl endopeptidase (WAKO) was added, and the mixture was left to stand at 37°C for 60 minutes. The solution was then collected in a new centrifuge tube. 20 μL of 20 mM NH₄HCO₃·70% acetonitrile was added to the membrane, which was then left to soak for 10 minutes at room temperature. The membrane was then further collected. The membrane was then dissolved in 10 μL of 0.1% formic acid and 4% acetonitrile, and transferred to a tube. (5) After drying the collected solution under reduced pressure, it was diluted with Solution A (0.1% formic acid, 4% acetonitrile solution). The solution was dissolved in 15 μl and subjected to mass spectrometry (ESI-TOF6600, manufactured by AB Sciex). (6) Proteins were identified based on mass data obtained from the mass spectrometer by searching NCBI or UniProt.

[0370] result Mass spectrometry was performed on each spot, and the following amino acid sequences were detected. Spot 1: amino acid sequence shown in SEQ ID NO: 3 Spot 2: Amino acid sequences shown in SEQ ID NOs: 6 to 8 Spot 3: Amino acid sequences shown in SEQ ID NOs: 11 to 17 Spot 4: Amino acid sequences shown in SEQ ID NOs: 20 to 24 Spot 5: Amino acid sequences shown in SEQ ID NOs: 27 to 31 Spot 6: Amino acid sequences shown in SEQ ID NOs: 34 to 41 Spot 7: Amino acid sequences shown in SEQ ID NOs: 44 to 54 Spot 8: Amino acid sequences shown in SEQ ID NOs: 57 to 59 Spot 9: Amino acid sequences shown in SEQ ID NOs: 62 to 68 Furthermore, the mass data obtained from the mass spectrometer for each spot was analyzed using NCBI for spot 1 and UniProt for spots 2 to 9, and the following proteins were identified as the respective spots. Spot 1: Alpha-actinin-3 (NCBI protein accession number 10 ... accession number XP_014051545.1, DNA accession number XM_014196070.1) (amino acid sequence: sequence No. 2, the base sequence encoding it: SEQ ID NO: 1) Spot 2: EEF1A2 binding protein-like (UniProt protein accession number B5RI29, DNA accession number ACH85270.1) (amino acid sequence: SEQ ID NO: 5, encoding nucleotide sequence: SEQ ID NO: 4) Spot 3: Alpha-1,4-glucan phosphorylase (UniProt protein accession number B5DG55, DNA accession number ACH70729.1) (amino acid sequence: SEQ ID NO: 10, encoding nucleotide sequence: SEQ ID NO: 9) Spot 4: Elongation factor 2 (UniProt protein accession number C0H9N2, DNA accession number ACN10751.1) (amino acid sequence: SEQ ID NO: 19, and the base sequence encoding it: SEQ ID NO: 18) Spot 5: Heat shock cognate 70 kDa protein (UniProt protein accession number B5X3U6, DNA accession number No. ACI33977.1) (amino acid sequence: SEQ ID NO: 26, base sequence encoding same: SEQ ID NO: 25) Spot 6: Serotransferrin (UniProt protein accession number P79815, DNA accession number BAA13759.1) (amino acid sequence: SEQ ID NO: 33 and the base sequence encoding it: SEQ ID NO: 32) Spot 7: Myosin binding protein H-like (UniProt protein accession number B5DG45, DNA accession number ACH70719.1) (amino acid sequence: SEQ ID NO: 43, encoding nucleotide sequence: SEQ ID NO: 42) Spot 8: Desmin (Fragment) (UniProt protein accession number Q8UWF1, DNA accession number CAC83053.1) (amino acid sequence: SEQ ID NO: No. 56, and the base sequence encoding it: SEQ ID NO: 55) Spot 9: Capping protein (Actin filament) muscle Z-line beta (UniProt protein accession number B5DFX6, DNA accession number ACH70650.1) (amino acid sequence: SEQ ID NO: No. 61, and the base sequence encoding it: SEQ ID NO: 60) Example 4: Antigen confirmation in other fish species (1) Using nine kinds of fish meat other than salmon, including horse mackerel, conger eel, black porgy, mackerel, sea bream, cod, yellowtail, eel, and flounder, the same procedures as those described in Examples 1 and 2 were carried out. The antigen was confirmed.

