Antigens of allergy and epitopes thereof

Abstract

The invention relates to allergy antigens and epitopes thereof. Provided are a novel antigen for egg allergy, a diagnostic method and diagnostic kit for egg allergy, a pharmaceutical composition containing the antigen, an egg in which the antigen has been removed or reduced, an egg processed product, or a bird produced or hatched from the egg, a method for producing the egg processed product in which the antigen has been removed or reduced, and a method for producing the egg processed product in which the antigen has been removed or reduced. And the detector is used for judging whether the egg antigen exists in the object or not. The present invention also relates to a polypeptide comprising the antigen epitope, a diagnostic kit, a diagnostic composition and a diagnostic method for allergy comprising the polypeptide, a pharmaceutical composition comprising the polypeptide, and a raw material or a processed product in which an antigen comprising the polypeptide has been removed or reduced. The present invention further relates to a detector for determining the presence or absence of an antigen in a subject.

Description

[0001] The present application is a divisional application of an application filed on December 6, 2017, with application number 201780093516.7 and invention name “Antigens and epitopes of allergic reactions”. Technical Field

[0002] The present invention relates to a novel antigen for egg allergy. The present invention also relates to a diagnostic kit, a diagnostic composition and a diagnostic method for egg allergy. The present invention also relates to a pharmaceutical composition containing the antigen, an egg from which the antigen is removed or reduced, an egg processed product or a bird produced by or hatched from the egg. The present invention also relates to a method for producing an egg processed product from which the antigen is removed or reduced. The present invention further relates to a detector for determining whether an object has an egg antigen.

[0003] The present invention also relates to a polypeptide containing an antigenic epitope. The present invention also relates to a diagnostic kit, a diagnostic composition and a diagnostic method for allergic reactions containing the polypeptide. The present invention also relates to a pharmaceutical composition containing the polypeptide, and a raw material or processed product in which the polypeptide is removed or reduced. The present invention also relates to a method for producing a processed product in which the polypeptide is removed or reduced. The present invention further relates to a detector for determining whether an antigen containing the polypeptide is present in an object. Background Art

[0004] IgE antibodies specific to specific antigens (hereinafter, also referred to as allergens) are produced in the serum and tissues of allergy patients. The physiological consequences of the interaction between the IgE antibodies and the specific antigens cause allergic reactions. Antigens in a broad sense refer to foods / ingredients that cause allergic symptoms; in a narrow sense, they refer to proteins contained in foods / ingredients that are bound by specific IgE antibodies (hereinafter, also referred to as allergen components).

[0005] Most of the existing allergy detection reagents are prepared by simply grinding allergen candidate foods / ingredients to prepare antigen reagents (Patent Document 1). Therefore, among the allergen components contained in the existing antigen reagents, a positive reaction can only be detected in the allergy test when the binding with IgE antibodies exceeds the threshold content for determining a positive reaction, and the diagnostic efficiency is not yet sufficiently high.

[0006] As egg allergens, the substances shown in the following table are currently known (Non-Patent Document 1).

[0007] [Table 1]

[0008]

[0009] Several allergen components have been suggested in food / ingredients that are candidates for allergens and have been commercialized as test kits. However, in order to improve the reliability of allergy detection, it is necessary to cover specific allergen components, but the patient detection rate by measuring the above allergen components is still insufficient. Identification of new egg allergens is not only necessary to improve the accuracy of diagnostic reagents, but is also very important as a target for hypoallergenic foods, hypoallergenic ingredients, and therapeutic drugs.

[0010] On the other hand, regarding the separation / purification of proteins, in recent years, as a method for separating and purifying a plurality of proteins from a small amount of sample, a two-dimensional electrophoresis method is used, in which isoelectric electrophoresis is performed in the first dimension and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is performed in the second dimension. The applicant has so far developed a two-dimensional electrophoresis method with high separation capability (Patent Documents 2 to 5).

[0011] Allergen-specific IgE antibodies recognize and bind to an epitope that is a specific amino acid sequence in an allergen component. However, the current situation is that there are only a few examples (non-patent literature 2) in which allergen components are analyzed to epitopes, and analysis is extremely rare. In addition, there is no diagnostic kit for allergic reactions that utilizes a polypeptide containing an epitope in the current market.

[0012] Prior art literature

[0013] Patent Literature

[0014] Patent Document 1: Japanese Patent Application Laid-Open No. 2002-286716

[0015] Patent Document 2: Japanese Patent Application Laid-Open No. 2011-33544

[0016] Patent Document 3: Japanese Patent Application Laid-Open No. 2011-33546

[0017] Patent Document 4: Japanese Patent Application Laid-Open No. 2011-33547

[0018] Patent Document 5: Japanese Patent Application Laid-Open No. 2011-33548

[0019] Non-patent literature

[0020] Non-patent document 1: Allergen Nomenclature, WHO / IUIS Allergen Nomenclature Sub-Committee, [May 31, 2018], インターネット<URL: http: / / www.allergen.org / search.php? allergenname=&allergensource=gallus+dome sticus&TaxSource=&TaxOrder=&foodallerg=all&bioname=>

[0021] Non-patent document 2: Matsuo, H., et al., J. Biol. Chem., (2004), Vol. 279, No. 13, pp. 12135-12140 Summary of the invention

[0022] Problem that the invention aims to solve

[0023] The present invention provides a novel antigen for egg allergy. The present invention also provides a method for diagnosing egg allergy and a diagnostic kit. The present invention also provides a pharmaceutical composition containing the antigen, an egg or egg processed product in which the antigen is removed or reduced, or a bird that produces the egg or is hatched from the egg. The present invention further provides a detector for determining whether an object has an egg antigen.

[0024] The present invention also provides a polypeptide containing an antigenic epitope. The present invention also provides a diagnostic kit, a diagnostic composition and a diagnostic method for allergic reactions containing the polypeptide. The present invention also provides a pharmaceutical composition containing the polypeptide, and a raw material or processed product in which the antigen containing the polypeptide is removed or reduced. The present invention also relates to a method for manufacturing the processed product in which the antigen is removed or reduced. The present invention further provides a detector for determining whether an antigen containing the polypeptide is present in an object.

[0025] Solutions for solving problems

[0026] The present inventors have conducted intensive research on the identification of antigens that are the cause of egg allergy in order to solve the above problems. As a result, they have successfully identified a novel antigen that is specifically bound by IgE antibodies in the serum of patients with egg allergy. Based on this finding, the present invention has been completed.

[0027] That is, in one embodiment, the present invention is as follows.

[0028] [1] A diagnostic kit for egg allergy, comprising at least one of the following proteins (4) to (10) as an antigen:

[0029] (4) (4A) A protein comprising vitellogenin-3 or a mutant thereof, which is any one of the following proteins (4A-a) to (4A-e) as an egg allergy antigen:

[0030] (4A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:47;

[0031] (4A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 47;

[0032] (4A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 46;

[0033] (4A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 46; or

[0034] (4A-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 represented by SEQ ID NO:46; or

[0035] (4B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 47 to 57;

[0036] (5) (5A) A protein comprising vitellogenin-2 precursor (hereinafter, also referred to as vitellogenin-2) or a mutant thereof, which is any one of the following (5A-a) to (5A-e) as an egg allergy antigen:

[0037] (5A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 59;

[0038] (5A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 59;

[0039] (5A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 58;

[0040] (5A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 58; or

[0041] (5A-e) a protein containing 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 set forth in SEQ ID NO: 58; or

[0042] (5B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 59 to 104;

[0043] (6) (6A) Vitellogenin-2 precursor (hereinafter, also referred to as vitellogenin-2) or a mutant thereof, which is any one of the following proteins (6A-a) to (6A-e) as an egg allergy antigen:

[0044] (6A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:106;

[0045] (6A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 106;

[0046] (6A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 105;

[0047] (6A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 105; or

[0048] (6A-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 represented by SEQ ID NO: 105; or

[0049] (6B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 106 to 150;

[0050] (7) (7A) Apolipoprotein B precursor (hereinafter, also referred to as apolipoprotein B) or a mutant thereof, which is any one of the following proteins (7A-a) to (7A-e) as an egg allergy antigen:

[0051] (7A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:152;

[0052] (7A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 152;

[0053] (7A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 151;

[0054] (7A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 151; or

[0055] (7A-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 represented by SEQ ID NO: 151; or

[0056] (7B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 152 to 235;

[0057] (8) (8A) Apolipoprotein B precursor (hereinafter, also referred to as apolipoprotein B) or a mutant thereof, which is any one of the following proteins (8A-a) to (8A-e) as an egg allergy antigen:

[0058] (8A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 237;

[0059] (8A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 237;

[0060] (8A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 236;

[0061] (8A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 236; or

[0062] (8A-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 set forth in SEQ ID NO: 236; or

[0063] (8B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 237 to 249;

[0064] (9) (9A) Apolipoprotein B precursor (hereinafter, also referred to as apolipoprotein B) or a mutant thereof, which is any one of the following proteins (9A-a) to (9A-e) as an egg allergy antigen:

[0065] (9A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 251.

[0066] (9A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 251;

[0067] (9A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 250;

[0068] (9A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 250; or

[0069] (9A-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 represented by SEQ ID NO: 250; or

[0070] (9B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 251 to 260;

[0071] (10) (10A) Vitellogenin-2 precursor (hereinafter, also referred to as vitellogenin-2) or a mutant thereof, which is any one of the following proteins (10A-a) to (10A-e) as an egg allergy antigen:

[0072] (10A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 262;

[0073] (10A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 262;

[0074] (10A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 261;

[0075] (10A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 261; or

[0076] (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 represented by SEQ ID NO: 261; or

[0077] (10B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 262 to 271.

[0078] [2] A composition for diagnosing egg allergy, comprising at least one of the proteins specified in (4) to (10) in [1] above as an antigen.

[0079] [3] A method for providing an index for diagnosing egg allergy in a subject, comprising the following steps:

[0080] (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies;

[0081] (ii) detecting the binding of IgE antibodies in a sample obtained from the subject to the antigen;

[0082] (iii) when the subject's IgE antibody is detected to be bound to the antigen, providing an indication that the subject has an egg allergy;

[0083] Here, the antigen is at least one of the proteins defined as (4) to (10) in [1] above.

[0084] [4] A pharmaceutical composition comprising at least one of the proteins defined in (4) to (10) in [1] above.

[0085] [5] The pharmaceutical composition according to [4] above, which is used for treating egg allergy.

[0086] [6] An egg, an egg product or a bird, characterized in that it is an egg, an egg product or a bird that produces or is hatched from an antigen that has been removed or reduced, and the antigen is at least one of the proteins defined as (4) to (10) in [1] above.

[0087] [7] A detector for determining the presence or absence of an egg antigen in an object, characterized in that it contains an antibody that binds to at least one of the proteins specified as (4) to (10) in [1] above.

[0088] [8] A detector for determining the presence or absence of egg allergic antigens in an object, characterized in that it comprises a primer having a base sequence complementary to at least one of the base sequences shown in sequence numbers 46, 58, 105, 151, 236, 250 or 261.

[0089] [9] A method for producing an egg processed product from which antigens have been removed or reduced, comprising a step of confirming that the antigens have been removed or reduced during the production of the processed product, wherein the antigen is at least one of the proteins defined as (4) to (10) in [1] above.

[0090]

[10] An egg-derived antigen which is at least one of the proteins specified in (4) to (10) in [1] above and causes an egg allergy.

[0091] The present inventors have also successfully discovered epitopes of egg-derived antigens including the above-mentioned antigens.

[0092] Epi-position has shorter amino acid sequence, therefore as long as different allergen compositions also have same amino acid sequence, then this IgE antibody can be combined with multiple allergen compositions.There is common epi-position in different allergen compositions, and as a result, because both are combined with the IgE antibody of allergy patient, so this antigen has intersectionality.Therefore, the epi-position limited by the application can make the diagnosis of allergy, treatment and the detection of multiple allergen compositions that comprise this epi-position etc. that comprise intersectionality become possibility.

[0093] The present invention has been completed based on this finding. That is, in another embodiment, the present invention is as follows.

[0094]

[11] A polypeptide that specifically binds to IgE antibodies of allergy patients, which is any one of the following:

[0095] (1β) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 272 to 295 and 319 to 525;

[0096] (2β) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 296 to 303 and 526 to 552;

[0097] (3β) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 304 to 311 and 553 to 625;

[0098] (4β) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 312 to 318 and 626 to 682.

[0099]

[12] The polypeptide according to

[11] above, wherein the number of amino acid residues is 500 or less.

[0100]

[13] A diagnostic kit for allergy, comprising at least one of the polypeptides described in

[11] or

[12] above.

[0101]

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

[11] or

[12] above as an antigen.

[0102]

[15] A method for providing an index for diagnosing an allergic reaction in a subject, comprising the following steps:

[0103] (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies;

[0104] (ii) detecting the binding of IgE antibodies in a sample obtained from the subject to the antigen;

[0105] (iii) when the binding of the subject's IgE antibody to the antigen is detected, providing an indication that the subject has an allergic reaction;

[0106] Here, the antigen is at least one of the polypeptides described in

[11] or

[12] above.

[0107]

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

[11] or

[12] above.

[0108]

[17] The pharmaceutical composition according to the above-mentioned

[16] , which is used for treating allergic reactions.

[0109]

[18] A detector for determining the presence or absence of an antigen in an object, characterized in that it comprises an antibody that binds to at least one of the polypeptides described in

[11] or

[12] above.

[0110]

[19] A detector for determining whether an antigen is present in an object, characterized in that it comprises any of the following primers:

[0111] (a) a primer comprising a portion of the base sequence of a nucleic acid encoding the polypeptide described in

[11] or

[12] above and / or its complementary strand; or

[0112] (b) A primer that is a portion of at least one of the base sequences shown in SEQ ID NOs: 46, 58, 105, 151, 236, 250 or 261; and / or a primer that is a portion of a sequence complementary to at least one of the base sequences shown in SEQ ID NOs: 46, 58, 105, 151, 236, 250 or 261.

[0113]

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

[11] or

[12] above.

[0114]

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

[11] or

[12] above.

[0115] Effects of the Invention

[0116] According to the present invention, a novel antigen for egg allergy can be provided. In the present invention, since a novel antigen (allergen component) causing egg allergy is identified, a highly sensitive diagnosis method and a diagnosis kit for egg allergy, a pharmaceutical composition containing the antigen, an egg or egg processed product from which the antigen is removed or reduced, or a bird that produces the egg or hatches from the egg, a method for producing an egg processed product from which the antigen is removed or reduced, and a detector for determining whether the egg antigen is present in an object can be provided.

[0117] In addition, the present invention can provide a novel polypeptide containing an antigenic epitope. By using the polypeptide of the present invention, it is possible to provide a highly sensitive diagnostic kit, diagnostic composition and diagnostic method for allergic reactions, a pharmaceutical composition containing the polypeptide, a detector for determining whether an antigen containing the polypeptide is present in an object, a raw material or processed product in which the polypeptide is removed or reduced, and a method for producing the processed product. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 This is a gel photo showing a protein electrophoresis diagram of proteins contained in the yolk of an egg (raw egg) using two-dimensional electrophoresis. The bands on the left side of the photo are bands of molecular weight markers, and the values ​​on the left side of the photo are the molecular weights (KDa) of each molecular weight marker. The values ​​on the top of the photo indicate the isoelectric point.

[0119] Figure 2 This is a photograph of a two-dimensional electrophoresis diagram of proteins contained in the yolk of an egg (raw egg), obtained by immunoblotting using the serum of the egg allergy patient 1.

[0120] Figure 3 This is a gel photograph showing a two-dimensional electrophoresis pattern of proteins contained in egg yolk (raw egg). Spots 1, 2, and 3 to which the serum of egg allergy patient 1 specifically reacted are shown enclosed by squares.

[0121] Figure 4This is a photograph of a two-dimensional electrophoresis diagram of proteins contained in egg yolk (raw egg) and immunoblotting using the serum of egg allergy patient 2. Spots 4 to 8 where the serum of egg allergy patient 2 specifically reacted are shown enclosed by squares.

[0122] Figure 5 This is a photograph of a two-dimensional electrophoresis diagram of proteins contained in egg yolk (raw egg), obtained by immunoblotting using the serum of egg allergy patient 3. Spots 9 and 10, where the serum of egg allergy patient 3 specifically reacted, are respectively indicated by being enclosed by squares.

[0123] Figure 6 This is a photograph of a two-dimensional electrophoresis diagram of proteins contained in egg yolk (raw egg), obtained by immunoblotting using the serum of egg allergy patient 4. The spot 11 to which the serum of egg allergy patient 4 specifically reacted is indicated by a frame. DETAILED DESCRIPTION

[0124] The present invention will be described in detail below, but the present invention is not limited to these contents.

[0125] In this specification, unless otherwise defined, scientific terms and technical terms used in connection with the present invention have the meanings generally understood by those skilled in the art.

[0126] In this specification, allergic reaction refers to: in an organism sensitive to a certain antigen, when the antigen enters again, the state of allergic reaction that shows discomfort to the organism. Allergic reaction can occur when contacting with an antigen or taking the antigen. Here, contact refers to feeling an object, especially, for the human body, it refers to attachment to the skin, mucous membranes (eyes, lips, etc.). In addition, ingestion refers to being absorbed into the body, and is absorbed by means such as inhalation and oral administration. Allergic reactions produced when taking food are usually particularly referred to as food allergies. In a preferred embodiment, allergic reactions can also be food allergies. Most allergic diseases in food produce IgE antibodies specific to antigens in blood and tissues. IgE antibodies bind to mast cells or basophils. When the antigen specific to the IgE antibody enters the body of an allergic disease patient again, the antigen is combined with the IgE antibody bound to mast cells or basophils, and then the IgE antibody is cross-linked on the cell surface to produce the physiological effect of IgE antibody-antigen interaction. As their physiological effects, the release of histamine, 5-hydroxytryptamine, heparin, eotaxin or various leukotrienes can be listed. These released substances cause allergic reactions produced by the combination of IgE antibodies and specific antigens. In detail, IgE antibodies recognize and bind to epitopes that are specific amino acid sequences in specific antigens, and the allergic reactions produced by specific antigens are achieved through the above-mentioned pathways.

[0127] The allergic reaction targeted by the present invention is not particularly limited as long as it is an allergic reaction to an allergen containing the epitope used. Such allergic reactions may also include: allergic reactions to plants of the family Oleaceae, Asteraceae, Gramineae, Bromeliaceae, Juglandaceae, Cucurbitaceae, Fabaceae, etc.; allergic reactions to animals of the family Phasianidae, Bovidae, etc.; allergic reactions to seafood of the family Carangidae, Sparidae, Salmonidae, Penaeidae, etc. Examples of plants of the Oleaceae family include olives (scientific name: oleaeuropaea), plants of the Asteraceae family include artichokes (scientific name: Cynara scolymus), plants of the Gramineae family include wheat (scientific name: Triticum aestivum) and Aegilops tauschii, plants of the Bromeliaceae family include pineapples (scientific name: Ananas comosus), plants of the Juglandaceae family include walnuts (scientific name: Juglans regia), plants of the Cucurbitaceae family include pumpkins (scientific name: Cucurbita moschata), and plants of the Leguminosae family include soybeans (scientific name: Glycine max). Animals of the Phasianidae family include quails (scientific name: Conturnix japonica) and turkeys (scientific name: Meleagris gallopavo), and animals of the Bovidae family include cattle (scientific name: Bos taurus). Examples of seafood from the family Carangidae include greater amberjack (scientific name: Serioladumerili); examples of seafood from the family Sparidae include red sea bream (scientific name: Pagrus major); examples of seafood from the family Salmonidae include rainbow trout (scientific name: Oncorhynchus mykiss); examples of seafood from the family Penaeidae include Litopenaeus vannamei.

[0128] In this specification, eggs refer to bird eggs, preferably chicken eggs, unless otherwise specified. It should be noted that in this specification, fish eggs are marked as "fish eggs" to distinguish them from "eggs" which mainly refer to bird eggs.

[0129] In this specification, egg allergy refers to a state in which an allergic reaction occurs when a protein or the like contained in an egg is used as an antigen. Egg allergy refers to an allergic reaction that occurs when an antigen contained in an egg is contacted or ingested. Generally, an allergic reaction that occurs when a food is ingested is particularly referred to as a food allergy. Egg allergy may also be a food allergy.

[0130] In this specification, an antigen refers to a substance that causes an allergic reaction and is also called an allergen component. The antigen is preferably a protein.

[0131] In this specification, protein is a molecule having a structure in which natural amino acids are connected by peptide bonds. There is no particular limitation on the number of amino acids contained in the protein. In this specification, the term "polypeptide" also means: a molecule having a structure in which natural amino acids are connected by peptide bonds. There is no particular limitation on the number of amino acids contained in the polypeptide. "Polypeptide" is a concept that includes "protein". In addition, polypeptides formed by connecting 2 to 50 amino acids by peptide bonds are sometimes particularly referred to as peptides. In addition, with regard to amino acids, when optical isomers may exist, L-forms are indicated unless otherwise specified. The notation of the amino acid sequence of proteins, polypeptides or peptides used in this specification is as follows: Based on standard usage and the notation commonly used in the art, amino acids are indicated by single-letter notations, the left direction is the amino terminal direction and the right direction is the carboxyl terminal direction. It should be noted that in the single-letter notation of amino acids, X means: any substance having an amino group and a carboxyl group that can bind to the amino acids at both ends can be used, and in particular, any of the 20 natural amino acids can be used. The residue represented by X is an amino acid residue at a site where binding to IgE antibodies of allergy patients is maintained even when substituted with alanine by alanine scanning as shown in Example 6. It is well known to those skilled in the art that there is a high probability that such a site can maintain binding to the IgE antibody even when substituted with any other amino acid.

[0132] Antigen limitation

[0133] The above-mentioned method was used to define the antigens of egg allergy among the proteins contained in the egg. Specifically, the raw eggs were separated into yolk and white, and the egg proteins contained therein were subjected to two-dimensional electrophoresis under the following conditions.

[0134] The first dimension electrophoresis uses a gel with a gel length of 5 to 10 cm and a pH range of 3 to 10 as a gel for isoelectric point electrophoresis, and a pH gradient of the gel relative to the electrophoresis direction, when the total length of the gel is set as 1, the gel length to pH 5 is set as a, the gel length of pH 5 to 7 is set as b, and the gel length above pH 7 is set as c, 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. Specifically, IPG gel Immobiline Drystrip (pH 3-10 NL) manufactured by GE Healthcare Bioscience (hereinafter referred to as GE) is used for isoelectric point electrophoresis. The electrophoresis apparatus used is IPGphor manufactured by GE. The upper limit of the electrophoresis instrument current value was set to 75 μA per gel, and the voltage program was set as follows: (1) the constant voltage was increased from 300 V to 750 Vhr to perform a constant voltage process (the current variation amplitude during 30 minutes of electrophoresis before the end of this process was 5 μA); (2) the voltage was gradually increased from 300 Vhr to 1000 V; (3) further, the voltage was gradually increased from 4500 Vhr to 5000 V; (4) thereafter, the first-dimensional isoelectric point electrophoresis was performed at a constant voltage of 5000 V until the total Vhr reached 12000.

[0135] The second dimension electrophoresis uses a polyacrylamide gel in which the gel concentration at the base end of the swimming direction is set to 3-6% and the gel concentration at the front end of the swimming direction is set to be higher than the gel concentration at the base end of the swimming direction. Specifically, NuPAGE 4-12% Bris-Tris Gels IPG well mini 1mm manufactured by Life Technologies was used for SDS-PAGE. The electrophoresis apparatus used was XCell SureLock Mini-Cell manufactured by Life Technologies. As the swimming buffer, 50mM MOPS, 50mM Tris base, 0.1% (w / v) SDS, 1mM EDTA were used, and the swimming was carried out for about 45 minutes at a constant voltage of 200V.

[0136] The results showed that when the egg protein was subjected to two-dimensional electrophoresis under the above conditions, the antigens in spots 1 to 3 specifically bound to the IgE antibodies of patients with egg allergy who were diagnosed with food-dependent exercise-induced allergy (Food-Dependent Exercise-Induced Anaphylaxis). In addition, it was found that in patients with other egg allergies, the antigens in spots 4 to 11 specifically bound to the IgE antibodies of the patients ( Figures 4 to 6 ).

[0137] antigen

[0138] (4) Spot 4 antigen

[0139] As a result of sequence identification of spot 4 by mass spectrometry, amino acid sequences of SEQ ID NOs: 48 to 57 were detected.

[0140] In addition, for spot 4, the mass spectrum data (SEQ ID NOs. 48 to 57) obtained by the mass spectrometer were compared and analyzed with the protein data of NCBI, and it was identified as vitellogenin-3 (amino acid sequence: SEQ ID NO. 47, base sequence encoding it: SEQ ID NO. 46).

[0141] Therefore, in the present application, the antigen of spot 4 is any of the following (4A-a) to (4A-e) and (4B).

[0142] (4A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 47.

[0143] (4A-b) A protein comprising an amino acid sequence having 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 with the amino acid sequence shown in SEQ ID NO: 47.

[0144] (4A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 46.

[0145] (4A-d) A protein comprising an amino acid sequence encoded by a base sequence having 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 base sequence shown in SEQ ID NO: 46.

[0146] (4A-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 represented by SEQ ID NO: 46.

[0147] (4B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 47 to 57, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 47, 50, 51, 53 and 54, preferably a protein comprising at least 2, 3, 4 or all of the amino acid sequences. The amino acid sequence represented by any of SEQ ID NOs: 47 to 57 may have one or more amino acids deleted, substituted, inserted or added.

[0148] The proteins of (4A-a) to (4A-e) and (4B) mentioned above may also be proteins that appear as spots with a molecular weight of 20 kDa to 50 kDa, preferably a molecular weight of 25 kDa to 40 kDa, more preferably around a molecular weight of 30 kDa to 35 kDa, and an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, and further preferably 4.0 to 6.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0149] In addition, the epitopes of the antigens of (4A-a) to (4A-e) and (4B) described above are present in amino acids 14 to 28 (SEQ ID NO: 296), amino acids 164 to 178 (SEQ ID NO: 297), amino acids 204 to 218 (SEQ ID NO: 298), amino acids 214 to 228 (SEQ ID NO: 299), amino acids 224 to 238 (SEQ ID NO: 300), amino acids 244 to 258 (SEQ ID NO: 301), amino acids 254 to 268 (SEQ ID NO: 302), and amino acids 284 to 298 (SEQ ID NO: 303) of SEQ ID NO: 47. When the antigen of spot 4 contains a mutation relative to the amino acid sequence of SEQ ID NO: 47, the sequence of SEQ ID NO: 47 may be maintained for at least one amino acid corresponding to the epitope.

[0150] Furthermore, from the viewpoint that even if the amino acid sequence is mutated, the sensitivity (degree to which patients can be judged as positive) and specificity (degree to which healthy subjects are not judged as positive) of binding to IgE antibodies remain, the antigen of spot 4 may be the following mutant.

[0151] The site in the epitope having the sequence of SEQ ID NO: 296 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 526 or 527. Therefore, in another preferred embodiment, when the antigen of spot 4 contains a mutation relative to the amino acid sequence of SEQ ID NO: 47, amino acids 14 to 28 in SEQ ID NO: 47 of the antigen may also be the amino acid sequence of SEQ ID NO: 526 or 527.