[0371] The immunoblot using the serum of fish-allergic patient 1 was compared with the immunoblot using the serum of a control subject who was not allergic to fish. In the immunoblot performed using the serum of fish-allergic patient 1 for proteins contained in the flesh of each of the nine fish species (Fig. 2), unlike when the serum of a subject who was not allergic to fish was used (Fig. 3), spots corresponding to some of the nine spots obtained in salmon were detected.

[0372] The spots detected for each fish species are as follows: Horse mackerel: spots 1, 3-5, 8, and 9 Conger eel: spots 1, 3, 5, and 9 Black porgy: Spots 1-6, 8, and 9 Mackerel: Spots 1, 3, 5, 8, and 9 Thailand: Spots 1, 3-6, 8, and 9 Tara: Spots 1, 3, 5, 7-9 Yellowtail: Spots 1, 3-5, 8, 9 Eel: Spots 1, 3, 4, 6, 9 Flounder: Spots 1, 4, and 6 Example 5: Antigen confirmation by immunoblotting (2) As in Example 2, an immunoblot using serum from a fish-allergic patient was compared with an immunoblot using serum from a control subject without a fish allergy. In an immunoblot performed on salmon proteins using serum from fish-allergic patient 2 (Fig. 1B, left), seven of the nine spots detected in Example 2 (spots 1-6, 9) were detected, as well as three new spots (spots 11, 12, and 14) that were different from those detected when serum from a subject without a fish allergy was used (Fig. 3) and that were different from known salmon allergen proteins. A total of 10 spots detected are shown on the immunoblot (Fig. 1B, left).

[0373] The molecular weights and isoelectric points of the three newly detected spots are as follows: Spot 11: molecular weight 90-110, pI 6.5-7.0 Spot 12: molecular weight 120-140, pI 5.0-6.0 Spot 14: molecular weight 30-40, pI 6.5-7.5 Example 6: Mass spectrometry and antigen identification (2) As in Example 3, the amino acid sequences of the antigens that produced the three newly detected spots were identified by mass spectrometry.

[0374] Mass spectrometry was performed on each spot, and the following amino acid sequences were detected. Spot 11: Amino acid sequences represented by SEQ ID NOs: 111 to 119 Spot 12: Amino acid sequences represented by SEQ ID NOs: 122 to 136 Spot 14: Amino acid sequences represented by SEQ ID NOs: 145 to 149 Furthermore, the mass data obtained from the mass spectrometer for each spot was analyzed by NCBI, and each spot was identified as the following protein. Spot 11: glycogen phosphorylase, muscle form-like (NCBI protein accession number XP_013984904.1, DNA accession number XM_014129429.1) (amino acid sequence: SEQ ID NO: 110, cloned Base sequence to be loaded: SEQ ID NO: 109 Spot 12: myosin-binding protein C, fast-type-like (NCBI protein accession number XP_014014310.1, DNA accession number XM_014158835.1) (amino acid sequence: SEQ ID NO: 121 , and the base sequence encoding it: SEQ ID NO: 120) Spot 14: L-lactate dehydrogenase A chain-like (NCBI protein accession number XP_014003141.1, DNA accession number Accession number XM_014147666.1) (amino acid sequence: SEQ ID NO: 144, encoding nucleotide sequence: SEQ ID NO: 143) Example 7: Antigen confirmation by immunoblotting (3) As in Example 2, an immunoblot using serum from a fish-allergic patient was compared with an immunoblot using serum from a control subject without a fish allergy. In an immunoblot performed on salmon proteins using serum from fish-allergic patient 3 (Fig. 1B, right panel), two of the nine spots detected in Example 2 (spots 7 and 8) were detected, as well as two new spots (spots 10 and 13) that were different from those detected when serum from a subject without a fish allergy was used (Fig. 3) and that were different from known salmon allergen proteins. The four spots detected are shown on the immunoblot (Fig. 1B, right panel).