[0152] The site in the epitope having the sequence of SEQ ID NO: 297 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 529, 530 or 532. Therefore, in another preferred embodiment, when the antigen of spot 4 contains a mutation relative to the amino acid sequence of SEQ ID NO: 47, the amino acids 164 to 178 in SEQ ID NO: 47 of the antigen may also be the amino acid sequence of SEQ ID NO: 529, 530 or 532.

[0153] The site in the epitope having the sequence of SEQ ID NO: 298 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 536, 537, 545 or 546. Therefore, in another preferred embodiment, when the antigen of spot 4 contains a mutation relative to the amino acid sequence of SEQ ID NO: 47, the amino acids 204 to 218 in SEQ ID NO: 47 of the antigen may also be the amino acid sequence of SEQ ID NO: 536, 537, 545 or 546.

[0154] The site in the epitope having the sequence of SEQ ID NO: 299 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 547, 548 or 549. Therefore, in another preferred embodiment, when the antigen of spot 4 contains a mutation relative to the amino acid sequence of SEQ ID NO: 47, the amino acids 214 to 228 in SEQ ID NO: 47 of the antigen may also be the amino acid sequence of SEQ ID NO: 547, 548 or 549.

[0155] The site in the epitope having the sequence of SEQ ID NO: 301 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 552. Therefore, in another preferred embodiment, when the antigen of spot 4 contains a mutation relative to the amino acid sequence of SEQ ID NO: 47, the amino acids 244 to 258 in SEQ ID NO: 47 of the antigen may also be the amino acid sequence of SEQ ID NO: 552.

[0156] (5) Antigens of spots 5 and 6

[0157] As a result of sequence identification of spots 5 and 6 by mass spectrometry, amino acid sequences of SEQ ID NOs: 60 to 104 were detected.

[0158] In addition, for spots 5 and 6, the mass spectrum data (sequence numbers 60 to 104) obtained by the mass spectrometer were compared and analyzed with the protein data of NCBI, and identified as vitellogenin-2 precursor (amino acid sequence: sequence number 59, base sequence encoding it: sequence number 58).

[0159] Therefore, in the present application, the antigens of spots 5 and 6 are any of the following (5A-a) to (5A-e) and (5B).

[0160] (5A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 59.

[0161] (5A-b) A protein comprising an amino acid sequence having 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 with the amino acid sequence shown in SEQ ID NO: 59.

[0162] (5A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 58.

[0163] (5A-d) A protein comprising an amino acid sequence encoded by a base sequence having 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 base sequence shown in SEQ ID NO: 58.

[0164] (5A-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 represented by SEQ ID NO: 58.

[0165] (5B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 59 to 104, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 59, 60, 63, 65, 68, 69, 71, 73, 75 to 79, 81, 83 to 88, 91 to 94, 96 to 99 and 101 to 103, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or all of the amino acid sequences. The amino acid sequence represented by any of SEQ ID NOs: 59 to 104 may have one or more amino acids deleted, substituted, inserted or added.

[0166] The proteins of (5A-a) to (5A-e) and (5B) mentioned above may also be proteins that appear as spots with a molecular weight of 80 kDa to 260 kDa, preferably a molecular weight of 90 kDa to 200 kDa, and more preferably a molecular weight of about 110 kDa to 160 kDa, and an isoelectric point of 1.0 to 8.0, preferably 2.0 to 7.0, and even more preferably 3.0 to 6.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0167] In addition, the epitopes of the antigens of (5A-a) to (5A-e) and (5B) described above are present in amino acids 71 ​​to 85 (SEQ ID NO: 272), amino acids 141 to 155 (SEQ ID NO: 273), amino acids 471 to 485 (SEQ ID NO: 274), amino acids 521 to 535 (SEQ ID NO: 275), amino acids 611 to 625 (SEQ ID NO: 276), amino acids 651 to 665 (SEQ ID NO: 277), and amino acids 6 In the case where the antigens of spots 5 and 6 contain mutations relative to the amino acid sequence of SEQ ID NO: 59, the sequence of SEQ ID NO: 59 may be maintained for at least one amino acid corresponding to the epitope.

[0168] Furthermore, from the viewpoint that the sensitivity, specificity and the like of the binding property with IgE antibodies are retained even when the amino acid sequence is mutated, the antigens of spots 5 and 6 may be the following mutants.

[0169] The site in the epitope having the sequence of SEQ ID NO: 272 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 320 or 321. Therefore, in another preferred embodiment, when the antigens of spots 5 and 6 contain a mutation relative to the amino acid sequence of SEQ ID NO: 59, amino acids 71 ​​to 85 in SEQ ID NO: 59 of the antigen may also be the amino acid sequence of SEQ ID NO: 320 or 321.

[0170] The site in the epitope having the sequence of SEQ ID NO: 273 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 328. Therefore, in another preferred embodiment, when the antigens of spots 5 and 6 contain mutations relative to the amino acid sequence of SEQ ID NO: 59, amino acids 141 to 155 in SEQ ID NO: 59 of the antigen may also be the amino acid sequence of SEQ ID NO: 328.

[0171] The site in the epitope having the sequence of SEQ ID NO: 274 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 332 or 337. Therefore, in another preferred embodiment, when the antigens of spots 5 and 6 contain a mutation relative to the amino acid sequence of SEQ ID NO: 59, the amino acids 471 to 485 in SEQ ID NO: 59 of the antigen may also be the amino acid sequence of SEQ ID NO: 332 or 337.

[0172] The site in the epitope having the sequence of SEQ ID NO: 275 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 341, 348 or 352. Therefore, in another preferred embodiment, when the antigens of spots 5 and 6 contain a mutation relative to the amino acid sequence of SEQ ID NO: 59, the amino acids 521 to 535 in SEQ ID NO: 59 of the antigen may also be the amino acid sequence of SEQ ID NO: 341, 348 or 352.

[0173] The site in the epitope having the sequence of SEQ ID NO: 276 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 358. Therefore, in another preferred embodiment, when the antigens of spots 5 and 6 contain mutations relative to the amino acid sequence of SEQ ID NO: 59, the amino acids 611 to 625 in SEQ ID NO: 59 of the antigen may also be the amino acid sequence of SEQ ID NO: 358.

[0174] The site in the epitope having the sequence of SEQ ID NO: 277 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 361, 361, 370 or 371. Therefore, in another preferred embodiment, when the antigens of spots 5 and 6 contain a mutation relative to the amino acid sequence of SEQ ID NO: 59, the amino acids 651 to 665 in SEQ ID NO: 59 of the antigen may also be the amino acid sequence of SEQ ID NO: 361, 361, 370 or 371.

[0175] The site in the epitope having the sequence of SEQ ID NO: 278 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 376. Therefore, in another preferred embodiment, when the antigens of spots 5 and 6 contain mutations relative to the amino acid sequence of SEQ ID NO: 59, the amino acids 661 to 675 in SEQ ID NO: 59 of the antigen may also be the amino acid sequence of SEQ ID NO: 376.

[0176] The site in the epitope having the sequence of SEQ ID NO: 279 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 382, ​​383, 394, 395 or 403. Therefore, in another preferred embodiment, when the antigens of spots 5 and 6 contain a mutation relative to the amino acid sequence of SEQ ID NO: 59, the amino acids 711 to 725 in SEQ ID NO: 59 of the antigen may also be the amino acid sequence of SEQ ID NO: 382, ​​383, 394, 395 or 403.

[0177] The site in the epitope having the sequence of SEQ ID NO: 280 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 408. Therefore, in another preferred embodiment, when the antigens of spots 5 and 6 contain mutations relative to the amino acid sequence of SEQ ID NO: 59, the amino acids 741 to 755 in SEQ ID NO: 59 of the antigen may also be the amino acid sequence of SEQ ID NO: 408.

[0178] The site in the epitope having the sequence of SEQ ID NO: 281 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 415 or 416. Therefore, in another preferred embodiment, when the antigens of spots 5 and 6 contain a mutation relative to the amino acid sequence of SEQ ID NO: 59, the amino acids 751 to 765 in SEQ ID NO: 59 of the antigen may also be the amino acid sequence of SEQ ID NO: 415 or 416.

[0179] The site in the epitope having the sequence of SEQ ID NO: 282 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 420, 421, 427 or 428. Therefore, in another preferred embodiment, when the antigens of spots 5 and 6 contain a mutation relative to the amino acid sequence of SEQ ID NO: 59, the amino acids 881 to 895 in SEQ ID NO: 59 of the antigen may also be the amino acid sequence of SEQ ID NO: 420, 421, 427 or 428.

[0180] The site in the epitope having the sequence of SEQ ID NO: 285 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 431 or 432. Therefore, in another preferred embodiment, when the antigens of spots 5 and 6 contain a mutation relative to the amino acid sequence of SEQ ID NO: 59, the amino acids 1101 to 1115 in SEQ ID NO: 59 of the antigen may also be the amino acid sequence of SEQ ID NO: 431 or 432.

[0181] (6) Spot 7 antigen

[0182] As a result of sequence identification of spot 7 by mass spectrometry, an amino acid sequence of SEQ ID NOs: 107 to 150 was detected.

[0183] In addition, for spot 7, the mass spectrum data (SEQ ID NOs. 107 to 150) obtained by the mass spectrometer were compared and analyzed with the protein data of NCBI, and it was identified as vitellogenin-2 precursor (amino acid sequence: SEQ ID NO. 106, base sequence encoding it: SEQ ID NO. 105).

[0184] Therefore, in the present application, the antigen of spot 7 is any of the following (6A-a) to (6A-e) and (6B).

[0185] (6A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 106.

[0186] (6A-b) A protein comprising an amino acid sequence having 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 with the amino acid sequence shown in SEQ ID NO: 106.

[0187] (6A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 105.

[0188] (6A-d) A protein comprising an amino acid sequence encoded by a base sequence having 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 base sequence shown in SEQ ID NO: 105.

[0189] (6A-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 represented by SEQ ID NO: 105.

[0190] (6B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 106 to 150, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 44 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 106, 108, 110, 111, 113, 114, 116, 117, 120 to 124, 126 to 131, 133, 134, 136 to 138, 140 to 143, 145 to 147 and 149, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31 or all of the amino acid sequences. The amino acid sequence represented by any of SEQ ID NOs: 106 to 150 may have one or more amino acids deleted, substituted, inserted or added.

[0191] The proteins of (6A-a) to (6A-e) and (6B) mentioned above may also be proteins that appear as spots with a molecular weight of 110 kDa to 300 kDa, preferably a molecular weight of 130 kDa to 280 kDa, and more preferably a molecular weight of about 160 kDa to 260 kDa, and an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, and even more preferably 4.0 to 6.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0192] In addition, the epitopes of the antigens of (6A-a) to (6A-e) and (6B) described above are present in amino acids 71 ​​to 85 (SEQ ID NO: 272), amino acids 141 to 155 (SEQ ID NO: 273), amino acids 471 to 485 (SEQ ID NO: 274), amino acids 521 to 535 (SEQ ID NO: 275), amino acids 611 to 625 (SEQ ID NO: 276), amino acids 651 to 665 (SEQ ID NO: 277), amino acids 661 to 675 (SEQ ID NO: 278), amino acids 711 to 725 (SEQ ID NO: 279), amino acids 741 to 755 (SEQ ID NO: 280), amino acids 751 to 765 (SEQ ID NO: 281), amino acids 881 to 895 (SEQ ID NO: 282), amino acids 1011 to 1025 (SEQ ID NO: 283), 283), amino acids 1071-1085 (sequence number 284) and amino acids 1101-1115 (sequence number 285), amino acids 1207-1221 (sequence number 286), amino acids 1337-1351 (sequence number 287), amino acids 1417-1431 (sequence number 288), amino acids 1537-1551 (sequence number 289), amino acids 1587-1601 (sequence number 290), amino acids 1627-1641 (sequence number 291), amino acids 1687-1701 (sequence number 292), amino acids 1727-1741 (sequence number 293), amino acids 1757-1771 (sequence number 294) and amino acids 1797-1811 (sequence number 295). When the spot 7 antigen contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the sequence of SEQ ID NO: 106 may be maintained for at least one amino acid corresponding to the epitope.

[0193] Furthermore, from the viewpoint that the binding property with IgE antibody such as sensitivity and specificity is retained even if the amino acid sequence is mutated, the antigen of spot 7 may be the following mutant.

[0194] The site in the epitope having the sequence of SEQ ID NO: 272 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 320 or 321. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 71 ​​to 85 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 320 or 321.

[0195] The site in the epitope having the sequence of SEQ ID NO: 273 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 328. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 141 to 155 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 328.

[0196] The site in the epitope having the sequence of SEQ ID NO: 274 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 332 or 337. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 471 to 485 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 332 or 337.

[0197] The site in the epitope having the sequence of SEQ ID NO: 275 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 341, 348 or 352. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 521 to 535 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 341, 348 or 352.

[0198] The site in the epitope having the sequence of SEQ ID NO: 276 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 358. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 611 to 625 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 358.

[0199] The site in the epitope having the sequence of SEQ ID NO: 277 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 361, 361, 370 or 371. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 651 to 665 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 361, 361, 370 or 371.

[0200] The site in the epitope having the sequence of SEQ ID NO: 278 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 376. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 661 to 675 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 376.

[0201] The site in the epitope having the sequence of SEQ ID NO: 279 that is important for binding to the IgE antibody of the allergic patient is a site represented by an amino acid other than X in SEQ ID NO: 382, ​​383, 394, 395 or 403. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, amino acids 711 to 725 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 382, ​​383, 394, 395 or 403.

[0202] The site in the epitope having the sequence of SEQ ID NO: 280 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 408. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 741 to 755 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 408.

[0203] The site in the epitope having the sequence of SEQ ID NO: 281 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 415 or 416. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 751 to 765 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 415 or 416.

[0204] The site in the epitope having the sequence of SEQ ID NO: 282 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 420, 421, 427 or 428. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 881 to 895 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 420, 421, 427 or 428.

[0205] The site in the epitope having the sequence of SEQ ID NO: 285 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 431 or 432. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 1101 to 1115 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 431 or 432.

[0206] The site in the epitope having the sequence of SEQ ID NO: 286 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 437, 441 or 442. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 1207 to 1221 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 437, 441 or 442.

[0207] The site in the epitope having the sequence of SEQ ID NO: 287 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 450. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 1337 to 1351 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 450.

[0208] The site in the epitope having the sequence of SEQ ID NO: 288 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 459. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 1417 to 1431 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 459.

[0209] The site in the epitope having the sequence of SEQ ID NO: 290 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 467. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 1587 to 1601 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 467.

[0210] The site in the epitope having the sequence of SEQ ID NO: 291 that is important for binding to the IgE antibody of the allergic patient is a site represented by an amino acid other than X in SEQ ID NO: 474, 475, 477, 478, 488 or 489. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 1627 to 1641 in SEQ ID NO: 106 of the antigen may be the amino acid sequence of SEQ ID NO: 474, 475, 477, 478, 488 or 489.

[0211] The site in the epitope having the sequence of SEQ ID NO: 292 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 496, 501, 502 or 510. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 1687 to 1701 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 496, 501, 502 or 510.

[0212] The site in the epitope having the sequence of SEQ ID NO: 293 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 513 or 514. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 1727 to 1741 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 513 or 514.

[0213] The site in the epitope having the sequence of SEQ ID NO: 294 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 524 or 525. Therefore, in another preferred embodiment, when the antigen of spot 7 contains a mutation relative to the amino acid sequence of SEQ ID NO: 106, the amino acids 1757 to 1771 in SEQ ID NO: 106 of the antigen may also be the amino acid sequence of SEQ ID NO: 524 or 525.

[0214] (7) Spot 8 antigen

[0215] As a result of sequence identification of spot 8 by mass spectrometry, an amino acid sequence of SEQ ID NOs: 153 to 235 was detected.

[0216] In addition, for spot 8, mass spectrum data (SEQ ID NOs. 153 to 235) obtained by a mass spectrometer were compared and analyzed with protein data from NCBI, and it was identified as apolipoprotein B precursor (amino acid sequence: SEQ ID NO. 152, base sequence encoding it: SEQ ID NO. 151).

[0217] Therefore, in the present application, the antigen of spot 8 is any of the following (7A-a) to (7A-e) and (7B).

[0218] (7A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO:152.

[0219] (7A-b) A protein comprising an amino acid sequence having 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: 152.

[0220] (7A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 151.

[0221] (7A-d) A protein comprising an amino acid sequence encoded by a base sequence having 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 base sequence shown in SEQ ID NO: 151.

[0222] (7A-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 represented by SEQ ID NO: 151.

[0223] (7B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 152 to 235, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 83 sequences or all of the sequences. More preferably, it is a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 152, 154, 157-160, 163, 164, 167-172, 174, 175, 177, 178, 180, 182-185, 187, 189, 190, 192, 194-196, 198-200, 203-207, 209, 211, 212, 214-216, 220-229, 231 and 232, and preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 sequences or all of the sequences. The amino acid sequence represented by any of SEQ ID NOs: 152 to 235 may have one or more amino acids deleted, substituted, inserted or added.

[0224] The proteins of (7A-a) to (7A-e) and (7B) mentioned above may also be proteins that appear as spots with a molecular weight of 160 kDa to 550 kDa, preferably a molecular weight of 180 kDa to 400 kDa, and more preferably a molecular weight of around 200 kDa to 300 kDa, and an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, and even more preferably 4.0 to 6.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0225] In addition, the epitopes of the antigens of (7A-a) to (7A-e) and (7B) described above are present in amino acids 2556 to 2570 (SEQ ID NO: 304), amino acids 131 to 145 (SEQ ID NO: 305), amino acids 631 to 645 (SEQ ID NO: 306), amino acids 681 to 695 (SEQ ID NO: 307), amino acids 841 to 855 (SEQ ID NO: 308), amino acids 1131 to 1145 (SEQ ID NO: 309), amino acids 1201 to 1215 (SEQ ID NO: 310), and amino acids 1571 to 1585 (SEQ ID NO: 311) of SEQ ID NO: 152. When the antigen of spot 8 contains a mutation relative to the amino acid sequence of SEQ ID NO: 152, the sequence of SEQ ID NO: 152 may be maintained for at least one amino acid corresponding to the epitope.

[0226] Furthermore, from the viewpoint that the sensitivity, specificity and the like of the binding property with IgE antibody are retained even if the amino acid sequence is mutated, the antigen of spot 8 may be the following mutant.

[0227] The site in the epitope having the sequence of SEQ ID NO: 304 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 558. Therefore, in another preferred embodiment, when the antigen of spot 8 contains a mutation relative to the amino acid sequence of SEQ ID NO: 152, the amino acids 2556 to 2570 in SEQ ID NO: 152 of the antigen may also be the amino acid sequence of SEQ ID NO: 558.

[0228] The site in the epitope having the sequence of SEQ ID NO: 305 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 563, 564, 574 or 575. Therefore, in another preferred embodiment, when the antigen of spot 8 contains a mutation relative to the amino acid sequence of SEQ ID NO: 152, the amino acids 131 to 145 in SEQ ID NO: 152 of the antigen may also be the amino acid sequence of SEQ ID NO: 563, 564, 574 or 575.

[0229] The site in the epitope having the sequence of SEQ ID NO: 306 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 582. Therefore, in another preferred embodiment, when the antigen of spot 8 contains a mutation relative to the amino acid sequence of SEQ ID NO: 152, the amino acids 631 to 645 in SEQ ID NO: 152 of the antigen may also be the amino acid sequence of SEQ ID NO: 582.

[0230] The site in the epitope having the sequence of SEQ ID NO: 308 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 590, 596 or 603. Therefore, in another preferred embodiment, when the antigen of spot 8 contains a mutation relative to the amino acid sequence of SEQ ID NO: 152, the amino acids 841 to 855 in SEQ ID NO: 152 of the antigen may also be the amino acid sequence of SEQ ID NO: 590, 596 or 603.

[0231] The site in the epitope having the sequence of SEQ ID NO: 309 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 610, 611 or 612. Therefore, in another preferred embodiment, when the antigen of spot 8 contains a mutation relative to the amino acid sequence of SEQ ID NO: 152, the amino acids 1131 to 1145 in SEQ ID NO: 152 of the antigen may also be the amino acid sequence of SEQ ID NO: 610, 611 or 612.

[0232] The site in the epitope having the sequence of SEQ ID NO: 310 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 620 or 621. Therefore, in another preferred embodiment, when the antigen of spot 8 contains a mutation relative to the amino acid sequence of SEQ ID NO: 152, the amino acids 1201 to 1215 in SEQ ID NO: 152 of the antigen may also be the amino acid sequence of SEQ ID NO: 620 or 621.

[0233] The site in the epitope having the sequence of SEQ ID NO: 311 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 625. Therefore, in another preferred embodiment, when the antigen of spot 8 contains a mutation relative to the amino acid sequence of SEQ ID NO: 152, the amino acids 1571 to 1585 in SEQ ID NO: 152 of the antigen may also be the amino acid sequence of SEQ ID NO: 625.

[0234] (8) Spot 9 antigen

[0235] As a result of sequence identification of spot 9 by mass spectrometry, amino acid sequences of SEQ ID NOs: 238 to 249 were detected.

[0236] In addition, for spot 9, mass spectrum data (SEQ ID NOs. 238 to 249) obtained by a mass spectrometer were compared and analyzed with protein data from NCBI, and it was identified as apolipoprotein B precursor (amino acid sequence: SEQ ID NO. 237, base sequence encoding it: SEQ ID NO. 236).

[0237] Therefore, in the present application, the antigen of spot 9 is any of the following (8A-a) to (8A-e) and (8B).

[0238] (8A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 237.

[0239] (8A-b) A protein comprising 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: 237.

[0240] (8A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 236.

[0241] (8A-d) A protein comprising an amino acid sequence encoded by a base sequence having 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 base sequence shown in SEQ ID NO: 236.

[0242] (8A-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 represented by SEQ ID NO: 236.

[0243] (8B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 237 to 249, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 237 to 239, 241, 242, 244 and 246 to 249, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9 or all of the amino acid sequences. The amino acid sequence represented by any of SEQ ID NOs: 237 to 249 may have one or more amino acids deleted, substituted, inserted or added.

[0244] The proteins of (8A-a) to (8A-e) and (8B) mentioned above may also be proteins that appear as spots with a molecular weight of 20 kDa to 50 kDa, preferably a molecular weight of 25 kDa to 45 kDa, more preferably around a molecular weight of 30 kDa to 40 kDa, and an isoelectric point of 7.0 to 11.0, preferably 8.0 to 10.0, and even more preferably 9.0 to 10.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0245] In addition, the epitopes of the above-mentioned (8A-a) to (8A-e) and (8B) antigens are present in amino acids 131 to 145 (SEQ ID NO: 305) of SEQ ID NO: 237. When the spot 9 antigen contains a mutation relative to the amino acid sequence of SEQ ID NO: 237, the sequence in SEQ ID NO: 237 may be maintained for at least one amino acid corresponding to the epitope.

[0246] Furthermore, from the viewpoint that the binding property with IgE antibody such as sensitivity and specificity is retained even if the amino acid sequence is mutated, the antigen of spot 9 may be the following mutant.

[0247] The site in the epitope having the sequence of SEQ ID NO: 305 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 563, 564, 574 or 575. Therefore, in another preferred embodiment, when the antigen of spot 9 contains a mutation relative to the amino acid sequence of SEQ ID NO: 237, the amino acids 131 to 145 in SEQ ID NO: 237 of the antigen may also be the amino acid sequence of SEQ ID NO: 563, 564, 574 or 575.

[0248] (9) Spot 10 antigen

[0249] As a result of sequence identification of spot 10 by mass spectrometry, an amino acid sequence of SEQ ID NOs: 252 to 260 was detected.

[0250] In addition, the mass spectrum data (SEQ ID NOs. 252 to 260) obtained by the mass spectrometer were compared and analyzed with the protein data of NCBI, and spot 10 was identified as apolipoprotein B precursor (amino acid sequence: SEQ ID NO. 251, base sequence encoding it: SEQ ID NO. 250).

[0251] Therefore, in the present application, the antigen of spot 10 is any of the following (9A-a) to (9A-e) and (9B).

[0252] (9A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 251.

[0253] (9A-b) A protein comprising 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: 251.

[0254] (9A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 250.

[0255] (9A-d) A protein comprising an amino acid sequence encoded by a base sequence having 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 base sequence shown in SEQ ID NO: 250.

[0256] (9A-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 represented by SEQ ID NO: 250.

[0257] (9B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 251 to 260, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 251 to 254, 256, 257 and 260, preferably a protein comprising at least 2, 3, 4, 5, 6 or all of the amino acid sequences. The amino acid sequence represented by any of SEQ ID NOs: 251 to 260 may have one or more amino acids deleted, substituted, inserted or added.

[0258] The proteins of (9A-a) to (9A-e) and (9B) mentioned above may also be proteins that appear as spots with a molecular weight of 20 kDa to 50 kDa, preferably a molecular weight of 25 kDa to 45 kDa, more preferably around a molecular weight of 30 kDa to 40 kDa, and an isoelectric point of 7.0 to 11.0, preferably 8.0 to 10.0, and even more preferably 9.0 to 10.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0259] (10) Antigen of spot 11

[0260] As a result of sequence identification of spot 11 by mass spectrometry, the amino acid sequence of SEQ ID NOs: 263 to 271 was detected.

[0261] In addition, for spot 11, the mass spectrum data (sequence numbers 263 to 271) obtained by the mass spectrometer were compared and analyzed with the protein data of NCBI, and it was identified as vitellogenin-2 precursor (amino acid sequence: sequence number 262, base sequence encoding it: sequence number 261).

[0262] Therefore, in the present application, the antigen of spot 11 is any of the following (10A-a) to (10A-e) and (10B).

[0263] (10A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 262.

[0264] (10A-b) A protein comprising an amino acid sequence having 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: 262.

[0265] (10A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 261.

[0266] (10A-d) A protein comprising an amino acid sequence encoded by a base sequence having 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 base sequence shown in SEQ ID NO: 261.

[0267] (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 represented by SEQ ID NO: 261.

[0268] (10B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 262 to 271, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 262 to 265, 268 and 269, preferably a protein comprising at least 2, 3, 4, 5 or all of the amino acid sequences. The amino acid sequence represented by any of SEQ ID NOs: 262 to 271 may have one or more amino acids deleted, substituted, inserted or added.

[0269] The proteins (10A-a) to (10A-e) and (10B) mentioned above may also be proteins that appear as spots with a molecular weight of 15 kDa to 40 kDa, preferably a molecular weight of 17 kDa to 35 kDa, more preferably around a molecular weight of 20 kDa to 30 kDa, and an isoelectric point of 6.0 to 11.0, preferably 7.0 to 10.0, and even more preferably 8.0 to 10.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0270] In addition, the epitopes of the antigens of (10A-a) to (10A-e) and (10B) described above are present in amino acids 41 to 55 (SEQ ID NO: 288), amino acids 161 to 175 (SEQ ID NO: 289), and amino acids 211 to 255 (SEQ ID NO: 290) of SEQ ID NO: 262. When the antigen of spot 11 contains a mutation relative to the amino acid sequence of SEQ ID NO: 262, the sequence of SEQ ID NO: 262 may be maintained for at least one region of the amino acids corresponding to the epitope.

[0271] Furthermore, from the viewpoint that the sensitivity, specificity and the like of the binding property with IgE antibody are retained even if the amino acid sequence is mutated, the antigen of spot 11 may be the following mutant.

[0272] The site in the epitope having the sequence of SEQ ID NO: 288 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 459. Therefore, in another preferred embodiment, when the antigen of spot 11 contains a mutation relative to the amino acid sequence of SEQ ID NO: 262, the amino acids 41 to 55 in SEQ ID NO: 262 of the antigen may also be the amino acid sequence of SEQ ID NO: 459.

[0273] The site in the epitope having the sequence of SEQ ID NO: 290 that is important for binding to the IgE antibody of the allergic patient is the site represented by the amino acid other than X in SEQ ID NO: 467. Therefore, in another preferred embodiment, when the antigen of spot 11 contains a mutation relative to the amino acid sequence of SEQ ID NO: 262, the amino acids 211 to 255 in SEQ ID NO: 262 of the antigen may also be the amino acid sequence of SEQ ID NO: 467.

[0274] The proteins as antigens described in (4) to (10) above also include proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring opening, deamination, etc.

[0275] Preferably, the protein as an antigen in (4) to (10) above is an antigen for egg allergy.

[0276] In the present specification, the situation of "one or more amino acids are deleted, substituted, inserted or added" in an amino acid sequence means that in the amino acid sequence of interest, one or more amino acids are deleted (for example, 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1% of the amino acids relative to the total length of the amino acid sequence), or are substituted with other amino acids, or other amino acids are inserted, and / or other amino acids are added to form an amino acid sequence.

[0277] In the above, the substitution is preferably a conservative substitution. Conservative substitution refers to replacing a specific amino acid residue with a residue having similar physicochemical characteristics; any substitution can be made as long as the characteristics related to the original sequence structure are not substantially changed. For example, the substitution of amino acids can be made as long as the helix present in the original sequence is not destroyed, or other types of secondary structures that are characteristics of the original sequence are not destroyed. In the following, conservative substitutions of amino acid residues are exemplified according to the classification of residues that can be substituted, but the amino acid residues that can be substituted are not limited to the substances described below.

[0278] Group A: leucine, isoleucine, valine, alanine, methionine

[0279] Group B: Aspartic acid, Glutamic acid

[0280] Group C: Asparagine, Glutamine

[0281] Group D: lysine, arginine

[0282] Group E: Serine, Threonine

[0283] Group F: Phenylalanine, Tyrosine

[0284] In the case of non-conservative substitutions, one of the above-mentioned types can also be replaced with a member of another type. For example, in order to exclude unexpected sugar chain modifications, the amino acids in the above-mentioned B, D, and E groups can also be replaced with amino acids in other groups. Alternatively, in order to prevent folding in the three-dimensional protein, cysteine ​​can be deleted or replaced with other amino acids. Alternatively, in order to maintain the hydrophilicity / hydrophobicity balance, or to facilitate synthesis, the hydrophilicity index (hydropathy index) of amino acids related to amino acids as a hydrophobicity / hydrophilicity index (J. Kyte and R. Doolittle, J. Mol. Biol., Vol. 157, p. 105-132, 1982) can also be replaced with amino acids in order to increase the hydrophilicity.

[0285] As another embodiment, the substitution may be to an amino acid with less steric hindrance than the original amino acid, for example, substitution from group F to group A, B, C, D, or E; or substitution may be from a charged amino acid to an uncharged amino acid, for example, substitution from group B to group C. Thus, the binding property to the IgE antibody is improved.

[0286] In this specification, the % identity of two amino acid sequences can be determined by visual inspection and mathematical calculation. In addition, computer programs can also be used to determine the % identity. As such computer programs, for example, BLAST and ClustalW can be listed. In particular, the various conditions (parameters) of the identity search using the BLAST program can be recorded according to Altschul et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997), from the National Center for Biotechnology Information (NCBI), DNA Data Bank of Japan (DDBJ) website, etc., publicly obtained (BLAST manual, Altschul et al. NCB / NLM / NIH Bethesda, MD 20894; Altschul et al.). In addition, programs such as genetic information processing software GENETYX Ver. 7 (GENETYX), DINASIS Pro (Hitachi Soft, Ltd.), Vector NTI (Infomax) can also be used to determine.

[0287] In this specification, the situation of "deletion, substitution, insertion or addition of one or more nucleotides" in the base sequence means that in the base sequence to be targeted, one or more nucleotides are deleted (for example, 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1% of the amino acids relative to the total length of the base sequence), or replaced with other nucleotides, or inserted with other nucleotides, and / or the base sequence formed by the addition of other nucleotides. It is preferred that no frame shift is generated in the sequence encoding the amino acid by the deletion, substitution, insertion or addition of the above nucleotides.

[0288] In this specification, the % identity of two base sequences can be determined by visual inspection and mathematical calculation. In addition, the % identity can also be determined using a computer program. As such a sequence comparison computer program, for example, the BLASTN program (Altschul et al. (1990) J. Mol. Biol. 215: 403-10) available from the website of the National Library of Medicine of the United States https: / / blast.ncbi.nlm.nih.gov / Blast.cgi can be used, and the default parameters (DefaultParameter) are used to determine.

[0289] In this specification, "stringent conditions" refers to hybridization under moderate or highly stringent conditions. Specifically, as moderately stringent conditions, for example, a person skilled in the art with general skills can easily determine based on the length of the DNA. Basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Chapter 6-7, Cold Spring Harbor Laboratory Press, 2001. Preferably, moderately stringent conditions as hybridization conditions include: 1×SSC to 6×SSC, 42°C to 55°C; more preferably 1×SSC to 3×SSC, 45°C to 50°C; most preferably 2×SSC, 50°C. When the hybridization solution contains, for example, about 50% formamide, a temperature 5 to 15°C lower than the above temperature can be used. Washing conditions include: 0.5×SSC to 6×SSC, 40°C to 60°C. During hybridization and washing, 0.05% to 0.2%, preferably about 0.1% SDS is usually added. As highly stringent conditions, it can also be easily determined by a person skilled in the art based on the length of the DNA, for example. Generally, highly stringent conditions include hybridization and / or washing with a higher temperature and / or a lower salt concentration than moderately stringent conditions. For example, as hybridization conditions, there can be listed: 0.1×SSC to 2×SSC, 55°C to 65°C; more preferably 0.1×SSC to 1×SSC, 60°C to 65°C; most preferably 0.2×SSC, 63°C. As washing conditions, there can be listed: 0.2×SSC to 2×SSC, 50°C to 68°C, more preferably 0.2×SSC, 60 to 65°C.

[0290] Antigens can also be obtained by separation / purification from eggs (preferably chicken eggs) by combining protein purification methods known to those skilled in the art. Alternatively, antigens can also be obtained by expressing antigens as recombinant proteins using gene recombination techniques known to those skilled in the art, and then by separation / purification using protein purification methods known to those skilled in the art.

[0291] Examples of protein purification methods include methods utilizing solubility such as salting out and solvent precipitation; methods utilizing differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE; methods utilizing charge such as ion exchange chromatography and hydroxyapatite chromatography; methods utilizing specific affinity such as affinity chromatography; methods utilizing differences in hydrophobicity such as reversed-phase high performance liquid chromatography; and methods utilizing differences in isoelectric points such as isoelectric point electrophoresis.

[0292] The preparation of proteins using genetic recombination technology can be carried out as follows: prepare an expression vector containing a nucleic acid encoding an antigen, introduce the expression vector into a suitable host cell by gene introduction or transformation, culture the host cell under conditions suitable for the expression of the recombinant protein, and then recover the recombinant protein expressed in the host cell.

[0293] A "vector" is a nucleic acid that can be used to introduce a nucleic acid connected to it into a host cell; an "expression vector" is a vector that can guide 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 expression vector suitable for recombinant protein expression according to the type of host cell used.

[0294] "Host cell" is a cell into which a gene has been introduced or transformed by a vector. The host cell can be appropriately selected by a person skilled in the art according to the vector used. The host cell can be derived from a prokaryotic organism such as Escherichia coli (E. coli). When a prokaryotic cell such as Escherichia coli is used as a host, in order to facilitate the expression of the recombinant protein in the prokaryotic cell, the antigen of the present invention preferably contains an N-terminal methionine residue. The N-terminal methionine can also be cut off from the recombinant protein after expression. Alternatively, it can be a unicellular eukaryotic organism such as yeast; a cell from a eukaryotic organism such as a plant cell, an animal cell (for example, a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell or an insect cell), or a silkworm.

[0295] The gene introduction or transformation of the expression vector to the host cell can be suitably carried out by methods well known to those skilled in the art. In addition, those skilled in the art will suitably select conditions suitable for expressing the recombinant protein to cultivate the host cell according to the type of the host cell, so that the recombinant protein can be expressed. Then, the host cell expressing the recombinant protein is homogenized, and the method of purifying the above-mentioned protein by the homogenous mixture obtained is suitably combined, so that the antigen expressed as the recombinant protein can be separated / purified.

[0296] Diagnostic Kits / Diagnostic Methods(1)

[0297] The present invention provides a method for providing an indicator for diagnosing egg allergy in a subject, comprising the following steps:

[0298] (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies;

[0299] (ii) detecting the binding of IgE antibodies in a sample obtained from the subject to the antigen;

[0300] (iii) when the subject's IgE antibody is detected to be bound to the antigen, providing an indication that the subject has an egg allergy;

[0301] Here, the antigen is at least one of the proteins defined as the antigens in (4) to (10) above.

[0302] The sample obtained from the object refers to a solution containing IgE antibodies collected from the object. Such a solution contains, for example, blood, saliva, sputum, nasal mucus, urine, sweat, and tears. Before contacting with the antigen, the sample obtained from the object may also be subjected to a pretreatment for increasing the concentration of IgE antibodies in the sample. As a pretreatment of the sample, it may also include, for example, obtaining serum or plasma from blood. In addition, the Fab portion as the antigen-binding portion may also be further purified. In a particularly preferred embodiment, the above-mentioned step (i) is performed by contacting the IgE antibodies in the serum obtained from the object with the antigen.

[0303] The IgE antibody may be the IgE antibody itself, or may be a mast cell or the like to which the IgE antibody binds.

[0304] The contact of the sample obtained from the object with the antigen and the detection of their combination can be carried out by known methods. As such a method, for example, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, protein immunoblotting, immunoprecipitation, and immunochromatography can be used. These are all methods of contacting and combining the antigen with the object IgE antibody, causing it to act on the secondary antibody formed by enzyme labeling of the IgE antibody specifically bound to the antigen, adding the substrate of the enzyme (usually a color developing or luminescent reagent) to detect the product of the enzyme reaction, thereby being able to detect the method of binding the antigen to the object IgE antibody. Or a method for detecting a fluorescently labeled secondary antibody. Alternatively, it is also possible to use surface plasmon resonance (SPR) and other assays that can evaluate the binding of antigens to IgE antibodies for detection. It is also possible to mix a variety of antigen-specific IgE antibodies.

[0305] The antigen may also be in a state where the separated antigen is fixed to a carrier. In this case, ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance may be used in the above steps (i) and (ii); in addition, in the above step (i), the sample obtained from the object is contacted with the surface on which the antigen is fixed. The separated antigen can be obtained by separation / purification from eggs (preferably chicken eggs) in combination with a protein purification method known to those skilled in the art, or it can also be prepared by genetic recombination technology. In addition, the antibody may be fixed.

[0306] The antigen may also be in a state where it is not fixed to a carrier. In this case, in the above steps (i) and (ii), a flow cytometer or the like can be used, and the presence of the antigen bound to the antibody can be confirmed by laser. Examples thereof include a basophil activation test (BAT) and the like. In addition, a histamine free test (HRT) can also be used to confirm whether histamine is freed by further contacting the antigen with the blood cells in the sample.

[0307] In addition, the antigen can also be transferred from a state separated by two-dimensional electrophoresis and detected by protein immunoblotting. Two-dimensional electrophoresis is a method of separating protein samples by performing isoelectric point electrophoresis in the first dimension and SDS-PAGE (SDS-polyacrylamide gel electrophoresis) in the second dimension. In this case, the conditions of two-dimensional electrophoresis are not particularly limited as long as they are conditions that can separate the antigens of the present invention. For example, the conditions of two-dimensional electrophoresis described in the above-mentioned "Limitation of Antigens" item can be used. Alternatively, the electrophoresis conditions can be set with reference to the records of the above-mentioned patent documents 1 to 4, for example, two-dimensional electrophoresis is performed under at least one condition selected from the group consisting of the following conditions:

[0308] (A) As a first-dimensional isoelectric point electrophoresis gel, when the gel length is in the range of 5 to 10 cm and the pH range of the gel is 3 to 10, the gel length of the pH gradient of the gel relative to the migration direction up to pH 5 is set as a, the gel length of pH 5 to 7 is set as b, and the gel length of pH 7 or above is set as c, the relationship of "a < b" and "b > c" is satisfied;

[0309] (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;

[0310] (C) in the first-dimensional isoelectric point electrophoresis, a constant voltage of 100 V to 600 V is applied to each gel containing the sample to perform a constant voltage step, and after the electrophoresis amplitude is within the range of 5 μA for every 30 minutes of electrophoresis, a voltage increase step is started in which the voltage is increased from the constant voltage;

[0311] (D) In ​​the case of (C), the final voltage of the voltage increasing step is set within the range of 3000V to 6000V;

[0312] (E) the length of the first-dimension isoelectric point electrophoresis gel in the long-side direction is set to 5 to 10 cm, and the gel concentration at the base end of the second-dimension electrophoresis gel in the migration direction is set to 3 to 6%; and

[0313] (F) In the case of (E), the gel concentration at the front end side portion in the migration direction of the second-dimensional electrophoresis gel is set higher than the gel concentration at the base end portion in the migration direction.

[0314] The antigens of (4) to (10) above are antigens that specifically bind to IgE antibodies of patients with egg allergy. Therefore, if the binding of the subject's IgE antibodies to the antigen is detected, it provides an indicator that the subject is egg allergic.

[0315] The present invention also provides a diagnostic kit for egg allergy comprising at least one of the above-mentioned antigens (4) to (10). The diagnostic kit of the present invention can be used in the above-mentioned method for providing indicators for diagnosing egg allergy or the following diagnostic method. In addition to containing at least one of the above-mentioned antigens (4) to (10), the diagnostic kit of the present invention may also contain an enzyme-labeled anti-IgE antibody and a colorimetric substrate or a luminescent substrate that serves as a substrate for the enzyme. In addition, a fluorescently labeled anti-IgE antibody may also be used. In the diagnostic kit of the present invention, the antigen may also be provided in a state where the antigen is immobilized on a carrier. The diagnostic kit of the present invention may also be provided together with instructions regarding the steps used for diagnosis and a method package containing the instructions.

[0316] In another embodiment, the diagnostic kit includes a companion diagnostic drug for egg allergy. A companion diagnostic drug is a substance used for the following purposes: identifying patients who expect the drug to be effective or patients who are at risk of severe side effects of the drug, or studying the reactivity of the drug in order to optimize the treatment using the drug. Here, the optimization of treatment includes, for example, determining the dosage volume, determining the suspension of drug administration, and confirming whether immune tolerance is obtained by using which allergen component.

[0317] The present invention also provides a composition for diagnosing egg allergy, comprising at least one of the above-mentioned antigens (4) to (10). The composition for diagnosing the present invention can be used in the following diagnostic method. The composition for diagnosing the present invention can also contain pharmaceutically acceptable carriers and additives that are usually used together with the antigen of the present invention as needed.

[0318] In one aspect, the present invention is a method for diagnosing egg allergy in a subject, comprising the following steps:

[0319] (i) contacting a sample obtained from a subject with an antigen;

[0320] (ii) detecting the binding of IgE antibodies in a sample obtained from the subject to the antigen;

[0321] (iii) when the subject's IgE antibody is detected to be bound to the antigen, the subject is judged to have an egg allergy;

[0322] Here, the antigen is at least one of the proteins defined as the antigens in (4) to (10) above. The steps (i) and (ii) are performed according to the description of the steps of the method for providing an indicator for diagnosing egg allergy.

[0323] In another embodiment, the present invention provides a method for diagnosing an egg allergy in a subject, comprising administering to the subject at least one of the antigens of (4) to (10) above. The method may also be performed in the form of a skin test characterized by administering an antigen to the skin. The skin test includes the following forms: a prick test in which a diagnostic composition is applied to the skin, the skin is lightly scratched to a level that does not bleed, the skin is penetrated with the antigen, and then the skin reaction is observed; a scratch test in which the skin is lightly scratched after the diagnostic composition is applied and then the reaction is observed; a patch test in which a diagnostic composition in the form of a cream, ointment, etc. is applied to the skin and then the reaction is observed; an intradermal test in which the antigen is administered intradermally and then the reaction is observed; etc. In the case where a skin reaction such as swelling occurs on the part of the skin to which the antigen is applied, the subject is diagnosed as having an egg allergy. Here, the amount of antigen applied to the skin may also be, for example, an amount of 100 μg or less per time.

[0324] In the diagnosis of allergy, tolerance tests for the purpose of limiting antigens are often performed. At least one of the antigens (4) to (10) above can be used as an active ingredient in tolerance tests for diagnosing egg allergy. Here, the antigen protein used in the tolerance test can be a purified protein or a protein expressed in food / ingredients, such as pollen rice in which a gene for a Chinese fir pollen antigen is transformed into rice and the antigen protein is expressed in rice.

[0325] In another embodiment, the present invention provides at least one of the antigens (4) to (10) for use in diagnosing egg allergy. This includes providing at least one of the antigens (4) to (10) mixed with a known antigen.

[0326] In yet another embodiment, the present invention provides use of at least one of the antigens of (4) to (10) above in the preparation of a diagnostic drug for egg allergy.

[0327] Pharmaceutical composition / treatment method (1)

[0328] The present invention provides a pharmaceutical composition comprising at least one of the antigens described in (4) to (10) above.

[0329] In one embodiment, the pharmaceutical composition is used to treat egg allergy. The purpose of treating allergy is to increase the limit amount of antigen that will not cause disease even if ingested, and ultimately to achieve a state (remission) in which no disease will occur after normal antigen intake.

[0330] The present invention also provides a method for treating egg allergy, comprising: administering at least one of the antigens of (4) to (10) above to a patient in need of treatment for egg allergy.

[0331] In another embodiment, the present invention provides at least one of the antigens (4) to (10) for use in the treatment of egg allergy. In yet another embodiment, the present invention provides use of at least one of the antigens (4) to (10) for the preparation of a therapeutic drug for egg allergy.

[0332] In the treatment of allergies, desensitization therapy is often performed in which the purpose is to induce immune tolerance by administering antigens to patients. At least one of the antigens (4) to (10) above can be used as an active ingredient in the desensitization therapy for treating egg allergies. Here, the antigen protein used in the desensitization therapy may be a purified protein or a protein expressed in food / ingredients, such as pollen rice in which a gene for a cedar pollen antigen is transformed into rice and the antigen protein is expressed in rice.

[0333] The pharmaceutical composition of the present invention can be administered via common administration routes, including, for example, oral, sublingual, transdermal, intradermal, subcutaneous, intravascular, intranasal, intramuscular, intraperitoneal, and intrarectal administration.

[0334] The pharmaceutical composition of the present invention can be used as needed together with the antigen of the present invention by conventional methods to add commonly used pharmaceutically permitted adjuvants, excipients, or various additives (such as stabilizers, cosolvents, emulsifiers, buffers, storage agents, colorants, etc.) to the pharmaceutical composition. The dosage form of the pharmaceutical composition can be appropriately selected by a person skilled in the art according to the route of administration. It can also be in the form of, for example, tablets, capsules, syrups, sublingual tablets, injections, nasal sprays, ointments, solutions, creams, dews, suppositories, etc. The dosage, number of administrations, and / or administration period of the pharmaceutical composition of the present invention can be appropriately selected by a physician according to the characteristics of the patient such as the route of administration, symptoms, age, and weight. For example, in the case of adults, it can also be administered at a dosage of less than 100 μg per time. The dosing interval can be, for example, once a day, once a week, twice a month, or once every three months. The administration period can be, for example, several weeks to several years. During the administration period, it can also be an administration method that increases the dosage in a gradient.

[0335] Detector(1)

[0336] The present invention provides a detector comprising an antibody against at least one of the antigens of (4) to (10) above.

[0337] The antibody can be prepared by conventional methods. For example, a mammal such as a rabbit can be immunized with the antigens of (4) to (10) above. The antibody can be an IgE antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (e.g., Fab, F(ab')2, Fab').

[0338] In addition, in the above-mentioned detector, the antibody can be provided in the form of being bound to a carrier. The carrier is not particularly limited as long as it can be used in the detection of antibody-antigen binding. Any carrier known to those skilled in the art can be used.

[0339] Examples of methods for examining whether an antigen is contained include the following methods.

[0340] A method in which a detector containing the prepared IgE antibody is brought into contact with a sample obtained from a food / ingredient, etc., and the binding of the IgE antibody to an antigen in the sample is detected using, for example, an ELISA method, and when the binding of the IgE antibody to the antigen is detected, it is determined that the antigen remains in the target food / ingredient, etc.

[0341] A method in which food / ingredients are wrapped with filter paper or the like and reacted with an antibody solution to detect antigens contained therein.

[0342] Another embodiment of the present invention includes a detector for determining the presence or absence of an antigen causing an egg allergy in an object, characterized in that it includes a primer having a base sequence complementary to at least a portion of the base sequence shown in SEQ ID NO: 46, 58, 105, 151, 236, 250, or 261. In a non-limiting manner, the primer has, for example, a base sequence complementary to a portion of the 3' end or a central portion of at least one of the base sequences shown in SEQ ID NO: 46, 58, 105, 151, 236, 250, or 261, preferably 12 residues, 15 bases, 20 bases, or 25 bases. In particular, when mRNA is used as the object, the primer has a complementary primer of a poly(A) tail. In a preferred embodiment, the detector comprising the above-mentioned primers further comprises a primer comprising a base sequence at the 5' end portion of at least one of the base sequences shown in sequence numbers 46, 58, 105, 151, 236, 250 or 261, preferably a base sequence consisting of 12 bases, 15 bases, 20 bases or 25 bases.

[0343] For example, using DNA or mRNA obtained from eggs or birds as a template and the complementary primers, cDNA is amplified by PCR (polymerase chain reaction) including RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and the sequence of the amplified cDNA is compared with SEQ ID NOs: 46, 58, 105, 151, 236, 250 or 261 to determine the presence or absence of the antigen. The method of amplification by PCR can be exemplified by RACE (Rapid Amplification of cDNA Ends) and the like. At this time, when a point mutation encoding the same amino acid exists in the comparison between the amplified cDNA and sequence numbers 46, 58, 105, 151, 236, 250 or 261; or, even if there is a base insertion, deletion, substitution or addition in the base sequence of the amplified cDNA relative to the base sequence of sequence numbers 46, 58, 105, 151, 236, 250 or 261, the antigen is determined to be present when the amino acid sequence encoded by the cDNA has an identity of 70% or more, preferably 80, 90, 95, 98, 99% or more to the amino acid sequence of sequence numbers 47, 59, 106, 152, 237, 251 or 262.

[0344] In one embodiment, the detector can be used to detect whether an antigen is contained in food (eggs or birds) or in an object in a food production line. The detector can be used for quality inspection of production lines and pre-shipment products by manufacturers; it can also be used by tasters to check whether the target food / food contains antigens.

[0345] Antigen-removed foods, etc. (1)

[0346] The present invention provides an egg, an egg processed product, or a bird producing the egg or hatched from the egg, characterized in that at least one of the antigens (4) to (10) above is removed or reduced.

[0347] The method for removing or reducing antigens of the present invention is not limited to eggs, processed egg products, or birds that produce or hatch eggs. The removal or reduction of antigens is not limited to the removal or reduction of antigens of the present invention, and any method may be used.

[0348] For example, the antigen-deleted or -reduced eggs of the present invention can be prepared using gene knockout technology to produce eggs in which the expression of the antigen of the present invention is knocked out.

[0349] The gene knockout technology can use any of the methods known to those skilled in the art. For example, Oishi, et al. (Scientific Reports, Vol. 6, Article number: 23980, 2016, doi: 10.1038 / srep23980) describes the application of CRISPER / Cas9 to primordial germ cells of chickens as a genome editing technology to obtain individuals with ovomucoid gene deletions. The same method can also be used to obtain eggs from which antigens have been removed according to the present invention. In addition, birds or eggs that do not contain antigens or have low antigen content can also be mated through artificial insemination to obtain birds or eggs from which antigens have been removed or reduced according to the present invention. Artificial mating of birds or eggs can be performed by conventional methods.

[0350] The processed egg product from which the antigens of the present invention have been removed or reduced may also be a processed product using the eggs from which the antigens of the present invention have been removed or reduced as a raw material. When ordinary eggs are used as raw materials, the treatment for removing or reducing the antigens of the present invention is performed before or after the preparation of the processed egg product. In the processed egg product using ordinary eggs as raw materials, the methods for removing or reducing the antigens of the present invention include: methods for removing protein components in food / ingredients by high pressure treatment and elution with neutral salt solutions, high temperature steam, etc.; methods for hydrolysis, denaturation, and amino acid denaturation (chemical modification / detachment of side chains, etc.) by heat treatment and acid treatment.

[0351] The bird from which the antigen of the present invention is removed or reduced can also be a bird hatched and grown from the egg from which the antigen is removed or reduced as described above. Birds include birds at various growth stages, including chicks, young birds, young birds, adult birds, and old birds. In this specification, a bird refers to an animal belonging to the avian species, preferably a chicken.

[0352] Method for producing antigen-removed egg processed product (1)

[0353] The present invention provides a method for producing an egg processed product from which antigens have been removed or reduced, comprising the step of confirming that antigens have been removed or reduced during the production process of the processed product, wherein the antigen is at least one of the antigens described in (4) to (10).

[0354] In the production process of the antigen-removed or reduced egg processed product, the step of confirming that the antigen has been removed or reduced can be performed by confirming whether the antigen is contained by the method described in the above-mentioned item "Detector (1)".

[0355] Alternatively, the production of an antigen-removed or reduced egg processed product can be carried out by the method described in the above-mentioned section "Antigen-removed food, etc. (1)".

[0356] Epitope of antigen

[0357] As shown in Examples 2 to 5, for a defined antigen, as shown in Example 6, an epitope and amino acids within the epitope that are important for binding to IgE antibodies of allergy patients are defined.

[0358] The present invention provides the following polypeptides (1β) to (4β) as polypeptides comprising an amino acid sequence that specifically binds to IgE antibodies of allergy patients.

[0359] (1β) Epitope of vitellogenin-2

[0360] In the present invention, the polypeptide (1β) is any polypeptide selected from the group consisting of the following (1β-1) to (1β-21).

[0361] (1β-1) comprises a member selected from the group consisting of SEQ ID NOs: 272 to 295, 319, 323, 324, 327, 330, 331, 333, 335, 336, 339, 340, 342, 343, 344, 346, 347, 350, 351, 353, 355, 356, 357, 359, 360, 363, 366, 369, 374, 375, 377, 379, 380, 381, 384, 385, 388, 391, 392, 393, 397, 400, 401, 402, 404, 405, 406, 407, 409, 412, 414, 419, 422, 425, 426, 430, 434, 436, 440, 444, 446, 449, 451, 452, 455, 458, 461, 462, 464, 465, 466, 470, 471, 473, 476, 479, 480, 482, 483, 486, 487, 491, 492, 494, 495, 497, 498, 500, 503, 505, 507, 509, 510, 512, 515, 516, 517, 520, 522, and 523.