[0375] The molecular weights and isoelectric points of the two newly detected spots are as follows: Spot 10: molecular weight 200-230, pI 4.5-5.5 Spot 13: molecular weight 45-55, pI 4.0-5.5 Example 8: Mass spectrometry and antigen identification (3) As in Example 3, the amino acid sequences of the antigens that produced the two newly detected spots were identified by mass spectrometry.

[0376] Mass spectrometry was performed on each spot, and the following amino acid sequences were detected. Spot 10: Amino acid sequences shown in SEQ ID NOs: 71 to 108 Spot 13: Amino acid sequences represented by SEQ ID NOs: 139 to 142 Furthermore, the mass data obtained from the mass spectrometer for each spot was analyzed by NCBI, and each spot was identified as the following protein. Spot 10: Myosin heavy chain, fast skeletal muscle-like (myosin heavy chain, fast skeletal muscle-like) (NCBI protein accession number XP_014039990.1, DNA accession number XM_014184515.1) (amino acid sequence: SEQ ID NO: 70, encoding nucleotide sequence: SEQ ID NO: 69) Spot 13: ATP synthase subunit beta, mitochondrial (NCBI protein accession number XP_014007238.1, DNA accession number XM_014151763.1) (amino acid sequence: SEQ ID NO: 138, coding for (SEQ ID NO: 137) Example 9: Antigen confirmation in other fish species (2) Antigens were confirmed in the same manner as in Example 4 using the flesh of six types of fish: horse mackerel, conger eel, salmon, mackerel, sea bream, and cod. Immunoblots using the serum of fish allergy patient 4 were compared with immunoblots using the serum of a control subject without fish allergy. In immunoblots (Figure 4) performed on proteins contained in the flesh of each of the six types of fish using the serum of fish allergy patient 4, unlike when the serum of a subject without fish allergy was used (Figure 3), spots corresponding to some of the total 14 spots obtained with the serum of fish allergy patients 1 to 3 were detected in salmon.

[0377] The spots detected for each fish species are as follows: Horse mackerel: Spots 1, 3, 4, 8, 10, 11, and 14 Conger eel: spots 1, 4, 10, and 11 Salmon: Spots 2, 8, and 10-14 Mackerel: Spots 4, 8, 10, 11, 13, and 14 Ties: Spots 1, 4, 6, 8, 10, and 13 Tara: Spots 1, 5, 8, 10, 11, and 13 Example 10: Epitope Identification Epitopes of fish allergen components Epitopes were identified for fish allergen components using the following procedure.

[0378] (A) Epitope mapping (1) Epitope mapping was performed using a library of overlapping peptides (15 amino acids in length) corresponding to amino acid sequences identified as fish allergens and known fish allergens such as aldolase, beta-enolase, and glyceraldehyde-3-phosphate dehydrogenase (Non-Patent Documents 2-4). Specifically, the sequences were SEQ ID NO: 2 for α-actinin-3, SEQ ID NO: 5 for EEF1A2 binding protein-like, SEQ ID NO: 10 for α-1,4-glucan phosphorylase, SEQ ID NO: 19 for elongation factor 2, SEQ ID NO: 26 for heat shock cognate 70 kDa protein, SEQ ID NO: 33 for serotransferrin, SEQ ID NO: 43 for myosin binding protein H-like, SEQ ID NO: 56 for desmin (fragment), SEQ ID NO: 61 for capping protein (actin filament) muscle Z-line beta, and SEQ ID NO: 10 for myosin heavy chain, fast skeletal muscle. A library of overlapping peptides was prepared based on the amino acid sequences of SEQ ID NO: 70 for muscle-like, SEQ ID NO: 110 for glycogen phosphorylase, muscle form-like, SEQ ID NO: 121 for myosin-binding protein C, fast-type-like, SEQ ID NO: 138 for ATP synthase subunit β, mitochondrial, SEQ ID NO: 144 for L-lactate dehydrogenase A chain-like, SEQ ID NO: 445 for aldolase, SEQ ID NO: 446 for beta-enolase, and SEQ ID NO: 447 for glyceraldehyde-3-phosphate dehydrogenase.

[0379] Each synthesized peptide was shifted by 10 amino acids, i.e., each peptide overlaps with the previous and subsequent peptides by 5 amino acids each.

[0380] For the preparation of peptide arrays, the Intavis CelluSpots™ technology was used, which follows the procedure: (1) Peptide arrays were deposited on amino-modified cellulose discs using an automated synthesizer (Intavis MultiPep (2) synthesize the target peptide using the amino-modified cellulose disc. (3) The cellulose-binding peptides were spotted onto a coated glass slide to prepare a peptide array. (1) Peptide synthesis Peptide synthesis was performed stepwise on amino-modified cellulose discs in a 384-well synthesis plate using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Specifically, amino acids bearing Fmoc groups were activated with a solution of N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in dimethylformamide (DMF) and added dropwise to the cellulose discs to couple the Fmoc-conjugated amino acids to the amino groups on the cellulose discs. The unreacted amino groups were capped with acetic anhydride and washed with DMF. The Fmoc groups were then removed from the amino groups of the amino acids bound to the cellulose discs by treating with piperidine and washing with DMF. The amino termini were then extended by repeating the coupling, capping, and Fmoc removal steps. (2) Dissolution of amino-modified cellulose discs The cellulose disk with the peptide of interest bound thereto obtained in the above "(1) Peptide synthesis" was transferred to a 96-well plate, and trifluoroacetic acid (T The peptides were treated with a side-chain deprotection mixture of TFA, dichloromethane, triisopropylsilane (TIPS), and water. The deprotected cellulose-bound peptides were then dissolved in a mixture of TFA, perfluoromethanesulfonic acid (TFMSA), TIPS, and water, precipitated with tetrabutyl methyl ether (TBME), resuspended in dimethyl sulfoxide (DMSO), and mixed with a mixture of NaCl, Na citrate, and water to obtain a peptide solution for slide spotting. (3) Spotting of cellulose-binding peptide solution The peptide solution for slide spotting obtained in the above "(2) Dissolution of amino-modified cellulose disc" was transferred to the Intavis CelluSpots using the Intavis slide spotting robot. (trademark) slides and dried to prepare peptide arrays.

[0381] Using the peptide array, whether or not each peptide fragment binds to IgE antibodies in the serum of fish allergy patients through an antigen-antibody reaction was measured according to the following procedure. (1) The peptide was shaken in PBST at room temperature for 1 hour. (2) The cells were shaken overnight at room temperature in 5% serum / PBST. (3) The sections were washed with PBST solution (PBS buffer containing 0.1% of the nonionic surfactant Tween 20) for 5 minutes (×3). (4) Anti-human IgE antibody-HRP (1:5,000, PBST) was added and the mixture was shaken at room temperature for 1 hour. (5) Wash with PBST solution for 5 minutes (x 3 times). (6) Pierce ECL Plus Western Blotting Substrate (Thermo) was added and allowed to stand at room temperature for 5 minutes. (7) Using an Amersham Imager 600, the peptides treated in (1) to (6) above were analyzed. Chemiluminescence was measured.

[0382] The images obtained by the measurement in (7) above were analyzed using ImageQuant TL (GE Healthcare). The amount of chemiluminescence was quantified using the serum of four non-fish allergic subjects. The results were obtained using the serum of five patients. The value obtained by dividing the average value digitized from the image obtained from the fish allergy (average value of patient group / non-fish allergy) Peptides with an IgE value (average value of the subject group) of 1.5 or higher and a significant difference of 0.05 or less by the Mann-Whitney U test were determined to be peptides that bound specifically to IgE antibodies in patients.