[0362] (1β-2) A polypeptide comprising the amino acid sequence of XXXXXQXEEYNGXWP (SEQ ID NO: 320). Preferably, it is a polypeptide comprising the amino acid sequence of XXXSPQVEEYNGVWP (SEQ ID NO: 321). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 amino acids of amino acids 1 to 5, 7 and 13 of SEQ ID NO: 272 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 amino acids of amino acids 1 to 3 of SEQ ID NO: 272 are replaced by other amino acids, preferably alanine.

[0363] In another embodiment, the polypeptide comprises the amino acid sequence of XEEYNG (SEQ ID NO: 322). More preferably, the polypeptide comprises the amino acid sequence in which the amino acid at position 1 of SEQ ID NO: 323 is substituted with another amino acid, preferably alanine.

[0364] (1β-3) A polypeptide comprising the amino acid sequence of XXXXXXXXNVYEXXX (SEQ ID NO: 328). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1 to 8 and 13 to 15 of SEQ ID NO: 273 are substituted with other amino acids, preferably alanine.

[0365] In another embodiment, it is a polypeptide comprising the amino acid sequence of XXXXXXXXNV (SEQ ID NO: 325). Preferably, it is a polypeptide comprising the amino acid sequence of XXXIKXXXNV (SEQ ID NO: 326). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 amino acids of the amino acids at positions 1 to 8 of SEQ ID NO: 327 are replaced by other amino acids, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 amino acids of the amino acids at positions 1 to 3 and 6 to 8 of SEQ ID NO: 327 are replaced by other amino acids, preferably alanine.

[0366] (1β-4) A polypeptide comprising the amino acid sequence of RXRXXKXXXXXXXXX (SEQ ID NO: 332). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the amino acids at positions 2, 4, 5, 7 to 15 of SEQ ID NO: 274 are substituted with other amino acids, preferably alanine.

[0367] In another embodiment, the polypeptide comprises an amino acid sequence of RXXXXKXXXALXXXX (SEQ ID NO: 337). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 2 to 5, 7 to 9, and 12 to 15 of SEQ ID NO: 274 are substituted with other amino acids, preferably alanine.

[0368] In another embodiment, the polypeptide comprises the amino acid sequence of RXRXXKXX (SEQ ID NO: 329). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 2, 4, 5, 7 and 8 of SEQ ID NO: 330 are substituted with other amino acids, preferably alanine.

[0369] In another embodiment, the polypeptide comprises the amino acid sequence of KXXXALXXXX (SEQ ID NO: 334). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 2 to 4 and 7 to 10 of SEQ ID NO: 335 are substituted with other amino acids, preferably alanine.

[0370] (1β-5) A polypeptide comprising the amino acid sequence of AXXKIAXXXPKTVXX (SEQ ID NO: 341). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 2, 3, 7 to 9, 14 and 15 of SEQ ID NO: 275 are substituted with other amino acids, preferably alanine.

[0371] In another embodiment, the polypeptide comprises the amino acid sequence of AXXXXAXXXXXTVQX (SEQ ID NO: 348). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 2 to 5, 7 to 11 and 15 of SEQ ID NO: 275 are substituted with other amino acids, preferably alanine.

[0372] In another embodiment, the polypeptide comprises the amino acid sequence of AXXXXAXKXXXXXQG (SEQ ID NO: 352). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 2 to 5, 7, 9 to 13 of SEQ ID NO: 275 are substituted with other amino acids, preferably alanine.

[0373] In another embodiment, the polypeptide comprises the amino acid sequence of AXXKIAXXXP (SEQ ID NO: 338). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 2, 3, 7 to 9 of SEQ ID NO: 339 are substituted with other amino acids, preferably alanine.

[0374] In another embodiment, the polypeptide comprises the amino acid sequence of AWKDPKXXXG (SEQ ID NO: 345). More preferably, the polypeptide comprises the amino acid sequence in which any one, two or three of the amino acids at positions 7 to 9 of SEQ ID NO: 346 are substituted with other amino acids, preferably alanine.

[0375] In another embodiment, the polypeptide comprises the amino acid sequence of AXKXXXXX (SEQ ID NO: 349). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 2, 4 to 8 of SEQ ID NO: 350 are substituted with other amino acids, preferably alanine.

[0376] (1β-6) A polypeptide comprising the amino acid sequence of XALXXLSPKLDSMSY (SEQ ID NO: 358). More preferably, a polypeptide comprising an amino acid sequence in which any one, two or three of the amino acids at positions 1, 4 and 5 of SEQ ID NO: 276 are substituted with other amino acids, preferably alanine.

[0377] In another embodiment, the polypeptide comprises the amino acid sequence of XLSPKLDS (SEQ ID NO: 354). More preferably, the polypeptide comprises the amino acid sequence in which the amino acid at position 1 of SEQ ID NO: 655 is substituted with another amino acid, preferably alanine.

[0378] (1β-7) A polypeptide comprising the amino acid sequence of VXXXRTMFPXAXISK (SEQ ID NO: 361). Preferably, it is a polypeptide comprising the amino acid sequence of VNXXRTMFPSAXISK (SEQ ID NO: 362). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 amino acids of the amino acids at positions 2 to 4, 10 and 12 of SEQ ID NO: 277 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 amino acids of the amino acids at positions 3, 4 and 12 of SEQ ID NO: 277 are replaced by other amino acids, preferably alanine.

[0379] In another embodiment, it is a polypeptide comprising the amino acid sequence of XXSXXXMXPXAXXXK (SEQ ID NO: 370). Preferably, it is a polypeptide comprising the amino acid sequence of VNSPRTMXPXAXXXK (SEQ ID NO: 371). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the amino acids at positions 1, 2, 4 to 6, 8, 10, 12 to 14 of SEQ ID NO: 277 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 amino acids of the amino acids at positions 8, 10, 12 to 14 of SEQ ID NO: 277 are replaced with other amino acids, preferably alanine.

[0380] In another embodiment, the polypeptide comprises the amino acid sequence of SXXXMXPXAX (SEQ ID NO: 364), and preferably the polypeptide comprises the amino acid sequence of SPRTMXPXAX (SEQ ID NO: 365).

[0381] More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 2 to 4, 6, 8 and 10 of SEQ ID NO: 366 are substituted with other amino acids, preferably alanine. Still more preferably, a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 3, 4 and 12 of SEQ ID NO: 366 are substituted with other amino acids, preferably alanine.

[0382] In another embodiment, the polypeptide comprises the amino acid sequence of XMXPXAXXXK (SEQ ID NO: 367), and preferably the polypeptide comprises the amino acid sequence of TMXPXAXXXK (SEQ ID NO: 368).

[0383] More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1, 3, 5, 7 to 9 of SEQ ID NO: 369 are substituted with other amino acids, preferably alanine. Still more preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 3, 5, 7 to 9 of SEQ ID NO: 369 are substituted with other amino acids, preferably alanine.

[0384] (1β-8) A polypeptide comprising the amino acid sequence of AIISKLXAXXAXSXA (SEQ ID NO: 376). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four or five of the amino acids at positions 7, 9, 10, 12 and 14 of SEQ ID NO: 278 are substituted with other amino acids, preferably alanine.

[0385] In another embodiment, the polypeptide comprises the amino acid sequence of IXKLXAXXA (SEQ ID NO: 372). Preferably, the polypeptide comprises the amino acid sequence of ISKLXAXXA (SEQ ID NO: 373). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3 or 4 amino acids at positions 2, 5, 7 and 8 of SEQ ID NO: 374 are replaced with other amino acids, preferably alanine. Further preferably, the polypeptide comprises an amino acid sequence in which any 1, 2 or 3 amino acids at positions 5, 7 and 8 of SEQ ID NO: 374 are replaced with other amino acids, preferably alanine.

[0386] (1β-9) A polypeptide comprising the amino acid sequence of GKALQXXKELPTXTP (SEQ ID NO: 382). Preferably, it is a polypeptide comprising the amino acid sequence of GKALQXXKELPTETP (SEQ ID NO: 383). More preferably, it is a polypeptide comprising an amino acid sequence in which any one, two or three of the amino acids at positions 6, 7 and 13 of SEQ ID NO: 279 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any one or two of the amino acids at positions 6 and 7 of SEQ ID NO: 279 are replaced by other amino acids, preferably alanine.

[0387] In another embodiment, it is a polypeptide comprising the amino acid sequence of XAXXXXXXEXXXXXP (SEQ ID NO: 394). Preferably, it is a polypeptide comprising the amino acid sequence of XXALXXXXEXXXXXP (SEQ ID NO: 395). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids of the amino acids at positions 1, 2, 4 to 8, 10 to 14 of SEQ ID NO: 279 are replaced with other amino acids, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids of the amino acids at positions 1, 2, 5 to 8, 10 to 14 of SEQ ID NO: 279 are replaced with other amino acids, preferably alanine.

[0388] In another embodiment, the polypeptide comprises the amino acid sequence of GKAXXXXKEXXXXXX (SEQ ID NO: 403). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 4 to 7 and 10 to 15 of SEQ ID NO: 279 are substituted with other amino acids, preferably alanine.

[0389] In another embodiment, the polypeptide comprises the amino acid sequence of KALQXXKELP (SEQ ID NO: 378). More preferably, the polypeptide comprises the amino acid sequence in which any one or two of the amino acids at positions 5 and 6 of SEQ ID NO: 379 are substituted with other amino acids, preferably alanine.

[0390] In another embodiment, it is a polypeptide comprising the amino acid sequence of XAXXXXXEX (SEQ ID NO: 386). Preferably, it is a polypeptide comprising the amino acid sequence of XALXXXXEX (SEQ ID NO: 387). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 amino acids of the amino acids at positions 1, 3 to 7 and 9 of SEQ ID NO: 388 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 amino acids of the amino acids at positions 1, 4 to 7 and 9 of SEQ ID NO: 388 are replaced with other amino acids, preferably alanine.

[0391] In another embodiment, it is a polypeptide comprising the amino acid sequence of XXXEXXXXXP (SEQ ID NO: 389). Preferably, it is a polypeptide comprising the amino acid sequence of XXXEXXXEXP (SEQ ID NO: 390). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 amino acids of the amino acids at positions 1 to 3 and 5 to 9 of SEQ ID NO: 392 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 amino acids of the amino acids at positions 1 to 3, 5 to 7 and 9 of SEQ ID NO: 392 are replaced with other amino acids, preferably alanine.

[0392] In another embodiment, the polypeptide comprises the amino acid sequence of KAXXXXKEXX (SEQ ID NO: 396). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 3 to 6, 9 and 10 of SEQ ID NO: 397 are substituted with other amino acids, preferably alanine.

[0393] In another embodiment, it is a polypeptide comprising the amino acid sequence of XXKEXXXXXX (SEQ ID NO: 398). Preferably, it is a polypeptide comprising the amino acid sequence of GXKEXPXXTX (SEQ ID NO: 399). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 amino acids of the amino acids at positions 1, 2, 5 to 10 of SEQ ID NO: 400 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 amino acids of the amino acids at positions 2, 5, 7, 8 and 10 of SEQ ID NO: 400 are replaced with other amino acids, preferably alanine.

[0394] (1β-10) A polypeptide comprising the amino acid sequence of XXXNKELXQQVMXXV (SEQ ID NO: 408). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four, five or six of amino acids 1 to 3, 8, 13 and 14 of SEQ ID NO: 280 are substituted with other amino acids, preferably alanine.

[0395] In another embodiment, the polypeptide comprises the amino acid sequence of ELXQQ (SEQ ID NO: 406). More preferably, the polypeptide comprises the amino acid sequence in which the amino acid at position 3 of SEQ ID NO: 407 is substituted with another amino acid, preferably alanine.

[0396] (1β-11) A polypeptide comprising the amino acid sequence of XXXXVVXPADXNAAI (SEQ ID NO: 415). Preferably, it is a polypeptide comprising the amino acid sequence of XXXTVVEPADXNAAI (SEQ ID NO: 416). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 amino acids of the amino acids at positions 1 to 4, 7 and 11 of SEQ ID NO: 281 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 amino acids of the amino acids at positions 1 to 3 and 11 of SEQ ID NO: 281 are replaced by other amino acids, preferably alanine.

[0397] In another embodiment, the polypeptide comprises the amino acid sequence of XXXVVXPA (SEQ ID NO: 410). Preferably, the polypeptide comprises the amino acid sequence of XXTVVEPA (SEQ ID NO: 411). More preferably, the polypeptide comprises the amino acid sequence in which any one, two, three or four of the amino acids at positions 1 to 3 and 6 of SEQ ID NO: 412 are substituted with other amino acids, preferably alanine.

[0398] In another embodiment, a polypeptide comprising the amino acid sequence of ADXNA (SEQ ID NO: 413) is preferred, and a polypeptide comprising an amino acid sequence in which the amino acid at position 3 of SEQ ID NO: 413 is substituted with another amino acid, preferably alanine, is more preferred.

[0399] (1β-12) A polypeptide comprising the amino acid sequence of KXXAKXXXXXKNXXX (SEQ ID NO: 420). Preferably, it is a polypeptide comprising the amino acid sequence of KXXAKXDVXXKNLXX (SEQ ID NO: 421). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the amino acids at positions 2, 3, 6 to 10, 13 to 15 of SEQ ID NO: 282 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 amino acids of the amino acids at positions 2, 3, 6, 9, 10, 14 and 15 of SEQ ID NO: 282 are replaced by other amino acids, preferably alanine.

[0400] In another embodiment, it is a polypeptide comprising the amino acid sequence of KFXAKXDXKXKNXKX (SEQ ID NO: 427). Preferably, it is a polypeptide comprising the amino acid sequence of KFDAKXDVKXKNXKX (SEQ ID NO: 428). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 amino acids of the amino acids at positions 3, 6, 8, 10, 13 and 15 of SEQ ID NO: 282 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 amino acids of the amino acids at positions 6, 10, 13 and 15 of SEQ ID NO: 282 are replaced with other amino acids, preferably alanine.

[0401] In another embodiment, the polypeptide comprises an amino acid sequence of XXXXKNLX (SEQ ID NO: 417). Preferably, the polypeptide comprises an amino acid sequence of DVXXKNLX (SEQ ID NO: 418). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4 or 5 amino acids of the amino acids at positions 1 to 4 and 8 of SEQ ID NO: 419 are replaced with other amino acids, preferably alanine. Further preferably, the polypeptide comprises an amino acid sequence in which any 1, 2 or 3 amino acids of the amino acids at positions 3, 4 and 8 of SEQ ID NO: 419 are replaced with other amino acids, preferably alanine.

[0402] In another embodiment, the polypeptide comprises an amino acid sequence of DXKXKNXKX (SEQ ID NO: 423). Preferably, the polypeptide comprises an amino acid sequence of DVKXKNXKX (SEQ ID NO: 424). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3 or 4 amino acids of the amino acids at positions 2, 4, 7 and 9 of SEQ ID NO: 425 are replaced with other amino acids, preferably alanine. Further preferably, the polypeptide comprises an amino acid sequence in which any 1, 2 or 3 amino acids of the amino acids at positions 4, 7 and 9 of SEQ ID NO: 425 are replaced with other amino acids, preferably alanine.

[0403] (1β-13) A polypeptide comprising the amino acid sequence of NXXXKKXNXVXAXXX (SEQ ID NO: 431). Preferably, it is a polypeptide comprising the amino acid sequence of NRXXKKXNTVLAXXX (SEQ ID NO: 432). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids of the amino acids at positions 2 to 4, 7, 9, 11, 13 to 15 of SEQ ID NO: 285 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 amino acids of the amino acids at positions 3, 4, 7, 13 to 15 of SEQ ID NO: 285 are replaced by other amino acids, preferably alanine.

[0404] In another embodiment, the polypeptide comprises the amino acid sequence of XXKKXNTVLA (SEQ ID NO: 429). More preferably, the polypeptide comprises the amino acid sequence in which any one, two or three of the amino acids at positions 1, 2 and 5 of SEQ ID NO: 430 are substituted with other amino acids, preferably alanine.

[0405] (1β-14) A polypeptide comprising the amino acid sequence of SSSSSSSXSNSKSSS (SEQ ID NO: 437). More preferably, a polypeptide comprising the amino acid sequence in which the amino acid at position 8 of SEQ ID NO: 286 is substituted with another amino acid, preferably alanine.

[0406] In another embodiment, it is a polypeptide comprising an amino acid sequence of XXSXSSSXXXSKSSS (SEQ ID NO. 441). Preferably, it is a polypeptide comprising an amino acid sequence of XXSSSSSXXNSKSSS (SEQ ID NO. 442). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 amino acids of the amino acids at positions 1, 2, 4, 8 to 10 of SEQ ID NO. 286 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 amino acids of the amino acids at positions 1, 2, 8 and 9 of SEQ ID NO. 286 are replaced with other amino acids, preferably alanine.

[0407] In another embodiment, the polypeptide comprises the amino acid sequence of SSSXSNSKS (SEQ ID NO: 433), and more preferably, the polypeptide comprises the amino acid sequence in which the amino acid at position 4 of SEQ ID NO: 434 is substituted with another amino acid, preferably alanine.

[0408] In another embodiment, the polypeptide comprises the amino acid sequence of SXSNSKSSSS (SEQ ID NO: 435). More preferably, the polypeptide comprises the amino acid sequence in which the amino acid at position 2 of SEQ ID NO: 436 is substituted with another amino acid, preferably alanine.

[0409] In another embodiment, the polypeptide comprises an amino acid sequence of XXXSKSS (SEQ ID NO. 438). Preferably, the polypeptide comprises an amino acid sequence of XXNSKSS (SEQ ID NO. 439). More preferably, the polypeptide comprises an amino acid sequence in which any one, two or three amino acids of the amino acids at positions 1 to 3 of SEQ ID NO. 440 are replaced with other amino acids, preferably alanine. Further preferably, the polypeptide comprises an amino acid sequence in which any one or two amino acids of the amino acids at positions 1 and 2 of SEQ ID NO. 400 are replaced with other amino acids, preferably alanine.

[0410] (1β-15) A polypeptide comprising the amino acid sequence of XEXXXXKXPXXXAXX (SEQ ID NO: 450). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1, 3 to 6, 8, 10 to 12, 14 and 15 of SEQ ID NO: 287 are substituted with other amino acids, preferably alanine.

[0411] In another embodiment, the polypeptide comprises an amino acid sequence of XEXXXXKXPX (SEQ ID NO: 443). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1, 3 to 6, 8 and 10 of SEQ ID NO: 444 are substituted with other amino acids, preferably alanine.

[0412] In another embodiment, the polypeptide comprises the amino acid sequence of XXXKXPXXXA (SEQ ID NO: 445). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids 1 to 3, 5, 7 to 9 of SEQ ID NO: 446 are substituted with other amino acids, preferably alanine.

[0413] In another embodiment, it is a polypeptide comprising an amino acid sequence of XKXPXXXAXX (SEQ ID NO: 447). Preferably, it is a polypeptide comprising an amino acid sequence of XKXPXXXAXS (SEQ ID NO: 448). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 amino acids of amino acids at positions 1, 3, 5 to 7, 9 and 10 of SEQ ID NO: 449 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 amino acids of amino acids at positions 1, 3, 5 to 7 and 9 of SEQ ID NO: 449 are replaced with other amino acids, preferably alanine.

[0414] (1β-16) A polypeptide comprising the amino acid sequence of XXTXXXKXXLXADAX (SEQ ID NO: 459). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of amino acids 1, 2, 4 to 6, 8, 9, 11 and 15 of SEQ ID NO: 288 are substituted with other amino acids, preferably alanine.

[0415] In another embodiment, the polypeptide comprises the amino acid sequence of TXXXKXXLXA (SEQ ID NO: 453). Preferably, the polypeptide comprises the amino acid sequence of TXXXKXKLCA (SEQ ID NO: 454). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 2 to 4, 6, 7 and 9 of SEQ ID NO: 455 are substituted with other amino acids, preferably alanine.

[0416] In another embodiment, it is a polypeptide comprising the amino acid sequence of KXXLXADAX (SEQ ID NO: 456). Preferably, it is a polypeptide comprising the amino acid sequence of KXKLCADAX (SEQ ID NO: 457). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 amino acids of the amino acids at positions 2, 3, 5 and 9 of SEQ ID NO: 458 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1 or 2 amino acids of the amino acids at positions 2 and 9 of SEQ ID NO: 458 are replaced with other amino acids, preferably alanine.

[0417] (1β-17) A polypeptide comprising the amino acid sequence of KIKTXNEXXFXXSMP (SEQ ID NO: 467). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three, four or five of the amino acids at positions 5, 8, 9, 11 and 12 of SEQ ID NO: 290 are substituted with other amino acids, preferably alanine.

[0418] In another embodiment, the polypeptide comprises the amino acid sequence of KIKTX (SEQ ID NO: 460), and more preferably comprises the amino acid sequence in which the amino acid at position 5 of SEQ ID NO: 461 is substituted with another amino acid, preferably alanine.

[0419] In another embodiment, the polypeptide comprises the amino acid sequence of XNEXXFNYSM (SEQ ID NO: 463). More preferably, the polypeptide comprises the amino acid sequence in which any one, two or three of the amino acids at positions 1, 4 and 5 of SEQ ID NO: 464 are substituted with other amino acids, preferably alanine.

[0420] (1β-18) A polypeptide comprising the amino acid sequence of XAXXXXAXXIXIGXH (SEQ ID NO: 474). Preferably, it is a polypeptide comprising the amino acid sequence of EAXXXXAXXIXIGXH (SEQ ID NO: 475). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1, 3 to 6, 8, 9, 11 and 14 of SEQ ID NO: 291 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of the amino acids at positions 3 to 6, 8, 9, 11 and 14 of SEQ ID NO: 291 are replaced by other amino acids, preferably alanine.

[0421] In another embodiment, it is a polypeptide comprising the amino acid sequence of XAXNXXAXXXKIGXH (SEQ ID NO: 477). Preferably, it is a polypeptide comprising the amino acid sequence of XAXNXKAXXXKIGXH (SEQ ID NO: 478). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 amino acids of the amino acids at positions 1, 3, 5, 6, 8 to 10, and 14 of SEQ ID NO: 291 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 amino acids of the amino acids at positions 1, 3, 5, 8 to 10, and 14 of SEQ ID NO: 291 are replaced with other amino acids, preferably alanine.

[0422] In another embodiment, it is a polypeptide comprising the amino acid sequence of XAXXLXAXNIXXGXH (SEQ ID NO: 488). Preferably, it is a polypeptide comprising the amino acid sequence of EAXXLKAXNIXXGXH (SEQ ID NO: 489). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 amino acids of the amino acids at positions 1, 3, 4, 6, 11, 12 and 14 of SEQ ID NO: 291 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 amino acids of the amino acids at positions 3, 4, 8, 11, 12 and 14 of SEQ ID NO: 291 are replaced with other amino acids, preferably alanine.

[0423] In another embodiment, it is a polypeptide comprising an amino acid sequence of XAXNXXAXX (SEQ ID NO: 468). Preferably, it is a polypeptide comprising an amino acid sequence of EAXNLXAXX (SEQ ID NO: 469). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 amino acids of the amino acids at positions 1, 3, 5, 6, 8 and 9 of SEQ ID NO: 470 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 amino acids of the amino acids at positions 3, 6, 8 and 9 of SEQ ID NO: 470 are replaced with other amino acids, preferably alanine.

[0424] In another embodiment, the polypeptide comprises the amino acid sequence of XXIXIGXH (SEQ ID NO: 472). More preferably, the polypeptide comprises the amino acid sequence in which any one, two, three or four of the amino acids at positions 1, 2, 4 and 7 of SEQ ID NO: 473 are substituted with other amino acids, preferably alanine.

[0425] In another embodiment, the polypeptide comprises the amino acid sequence of AXXLKAXNI (SEQ ID NO: 481). More preferably, the polypeptide comprises the amino acid sequence in which any one, two or three of the amino acids at positions 2, 3 and 7 of SEQ ID NO: 482 are substituted with other amino acids, preferably alanine.

[0426] In another embodiment, it is a polypeptide comprising the amino acid sequence of XAXNIXXGX (SEQ ID NO. 484). Preferably, it is a polypeptide comprising the amino acid sequence of KAXNIXXGX (SEQ ID NO. 485). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 amino acids of the amino acids at positions 1, 3, 6, 7 and 9 of SEQ ID NO. 486 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 amino acids of the amino acids at positions 3, 6, 7 and 9 of SEQ ID NO. 486 are replaced with other amino acids, preferably alanine.

[0427] (1β-19) A polypeptide comprising the amino acid sequence of AAPXXGXDKXXXXGK (SEQ ID NO: 496). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 4, 5, 7, 10 to 13 of SEQ ID NO: 292 are substituted with other amino acids, preferably alanine.

[0428] In another embodiment, the polypeptide comprises the amino acid sequence of AAPXXGIDKLXFDGK (SEQ ID NO: 501). Preferably, the polypeptide comprises the amino acid sequence of AAPXXGIDKLYFDGK (SEQ ID NO: 502). More preferably, the polypeptide comprises an amino acid sequence in which any one, two or three of the amino acids at positions 4, 5 and 11 of SEQ ID NO: 292 are replaced with other amino acids, preferably alanine. Further preferably, the polypeptide comprises an amino acid sequence in which any one or two of the amino acids at positions 4 and 5 of SEQ ID NO: 292 are replaced with other amino acids, preferably alanine.

[0429] In another embodiment, the polypeptide comprises the amino acid sequence of AAXXXXIDKXXXXXK (SEQ ID NO: 510). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 3 to 6 and 10 to 14 of SEQ ID NO: 292 are substituted with other amino acids, preferably alanine.

[0430] In another embodiment, the polypeptide comprises the amino acid sequence of AAPXXG (SEQ ID NO: 490). More preferably, the polypeptide comprises the amino acid sequence in which any one or two of the amino acids at positions 4 and 5 of SEQ ID NO: 491 are substituted with other amino acids, preferably alanine.

[0431] In another embodiment, the polypeptide comprises the amino acid sequence of DKXXXX (SEQ ID NO: 493). More preferably, the polypeptide comprises the amino acid sequence in which any one, two, three or four of the amino acids at positions 3 to 6 of SEQ ID NO: 494 are substituted with other amino acids, preferably alanine.

[0432] In another embodiment, the polypeptide comprises the amino acid sequence of DKLXFDG (SEQ ID NO: 499). More preferably, the polypeptide comprises the amino acid sequence in which the amino acid at position 4 of SEQ ID NO: 500 is substituted with another amino acid, preferably alanine.

[0433] In another embodiment, the polypeptide comprises the amino acid sequence of AAXXXXID (SEQ ID NO: 504). More preferably, the polypeptide comprises the amino acid sequence in which any one, two, three or four of the amino acids at positions 3 to 6 of SEQ ID NO: 505 are substituted with other amino acids, preferably alanine.

[0434] In another embodiment, the polypeptide comprises the amino acid sequence of XXXXIDKXXX (SEQ ID NO: 506). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids 1 to 4 and 8 to 10 of SEQ ID NO: 507 are substituted with other amino acids, preferably alanine.