[0383] (B) Epitope mapping (2): overlapping The sequences of peptides to which patient-specific IgE antibodies in serum bound by the above (A) (sequence numbers 150 to 154 for α-actinin-3, sequence number 155 for EEF1A2 binding protein-like, sequence numbers 156 and 157 for α-1,4-glucan phosphorylase, sequence numbers 158 and 159 for elongation factor 2, sequence numbers 160 to 162 for heat shock cognate 70 kDa protein, sequence numbers 163 to 167 for serotransferrin, sequence numbers 168 to 171 for myosin binding protein H-like, sequence numbers 172 to 174 for desmin (fragment), and sequence numbers 173 to 175 for capping protein (actin filament) muscle Z- SEQ ID NOs: 175 to 178 for myosin heavy chain, fast skeletal muscle-like, SEQ ID NOs: 179 to 185 for glycogen phosphorylase, muscle form-like, SEQ ID NO: 186 for glycogen phosphorylase, muscle form-like, SEQ ID NOs: 187 to 196 for myosin-binding protein C, fast-type-like, SEQ ID NO: 197 for ATP synthase subunit β, mitochondrial, SEQ ID NO: 198 for L-lactate dehydrogenase A chain-like, SEQ ID NO: 199 for aldolase, SEQ ID NOs: 200 to 202 for beta-enolase, and SEQ ID NOs: 203 and 204 for glyceraldehyde-3-phosphate dehydrogenase. Peptide sequences and amino acid sequences of allergen components containing the peptide sequences (SEQ ID NO: 2 for α-actinin-3, SEQ ID NO: 5 for EEF1A2 binding protein-like, SEQ ID NO: 10 for α-1,4-glucan phosphorylase, SEQ ID NO: 19 for elongation factor 2, SEQ ID NO: 26 for heat shock cognate 70 kDa protein, SEQ ID NO: 33 for serotransferrin, SEQ ID NO: 43 for myosin binding protein H-like, SEQ ID NO: 56 for desmin (fragment), SEQ ID NO: 61 for capping protein (actin filament) muscle Z-line beta, myosin heavy chain, fast skeletal muscle) A library of overlapping peptide fragments (length: 10 amino acids) was prepared using sequences obtained by adding the sequences before and after the peptides in SEQ ID NO: 70 for glycogen phosphorylase, muscle form-like, SEQ ID NO: 110 for myosin-binding protein C, fast-type-like, SEQ ID NO: 121 for ATP synthase subunit β, mitochondrial, SEQ ID NO: 138 for L-lactate dehydrogenase A chain-like, SEQ ID NO: 144 for L-lactate dehydrogenase A chain-like, SEQ ID NO: 445 for aldolase, SEQ ID NO: 446 for beta-enolase, and SEQ ID NO: 447 for glyceraldehyde-3-phosphate dehydrogenase, and epitope mapping was performed.

[0384] Each synthesized peptide was shifted by one amino acid, i.e., each peptide overlaps with the previous and subsequent peptides by 9 amino acids.

[0385] The library was prepared using the same procedure as in (A) above, and each peptide fragment was assayed for binding to IgE antibodies in the serum of patients and non-fish allergic subjects using the same method. Peptides shifted by one amino acid at a time lost or reduced binding to patient IgE antibodies, and the amino acids at positions where binding to IgE antibodies in non-fish allergic subjects occurred were determined to be amino acids that adversely affected the specificity of IgE antibody binding.

[0386] As in (A) above, the images obtained by the measurement were analyzed using ImageQuant TL (GE Healthcare). The amount of chemiluminescence was quantified using a chemiluminescence analyzer (manufactured by NIH Inc.). Peptides were determined to have bound to IgE antibodies in a patient-specific manner based on images obtained using the serum of five patients. When the value obtained using the sequences (SEQ ID NOS: 150-204) on which the overlapping was based was taken as 100%, peptides with a binding affinity of less than 20% were determined to have no binding affinity to IgE antibodies, peptides with a binding affinity of 20% to less than 50% were determined to have poor binding affinity to IgE antibodies but still had binding affinity, peptides with a binding affinity of 50% to less than 70% were determined to have some poor binding affinity to IgE antibodies but still had binding affinity, peptides with a binding affinity of 70% to less than 90% were determined to have almost no difference in binding affinity to IgE antibodies, and peptides with a binding affinity of 90% or more were determined to have no difference in binding affinity to IgE antibodies or good binding affinity to IgE antibodies. These peptides were determined to have retained binding affinity to IgE antibodies.

[0387] This analysis identified regions in the overlapping sequences that were important for binding to the patient's IgE antibodies.