[0435] In another embodiment, the polypeptide comprises the amino acid sequence of DKXXXXXKT (SEQ ID NO: 508). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids 3 to 7 of SEQ ID NO: 509 are substituted with other amino acids, preferably alanine.

[0436] (1β-20) A polypeptide comprising the amino acid sequence of XXXEXXMXNGYXAKNA (SEQ ID NO: 513). Preferably, it is a polypeptide comprising the amino acid sequence of XQEXXMXNGYLAKNA (SEQ ID NO: 514). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1, 2, 4, 5, 7 and 11 of SEQ ID NO: 293 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 1, 4, 5 and 7 of SEQ ID NO: 293 are replaced by other amino acids, preferably alanine.

[0437] In another embodiment, the polypeptide comprises the amino acid sequence of MXNGYLAKNA (SEQ ID NO: 511). More preferably, the polypeptide comprises the amino acid sequence in which the amino acid at position 2 of SEQ ID NO: 512 is substituted with another amino acid, preferably alanine.

[0438] (1β-21) A polypeptide comprising the amino acid sequence of XAXXLXXXFVKLXXX (SEQ ID NO: 524). Preferably, it is a polypeptide comprising the amino acid sequence of XAXXLXXSFVKLXXX (SEQ ID NO: 525). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids of the amino acids at positions 1, 3, 4, 6 to 8, 13 to 15 of SEQ ID NO: 294 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 amino acids of the amino acids at positions 1, 3, 4, 6, 7, 13 to 15 of SEQ ID NO: 294 are replaced by other amino acids, preferably alanine.

[0439] In another embodiment, it is a polypeptide comprising the amino acid sequence of AXXLXXXFVK (SEQ ID NO: 518). Preferably, it is a polypeptide comprising the amino acid sequence of AXXLXXSFVK (SEQ ID NO: 519). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 amino acids of the amino acids at positions 2, 3, 5 to 7 of SEQ ID NO: 520 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 amino acids of the amino acids at positions 2, 3, 5 and 6 of SEQ ID NO: 520 are replaced with other amino acids, preferably alanine.

[0440] In another embodiment, the polypeptide comprises the amino acid sequence of XSFVKLX (SEQ ID NO: 521). More preferably, the polypeptide comprises the amino acid sequence in which any one or two of the amino acids at positions 1 and 7 of SEQ ID NO: 522 are substituted with other amino acids, preferably alanine.

[0441] (2β) Epitope of vitellogenin-3

[0442] In the present invention, the polypeptide (2β) is any polypeptide selected from the group consisting of the following (2β-1) to (2β-6).

[0443] (2β-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 296 to 303, 528, 531, 533, 534, 535, 539, 541, 542, 544, 550 and 551.

[0444] (2β-2) A polypeptide comprising the amino acid sequence of XPRTCDXXXKXXXXX (SEQ ID NO: 526). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1, 7 to 9, and 11 to 15 of SEQ ID NO: 296 are substituted with other amino acids, preferably alanine.

[0445] In another embodiment, the polypeptide comprises the amino acid sequence of SPRXXXXXLXLPXXX (SEQ ID NO: 527). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 4 to 8, 10, 13 to 15 of SEQ ID NO: 296 are substituted with other amino acids, preferably alanine.

[0446] (2β-3) A polypeptide comprising the amino acid sequence of SXXXXXXEXXXXXXX (SEQ ID NO: 529). Preferably, it is a polypeptide comprising the amino acid sequence of SXXXXSXEXXXXXXX (SEQ ID NO: 530). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acids of amino acids 2 to 7 and 9 to 15 of SEQ ID NO: 297 are replaced by other amino acids, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids of amino acids 2 to 5, 7 and 9 to 15 of SEQ ID NO: 297 are replaced by other amino acids, preferably alanine.

[0447] In another embodiment, the polypeptide comprises an amino acid sequence of XXXLXSXXXXXXXXX (SEQ ID NO: 532). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the amino acids 1 to 3, 5, 7 to 15 of SEQ ID NO: 297 are substituted with other amino acids, preferably alanine.

[0448] (2β-4) A polypeptide comprising the amino acid sequence of GKXXXXXXXXXXXKK (SEQ ID NO: 536). Preferably, it is a polypeptide comprising the amino acid sequence of GKXXXXXXXXXXXXEKK (SEQ ID NO: 537). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 3 to 13 of SEQ ID NO: 298 are replaced by other amino acids, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 3 to 12 of SEQ ID NO: 298 are replaced by other amino acids, preferably alanine.

[0449] In another embodiment, it is a polypeptide comprising the amino acid sequence of XXTCXXXXXXXXEKK (SEQ ID NO: 545). Preferably, it is a polypeptide comprising the amino acid sequence of XKTCXIXXXXXXEKK (SEQ ID NO: 546). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the amino acids at positions 1, 2, 5 to 12 of SEQ ID NO: 298 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 amino acids of the amino acids at positions 1, 5, 7 to 12 of SEQ ID NO: 298 are replaced with other amino acids, preferably alanine.

[0450] In another embodiment, the polypeptide comprises the amino acid sequence of CXIXXXXXX (SEQ ID NO: 538). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 amino acids at positions 2, 4 to 9 of SEQ ID NO: 539 are substituted with other amino acids, preferably alanine.

[0451] In another embodiment, the polypeptide comprises the amino acid sequence of IXXXXXXEKK (SEQ ID NO: 540). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 2 to 7 of SEQ ID NO: 541 are substituted with other amino acids, preferably alanine.

[0452] In another embodiment, the polypeptide comprises the amino acid sequence of XXXEKK (SEQ ID NO: 543). More preferably, the polypeptide comprises the amino acid sequence in which any one, two or three of the amino acids 1 to 3 of SEQ ID NO: 544 are substituted with other amino acids, preferably alanine.

[0453] (2β-5) A polypeptide comprising the amino acid sequence of XAXXXREYQMPSGYL (SEQ ID NO: 547). More preferably, a polypeptide comprising an amino acid sequence in which any one, two, three or four of the amino acids at positions 1, 3 to 5 of SEQ ID NO: 299 are substituted with other amino acids, preferably alanine.

[0454] In another embodiment, the polypeptide comprises the amino acid sequence of XAXXXXXXQMPSXXL (SEQ ID NO: 548). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1, 3 to 8, 13 and 14 of SEQ ID NO: 299 are substituted with other amino acids, preferably alanine.

[0455] In another embodiment, the polypeptide comprises the amino acid sequence of DAXXXXXXQXXXXYL (SEQ ID NO: 549). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 3 to 8 and 10 to 13 of SEQ ID NO: 299 are substituted with other amino acids, preferably alanine.

[0456] (2β-6) A polypeptide comprising the amino acid sequence of XTXXXACKLXXXXXX (SEQ ID NO: 552). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1, 3 to 5, and 10 to 15 of SEQ ID NO: 301 are substituted with other amino acids, preferably alanine.

[0457] In one embodiment, the polypeptide of (2β) may not include the C-terminal portion of vitellogenin-3 (SEQ ID NOs. 2 and 13) or a mutant or homolog that is identical to or has an identity of 90%, 80% or 70% or more with respect to the entire length of the amino acid sequence.

[0458] (3β) Apolipoprotein B epitope

[0459] In the present invention, the polypeptide (3β) is any polypeptide selected from the group consisting of the following (3β-1) to (3β-8).

[0460] (3β-1) A polypeptide comprising an amino acid sequence of at least one selected from the group consisting of SEQ ID NOs: 304 to 311, 553, 555, 556, 557, 561, 562, 565, 569, 571, 573, 576, 577, 579, 581, 583, 584, 585, 587, 589, 592, 594, 595, 598, 600, 602, 304, 605, 608, 609, 613, 616, 617, 619, 622, 623 and 624.

[0461] (3β-2) A polypeptide comprising the amino acid sequence of AEXXXXXXXXXXLNX (SEQ ID NO: 558). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 3 to 12 and 15 of SEQ ID NO: 304 are substituted with other amino acids, preferably alanine.

[0462] In another embodiment, the polypeptide comprises the amino acid sequence of AEXXXXXX (SEQ ID NO: 554). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids 3 to 8 of SEQ ID NO: 555 are substituted with other amino acids, preferably alanine.

[0463] (3β-3) A polypeptide comprising the amino acid sequence of XXXXXXXXXXXXXNV (SEQ ID NO: 563). Preferably, it is a polypeptide comprising the amino acid sequence of XXXXXXXXXDEPLNV (SEQ ID NO: 564). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acids of the amino acids at positions 1 to 13 of SEQ ID NO: 305 are replaced by other amino acids, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids of the amino acids at positions 1 to 9 of SEQ ID NO: 305 are replaced by other amino acids, preferably alanine.

[0464] In another embodiment, it is a polypeptide comprising an amino acid sequence of XXXXXXXXXKXEXXNX (SEQ ID NO: 574). Preferably, it is a polypeptide comprising an amino acid sequence of XXXKLYPXKXEXXNX (SEQ ID NO: 575). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids of amino acids at positions 1 to 8, 10, 12, 13, and 15 of SEQ ID NO: 305 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 amino acids of amino acids at positions 1 to 3, 8, 10, 12, 13, and 15 of SEQ ID NO: 305 are replaced with other amino acids, preferably alanine.

[0465] In another embodiment, the polypeptide comprises the amino acid sequence of XEPLN (SEQ ID NO: 559), and more preferably, the polypeptide comprises the amino acid sequence in which the amino acid at position 1 of SEQ ID NO: 561 is substituted with another amino acid, preferably alanine.

[0466] In another embodiment, the polypeptide comprises the amino acid sequence of XEPLNV (SEQ ID NO: 560), and more preferably, the polypeptide comprises the amino acid sequence in which the amino acid at position 1 of SEQ ID NO: 562 is substituted with another amino acid, preferably alanine.

[0467] In another embodiment, it is a polypeptide comprising an amino acid sequence of XXXXXXKXEX (SEQ ID NO: 567). Preferably, it is a polypeptide comprising an amino acid sequence of XKLYPXKXXX (SEQ ID NO: 568). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 amino acids of the amino acids at positions 1 to 6, 8 and 10 of SEQ ID NO: 569 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 amino acids of the amino acids at positions 1, 6, 8 to 10 of SEQ ID NO: 569 are replaced with other amino acids, preferably alanine.

[0468] In another embodiment, the polypeptide comprises an amino acid sequence of KXEXXNX (SEQ ID NO: 570). More preferably, the polypeptide comprises an amino acid sequence in which any one, two, three or four of the amino acids at positions 2, 4, 5 and 7 of SEQ ID NO: 571 are substituted with other amino acids, preferably alanine.

[0469] In another embodiment, the polypeptide comprises the amino acid sequence of KXEX (SEQ ID NO: 572), and more preferably comprises a polypeptide comprising an amino acid sequence in which any one or two of the amino acids at positions 2 and 4 of SEQ ID NO: 573 are substituted with other amino acids, preferably alanine.

[0470] (3β-4) A polypeptide comprising the amino acid sequence of VSKRXXVXXXXXXXX (SEQ ID NO: 582). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 5, 6, 8 to 15 of SEQ ID NO: 306 are substituted with other amino acids, preferably alanine.

[0471] In another embodiment, the polypeptide comprises the amino acid sequence of VSKRXX (SEQ ID NO: 578). More preferably, the polypeptide comprises the amino acid sequence in which any one or two of the amino acids at positions 5 and 6 of SEQ ID NO: 579 are substituted with other amino acids, preferably alanine.

[0472] In another embodiment, the polypeptide comprises the amino acid sequence of SVPXXXX (SEQ ID NO: 580), and more preferably comprises a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids 4 to 7 of SEQ ID NO: 581 are substituted with other amino acids, preferably alanine.

[0473] (3β-5) A polypeptide comprising the amino acid sequence of XXXGXXXPXAXXAXX (SEQ ID NO: 590). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of amino acids 1 to 3, 5 to 7, 9, 11 and 12 of SEQ ID NO: 308 are substituted with other amino acids, preferably alanine.

[0474] In another embodiment, the polypeptide comprises an amino acid sequence of XXXGXXXXGAKVAVX (SEQ ID NO: 596). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of amino acids 1 to 3, 5 to 8 and 15 of SEQ ID NO: 308 are substituted with other amino acids, preferably alanine.

[0475] In another embodiment, the polypeptide comprises the amino acid sequence of XXXXIXPXAXXAXK (SEQ ID NO: 603). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1 to 4, 6, 7, 9, 11, 12 and 14 of SEQ ID NO: 308 are substituted with other amino acids, preferably alanine.

[0476] In another embodiment, the polypeptide comprises the amino acid sequence of SGXX (SEQ ID NO: 586). More preferably, the polypeptide comprises the amino acid sequence in which any one or two of the amino acids at positions 3 and 4 of SEQ ID NO: 587 are substituted with other amino acids, preferably alanine.

[0477] In another embodiment, the polypeptide comprises the amino acid sequence of XXXPXAXXAX (SEQ ID NO: 588). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1 to 3, 5, 7, 8 and 10 of SEQ ID NO: 589 are substituted with other amino acids, preferably alanine.

[0478] In another embodiment, the polypeptide comprises the amino acid sequence of XGXXXXGAK (SEQ ID NO: 591). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1, 3 to 6 of SEQ ID NO: 592 are substituted with other amino acids, preferably alanine.

[0479] In another embodiment, the polypeptide comprises the amino acid sequence of XXXXGAKVAV (SEQ ID NO: 593). More preferably, the polypeptide comprises the amino acid sequence in which any one, two, three or four of the amino acids 1 to 4 of SEQ ID NO: 594 are substituted with other amino acids, preferably alanine.

[0480] In another embodiment, the polypeptide comprises the amino acid sequence of XXIXXPXA (SEQ ID NO: 597). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1, 2, 4, 5 and 7 of SEQ ID NO: 598 are substituted with other amino acids, preferably alanine.

[0481] In another embodiment, the polypeptide comprises the amino acid sequence of IXXPXAXXAX (SEQ ID NO: 599). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 2, 3, 5, 7, 8 and 10 of SEQ ID NO: 600 are substituted with other amino acids, preferably alanine.

[0482] In another embodiment, the polypeptide comprises the amino acid sequence of XPXAXXAX (SEQ ID NO: 601). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1, 3, 5, 6 and 8 of SEQ ID NO: 602 are substituted with other amino acids, preferably alanine.

[0483] (3β-6) A polypeptide comprising the amino acid sequence of XXXXPXXXYXXMSSS (SEQ ID NO: 610). Preferably, it is a polypeptide comprising the amino acid sequence of XXXXPXXXYLXMSSS (SEQ ID NO: 611). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids of amino acids 1 to 4, 6 to 8, 10 and 11 of SEQ ID NO: 309 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 amino acids of amino acids 1 to 4, 6 to 8 and 11 of SEQ ID NO: 309 are replaced by other amino acids, preferably alanine.

[0484] In another embodiment, the polypeptide comprises the amino acid sequence of XXXXXXXKXYXQMSSX (SEQ ID NO: 612). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids 1 to 6, 8, 10 and 15 of SEQ ID NO: 309 are substituted with other amino acids, preferably alanine.

[0485] In another embodiment, the polypeptide comprises an amino acid sequence of PXXXYXXMSS (SEQ ID NO: 606). Preferably, the polypeptide comprises an amino acid sequence of PXXXYLXMSS (SEQ ID NO: 607). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4 or 5 amino acids of the amino acids at positions 2 to 4, 6 and 7 of SEQ ID NO: 608 are replaced with other amino acids, preferably alanine. Further preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3 or 4 amino acids of the amino acids at positions 2 to 4 and 7 of SEQ ID NO: 608 are replaced with other amino acids, preferably alanine.

[0486] (3β-7) A polypeptide comprising the amino acid sequence of XXLXDXXXXXXXXXX (SEQ ID NO: 620). Preferably, it is a polypeptide comprising the amino acid sequence of XXLXDXXXXLXXMXX (SEQ ID NO: 621). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the amino acids at positions 1, 2, 4, 6 to 15 of SEQ ID NO: 310 are replaced with other amino acids, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1, 2, 4, 6 to 9, 11, 12, 14, and 15 of SEQ ID NO: 310 are replaced with other amino acids, preferably alanine.

[0487] In another embodiment, it is a polypeptide comprising the amino acid sequence of LXDXXXXXX (SEQ ID NO: 614). Preferably, it is a polypeptide comprising the amino acid sequence of LXDXXXXLX (SEQ ID NO: 615). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 amino acids of the amino acids at positions 2, 4 to 9 of SEQ ID NO: 616 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 amino acids of the amino acids at positions 2, 4 to 7 and 9 of SEQ ID NO: 616 are replaced with other amino acids, preferably alanine.

[0488] In another embodiment, the polypeptide comprises an amino acid sequence of DXXXXLXXMX (SEQ ID NO: 618). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 2 to 5, 7, 8 and 10 of SEQ ID NO: 619 are substituted with other amino acids, preferably alanine.

[0489] (3β-8) A polypeptide comprising the amino acid sequence of LHLXXXXXXXXXXXX (SEQ ID NO: 625). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the amino acids at positions 4 to 15 of SEQ ID NO: 311 are substituted with other amino acids, preferably alanine.

[0490] (4β) Epitopes of vitellogenin-1 / yolk lipophosphoprotein-1

[0491] In the present invention, the polypeptide (4β) is any polypeptide selected from the group consisting of the following (4β-1) to (4β-7).

[0492] (4β-1) A polypeptide comprising at least one amino acid sequence selected from the group consisting of sequence numbers 312 to 318, 627, 630, 631, 632, 636, 637 (amino acid sequence: ELL), 641, 642, 644, 647, 649, 657, 658, 659, 660, 663, 664, 665, 668, 669, 672, 674, 676, 680 and 681.

[0493] (4β-2) A polypeptide comprising the amino acid sequence of XXXXXMFXQELLFGX (SEQ ID NO: 633). Preferably, it is a polypeptide comprising the amino acid sequence of XXXXXMFGQELLFGX (SEQ ID NO: 634). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 amino acids of amino acids 1 to 5, 8 and 15 of SEQ ID NO: 312 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 amino acids of amino acids 1 to 5 and 15 of SEQ ID NO: 312 are replaced by other amino acids, preferably alanine.

[0494] In another embodiment, it is a polypeptide comprising an amino acid sequence of XXXXXXMXXXXXLXXX (SEQ ID NO: 639). Preferably, it is a polypeptide comprising an amino acid sequence of SXXXXMFXXXXLXXX (SEQ ID NO: 638). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids of amino acids 1 to 5, 8 to 11, 13 to 15 of SEQ ID NO: 312 are replaced with other amino acids, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids of amino acids 2 to 5, 7 to 11, 13 to 15 of SEQ ID NO: 312 are replaced with other amino acids, preferably alanine.

[0495] In another embodiment, the polypeptide comprises an amino acid sequence of XXYXXMFXXXXXFXX (SEQ ID NO: 643). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the amino acids at positions 1, 2, 4, 5, 8 to 12, 14 and 15 of SEQ ID NO: 312 are substituted with other amino acids, preferably alanine.

[0496] In another embodiment, the polypeptide comprises the amino acid sequence of XXXXXMFG (SEQ ID NO: 626). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids 1 to 5 of SEQ ID NO: 627 are substituted with other amino acids, preferably alanine.

[0497] In another embodiment, the polypeptide comprises the amino acid sequence of XXXMFXQELL (SEQ ID NO: 628). Preferably, the polypeptide comprises the amino acid sequence of XXXMFGQELL (SEQ ID NO: 629). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3 or 4 amino acids of the amino acids at positions 1 to 6 of SEQ ID NO: 630 are replaced with other amino acids, preferably alanine. Further preferably, the polypeptide comprises an amino acid sequence in which any 1, 2 or 3 amino acids of the amino acids at positions 1 to 3 of SEQ ID NO: 630 are replaced with other amino acids, preferably alanine.

[0498] In another embodiment, the polypeptide comprises the amino acid sequence of SXXXXMFX (SEQ ID NO: 635). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 2 to 5 and 8 of SEQ ID NO: 636 are substituted with other amino acids, preferably alanine.

[0499] In another embodiment, the polypeptide comprises the amino acid sequence of XXYXXMF (SEQ ID NO: 640). More preferably, the polypeptide comprises an amino acid sequence in which any one, two, three or four of the amino acids at positions 1, 2, 4 and 5 of SEQ ID NO: 641 are substituted with other amino acids, preferably alanine.

[0500] (4β-3) A polypeptide comprising the amino acid sequence of XXXXXXXXDTLXXXX (SEQ ID NO: 650). Preferably, it is a polypeptide comprising the amino acid sequence of XXXXRXXXDTLXXXX (SEQ ID NO: 651). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the amino acids at positions 1 to 8 and 12 to 15 of SEQ ID NO: 313 are replaced by other amino acids, preferably alanine. More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1 to 4, 6 to 8, 12 to 15 of SEQ ID NO: 313 are replaced by other amino acids, preferably alanine.

[0501] In another embodiment, it is a polypeptide comprising an amino acid sequence of XXXXDTLX (SEQ ID NO: 645). Preferably, it is a polypeptide comprising an amino acid sequence of RXXXDTLX (SEQ ID NO: 646). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 amino acids of the amino acids at positions 1 to 4 and 8 of SEQ ID NO: 647 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 amino acids of the amino acids at positions 2 to 4 and 8 of SEQ ID NO: 647 are replaced with other amino acids, preferably alanine.

[0502] In another embodiment, the polypeptide comprises the amino acid sequence of DTLXXXXX (SEQ ID NO: 648). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids 4 to 8 of SEQ ID NO: 649 are substituted with other amino acids, preferably alanine.

[0503] (4β-4) A polypeptide comprising the amino acid sequence of XXXSKTAXVXXXXXX (SEQ ID NO: 652). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1 to 3, 8, 10 to 15 of SEQ ID NO: 315 are substituted with other amino acids, preferably alanine.

[0504] In another embodiment, the polypeptide comprises an amino acid sequence of XXXSXXFAXXRXXXX (SEQ ID NO: 653). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1 to 3, 5, 6, 9, 10, 12 to 15 of SEQ ID NO: 315 are substituted with other amino acids, preferably alanine.

[0505] In another embodiment, the polypeptide comprises the amino acid sequence of XXXSXXFAXXXXIXD (SEQ ID NO: 654). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1 to 3, 5, 6, 9 to 12, and 14 of SEQ ID NO: 315 are substituted with other amino acids, preferably alanine.

[0506] (4β-5) A polypeptide comprising the amino acid sequence of XNIXXXAAXXXXPXX (SEQ ID NO: 655). More preferably, a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1, 4 to 6, 9 to 12, 14 and 15 of SEQ ID NO: 316 are substituted with other amino acids, preferably alanine.

[0507] In another embodiment, the polypeptide comprises the amino acid sequence of XXXXXXAASXMXPXX (SEQ ID NO: 656). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids 1 to 6, 10, 12, 14 to 15 of SEQ ID NO: 316 are substituted with other amino acids, preferably alanine.

[0508] (4β-6) A polypeptide comprising the amino acid sequence of PXXXXMXXDXXXASG (SEQ ID NO: 661). Preferably, it is a polypeptide comprising the amino acid sequence of PXIMXMXFDSXSASG (SEQ ID NO: 662). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids at positions 2 to 5, 7, 8, 10 to 12 of SEQ ID NO: 317 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 amino acids at positions 2, 5, 7 and 11 of SEQ ID NO: 317 are replaced by other amino acids, preferably alanine.

[0509] In another embodiment, the polypeptide comprises the amino acid sequence of XXXXXXXXXXXSASG (SEQ ID NO: 666). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids 1 to 11 of SEQ ID NO: 317 are substituted with other amino acids, preferably alanine.

[0510] (4β-7) A polypeptide comprising the amino acid sequence of XXXXXXXDVPIEKIQ (SEQ ID NO: 670). Preferably, it is a polypeptide comprising the amino acid sequence of XXXXXYSDVPIEKIQ (SEQ ID NO: 671). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 amino acids of the amino acids at positions 1 to 7 of SEQ ID NO: 318 are replaced by other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 amino acids of the amino acids at positions 1 to 5 of SEQ ID NO: 318 are replaced by other amino acids, preferably alanine.

[0511] In another embodiment, the polypeptide comprises the amino acid sequence of XFXXVXSXXXXXXXQ (SEQ ID NO: 675). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1, 3, 4, 6, 8 to 14 of SEQ ID NO: 318 are substituted with other amino acids, preferably alanine.

[0512] In another embodiment, it is a polypeptide comprising an amino acid sequence of XXXXXXXSXVXIXKXQ (SEQ ID NO: 677). Preferably, it is a polypeptide comprising an amino acid sequence of XXXXXXXSDVXIXKIQ (SEQ ID NO: 678). More preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids of the amino acids at positions 1 to 6, 8, 10 and 12 of SEQ ID NO: 318 are replaced with other amino acids, preferably alanine. Further preferably, it is a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 amino acids of the amino acids at positions 1 to 6, 10 and 12 of SEQ ID NO: 318 are replaced with other amino acids, preferably alanine.

[0513] In another embodiment, the polypeptide comprises the amino acid sequence of SFKPVXXXXXXXXXX (SEQ ID NO: 682). More preferably, the polypeptide comprises the amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids 6 to 15 of SEQ ID NO: 318 are substituted with other amino acids, preferably alanine.

[0514] In another embodiment, the polypeptide comprises the amino acid sequence of XXDVPIEK (SEQ ID NO: 667). More preferably, the polypeptide comprises the amino acid sequence in which any one or two of the amino acids at positions 1 and 2 of SEQ ID NO: 668 are substituted with other amino acids, preferably alanine.

[0515] In another embodiment, the polypeptide comprises the amino acid sequence of XSXVXXEX (SEQ ID NO: 673). More preferably, the polypeptide comprises an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1, 3, 5, 6 and 8 of SEQ ID NO: 674 are substituted with other amino acids, preferably alanine.

[0516] In another embodiment, the polypeptide comprises the amino acid sequence of SFKPVX (SEQ ID NO: 679), and more preferably comprises the amino acid sequence in which the amino acid at position 6 of SEQ ID NO: 680 is substituted with another amino acid, preferably alanine.

[0517] In one embodiment, the polypeptide of (4β) may also not contain the central portion of vitellogenin-1 or the C-terminal portion of yolk lipophosphoprotein-1 (SEQ ID NO: 18) or a mutant or homolog that is identical to or has an identity of more than 90%, more than 80% or more than 70% relative to the full length of the amino acid sequence.

[0518] There is no particular limitation on the length of the polypeptides of (1β) to (4β). In a preferred embodiment, the length of the polypeptides of (1β) to (4β) 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, 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, or 5 amino acids or less.

[0519] The polypeptides of (1β) to (4β) can also be prepared by chemical synthesis methods such as solid phase synthesis of peptides. Alternatively, the polypeptide containing the epitope can be expressed as a recombinant protein by a gene recombination technique known to those skilled in the art, and then isolated / produced by a protein production method known to those skilled in the art.