[0388] (C) Epitope mapping (3): Alanine scanning For the amino acid sequence identified in (A) above, a library of peptide fragments was prepared by substituting one amino acid at a time with alanine from the amino-terminus using a method called alanine scan (Non-Patent Document 5), using the same method as above, and each peptide fragment was assayed for binding to IgE antibodies in the serum of patients and non-fish allergic subjects using the same method as above. The amino acids at positions where alanine substitution resulted in loss or reduction of binding to IgE antibodies in patients and binding to IgE antibodies in non-fish allergic subjects 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. Furthermore, alanine substitution maintained binding to IgE antibodies in patients, and reduced binding to IgE antibodies in non-fish allergic subjects. The amino acids at positions where no binding to IgE antibodies occurred were determined to be unimportant for expressing the original antigenicity and thus could be substituted.

[0389] As in (A) above, the images obtained by the measurement were analyzed using ImageQuant TL (GE Healthcare). The amount of chemiluminescence was quantified using a chemiluminescence analyzer (manufactured by NIH Inc.). Peptides were determined to have bound to IgE antibodies in a patient-specific manner based on images obtained using the serum of five patients. When the value obtained using the sequences (SEQ ID NOS: 150-204) on which the overlapping was based was taken as 100%, peptides with a binding affinity of less than 20% were determined to have no binding affinity to IgE antibodies, peptides with a binding affinity of 20% to less than 50% were determined to have poor binding affinity to IgE antibodies but still had binding affinity, peptides with a binding affinity of 50% to less than 70% were determined to have some poor binding affinity to IgE antibodies but still had binding affinity, peptides with a binding affinity of 70% to less than 90% were determined to have almost no difference in binding affinity to IgE antibodies, and peptides with a binding affinity of 90% or more were determined to have no difference in binding affinity to IgE antibodies or good binding affinity to IgE antibodies. These peptides were determined to have retained binding affinity to IgE antibodies.

[0390] This analysis revealed a consensus sequence important for the expression of original antigenicity in the region of the overlapping sequences that is important for binding to the patient's IgE antibody.

[0391] (D)Result As a result of the epitope mapping in (A) above, epitopes Nos. 1 to 5 (peptides having the amino acid sequences of SEQ ID NOs: 150 to 154, respectively) were identified for α-actinin-3, epitope No. 6 (peptide having the amino acid sequence of SEQ ID NO: 155) for EEF1A2 binding protein-like, epitope Nos. 7 and 8 (peptides having the amino acid sequences of SEQ ID NOs: 156 and 157, respectively) for α-1,4-glucan phosphorylase, epitope Nos. 9 and 10 (peptides having the amino acid sequences of SEQ ID NOs: 157 and 108, respectively) for elongation factor 2, and epitope Nos. 11 and 12 (peptides having the amino acid sequences of SEQ ID NOs: 158 and 159, respectively) for EEF1A2 binding protein-like. epitopes Nos. 11 to 13 for heat shock cognate 70 kDa protein (peptides having the amino acid sequences of SEQ ID NOs: 160 to 162, respectively); epitopes Nos. 14 to 18 for serotransferrin (peptides having the amino acid sequences of SEQ ID NOs: 163 to 167, respectively); epitopes Nos. 19 to 22 for myosin binding protein H-like (peptides having the amino acid sequences of SEQ ID NOs: 168 to 171, respectively); epitopes Nos. 23 to 25 (peptides having the amino acid sequences of SEQ ID NOs: 172 to 174, respectively) for desmin (fragment); epitopes Nos. 26 to 29 (peptides having the amino acid sequences of SEQ ID NOs: 175 to 178, respectively) for capping protein (actin filament) muscle Z-line beta; epitopes Nos. 30 to 36 (peptides having the amino acid sequences of SEQ ID NOs: 179 to 185, respectively) for myosin heavy chain, fast skeletal muscle-like; and epitopes Nos. 31 to 32 (peptides having the amino acid sequences of SEQ ID NOs: 179 to 185, respectively) for glycogen. Epitope No. 37 (peptide having the amino acid sequence of SEQ ID NO: 186) for phosphorylase, muscle form-like; epitopes Nos. 38 to 47 (peptides having the amino acid sequences of SEQ ID NOs: 187 to 196, respectively) for myosin-binding protein C, fast-type-like; epitope No. 48 (peptide having the amino acid sequence of SEQ ID NO: 197) for ATP synthase subunit β, mitochondrial; and epitope No.It was confirmed that patient-specific IgE antibodies bound to epitope No. 49 (peptide having the amino acid sequence of SEQ ID NO: 198) for aldolase, epitope No. 50 (peptide having the amino acid sequence of SEQ ID NO: 199) for aldolase, epitopes Nos. 51 to 53 (peptides having the amino acid sequences of SEQ ID NOs: 200 to 202) for beta-enolase, and epitopes Nos. 54 and 55 (peptides having the amino acid sequences of SEQ ID NOs: 203 and 204) for glyceraldehyde-3-phosphate dehydrogenase. The peptide sequences of these epitopes correspond to the allergen components identified in Examples 2, 3, and 5 to 8, as well as known allergen components. Table 2 shows which parts of the sequences of aldolase, beta-enolase, and glyceraldehyde-3-phosphate dehydrogenase correspond to which sequences.