[0520] Diagnostic kits / diagnostic methods (2)

[0521] The present invention provides a method for providing an index for diagnosing an allergic reaction of a subject, comprising the following steps:

[0522] (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies;

[0523] (ii) detecting the binding of IgE antibodies in a sample obtained from the subject to the antigen;

[0524] (iii) when the binding of the subject's IgE antibody to the antigen is detected, providing an indication that the subject has an allergic reaction;

[0525] Here, the antigen is a polypeptide that is at least one of the above-mentioned polypeptides (1β) to (4β), or a polypeptide in which two or more of the above-mentioned polypeptides (1β) to (4β) are linked via a spacer or without a spacer.

[0526] Hereinafter, in this specification, a polypeptide that is at least one of the polypeptides (1β) to (4β) or a polypeptide in which two or more of the polypeptides (1β) to (4β) are linked with or without a spacer is described as an "antigen comprising the above (1β) to (4β)". There is no particular limitation on the type of spacer, and substances commonly used by those skilled in the art can be used to link multiple peptides. The spacer may also be a hydrocarbon chain such as Acp(6)-OH.

[0527] The sample obtained from the subject is as described in the above-mentioned section "Diagnostic Kit / Diagnostic Method (1)".

[0528] The contact of the sample obtained from the control with the antigen and the detection of their binding can be carried out by known methods described in the above-mentioned "Diagnostic Kit / Diagnostic Method (1)", for example: ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassays, protein immunoblotting, immunoprecipitation, and immunochromatography.

[0529] The antigens containing the above (1β) to (4β) may also be in a state of being fixed to a carrier. In this case, the above steps (i) and (ii) may utilize ELISA, sandwich immunoassay, immunochromatography, or surface plasmon resonance; the above step (i) is performed by bringing a sample obtained from the subject into contact with a surface on which the antigens containing the above (1β) to (4β) are fixed. In addition, the binding of the IgE antibody of the subject to the antigens containing the above (1β) to (4β) may be detected by the above method using a substance in which the IgE antibody of the subject is fixed to a carrier.

[0530] The antigens containing the above (1β) to (4β) may also be in a state where they are not fixed to a carrier. In this case, in the above steps (i) and (ii), a flow cytometer or the like can be used to confirm the presence of the antigens containing the above (1β) to (4β) bound to the IgE antibody by laser. The method may include, for example, a basophil activation test (BAT) as a method for detecting the surface antigen CD203c that appears when basophils are activated by contacting the antigens containing the above (1β) to (4β). In addition, a histamine free test (HRT) may also be listed, which confirms whether histamine is freed by further contacting the antigens containing the above (1β) to (4β) with the blood cells in the sample.

[0531] The antigens including (1β) to (4β) are antigens that specifically bind to IgE antibodies of allergy patients. Therefore, if the binding of the subject's IgE antibodies to the antigen is detected, it provides an indicator that the subject has an allergy.

[0532] The present invention also provides a diagnostic kit for allergies comprising at least one of the above-mentioned antigens (1β) to (4β). The diagnostic kit of the present invention can be used in the above-mentioned method for providing an indicator for diagnosing allergies or the following diagnostic method. In addition to containing at least one of the above-mentioned antigens (1β) to (4β), the diagnostic kit of the present invention may also contain an anti-IgE antibody labeled with an enzyme and a colorimetric substrate or a luminescent substrate that serves as a substrate for the enzyme. In addition, a fluorescently labeled anti-IgE antibody may also be used. The diagnostic kit of the present invention may also be provided in a state where the antigens (1β) to (4β) are immobilized on a carrier. The diagnostic kit of the present invention may also be provided together with instructions regarding the steps used for diagnosis and a method package containing the instructions.

[0533] In another embodiment, the diagnostic kit includes a companion diagnostic drug for allergic reactions. The companion diagnostic drug refers to a substance used for the following purposes: specifying patients who expect the drug to be effective or patients who are at risk of severe side effects of the drug, or studying the reactivity of the drug in order to optimize the treatment using the drug. Here, the optimization of treatment includes, for example, determining the dosage volume, determining the suspension of drug administration, and confirming whether immune tolerance is obtained by using which allergen component.

[0534] The present invention also provides a composition for diagnosing allergic reactions comprising at least one of the above-mentioned antigens (1β) to (4β). The composition for diagnosing the present invention can be used in the following diagnostic method. The composition for diagnosing the present invention can also contain pharmaceutically acceptable carriers and additives that are usually used together with the antigen of the present invention as needed.

[0535] In one embodiment, the present invention is a method for diagnosing an allergic reaction in a subject, comprising the following steps:

[0536] (i) contacting a sample obtained from a subject with an antigen;

[0537] (ii) detecting the binding of IgE antibodies in a sample obtained from the subject to the antigen;

[0538] (iii) when the binding of the subject's IgE antibody to the antigen is detected, the subject is judged to have an allergic reaction;

[0539] Here, the antigen is at least one of the proteins defined as antigens including (1β) to (4β) above. Steps (i) and (ii) are performed according to the description of the steps of the method for providing an index for diagnosing an allergic reaction.

[0540] In another embodiment, the present invention provides a method for diagnosing an allergic reaction in a subject, comprising administering to the subject at least one of the antigens (1β) to (4β). The method may also be performed in the form of a skin test characterized in that the antigens (1β) to (4β) are administered to the skin. The skin test includes the following forms: a prick test in which, after applying a diagnostic composition to the skin, the skin is lightly scratched to a level that does not bleed, the skin is soaked with the antigens (1β) to (4β) and then the skin reaction is observed; a scratch test in which the skin is lightly scratched after the diagnostic composition is applied and then the reaction is observed; a patch test in which a diagnostic composition in the form of a cream, ointment, etc. is applied to the skin and then the reaction is observed; an intradermal test in which the antigens (1β) to (4β) are administered intradermally and then the reaction is observed; etc. In the case where a skin reaction such as swelling occurs on the part of the skin to which the antigens (1β) to (4β) are administered, the subject is diagnosed as having an allergic reaction. Here, the amount of the antigen containing (1β) to (4β) to be applied to the skin may be, for example, 100 μg or less per time.

[0541] In the diagnosis of allergy, tolerance tests for the purpose of limiting antigens are often performed. At least one of the above-mentioned antigens (1β) to (4β) can be used as an active ingredient in tolerance tests for diagnosing allergy. Here, the allergen component used in the tolerance test can be a purified polypeptide or a protein expressed in food / ingredients, such as pollen rice in which the gene of cedar pollen antigen is transformed into rice and the antigen protein is expressed in rice.

[0542] In another embodiment, the present invention provides at least one antigen comprising the above-mentioned (1β) to (4β) for use in diagnosis of allergy.

[0543] In yet another embodiment, the present invention provides use of at least one of the above antigens (1β) to (4β) for preparing a diagnostic drug for allergy.

[0544] In this item, the allergic reaction to be diagnosed or detected may be an allergic reaction to the antigens including (1β) to (4β). That is, the detection of allergic reaction may detect not only allergic reaction to a single antigen including (1β) to (4β) but also cross-reaction allergic reaction.

[0545] Pharmaceutical composition / treatment method (2)

[0546] The present invention provides a pharmaceutical composition comprising at least one of the above-mentioned antigens (1β) to (4β). In one embodiment, the above-mentioned pharmaceutical composition is used to treat allergies. The treatment of allergies is to increase the limit amount of antigen that will not cause disease even if ingested into the body, and ultimately aims to achieve a state (remission) in which no disease will occur after the usual antigen intake.

[0547] The present invention also provides a method for treating allergy, comprising: administering at least one of the antigens (1β) to (4β) to a patient in need of treatment for allergy.

[0548] In another embodiment, the present invention provides at least one of the antigens (1β) to (4β) for use in the treatment of allergies. In yet another embodiment, the present invention provides use of at least one of the antigens (1β) to (4β) for the preparation of a diagnostic drug for allergies.

[0549] In the treatment of allergies, desensitization therapy is often performed in which the purpose is to induce immune tolerance by administering antigens to patients. At least one of the above-mentioned antigens (1β) to (4β) can be used as an active ingredient in desensitization therapy for treating allergies. Here, the allergen component used in the desensitization therapy can be a purified polypeptide or a substance expressed in food / ingredients such as pollen rice.

[0550] The administration route, dosage, number of administrations and / or administration intervals of the pharmaceutical composition of the present invention, other ingredients contained in the pharmaceutical composition, and dosage form may be those described in the above-mentioned "Pharmaceutical composition / therapeutic method (1)". The dosage when using an antigen containing the above-mentioned (1β) to (4β), for example, in the case of adults, may be 100 μg or less per time.

[0551] In this item, the allergy to be treated may be an allergy to the antigens including (1β) to (4β). That is, the treatment of allergy can treat not only allergies to a single antigen including (1β) to (4β) but also cross-reaction allergies.

[0552] Detector(2)

[0553] The present invention provides a detector comprising an antibody against at least one of the antigens (1β) to (4β).

[0554] The antibody can be prepared by conventional methods. For example, a mammal such as a rabbit can be immunized with an antigen containing the above (1β) to (4β). The antibody can be an Ig antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (e.g., Fab, F(ab')2, Fab').

[0555] In addition, in the above-mentioned detector, the antibody can be provided in the form of being bound to a carrier. The carrier is not particularly limited as long as it can be used in the detection of the antibody binding to the above-mentioned (1β) to (4β) antigens. Any carrier known to those skilled in the art can be used. In addition, the antibody against the antigen containing the above-mentioned (1β) to (4β) is preferably an antibody against the epitope described in the above-mentioned "epitope of antigen". Thus, it can be a detector capable of detecting including cross-reactivity.

[0556] Examples of methods for examining whether or not the antigens including (1β) to (4β) are present include the following methods.

[0557] A method of bringing a detector containing the prepared antibody into contact with a sample obtained from a raw material / processed product, etc., and detecting the binding of the antibody to an antigen containing the above-mentioned (1β) to (4β) in the sample using, for example, an ELISA method, and, when the binding of the antibody to the antigen containing the above-mentioned (1β) to (4β) is detected, determining that the antigen remains in the target raw material / processed product, etc.

[0558] A method in which a raw material / processed product is wrapped with filter paper or the like and reacted with an antibody solution to detect the antigens containing the above (1β) to (4β) contained therein.

[0559] Another embodiment of the present invention includes a detector for determining whether an allergic reaction occurs in an object, comprising an antigen of (1β) to (4β), characterized in that it comprises a primer corresponding to an epitope. In a non-limiting manner, the primer can be designed to include, for example, a base sequence of a portion of a base sequence of a nucleic acid encoding an amino acid sequence defined by (1β) to (4β) or its complementary chain. Alternatively, the primer can be designed to include a base sequence of a complementary chain of a base sequence of a portion of an upstream region encoding an epitope or a base sequence of a portion of a downstream region encoding an epitope in a nucleic acid encoding a protein containing an epitope of an amino acid sequence defined by (1β) to (4β). Examples of such primers include primers that are part of at least one of the base sequences shown in SEQ ID NOs: 46, 58, 105, 151, 236, 250, or 261, and / or primers that are part of a sequence complementary to at least one of the base sequences shown in SEQ ID NOs: 46, 58, 105, 151, 236, 250, or 261. Here, the position of the epitope in the full-length sequence of the antigen is as defined in Table 2 of the following Example 6. In particular, when mRNA is used as the target, a complementary primer having a poly(A) tail may also be used.

[0560] For example, DNA or mRNA obtained from a sample is used as a template and the primers are used to amplify DNA by PCR (polymerase chain reaction) including RT-PCR, and by determining whether the amplified DNA sequence contains a nucleic acid encoding the amino acid sequence defined by the above (1β)-(4β), it is determined whether the antigen containing the above (1β)-(4β) exists. Examples of methods for amplifying mRNA by PCR include the RACE method and the like.

[0561] In one embodiment, the detector can be used to detect whether an object such as a raw material or processed product production line contains antigens including the above (1β) to (4β). The raw material can be food, cosmetic raw material, pharmaceutical raw material, etc. The processed product can be an edible processed product, cosmetic, pharmaceutical, etc. The detector can be used to explore the biological species contained in the raw materials, and can also be used for quality inspection of production lines and pre-shipment products conducted by manufacturers; it can also be used by tasters or users themselves to check whether the target raw materials / processed products contain antigens.

[0562] Allergen-free raw materials, etc. (2)

[0563] The present invention provides a raw material or processed product, characterized in that at least one of the antigens (1β) to (4β) is removed or reduced.

[0564] The method of removing or reducing antigens in raw materials or processed products of the present invention is not limited. The removal or reduction of antigens can be carried out by any method as long as the antigens containing the above-mentioned (1β) to (4β) can be removed or reduced, for example, the method described in the above-mentioned "Foods, etc. (1) from which allergens are removed" can be used.

[0565] The removal or reduction of at least one of the antigens (1β) to (4β) can be achieved by removing or reducing the antigen as a whole, or by cutting or removing the sequence portion defined by (1β) to (4β) from the antigen protein. "Removal" includes the deletion and modification of all or part of the sequence portion defined by (1) to (6).

[0566] For example, a material containing the above-mentioned (1β) to (4β) antigens can be deleted or reduced by using gene knockout technology to prepare a material containing the above-mentioned (1β) to (4β) antigens whose expression is knocked out. The gene knockout technology can use any method known to those skilled in the art.

[0567] Processed products from which the antigens (1β)-(4β) are removed or reduced, such as milk powder using purified peptides as raw materials, may be processed products using raw materials from which the antigens (1β)-(4β) are removed or reduced. When ordinary raw materials are used, treatment to remove or reduce the antigens (1β)-(4β) is performed before or after the preparation of the processed product. As a method for removing or reducing the antigens (1β)-(4β) in processed products using ordinary raw materials, the method described in the above-mentioned item "Allergen-removed foods, etc. (1)" can be used. As a method for cutting the antigens (1β)-(4β), a method of cutting with a specific digestive enzyme can be cited.

[0568] Method for producing processed products from which allergens are removed (2)

[0569] The present invention provides a method for producing a processed product in which an antigen has been removed or reduced, comprising the step of confirming that the antigen has been removed or reduced during the production of the processed product, wherein the antigen is at least one of the antigens (1β) to (4β) described above.

[0570] In this production method, antigen removal or reduction means that at least one of the antigens comprising the above (1β) to (4β) is removed or reduced; or, the sequence portion defined by the above (1β) to (4β) is cut off or removed from the antigen.

[0571] The method for confirming that the antigen has been removed or reduced in the manufacturing process of the processed product is not particularly limited, and any method capable of detecting at least one of the antigens (1β) to (4β) can be used. For example, the presence or absence of the polypeptide or antigen in the processed product can be confirmed by comparing the binding of a sample of a material produced in the manufacturing process of the processed product with an antibody against at least one of the antigens (1β) to (4β). The details of such a method are as described in the above-mentioned "diagnostic kit / diagnostic method (2)". That is, for the above-mentioned manufacturing method, the "IgE antibody of the object" in the above-mentioned "diagnostic kit / diagnostic method (2)" can be replaced with "antibody against at least one of the antigens (1β) to (4β)", and the "antigen" in the above-mentioned "diagnostic kit / diagnostic method (2)" can be replaced with "a sample of a material produced in the manufacturing process of the processed product", and the method described in the above-mentioned "diagnostic kit / diagnostic method (2)" can be used to confirm that the antigen has been removed or reduced in the manufacturing process of the processed product. In addition, the detector described in the above-mentioned "detector (2)" can also be used.

[0572] Example

[0573] Hereinafter, examples of the present invention will be described, but the technical scope of the present invention is not limited to these examples.

[0574] Example 1: Confirmation of protein profile

[0575] The proteins contained in the eggs were detected using the two-dimensional electrophoresis method described below.

[0576] Protein extraction

[0577] Separate raw eggs into egg whites and yolks.

[0578] The extraction and purification of proteins contained in egg white and egg yolk are performed as follows. After adding a solubilizing agent to the egg white to extract the protein, water or urea buffer is added to obtain a protein extract. The composition of the urea buffer is as follows.

[0579] 30mM Tris

[0580] 2M Thiourea

[0581] 7M Urea

[0582] 4% (w / v) CHAPS:

[0583] 3-[3-(Cholamidopropyl)dimethylamino]propanesulfonic acid inner salt

[0584] Appropriate amount of dilute hydrochloric acid

[0585] Distilled water was added to adjust the total volume to 100 mL and the pH to 8.5.

[0586] Then, 25 μg of each was mixed based on the weight of protein to obtain an extract. A surfactant buffer (Mammalian Lysis Buffer (MCLI), SIGMA) was added to the egg yolk as a solubilizing agent to extract the protein.

[0587] After that, two precipitation operations were performed using the 2D-CleanUP kit (manufactured by GE). In the first precipitation operation, TCA (trichloroacetic acid) was added to the recovered protein extract to precipitate, and the precipitate (TCA precipitate) produced by the operation was recovered. In the second precipitation operation, acetone was added to the recovered TCA precipitate to precipitate, and the precipitate (test sample) obtained by the operation was recovered.

[0588] Preparation of sample solution

[0589] A portion of the obtained sample (50 μg in terms of protein weight) was dissolved in 150 μl of DeStreak Rehydration Solution (GE), a swelling buffer for gel for first-dimensional isoelectric electrophoresis, to prepare a sample solution for first-dimensional isoelectric electrophoresis (swelling sample solution). The composition of DeStreak Rehydration Solution is as follows.

[0590] 7M Thiourea

[0591] 2M Urea

[0592] 4%(w / v)CHAPS

[0593] 0.5% (v / v) IPG buffer; manufactured by GE

[0594] Appropriate amount of BPB (bromophenol blue)

[0595] Penetration of the specimen into the first-dimension isoelectric electrophoresis gel

[0596] A gel for first-dimensional isoelectric electrophoresis (IPG gel Immobiline Drystrip (pH 3-10 NL, manufactured by GE) was immersed in 140 μl of the above-mentioned sample solution for first-dimensional isoelectric electrophoresis (sample solution for swelling) and allowed to permeate overnight at room temperature.

[0597] In this example, IPGphor manufactured by GE was used as the electrophoresis apparatus.

[0598] The swimming lane was filled with silicone oil. Filter papers moistened with water were placed at both ends of the gel soaked with the specimen, the swimming lane was set so that the gel was covered with silicone oil, and electrodes were placed between the gel and the filter paper.

[0599] The upper limit of the current value of the isoelectric point electrophoresis instrument is set to 75 μA per gel, and the voltage program is set as follows: (1) the constant voltage is increased from 300 V to 750 Vhr to perform a constant voltage process (the current variation amplitude during 30 minutes of electrophoresis before the end of this process is 5 μA); (2) the voltage is gradually increased from 300 Vhr to 1000 V; (3) further, the voltage is gradually increased from 4500 Vhr to 5000 V; (4) thereafter, the first dimension isoelectric point electrophoresis is performed at a constant voltage of 5000 V until Vhr reaches 12000.

[0600] SDS equilibration of isoelectric point electrophoresis gel

[0601] After the first-dimensional isoelectric electrophoresis, the gel was removed from the isoelectric electrophoresis 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 is as follows.

[0602] 100 mM Tris-HCl (pH 8.0)

[0603] 6M urea

[0604] 30% (v / v) glycerol

[0605] 2%(w / v)SDS

[0606] 1%(w / v)DTT

[0607] 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 an alkylating agent is as follows.

[0608] 100 mM Tris-HCl (pH 8.0)

[0609] 6M urea

[0610] 30% (v / v) glycerol

[0611] 2%(w / v)SDS

[0612] 2.5% (w / v) iodoacetamide

[0613] SDS-PAGE in the second dimension

[0614] In this example, the electrophoresis apparatus used was 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. In addition, a running buffer having the following composition was prepared and used.

[0615] 50mM MOPS

[0616] 50mM Tris base

[0617] 0.1%(w / v)SDS

[0618] 1mM EDTA

[0619] In this example, an agarose solution for bonding was used in which 0.5% (w / v) agarose S (manufactured by Nippongene Co., Ltd.) and an appropriate amount of BPB (bromophenol blue) were dissolved in a running buffer.

[0620] After washing the wells of SDS-PAGE with the above-mentioned electrophoresis buffer, remove the buffer used in the washing. Then, add a fully dissolved bonding agarose solution in the wells. Then, immerse the gel that has been subjected to SDS equilibration in agarose, and use tweezers to make the gel that has been subjected to SDS equilibration and the second dimension electrophoresis gel fit. Confirm that agarose is fully solidified under the state that the two gels are closely fitted, and electrophoresis is carried out for about 45 minutes with a constant voltage of 200V.

[0621] Fluorescent staining of gels

[0622] The gel was fluorescently stained using SYPRO Ruby (manufactured by Life Technologies).

[0623] First, the sealed container used was thoroughly washed with 98% (v / v) ethanol in advance. The following treatment was performed twice: the second-dimensional electrophoresis gel was removed from the SDS-PAGE instrument after electrophoresis, placed in a washed sealed container, and immersed in an aqueous solution containing 50% (v / v) methanol and 7% (v / v) acetic acid for 30 minutes. After that, the aqueous solution was replaced with water and immersed for 10 minutes. Next, the second-dimensional electrophoresis gel was immersed in 40 ml of SYPRO Ruby and shaken at room temperature overnight. Next, SYPRO Ruby was removed, the second-dimensional electrophoresis gel was washed with water, and then shaken with an aqueous solution containing 10% (v / v) methanol and 7% (v / v) acetic acid for 30 minutes. Furthermore, the aqueous solution was replaced with water and shaken for more than 30 minutes.

[0624] Analysis

[0625] The gel for second-dimensional electrophoresis subjected to the above series of treatments was subjected to scanning of a fluorescent image using Typhoon 9400 (manufactured by GE). The results of the two-dimensional electrophoresis of the proteins contained in the egg yolk are shown in FIG. Figure 1 The results for proteins are not shown. Molecular weight marker bands are visible on the left side of the gel photograph, and the positions of the bands indicate specific molecular weights (KDa).

[0626] Example 2: Confirmation of antigen by immunoblotting (1)

[0627] Antigen confirmation by immunoblotting was performed by performing the steps described in Example 1 up to "second-dimensional SDS-PAGE" and then performing the following operations of "transfer to membrane", "immunoblotting" and "analysis".

[0628] Transfer to membrane

[0629] Transfer to the membrane was performed using the following transfer apparatus and transfer buffer.

[0630] Blotting device: XCell SureLock Mini-Cell and XCell II blot module (Lifetechnologies)

[0631] Transfer buffer: NuPAGE transfer buffer (×20) (manufactured by Life Technologies) was diluted 20-fold with milliQ water and used.

[0632] Specifically, the proteins in the two-dimensional electrophoresis gel were transferred to a membrane (PVDF membrane) according to the following procedure.

[0633] (1) The PVDF membrane was immersed in 100% methanol, then immersed in milliQ water, and then transferred to a transfer buffer to perform a hydrophilization treatment on the PVDF membrane.

[0634] (2) The sponge, filter paper, second-dimensional SDS-PAGE gel, hydrophilized PVDF membrane, filter paper, and sponge were placed in the order described above and energized at a constant voltage of 30 V for 1 hour in a transfer device.

[0635] Immunoblotting

[0636] The serum of a patient with egg allergy (patient 1) or the serum of a non-egg allergy subject was used as the first antibody for immunoblotting of the membrane. The patient with egg allergy was diagnosed with food-dependent exercise-induced allergy (FDEIA) caused by eggs. It should be noted that for this patient, in the puncture test, raw eggs, boiled eggs, and hot spring eggs were all negative; in the specific IgE antibody test, the egg white, egg yolk, and ovomucoid were negative.

[0637] Immunoblotting of membranes was performed as follows.

[0638] (1) The transferred membrane was shaken in a 5% skim milk / PBST solution (PBS buffer containing 0.1% nonionic surfactant Tween 20) at room temperature for 1 hour.

[0639] (2) As the primary antibody, the cells were incubated in a 5% serum / 5% skim milk / PBST solution at room temperature for 1 hour.

[0640] (3) Wash with PBST solution (5 minutes x 3 times).

[0641] (4) As a secondary antibody, anti-human IgE-HRP (horseweed peroxidase) was diluted 5000 times with a 5% skim milk / PBST solution and allowed to stand at room temperature for 1 hour.

[0642] (5) Wash with PBST solution (5 minutes x 3 times).

[0643] (6) The sample was allowed to stand in Pierce Western Blotting Substrate Plus (manufactured by Thermo Fisher Scientific) for 5 minutes.

[0644] Analysis

[0645] The membrane subjected to the above series of treatments was subjected to scanning of a fluorescent image using Typhoon 9500 (manufactured by GE).

[0646] The immunoblot using the serum of the patient with egg allergy was compared with the immunoblot using the serum of the non-egg allergy subject as a control. The protein contained in the raw egg yolk was immunoblotted using the serum of the patient with egg allergy ( Figure 2 ), unlike the case where the serum of a non-egg allergic subject was used, three spots different from the known egg allergen proteins were detected.

[0647] The molecular weights and isoelectric points of the above three spots are as follows: Figure 3 ).

[0648] Spot 1: molecular weight 20-40 kDa, pI 4.0-10.0

[0649] Spot 2: molecular weight 30-60 kDa, pI 5.0-10.0

[0650] Spot 3: molecular weight 35-60 kDa, pI 5.0-10.0

[0651] Example 3: Mass spectrometry analysis and identification of antigens (1)

[0652] The amino acid sequence of the antigen producing three spots in Example 2 was identified by mass spectrometry.

[0653] Specifically, protein extraction and mass spectrometry analysis were performed according to the following steps.

[0654] (1) The yolk of raw eggs was subjected to protein extraction, two-dimensional electrophoresis and membrane transfer according to the procedures of Examples 1 and 2, and then stained with 0.008% direct blue / 40% ethanol / 10% acetic acid by shaking.

[0655] (2) Thereafter, the sample was treated with 40% ethanol / 10% acetic acid for 5 minutes three times for decolorization, washed with water for 5 minutes, and then air-dried.

[0656] (3) Cut the target spot with a clean cutter and add it to a centrifuge tube. After hydrophilizing the membrane with 50 μL of methanol, wash it twice with 100 μL of water and remove it by centrifugation. Then add 20 μL of 20 mM NH4HCO3·50% acetonitrile.

[0657] (4) Add 1 μL of 1 pmol / μL lysine-endo-peptidase (WAKO), let stand at 37°C for 60 minutes, and then recover the solution in a new centrifuge tube. Add 20 μL of 20 mM NH4HCO3·70% acetonitrile to the membrane, immerse it at room temperature for 10 minutes, and then recover it. Dissolve it with 10 μL of 0.1% formic acid and 4% acetonitrile, and transfer it to a test tube.

[0658] (5) The recovered solution was dried under reduced pressure, dissolved in 15 μl of solution A (0.1% formic acid, 4% acetonitrile solution), and subjected to mass spectrometry analysis (ESI-TOF6600, manufactured by AB Sciex).

[0659] (6) Protein identification based on mass spectrometry data obtained by a mass spectrometer was performed by searching NCBI or UniProt.

[0660] result

[0661] As a result of mass spectrometry analysis of each spot, the following amino acid sequences were detected.

[0662] Spot 1: Amino acid sequence shown in SEQ ID NOs: 3 to 11

[0663] Spot 2: amino acid sequence shown in SEQ ID NOs: 9 to 11, 14, and 16

[0664] Spot 3: Amino acid sequence shown in SEQ ID NOs: 19 to 45

[0665] Furthermore, for each spot, the mass spectrum data obtained by the mass spectrometer were analyzed using NCBI for spot 1 and spot 2, and the mass spectrum data obtained by the mass spectrometer were analyzed using UniProt for spot 3. As a result, each spot was identified as the following protein.

[0666] Spot 1: C-terminal portion of vitellogenin-3 (amino acid sequence: NCBI accession number XP_015146355, base sequence encoding the same: GenBank accession number XM_015290869.1) (amino acid sequence: sequence number 2, base sequence encoding the same: sequence number 1)

[0667] Spot 2: C-terminal portion of vitellogenin-3 (amino acid sequence: NCBI accession number XP_015146355, base sequence encoding the same: GenBank accession number XM_015290869.1) (amino acid sequence: sequence number 13, base sequence encoding the same: sequence number 12)

[0668] Spot 3: The central part of vitellogenin-1 (amino acid sequence: UniProt accession number P87498, base sequence encoding it: ENA (EMBL) accession number D89547.1), or the C-terminal part of lipovitelin-1 (amino acid sequence: sequence number 18, base sequence encoding it: sequence number 17)

[0669] Example 4: Confirmation of antigen by immunoblotting (2)

[0670] Similar to Example 2, antigen confirmation was performed by immunoblotting using sera from three other egg allergy patients (patient 2, patient 3, and patient 4). For proteins contained in raw egg yolk, immunoblotting using sera from egg allergy patients (patient 2 was Figure 4 For patient 3, Figure 5 For patient 4, Figure 6), unlike the case where the serum of a non-egg allergic subject was used, spots different from the known egg allergen protein were detected. Specifically, spots 4 to 8 were detected for patient 2, spots 9 and 10 were detected for patient 3, and spot 11 was detected for patient 4.

[0671] The molecular weights and isoelectric points of the above spots 4 to 11 are as follows: Figures 4 to 6 ).

[0672] Spot 4: molecular weight 20-50 kDa, pI 3.0-8.0

[0673] Spots 5 and 6: molecular weight 80-260 kDa, pI 1.0-8.0

[0674] Spot 7: molecular weight 110-300 kDa, pI 3.0-8.0

[0675] Spot 8: molecular weight 160-550 kDa, pI 3.0-8.0

[0676] Spot 9: molecular weight 20-50 kDa, pI 7.0-11.0

[0677] Spot 10: molecular weight 20-50 kDa, pI 7.0-11.0

[0678] Spot 11: molecular weight 15-40 kDa, pI 6.0-11.0

[0679] Example 5: Mass spectrometry analysis and identification of antigens (2)

[0680] In the same manner as in Example 3, the amino acid sequences of the antigens producing spots 4 to 11 in Example 4 were identified by mass spectrometry.

[0681] As a result of mass spectrometry analysis of each spot, the following amino acid sequences were detected.

[0682] Spot 4: amino acid sequence shown in SEQ ID NOs: 48 to 57

[0683] Spots 5 and 6: Amino acid sequences shown in SEQ ID NOs: 60 to 104

[0684] Spot 7: amino acid sequence shown in SEQ ID NOs: 107 to 150

[0685] Spot 8: Amino acid sequence shown in SEQ ID NOs: 153 to 235

[0686] Spot 9: amino acid sequence shown in SEQ ID NOs: 238 to 249

[0687] Spot 10: amino acid sequence shown in SEQ ID NOs: 252 to 260

[0688] Spot 11: Amino acid sequence shown in SEQ ID NOs: 263 to 271

[0689] Furthermore, as a result of analyzing the mass spectrum data obtained by the mass spectrometer for each spot using NCBI, each spot was identified as the following protein.

[0690] Spot 4: Vitellogenin-3 (amino acid sequence: NCBI accession number XP_015146355, base sequence encoding it: GenBank accession number XM_015290869.1) (amino acid sequence: sequence number 47, base sequence encoding it: sequence number 46)

[0691] Spots 5 and 6: Vitellogenin-2 precursor (amino acid sequence: NCBI accession number NP_001026447.1, base sequence encoding it: GenBank accession number NM_001031276.1) (amino acid sequence: sequence number 59, base sequence encoding it: sequence number 58)

[0692] Spot 7: Vitellogenin-2 precursor (amino acid sequence: NCBI accession number NP_001026447.1, base sequence encoding it: GenBank accession number NM_001031276.1) (amino acid sequence: sequence number 106, base sequence encoding it: sequence number 105)

[0693] Spot 8: Apolipoprotein B precursor (amino acid sequence: NCBI accession number NP_001038098.1, base sequence encoding it: GenBank accession number NM_001044633.1) (amino acid sequence: sequence number 152, base sequence encoding it: sequence number 151)

[0694] Spot 9: Apolipoprotein B precursor (amino acid sequence: NCBI accession number NP_001038098.1, base sequence encoding it: GenBank accession number NM_001044633.1) (amino acid sequence: sequence number 237, base sequence encoding it: sequence number 236)

[0695] Spot 10: Apolipoprotein B precursor (amino acid sequence: NCBI accession number NP_001038098.1, base sequence encoding it: GenBank accession number NM_001044633.1) (amino acid sequence: sequence number 251, base sequence encoding it: sequence number 250)

[0696] Spot 11: Vitellogenin-2 precursor (amino acid sequence: NCBI accession number NP_001026447.1, base sequence encoding it: GenBank accession number NM_001031276.1) (amino acid sequence: sequence number 262, base sequence encoding it: sequence number 261)

[0697] Example 6: Identification of epitopes

[0698] Epitopes of allergenic components of eggs

[0699] The epitopes of egg allergen components were identified according to the following procedure.

[0700] (A) Epitope mapping (1)

[0701] Epitope mapping was performed using a library of overlapping peptides (length: 15 amino acids) corresponding to amino acid sequences defined as allergic components of eggs. Specifically, a library of overlapping peptides was prepared for the amino acid sequences of sequence numbers 59, 106, and 262 for vitellogenin-2 precursor, the amino acid sequences of sequence numbers 2, 13, and 47 for vitellogenin-3, the amino acid sequences of sequence numbers 152, 237, and 251 for apolipoprotein B precursor, and the amino acid sequence of sequence number 18 for vitellogenin-1 or yolk lipophosphoprotein-1.

[0702] Each synthesized peptide was shifted by 10 amino acids, that is, each peptide had a repeat of 5 amino acids each with the pre-peptide and post-peptide.

[0703] In order to prepare the peptide array, the Intavis CelluSpots (trademark) technology was used. Specifically, the following steps were followed: (1) synthesizing the target peptide on an amino-modified cellulose disk using an automated synthesis device (Intavis MultiPep RS); (2) dissolving the amino-modified cellulose disk to obtain a cellulose-bound peptide solution; (3) spotting the cellulose-bound peptide on a coated cover glass to prepare a peptide array. The details of each step are as follows.

[0704] (1) Synthesis of peptides

[0705] On an amino-modified cellulose disk in a 384-well synthesis plate, peptide synthesis is performed in stages using 9-fluorenylmethoxycarbonyl (Fmoc) chemical reaction. That is, an amino acid to which an Fmoc group is bound is activated in a solution of N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in dimethylformamide (DMF), and then dropped onto the aforementioned cellulose disk to allow the amino group on the cellulose disk to bind (couple) to the aforementioned Fmoc-bound amino acid, and unreacted amino groups are capped with anhydrous acetic acid, and washed with DMF, and then treated with piperidine and washed with DMF to remove the Fmoc group from the amino group of the amino acid to which the amino group on the cellulose disk is bound. The above coupling, capping reaction, and removal of the Fmoc group are repeated for the amino acid to which the amino group on the aforementioned cellulose disk is bound, and the amino terminal is extended to perform peptide synthesis.

[0706] (2) Dissolution of amino-modified cellulose disks

[0707] The cellulose disk bound with the target peptide obtained in the above "(1) Synthesis of peptide" was transferred to a 96-well plate and treated with a side chain deprotection mixed solution of trifluoroacetic acid (TFA), dichloromethane, triisopropylsilane (TIPS) and water to deprotect the amino acid side chains. Thereafter, the deprotected cellulose-bound peptide was dissolved with a mixed solution of TFA, trifluoromethanesulfonic acid (TFMSA), TIPS and water, precipitated with tetrabutyl methyl ether (TBME), resuspended in dimethyl sulfoxide (DMSO), and mixed with a mixed solution of NaCl, sodium citrate and water to obtain a peptide solution for sliding spots.

[0708] (3) Spots of cellulose-bound peptide solution

[0709] The peptide solution for slide spotting obtained in the above "(2) Dissolution of amino-modified cellulose disk" was spotted on an Intavis CelluSpots (trademark) slide using an Intavis slide spotting robot and dried to prepare a peptide array.

[0710] Using the peptide fragments, each peptide fragment was tested for binding to IgE antibodies in the serum of WDEIA patients through an antigen-antibody reaction. The test was performed according to the following steps.

[0711] (1) The peptide was shaken in Pierce Protein-Free (PBS) Blocking Buffer (manufactured by Thermo) at room temperature for 1 hour.

[0712] (2) The cells were shaken overnight at room temperature in 2% serum / Pierce Protein-Free (PBS) Blocking Buffer (manufactured by Thermo Fisher Scientific).

[0713] (3) Wash with PBST solution (PBS buffer containing 0.1% nonionic surfactant Tween 20) for 5 minutes (× 3 times).

[0714] (4) Anti-human IgE antibody-HRP (1:10,000, Pierce Protein-Free (PBS) Blocking Buffer (manufactured by Thermo)) was added, and the mixture was shaken at room temperature for 1 hour.

[0715] (5) Wash with PBST solution for 5 minutes (× 3 times).

[0716] (6) Pierce ECL Plus Western Blotting Substrate (manufactured by Thermo Fisher Scientific) was added and the mixture was allowed to stand at room temperature for 5 minutes.

[0717] (7) Using Amersham Imager 600, the chemiluminescence of the peptides treated with (1) to (6) above was measured.

[0718] The chemiluminescence amount was quantified using ImageQuant TL (GE Healthcare) for the image obtained by the measurement in (7) above. The value of the non-egg allergy subject with the maximum quantified value from the image obtained using the sera of 5 non-egg allergy subjects was taken as the Nmax value, and the value obtained by dividing the quantified value of the image obtained using the sera of 6 patients by the Nmax value of each peptide was taken, and the peptide having a value greater than 0 and a value of 30,000 obtained by quantifying the image obtained using the patient's sera was judged to be a peptide that specifically binds to the patient's IgE antibody.

[0719] (B) Epitope mapping (2): Overlap

[0720] Based on the sequence of the peptide to which the IgE antibody in the serum of the patient specifically binds by the above (A) (SEQ ID NOs: 272 to 295 for vitellogenin-2 precursor; SEQ ID NOs: 296 to 303 for vitellogenin-3; SEQ ID NOs: 304 to 311 for apolipoprotein B precursor; and SEQ ID NOs: 312 to 318 for vitellogenin-1 or yolk lipophosphoprotein-1), the amino acid sequence of the peptide sequence and the allergen component containing the peptide sequence (SEQ ID NOs: 272 to 295 for vitellogenin-3; SEQ ID NOs: 296 to 303 for vitellogenin-3; SEQ ID NOs: 304 to 311 for apolipoprotein B precursor; and SEQ ID NOs: 312 to 318 for vitellogenin-1 or yolk lipophosphoprotein-1) A library of overlapping peptide fragments (length: 10 amino acids) is prepared from the sequence obtained by appending the preceding and following sequences of the peptide to the protein-2 precursor (the amino acid sequence of sequence numbers 59, 106 or 262, the amino acid sequence of sequence numbers 2, 13 or 47 for vitellogenin-3, the amino acid sequence of sequence numbers 152, 237 or 251 for apolipoprotein B, and the amino acid sequence of sequence number 18 for vitellogenin-1 or yolk lipophosphoprotein-1), and epitope mapping is performed.

[0721] Each synthesized peptide was shifted by 1 amino acid, that is, each peptide had a repeat of 9 amino acids each with the pre-peptide and post-peptide.

[0722] The library was prepared by the same procedure as in (A) above, and the binding of each peptide fragment to the IgE antibody in the serum of the patient and the non-egg allergy subject was determined by the same method as above. The amino acids at the positions where the binding to the patient's IgE antibody was lost or reduced by each peptide shifting one amino acid and the binding to the IgE antibody of the non-egg allergy subject was generated were judged as amino acids that adversely affected the binding specificity of the IgE antibody.

[0723] The chemiluminescence amount was quantified using ImageQuant TL (GE Healthcare) for the image obtained by measuring in the same manner as in (A) above. The value obtained by quantifying the image obtained by using only the anti-human IgE antibody-HRP antibody without performing the treatments of (2) and (3) in the spots of the cellulose-binding peptide solution in (3) above was divided by the value obtained by quantifying the image obtained by using the sera of 6 patients. Peptides having a value greater than 0 were judged to have residual binding to the IgE antibody, and peptides having a value less than the amino acid sequence of the sequence number 59, 106 or 262 for vitellogenin-2; the amino acid sequence of the sequence number 2, 13 or 47 for vitellogenin-3; the amino acid sequence of the sequence number 152, 237 or 269 for apolipoprotein B were judged to have residual binding to the IgE antibody. Peptides with a value of 20% or more obtained for the amino acid sequence of sequence number 251, and the amino acid sequence of sequence number 18 for vitellogenin-1 or vitellogenin-1) were judged to have no binding to IgE antibodies; peptides with a value of more than 20% and less than 50% were judged to have poor binding to IgE antibodies but have binding to IgE antibodies; peptides with a value of more than 50% and less than 70% were judged to have slightly poor binding to IgE antibodies but have binding to IgE antibodies; peptides with a value of more than 70% and less than 90% were judged to have almost no difference in binding to IgE antibodies; and peptides with a value of more than 90% were judged to have no difference in binding to IgE antibodies or to have good binding to IgE antibodies.

[0724] (C) Epitope mapping (3): Alanine scanning

[0725] For the amino acid sequence determined in (A) above, a library of peptide fragments obtained by replacing one amino acid with alanine from the amino acid terminal side is prepared in the same manner as above by a method called alanine scanning (non-patent document 1), and each peptide fragment is measured for binding to IgE antibodies in the serum of patients and non-egg allergy subjects by the same method as above. Amino acids at positions where the binding to the patient's IgE antibodies is lost or reduced by alanine substitution and the binding to the IgE antibodies of non-egg allergy subjects is generated are judged to be important amino acids for expressing the original antigenicity or amino acids that affect the expression of the original antigenicity. In addition, amino acids at positions where the binding to the patient's IgE antibodies is maintained by alanine substitution and the binding to the IgE antibodies of non-egg allergy subjects is not generated are judged to be amino acids that are not important for expressing the original antigenicity and can be replaced.

[0726] The chemiluminescence amount was quantified using ImageQuant TL (GE Healthcare) for the image obtained by measuring in the same manner as in (A) above. The value obtained by quantifying the image obtained by using only the anti-human IgE antibody-HRP antibody without performing the treatments of (2) and (3) in the spots of the cellulose-binding peptide solution in (3) above was divided by the value obtained by quantifying the image obtained by using the sera of 6 patients. Peptides having a value greater than 0 were judged to have residual binding to the IgE antibody, and peptides having a value less than the amino acid sequence of the sequence number 59, 106 or 262 for vitellogenin-2; the amino acid sequence of the sequence number 2, 13 or 47 for vitellogenin-3; the amino acid sequence of the sequence number 152, 237 or 269 for apolipoprotein B were judged to have residual binding to the IgE antibody. Peptides with a value of 20% or more obtained for the amino acid sequence of sequence number 251, and the amino acid sequence of sequence number 18 for vitellogenin-1 or vitellogenin-1) were judged to have no binding to IgE antibodies; peptides with a value of more than 20% and less than 50% were judged to have poor binding to IgE antibodies but have binding to IgE antibodies; peptides with a value of more than 50% and less than 70% were judged to have slightly poor binding to IgE antibodies but have binding to IgE antibodies; peptides with a value of more than 70% and less than 90% were judged to have almost no difference in binding to IgE antibodies; and peptides with a value of more than 90% were judged to have no difference in binding to IgE antibodies or to have good binding to IgE antibodies.

[0727] This analysis revealed common sequences important for expressing the original antigenicity.

[0728] (D) Results

[0729] As a result of the epitope mapping in (A) above, epitopes No. 1 to 24 (peptides having amino acid sequences of SEQ ID NOs. 272 ​​to 295, respectively) were confirmed for vitellogenin-2 precursor, epitopes No. 25 to 32 (peptides having amino acid sequences of SEQ ID NOs. 296 to 303, respectively) were confirmed for vitellogenin-3, epitopes No. 33 to 40 (peptides having amino acid sequences of SEQ ID NOs. 304 to 311, respectively) were confirmed for apolipoprotein B precursor, and epitopes No. 41 to 47 (peptides having amino acid sequences of SEQ ID NOs. 312 to 318, respectively) were confirmed for vitellogenin-1 or vitellogenin-1, and IgE antibodies specifically bound to each of them. Table 2 shows which part of the allergen component sequences identified in Examples 2 to 5 the peptide sequences for these epitopes correspond to.

[0730] [Table 2-1]

[0731]

[0732] [Table 2-2]

[0733]

[0734] [Table 2-3]

[0735]

[0736] For each epitope, the results of (B) and (C) above identified amino acids important for binding of the following epitope sequences to IgE antibodies of allergic patients.

[0737] (1) Epitope 1

[0738] In epitope 1 (SEQ ID NO: 272), SEQ ID NO: 323 was identified as a region important for binding to the patient's IgE antibody. Sequence No. 319 was identified as a sequence that includes a region adjacent to epitope 1 on the amino terminal side and binds to the patient's IgE antibody.

[0739] Regarding sequence number 272, it was identified that amino acids at positions 6, 8 to 12, 14 and 15 are particularly important for binding to IgE antibodies of allergic patients, and amino acids at positions 4, 5, 7 and 13 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 1 to 3 of sequence number 272 are replaced with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0740] Regarding sequence number 323, it was identified that the amino acids at positions 2 to 6 are particularly important for binding to IgE antibodies of allergy patients, and the amino acid at position 1 is an amino acid that has an influence on binding to IgE antibodies of allergy patients.

[0741] (2) Epitope 2

[0742] In epitope 2 (SEQ ID NO: 273), SEQ ID NO: 327 was identified as a region important for binding to the patient's IgE antibody. Sequence No. 324 was identified as a sequence that includes a region adjacent to epitope 2 on the amino terminal side and binds to the patient's IgE antibody.

[0743] Regarding SEQ ID NO: 273, amino acids at positions 9 to 12 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 1 to 8 and 13 to 15 of SEQ ID NO: 273 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0744] Regarding sequence number 327, it was identified that the amino acids at positions 9 and 10 are particularly important for binding to IgE antibodies of allergic patients, and the amino acids at positions 4 and 5 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 1 to 3 and 6 to 8 of sequence number 327 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0745] (3) Epitope 3

[0746] In epitope 3 (SEQ ID NO: 274), SEQ ID NOs: 330, 331, 333, and 335 were identified as regions important for binding to the patient's IgE antibody. Sequence No. 336 was identified as a sequence that includes a region adjacent to epitope 3 on the carboxyl terminal side and binds to the patient's IgE antibody.

[0747] Regarding SEQ ID NO: 274, amino acids at positions 1, 3, and 6 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 2, 4, 5, and 7 to 15 of SEQ ID NO: 274 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0748] In addition, it was identified that amino acids at positions 1, 6, 10, and 11 are also particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 274. In this case, even if amino acids at positions 2 to 5, 7 to 9, and 12 to 15 of SEQ ID NO: 274 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0749] The amino acids at positions 1, 3, and 6 were identified as being particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 330. Even if the amino acids at positions 2, 4, 5, 7, and 8 of SEQ ID NO: 330 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0750] Regarding SEQ ID NO: 335, amino acids at positions 1, 5, and 6 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 2 to 4 and 7 to 10 of SEQ ID NO: 335 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0751] (4) Epitope 4

[0752] In epitope 4 (SEQ ID NO: 275), sequence numbers 339, 343, 344, 346, 347, and 350 were identified as regions important for binding to the patient's IgE antibody. In addition, sequence number 342 was identified as a sequence that includes a region adjacent to epitope 4 on the amino terminal side and binds to the patient's IgE antibody, and sequence number 351 was identified as a sequence that includes a region adjacent to epitope 4 on the carboxyl terminal side and binds to the patient's IgE antibody.

[0753] Regarding SEQ ID NO: 275, amino acids at positions 1, 4 to 6, and 10 to 13 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 2, 3, 7 to 9, 14, and 15 of SEQ ID NO: 275 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0754] In addition, amino acids at positions 1, 6, and 12 to 14 were identified as being particularly important for binding to allergic IgE antibodies in SEQ ID NO: 275. In this case, even if amino acids at positions 2 to 5, 7 to 11, and 15 of SEQ ID NO: 275 were substituted with alanine, the binding to allergic IgE antibodies was not affected.

[0755] Furthermore, it was identified that amino acids 1, 6, 8, 14, and 15 are also particularly important for binding to allergic IgE antibodies in SEQ ID NO: 275. In this case, even if amino acids 2 to 5, 7, 9 to 13 of SEQ ID NO: 275 are substituted with alanine, the binding to allergic IgE antibodies is not affected.

[0756] Regarding SEQ ID NO: 339, amino acids 1, 4 to 6, and 10 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even if amino acids 2, 3, 7 to 9 of SEQ ID NO: 339 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0757] Regarding SEQ ID NO: 346, amino acids 1 to 6 and 10 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids 7 to 9 of SEQ ID NO: 346 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0758] The amino acids at positions 1 and 3 of SEQ ID NO: 350 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 2 and 4 to 8 of SEQ ID NO: 350 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0759] (5) Epitope 5

[0760] In epitope 5 (SEQ ID NO: 276), sequence numbers 355 and 356 were identified as regions important for binding to the patient's IgE antibody. In addition, sequence number 353 was identified as a sequence that contains a region adjacent to epitope 5 on the amino terminal side and binds to the patient's IgE antibody, and sequence number 357 was identified as a sequence that contains a region adjacent to epitope 5 on the carboxyl terminal side and binds to the patient's IgE antibody.

[0761] Regarding SEQ ID NO: 276, amino acids at positions 2, 3, 6 to 15 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 1, 4, and 5 of SEQ ID NO: 276 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0762] Regarding SEQ ID NO: 355, amino acids 2 to 8 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when the amino acid 1 of SEQ ID NO: 355 was substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0763] (6) Epitope 6

[0764] In epitope 6 (SEQ ID NO: 277), sequence numbers 366 and 369 were identified as regions important for binding to the patient's IgE antibody. In addition, sequence numbers 359 and 363 were identified as sequences that contain a region adjacent to epitope 6 on the amino terminal side and bind to the patient's IgE antibody, and sequence number 360 was identified as a sequence that contains a region adjacent to epitope 6 on the carboxyl terminal side and binds to the patient's IgE antibody.

[0765] Regarding sequence number 277, it was identified that amino acids at positions 1, 5 to 9, 11, and 13 to 15 are particularly important for binding to IgE antibodies of allergic patients, and amino acids at positions 2 and 10 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 3, 4, and 12 of sequence number 277 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0766] In addition, it was identified that the amino acids at positions 3, 7, 9, 11, and 15 are particularly important for binding to IgE antibodies of allergic patients with SEQ ID NO: 277, and the amino acids at positions 1, 2, 4 to 6 are also amino acids that have an influence on binding to IgE antibodies of allergic patients. In this case, even if the amino acids at positions 8, 10, and 12 to 14 of SEQ ID NO: 277 are substituted with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0767] For sequence number 366, it was identified that the amino acids at positions 1, 5, 7, and 9 are particularly important for binding to IgE antibodies of allergic patients, and the amino acids at positions 2 to 4 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 6, 8, and 10 of sequence number 366 are replaced with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0768] Regarding sequence number 369, it was identified that the amino acids at positions 2, 4, 6, and 10 are particularly important for binding to IgE antibodies of allergic patients, and the amino acid at position 1 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 3, 5, 7 to 9 of sequence number 369 are replaced with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0769] (7) Epitope 7

[0770] In epitope 7 (SEQ ID NO: 278), SEQ ID NO: 374 was identified as a region important for binding to the patient's IgE antibody. Sequence No. 375 was identified as a sequence that includes a region adjacent to epitope 7 on the carboxyl terminal side and binds to the patient's IgE antibody.

[0771] Regarding SEQ ID NO: 278, amino acids 1 to 6, 8, 11, 13 and 15 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids 7, 9, 10, 12 and 14 of SEQ ID NO: 278 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0772] Regarding sequence number 374, it was identified that the amino acids at positions 1, 3, 4, 6, and 9 are particularly important for binding to IgE antibodies of allergic patients, and the amino acid at position 2 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 5, 7, and 8 of sequence number 374 are replaced with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0773] (8) Epitope 8

[0774] In epitope 8 (SEQ ID NO: 279), sequence numbers 379, 388, 391, 392, 397, and 400 were identified as regions important for binding to the patient's IgE antibody. In addition, sequence numbers 377, 384, and 385 were identified as sequences that contain a region adjacent to epitope 8 on the amino terminal side and bind to the patient's IgE antibody, and sequence numbers 380, 381, 393, 401, and 402 were identified as sequences that contain a region adjacent to epitope 8 on the carboxyl terminal side and bind to the patient's IgE antibody.

[0775] Regarding sequence number 279, it was identified that amino acids at positions 1 to 5, 8 to 12, 14 and 15 are particularly important for binding to IgE antibodies of allergic patients, and amino acid position 13 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 6 and 7 of sequence number 279 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0776] In addition, it was identified that the amino acids at positions 3, 9, and 15 are particularly important for binding to IgE antibodies of allergic patients with SEQ ID NO: 279, and the amino acid at position 4 is also an amino acid that has an influence on binding to IgE antibodies of allergic patients. In this case, even if the amino acids at positions 1, 2, 5 to 8, and 10 to 14 of SEQ ID NO: 279 are substituted with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0777] Furthermore, it was identified that amino acids 1 to 3, 8, and 9 are also particularly important for binding to allergic IgE antibodies in SEQ ID NO: 279. In this case, even if amino acids 4 to 7 and 10 to 15 of SEQ ID NO: 279 are substituted with alanine, the binding to allergic IgE antibodies is not affected.

[0778] Regarding SEQ ID NO: 379, amino acids 1 to 4 and 7 to 10 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids 5 and 6 of SEQ ID NO: 379 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0779] Regarding sequence number 388, it was identified that the amino acids at positions 2 and 8 are particularly important for binding to IgE antibodies of allergic patients, and the amino acid at position 3 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 1, 4 to 7, and 9 of sequence number 388 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0780] Regarding sequence number 392, it was identified that the amino acids at positions 4 and 10 are particularly important for binding to IgE antibodies of allergic patients, and the amino acid at position 8 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 1 to 3, 5 to 7, and 9 of sequence number 392 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0781] Regarding SEQ ID NO: 397, amino acids at positions 1, 2, 7, and 8 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 3 to 6, 9, and 10 of SEQ ID NO: 397 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0782] For SEQ ID NO: 400, the amino acids at positions 3 and 4 were identified as being particularly important for binding to IgE antibodies of allergic patients, and the amino acids at positions 1, 6, and 9 were identified as being amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 2, 5, 7, 8, and 10 of SEQ ID NO: 400 were replaced with alanine, the binding to IgE antibodies of allergic patients would not be affected.