[0392] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] For each epitope, as a result of (B) and (C) above, the following amino acids important for binding between the epitope sequence and IgE antibodies of allergic patients were identified.

[0393] (1) Epitope 1 Within epitope 1 (SEQ ID NO: 150), regions important for binding to the patient's IgE antibody were identified as SEQ ID NO: 207, which corresponds to amino acids 1 to 10, and SEQ ID NO: 210, which corresponds to amino acids 9 to 14.

[0394] It was identified that the amino acids 7 to 9 of SEQ ID NO: 207, which is the region important for binding, are particularly important for binding to IgE antibodies of allergy patients, and that the amino acids 1 and 10 are amino acids that affect binding to IgE antibodies of allergy patients. Substitution of amino acids 2 to 6 of SEQ ID NO: 207 with alanine did not affect binding to IgE antibodies of allergy patients.

[0395] Furthermore, the amino acids 1, 2, and 6 of SEQ ID NO: 210, which are the region important for binding, were identified as being particularly important for binding to IgE antibodies from allergy patients. Substitution of amino acids 3 to 5 of SEQ ID NO: 210 with alanine did not affect the binding to IgE antibodies from allergy patients.

[0396] Epitopes 2 to 55 were similarly identified, and the results are summarized in Table 3 below.

[0397] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] Example 11: Confirmation of epitope cross-reactivity Epitope No. 1 having the amino acid sequence "SMVLVKMKEIAEAYL" (SEQ ID NO: 160) found in the heat shock cognate 70 kDa protein of salmon in Table 3 above Regarding the amino acid sequence "EXXXXYL" (SEQ ID NO: 248) in which amino acids other than particularly important amino acids in the region important for binding (SEQ ID NO: 250) in 1 are replaced with arbitrary amino acids (X), As a result of searching for proteins with the same sequence on NCBI, we found the sequence "AMVLVKMKETAEAYL" (sequence number: XP_019944736.1) of the heat shock 70 kDa protein of Japanese flounder. The amino acid sequence of column number 451 was found.

[0398] The binding of peptides containing the amino acid sequences of SEQ ID NOs: 160 and 451 to IgE antibodies from one patient allergic to salmon and flounder was confirmed by ELISA. The peptides were synthesized using the Fmoc method so that they were biotinylated at the N-terminus.

[0399] Specifically, the ELISA was performed according to the following procedure. (1) The biotinylated peptide was prepared in PBST to a concentration of 1 μg / mL. (2) 40 μL of each peptide solution was added to each well of a streptavidin-coated 384-well plate, and the plate was shaken at room temperature for 1 hour. After recovering the solution, the plate was washed five times with PBS. (3) Add 40 μL of 5-fold diluted Blotting One (diluted with MQ) and shake at room temperature for 15 minutes. After removing the solution, the cells were washed five times with PBS. (4) 40 μL of serum dilution solution (1:10, dilution solution: Blocking One, manufactured by Nacalai Tesque) was added, and the mixture was shaken at room temperature for 1 hour. After removing the solution, the mixture was washed five times with PBS. (5) 40 μL of secondary antibody dilution solution (1:5000, dilution solution: Blocking One, manufactured by Nacalai Tesque) was added, and the mixture was shaken at room temperature for 1 hour. After removing the solution, the mixture was washed five times with PBS. (6) 40 μL of 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific) was added and shaken at room temperature for 15 minutes. (7) 40 μL of 2 M H2SO4 was added, and the absorbance at 450 nm was measured.

[0400] The amino acid "AMVLVKMKETAEAYL" of this flounder heat shock 70kDa protein A peptide having the amino acid sequence "SMVLVKMKEIAEAYL" of the salmon heat shock cognate 70 kDa protein was synthesized in the same manner as in Example 10 (A). The serum samples were prepared in the order of salmon and flounder, and the serum samples from four fish-allergic subjects (combined) were used in the same manner to measure whether IgE antibodies bound to the serum. The results are shown in Figure 5.

[0401] As is clear from Figure 5, patient-specific IgE antibodies bound to peptides having an amino acid sequence derived from flounder heat shock 70 kDa protein, as did peptides having an amino acid sequence derived from salmon heat shock cognate 70 kDa protein. Therefore, epitope No. 11 was confirmed to be cross-reactive between salmon and flounder. In addition to epitope No. 11, cross-reactivity was also confirmed with epitopes No. 1 to No. 55 (SEQ ID NOS: 150 to 204) identified in Example 10.

[0402] These epitopes also cross-react with horse mackerel, conger eel, black porgy, mackerel, sea bream, cod, yellowtail, and eel, in addition to flounder, indicating that these epitopes can be used to detect antigen cross-reactivity across fish species.< / url:>

Claims

1. 1. A polypeptide that specifically binds to IgE antibodies of an allergic patient, comprising: (3α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 156, 157, 231 to 237; (11α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 186, 366 to 370; (14α) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 198, 418 to 420; is one of the following: Here, the allergy is an allergy to an allergen containing the polypeptide.

2. The polypeptide of claim 1, which has 500 or fewer amino acid residues.

3. A diagnostic kit for allergies, comprising at least one polypeptide according to claim 1 or 2, wherein the allergy is an allergy to an allergen comprising the polypeptide.

4. A diagnostic composition for allergies, comprising at least one of the polypeptides described in claim 1 or 2 as an antigen, wherein the allergy is an allergy to an allergen containing the polypeptide.

5. 1. A method for providing an indicator for diagnosing allergy in a subject, comprising the steps of: (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting binding of IgE antibodies to the antigen in a sample obtained from the subject; (iii) if binding of the subject's IgE antibodies to the antigen is detected, an indication that the subject is allergic is provided; wherein the antigen is at least one of the polypeptides according to claim 1 or 2, and the allergy is an allergy to an allergen comprising said polypeptide.

6. A pharmaceutical composition comprising at least one of the polypeptides according to claim 1 or 2.

7. 7. The pharmaceutical composition according to claim 6 for treating allergies, wherein the allergy is an allergy to an allergen comprising the polypeptide.

8. A tester for determining the presence or absence of an antigen in a subject, characterized in that it comprises an antibody that binds to at least one of the polypeptides according to claim 1 or 2.

9. One of the following primers: (a) a primer comprising a portion of the base sequence of a nucleic acid encoding the polypeptide of claim 1 or 2 and / or a portion of its complementary strand; or (b) a primer which is a part of at least one of the nucleotide sequences represented by SEQ ID NO: 109 or 143 and / or a primer which is a part of a sequence complementary to at least one of the nucleotide sequences represented by SEQ ID NO: 109 or 143; A tester for determining the presence or absence of an antigen in a subject, comprising:

10. A raw material or processed product characterized in that an antigen has been removed or reduced, said raw material or processed product being at least one of the polypeptides described in claim 1 or 2.

11. A method for producing a processed product in which antigens have been removed or reduced, the method comprising a step of confirming that the antigens have been removed or reduced during the production process of the processed product, wherein the antigen is at least one of the polypeptides described in claim 1 or 2.

Images