[0783] (9) Epitope 9

[0784] In epitope 9 (SEQ ID NO: 280), SEQ ID NO: 407 was identified as a region important for binding to the patient's IgE antibody. Sequence No. 404 was identified as a sequence that includes a region adjacent to epitope 9 on the amino terminal side and binds to the patient's IgE antibody.

[0785] Regarding SEQ ID NO: 280, amino acids 4 to 7, 9 to 12, and 15 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even if amino acids 1 to 3, 8, 13, and 14 of SEQ ID NO: 280 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0786] The amino acids at positions 1, 2, 4, and 5 were identified as being particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 407. Even when the amino acid at position 3 of SEQ ID NO: 407 was substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0787] (10) Epitope 10

[0788] In epitope 10 (SEQ ID NO: 281), SEQ ID NOs: 412 and 414 were identified as regions important for binding to the patient's IgE antibody. Sequence No. 409 was identified as a sequence that includes a region adjacent to epitope 10 on the amino terminal side and binds to the patient's IgE antibody.

[0789] Regarding sequence number 281, it was identified that amino acids at positions 5, 6, 8 to 10, and 12 to 15 are particularly important for binding to IgE antibodies of allergic patients, and amino acids at positions 4 and 7 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 1 to 3 and 11 of sequence number 281 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0790] Regarding sequence number 412, it was identified that amino acids at positions 4 to 8 are particularly important for binding to IgE antibodies of allergic patients, and amino acid at position 3 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 1 and 2 of sequence number 412 are replaced with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0791] The amino acids at positions 1, 2, 4, and 5 were identified as being particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 414. Even when the amino acid at position 3 of SEQ ID NO: 414 was substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0792] (11) Epitope 11

[0793] In epitope 11 (SEQ ID NO: 282), sequence numbers 419 and 425 were identified as regions important for binding to the patient's IgE antibody. In addition, sequence number 422 was identified as a sequence that contains a region adjacent to epitope 11 on the amino terminal side and binds to the patient's IgE antibody, and sequence number 426 was identified as a sequence that contains a region adjacent to epitope 11 on the carboxyl terminal side and binds to the patient's IgE antibody.

[0794] Regarding sequence number 282, it was identified that the amino acids at positions 1, 4, 5, 11, and 12 are particularly important for binding to IgE antibodies of allergic patients, and the amino acids at positions 7, 8, and 13 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 2, 3, 6, 9, 10, 14, and 15 of sequence number 282 are replaced with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0795] In addition, it was identified that the amino acids at positions 1, 2, 4, 5, 7, 9, 11, 12, and 14 are particularly important for binding to IgE antibodies of allergic patients with SEQ ID NO: 282, and the amino acids at positions 3 and 8 are also amino acids that have an influence on binding to IgE antibodies of allergic patients. In this case, even if the amino acids at positions 6, 10, 13, and 15 of SEQ ID NO: 282 are substituted with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0796] For sequence number 419, it was identified that amino acids at positions 5 to 7 are particularly important for binding to IgE antibodies of allergic patients, and amino acids at positions 1 and 2 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 3, 4, and 8 of sequence number 419 are replaced with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0797] Regarding sequence number 425, it was identified that the amino acids at positions 1, 3, 5, 6, and 8 are particularly important for binding to IgE antibodies of allergic patients, and the amino acid at position 2 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 4, 7, and 9 of sequence number 425 are replaced with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0798] (14) Epitope 14

[0799] In epitope 14 (SEQ ID NO: 285), SEQ ID NO: 430 was identified as a region important for binding to the patient's IgE antibody.

[0800] Regarding sequence number 285, it was identified that the amino acids at positions 1, 5, 6, 8, 10, and 12 are particularly important for binding to IgE antibodies of allergic patients, and the amino acids at positions 2, 9, and 11 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 3, 4, 7, and 13 to 15 of sequence number 285 are replaced with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0801] Regarding SEQ ID NO: 430, amino acids at positions 3, 4, 6 to 9 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even if amino acids at positions 1, 2, and 5 of SEQ ID NO: 430 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0802] (15) Epitope 15

[0803] In epitope 15 (SEQ ID NO: 286), SEQ ID NOs: 434, 436, and 440 were identified as regions important for binding to the patient's IgE antibody.

[0804] Regarding SEQ ID NO: 286, amino acids 1 to 7 and 9 to 15 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when the amino acid at position 8 of SEQ ID NO: 286 was substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0805] In addition, it was identified that amino acids at positions 3, 5 to 7, and 11 to 15 are particularly important for binding to IgE antibodies of allergic patients for SEQ ID NO: 286, and amino acids at positions 4 and 10 are also amino acids that have an influence on binding to IgE antibodies of allergic patients. In this case, even if amino acids at positions 1, 2, 8, and 9 of SEQ ID NO: 286 are substituted with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0806] Regarding SEQ ID NO: 434, amino acids 1 to 3 and 5 to 9 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when the amino acid at position 4 of SEQ ID NO: 434 was substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0807] Regarding SEQ ID NO: 436, amino acids at positions 1, 3 to 10 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when the amino acid at position 2 of SEQ ID NO: 436 was substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0808] Regarding sequence number 440, it was identified that amino acids at positions 4 to 7 are particularly important for binding to IgE antibodies of allergic patients, and amino acid at position 3 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 1 and 2 of sequence number 440 are replaced with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0809] (16) Epitope 16

[0810] In epitope 16 (SEQ ID NO: 287), SEQ ID NOs: 444, 446, and 449 were identified as regions important for binding to the patient's IgE antibody.

[0811] Regarding SEQ ID NO: 287, amino acids at positions 2, 7, 9, and 13 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 1, 3 to 6, 8, 10 to 12, 14, and 15 of SEQ ID NO: 287 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0812] Regarding SEQ ID NO: 444, amino acids at positions 2, 7, and 9 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 1, 3 to 6, 8, and 10 of SEQ ID NO: 444 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0813] Regarding SEQ ID NO: 446, amino acids at positions 4, 6, and 10 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 1 to 3, 5, and 7 to 9 of SEQ ID NO: 446 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0814] Regarding sequence number 449, it was identified that the amino acids at positions 2, 4, and 8 are particularly important for binding to IgE antibodies of allergic patients, and the amino acid at position 10 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 1, 3, 5 to 7, and 9 of sequence number 449 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0815] (17) Epitope 17

[0816] In epitope 17 (SEQ ID NO: 288), SEQ ID NOs: 455 and 458 were identified as regions important for binding to the patient's IgE antibody. Sequence numbers 451 and 452 were identified as sequences that include a region adjacent to epitope 17 on the amino terminal side and bind to the patient's IgE antibody.

[0817] Regarding SEQ ID NO: 288, amino acids at positions 3, 7, 10, 12 to 14 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 1, 2, 4 to 6, 8, 9, 11 and 15 of SEQ ID NO: 288 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0818] Regarding sequence number 455, it was identified that amino acids at positions 1, 5, 8, and 10 are particularly important for binding to IgE antibodies of allergic patients, and amino acids at positions 7 and 9 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 2 to 4 and 6 of sequence number 455 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0819] Regarding sequence number 458, it was identified that amino acids at positions 1, 4, 6 to 8 are particularly important for binding to IgE antibodies of allergic patients, and amino acids at positions 3 and 5 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 2 and 9 of sequence number 458 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0820] (19) Epitope 19

[0821] In epitope 19 (SEQ ID NO: 290), sequence numbers 461 and 464 were identified as regions important for binding to the patient's IgE antibody. Sequence number 462 was identified as a sequence that contains a region adjacent to epitope 19 on the amino terminal side and binds to the patient's IgE antibody, and sequence numbers 465 and 466 were identified as sequences that contain a region adjacent to epitope 19 on the carboxyl terminal side and bind to the patient's IgE antibody.

[0822] Regarding SEQ ID NO: 290, amino acids 1 to 4, 6, 7, 10, and 13 to 15 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids 5, 8, 9, 11, and 12 of SEQ ID NO: 290 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0823] Regarding SEQ ID NO: 461, amino acids 1 to 4 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when the amino acid 5 of SEQ ID NO: 461 was substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0824] Regarding sequence number 464, amino acids at positions 2, 2, 6 to 10 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even if amino acids at positions 1, 4 and 5 of sequence number 464 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0825] (20) Epitope 20

[0826] In epitope 20 (SEQ ID NO: 291), sequence numbers 470, 473, 482, 483, and 486 were identified as regions important for binding to the patient's IgE antibody. In addition, sequence numbers 471, 479, and 480 were identified as sequences that contain a region adjacent to epitope 20 on the amino terminal side and bind to the patient's IgE antibody, and sequence numbers 476 and 487 were identified as sequences that contain a region adjacent to epitope 20 on the carboxyl terminal side and bind to the patient's IgE antibody.

[0827] Regarding sequence number 291, it was identified that the amino acids at positions 2, 7, 10, 12, 13, and 15 are particularly important for binding to IgE antibodies of allergic patients, and the amino acid at position 1 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 3 to 6, 8, 9, 11, and 14 of sequence number 291 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0828] In addition, it was identified that the amino acids at positions 2, 4, 7, 11 to 13, and 15 are particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 291, and the amino acid at position 6 is also an amino acid that has an influence on binding to IgE antibodies of allergic patients. In this case, even if the amino acids at positions 1, 3, 5, 8 to 10, and 14 of SEQ ID NO: 291 are substituted with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0829] In addition, it was identified that the amino acids at positions 2, 5, 7, 9, 10, 13, and 15 are particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 291, and the amino acids at positions 1 and 6 are also amino acids that have an influence on binding to IgE antibodies of allergic patients. In this case, even if the amino acids at positions 3, 4, 8, 11, 12, and 14 of SEQ ID NO: 291 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0830] Regarding sequence number 470, it was identified that the amino acids at positions 2, 4, and 7 are particularly important for binding to IgE antibodies of allergic patients, and the amino acids at positions 1 and 5 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 3, 6, 8, and 9 of sequence number 470 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0831] The amino acids at positions 3, 5, 6 and 8 were identified as being particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 473. Even if the amino acids at positions 1, 2, 4 and 7 of SEQ ID NO: 473 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0832] Regarding sequence number 482, amino acids at positions 1, 4 to 6, 8 and 9 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 2, 3 and 7 of sequence number 483 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0833] Regarding sequence number 486, it was identified that the amino acids at positions 2, 4, 5, and 8 are particularly important for binding to IgE antibodies of allergic patients, and the amino acid at position 1 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 3, 6, 7, and 9 of sequence number 486 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0834] (21) Epitope 21

[0835] In epitope 21 (SEQ ID NO: 292), sequence numbers 491, 494, 500, 505, 507, and 509 were identified as regions important for binding to the patient's IgE antibody. In addition, sequence numbers 492, 497, 498, and 503 were identified as sequences that contain a region adjacent to epitope 21 on the amino terminal side and bind to the patient's IgE antibody, and sequence number 495 was identified as a sequence that contains a region adjacent to epitope 21 on the carboxyl terminal side and binds to the patient's IgE antibody.

[0836] Regarding SEQ ID NO: 292, amino acids 1 to 3, 6, 8, 9, 14 and 15 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids 4, 5, 7 and 10 to 13 of SEQ ID NO: 292 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0837] In addition, it was identified that amino acids 1 to 3, 6 to 10, and 12 to 15 are particularly important for binding to IgE antibodies of allergic patients for SEQ ID NO: 292, and amino acids 1 to 3, 6 to 10, and 12 to 15 are amino acids that are particularly important for binding to IgE antibodies of allergic patients, and amino acid 11 is also an amino acid that has an influence on binding to IgE antibodies of allergic patients. In this case, even if amino acids 4 and 5 of SEQ ID NO: 292 are substituted with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0838] Furthermore, it was identified that amino acids 1, 2, 7 to 9, and 15 are also particularly important for binding to allergic IgE antibodies in SEQ ID NO: 292. In this case, even if amino acids 3 to 6 and 10 to 14 of SEQ ID NO: 292 are substituted with alanine, the binding to allergic IgE antibodies is not affected.

[0839] Regarding SEQ ID NO: 491, amino acids 1 to 3 and 6 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids 4 and 5 of SEQ ID NO: 491 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0840] Regarding SEQ ID NO: 494, amino acids at positions 1 and 2 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 3 to 6 of SEQ ID NO: 494 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0841] Regarding SEQ ID NO: 500, amino acids 1 to 3 and 5 to 7 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even if the amino acid at position 4 of SEQ ID NO: 500 was substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0842] Regarding SEQ ID NO: 505, amino acids at positions 1, 2, 7, and 8 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 3 to 6 of SEQ ID NO: 505 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0843] Regarding SEQ ID NO: 507, amino acids at positions 5 to 7 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 1 to 4 and 8 to 10 of SEQ ID NO: 507 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0844] Regarding SEQ ID NO: 509, amino acids at positions 1, 2, 8, and 9 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 3 to 7 of SEQ ID NO: 509 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0845] (22) Epitope 22

[0846] In epitope 22 (SEQ ID NO: 293), a region important for binding to the patient's IgE antibody was identified as SEQ ID NO: 512. Sequence No. 515 was identified as a sequence that includes a region adjacent to epitope 22 at the carboxyl terminal side and binds to the patient's IgE antibody.

[0847] Regarding sequence number 293, it was identified that amino acids at positions 3, 6, 8 to 10, and 12 to 15 are particularly important for binding to IgE antibodies of allergic patients, and amino acids at positions 2 and 11 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 1, 4, 5, and 7 of sequence number 293 are replaced with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0848] Regarding SEQ ID NO: 512, amino acids at positions 1, 3 to 10 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when the amino acid at position 2 of SEQ ID NO: 512 was substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0849] (23) Epitope 23

[0850] In epitope 23 (SEQ ID NO: 294), sequence numbers 520 and 522 were identified as regions important for binding to the patient's IgE antibody. In addition, sequence numbers 516 and 517 were identified as sequences that contain a region adjacent to epitope 23 on the amino terminal side and bind to the patient's IgE antibody, and sequence number 523 was identified as a sequence that contains a region adjacent to epitope 23 on the carboxyl terminal side and binds to the patient's IgE antibody.

[0851] Regarding sequence number 294, it was identified that the amino acids at positions 2, 5, and 9 to 12 are particularly important for binding to IgE antibodies of allergic patients, and the amino acid at position 8 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 1, 3, 4, 6, 7, and 13 to 15 of sequence number 294 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0852] Regarding sequence number 520, it was identified that amino acids at positions 1, 4, and 8 to 10 are particularly important for binding to IgE antibodies of allergic patients, and amino acid position 7 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 2, 3, 5, and 6 of sequence number 520 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0853] Regarding SEQ ID NO: 522, amino acids at positions 2 to 6 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 1 and 7 of SEQ ID NO: 522 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0854] (25) Epitope 25

[0855] Regarding sequence number 296 (epitope 25), amino acids at positions 2 to 6 and 10 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even if amino acids at positions 1, 7 to 9 and 11 to 15 of sequence number 296 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0856] In addition, amino acids 1 to 3, 9, 11, and 12 were identified as being particularly important for binding to allergic IgE antibodies in SEQ ID NO: 296. In this case, even if amino acids 4 to 8, 10, and 13 to 15 of SEQ ID NO: 296 were substituted with alanine, the binding to allergic IgE antibodies was not affected.

[0857] (26) Epitope 26

[0858] In epitope 26 (SEQ ID NO: 297), SEQ ID NOs: 528 and 531 were identified as regions important for binding to the patient's IgE antibody.

[0859] Regarding sequence number 297, it was identified that the amino acids at positions 1 and 8 are particularly important for binding to IgE antibodies of allergic patients, and the amino acid at position 6 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 2 to 5, 7, and 9 to 15 of sequence number 297 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0860] In addition, it was identified that amino acids at positions 4 and 6 are also particularly important for binding to allergic IgE antibodies in SEQ ID NO: 297. In this case, even if amino acids at positions 1 to 3, 5, and 7 to 15 of SEQ ID NO: 297 are substituted with alanine, the binding to allergic IgE antibodies is not affected.

[0861] (27) Epitope 27

[0862] In epitope 27 (SEQ ID NO: 298), sequence numbers 533, 534, 539, 541, and 544 were identified as regions important for binding to the patient's IgE antibody. In addition, sequence numbers 535 and 542 were identified as sequences that are included in the region adjacent to epitope 27 on the carboxyl terminal side and bind to the patient's IgE antibody.

[0863] Regarding sequence number 298, it was identified that amino acids at positions 1, 2, 14, and 15 are particularly important for binding to IgE antibodies of allergic patients, and amino acid 13 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 3 to 12 of sequence number 298 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0864] In addition, it was identified that the amino acids at positions 3, 4, and 13 to 15 are particularly important for binding to IgE antibodies of allergic patients with SEQ ID NO: 298, and the amino acids at positions 2 and 6 are also amino acids that have an influence on binding to IgE antibodies of allergic patients. In this case, even if the amino acids at positions 1, 5, and 7 to 12 of SEQ ID NO: 298 are substituted with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0865] The amino acids at positions 1 and 3 were identified as being particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 539. Even when amino acids at positions 2 and 4 to 9 of SEQ ID NO: 539 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0866] Regarding SEQ ID NO: 541, amino acids 1, 8 to 10 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids 2 to 7 of SEQ ID NO: 541 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0867] Regarding SEQ ID NO: 544, amino acids at positions 4 to 6 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 1 to 3 of SEQ ID NO: 544 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0868] (28) Epitope 28

[0869] Regarding sequence number 299 (epitope 28), amino acids at positions 2 and 6 to 15 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even if amino acids at positions 1 and 3 to 5 of sequence number 299 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0870] In addition, it was identified that amino acids at positions 2, 9 to 12, and 15 are also particularly important for binding to allergic IgE antibodies in SEQ ID NO: 299. In this case, even if amino acids at positions 1, 3 to 8, 13, and 14 of SEQ ID NO: 299 are substituted with alanine, the binding to allergic IgE antibodies is not affected.

[0871] Furthermore, it was identified that amino acids 1, 2, 9, 14, and 15 are also particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 299. In this case, even if amino acids 3 to 8 and 10 to 13 of SEQ ID NO: 299 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0872] (30) Epitope 30

[0873] In addition, sequence number 550 was identified as a sequence that contains a region adjacent to epitope 30 (sequence number 301) on the amino-terminal side and binds to the patient's IgE antibody; sequence number 551 was identified as a sequence that contains a region adjacent to epitope 30 on the carboxyl-terminal side and binds to the patient's IgE antibody.

[0874] Regarding SEQ ID NO: 301, amino acids at positions 2, 6 to 9 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even if amino acids at positions 1, 3 to 5, and 10 to 15 of SEQ ID NO: 301 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0875] (33) Epitope 33

[0876] In epitope 33 (SEQ ID NO: 304), sequence number 555 was identified as a region important for binding to the patient's IgE antibody. Sequence number 553 was identified as a sequence that contains a region adjacent to epitope 33 on the amino terminal side and binds to the patient's IgE antibody, and sequence numbers 556 and 557 were identified as sequences that contain a region adjacent to epitope 33 on the carboxyl terminal side and bind to the patient's IgE antibody.

[0877] Regarding SEQ ID NO: 304, amino acids at positions 1, 2, 13, and 14 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 3 to 12 and 15 of SEQ ID NO: 304 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0878] Regarding SEQ ID NO: 555, amino acids at positions 1 and 2 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 3 to 8 of SEQ ID NO: 555 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0879] (34) Epitope 34

[0880] In epitope 34 (SEQ ID NO: 305), sequence numbers 561, 562, 569, 571, and 573 were identified as regions important for binding to the patient's IgE antibody. Sequence number 565 was identified as a sequence that includes a region adjacent to epitope 34 on the amino terminal side and binds to the patient's IgE antibody.

[0881] For sequence number 305, it was identified that amino acids at positions 14 and 15 are particularly important for binding to IgE antibodies of allergic patients, and amino acids at positions 10 to 13 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 1 to 9 of sequence number 305 are replaced with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0882] In addition, for SEQ ID NO: 305, amino acids at positions 9, 11, and 14 were identified as being particularly important for binding to IgE antibodies of allergic patients, and amino acids at positions 4 to 7 were also identified as having an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 1 to 3, 8, 10, 12, 13, and 15 of SEQ ID NO: 305 were replaced with alanine, the binding to IgE antibodies of allergic patients would not be affected.

[0883] Regarding SEQ ID NO: 561, amino acids at positions 2 to 5 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when the amino acid at position 1 of SEQ ID NO: 561 was substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0884] Regarding SEQ ID NO: 562, amino acids 2 to 6 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when the amino acid 1 of SEQ ID NO: 562 was substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0885] For sequence number 569, it was identified that the amino acids at positions 7 and 9 are particularly important for binding to IgE antibodies of allergic patients, and the amino acids at positions 2 to 5 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 1, 6, 8, and 10 of sequence number 569 are replaced with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0886] The amino acids at positions 1, 3, and 6 were identified as being particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 571. Even if the amino acids at positions 2, 4, 5, and 7 of SEQ ID NO: 571 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0887] The amino acids at positions 1 and 3 were identified as being particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 573. Even when the amino acids at positions 2 and 4 of SEQ ID NO: 573 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0888] (35) Epitope 35

[0889] In epitope 35 (SEQ ID NO: 306), sequence numbers 579 and 581 were identified as regions important for binding to the patient's IgE antibody. In addition, sequence number 576 was identified as a sequence that contains a region adjacent to epitope 35 on the amino terminal side and binds to the patient's IgE antibody, and sequence number 577 was identified as a sequence that contains a region adjacent to epitope 35 on the carboxyl terminal side and binds to the patient's IgE antibody.

[0890] Regarding SEQ ID NO: 306, amino acids 1 to 4 and 7 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids 5, 6, 8 to 15 of SEQ ID NO: 306 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0891] Regarding SEQ ID NO: 579, amino acids 1 to 4 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids 5 and 6 of SEQ ID NO: 579 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0892] Regarding SEQ ID NO: 581, amino acids 1 to 3 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids 4 to 7 of SEQ ID NO: 581 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0893] (36) Epitope 36

[0894] In epitope 36 (SEQ ID NO: 307), sequence number 585 was identified as a region important for binding to the patient's IgE antibody. Sequence number 583 was identified as a sequence that includes a region adjacent to epitope 36 on the amino terminal side and binds to the patient's IgE antibody, and sequence number 584 was identified as a sequence that includes a region adjacent to epitope 36 on the carboxyl terminal side and binds to the patient's IgE antibody.

[0895] (37) Epitope 37

[0896] In epitope 37 (SEQ ID NO: 308), sequence numbers 587, 589, 592, 594, 598, 600, and 602 were identified as regions important for binding to the patient's IgE antibody. Sequence number 595 was identified as a sequence that includes a region adjacent to epitope 37 on the carboxyl terminal side and binds to the patient's IgE antibody.

[0897] Regarding sequence number 308, amino acids at positions 4, 8, 10, and 13 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 1 to 3, 5 to 7, 9, 11, 12, 14, and 15 of sequence number 308 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0898] In addition, amino acids at positions 4 and 9 to 14 were identified as being particularly important for binding to allergic IgE antibodies in SEQ ID NO: 308. In this case, even if amino acids at positions 1 to 3, 5 to 8, and 15 of SEQ ID NO: 308 were substituted with alanine, the binding to allergic IgE antibodies was not affected.

[0899] Furthermore, it was identified that amino acids at positions 5, 8, 10, 13, and 15 are also particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 308. In this case, even if amino acids at positions 1 to 4, 6, 7, 9, 11, 12, and 14 of SEQ ID NO: 308 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0900] The amino acids at positions 1 and 2 were identified as being particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 587. Even when the amino acids at positions 3 and 4 of SEQ ID NO: 587 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0901] Regarding SEQ ID NO: 589, amino acids at positions 4, 6, and 9 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 1 to 3, 5, 7, 8, and 10 of SEQ ID NO: 589 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0902] Regarding SEQ ID NO: 592, amino acids at positions 2 and 7 to 9 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even if amino acids at positions 1 and 3 to 6 of SEQ ID NO: 592 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0903] Regarding SEQ ID NO: 594, amino acids at positions 5 to 10 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 1 to 4 of SEQ ID NO: 594 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0904] The amino acids at positions 3, 6, and 8 were identified as being particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 598. Even if the amino acids at positions 1, 2, 4, 5, and 7 of SEQ ID NO: 598 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0905] The amino acids at positions 1, 4, 6 and 9 were identified as being particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 600. Even if the amino acids at positions 2, 3, 5, 7, 8 and 10 of SEQ ID NO: 600 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0906] The amino acids at positions 2, 4, and 7 were identified as being particularly important for binding to IgE antibodies of allergic patients in SEQ ID NO: 602. Even if the amino acids at positions 1, 3, 5, 6, and 8 of SEQ ID NO: 602 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0907] (38) Epitope 38

[0908] In epitope 38 (SEQ ID NO. 309), sequence number 608 was identified as a region important for binding to the patient's IgE antibody. In addition, sequence numbers 604 and 605 were identified as sequences that contain a region adjacent to epitope 38 on the amino terminal side and bind to the patient's IgE antibody, and sequence number 609 was identified as a sequence that contains a region adjacent to epitope 38 on the carboxyl terminal side and binds to the patient's IgE antibody.

[0909] Regarding sequence number 309, it was identified that amino acids at positions 5, 9, 12 to 15 are particularly important for binding to IgE antibodies of allergic patients, and amino acid position 10 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 1 to 4, 6 to 8, and 11 of sequence number 309 are substituted with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0910] In addition, amino acids at positions 7, 9, and 11 to 14 were identified as being particularly important for binding to allergic IgE antibodies in SEQ ID NO: 309. In this case, even if amino acids at positions 1 to 6, 8, 10, and 15 of SEQ ID NO: 309 were substituted with alanine, the binding to allergic IgE antibodies was not affected.

[0911] Regarding sequence number 608, it was identified that amino acids at positions 1, 5, and 8 to 10 are particularly important for binding to IgE antibodies of allergic patients, and amino acid at position 6 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 2 to 4 and 7 of sequence number 608 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0912] (39) Epitope 39

[0913] In epitope 39 (SEQ ID NO: 310), SEQ ID NO: 616, 617, and 619 were identified as regions important for binding to the patient's IgE antibody. Sequence No. 613 was identified as a sequence that includes a region adjacent to epitope 39 on the amino terminal side and binds to the patient's IgE antibody.

[0914] Regarding sequence number 310, it was identified that the amino acids at positions 3 and 5 are particularly important for binding to IgE antibodies of allergic patients, and the amino acids at positions 10 and 13 are amino acids that have an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 1, 2, 4, 6 to 9, 11, 12, 14, and 15 of sequence number 310 are replaced with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0915] Regarding sequence number 616, it was identified that the amino acids at positions 1 and 3 are particularly important for binding to IgE antibodies of allergic patients, and the amino acid at position 8 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if the amino acids at positions 2, 4 to 7, and 9 of sequence number 616 are replaced with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0916] Regarding SEQ ID NO: 619, amino acids at positions 1, 6, and 9 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids at positions 2 to 5, 7, 8, and 10 of SEQ ID NO: 619 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0917] (40) Epitope 40

[0918] Sequence numbers 622 and 623 were identified as sequences that contain a region adjacent to epitope 40 (sequence number 311) on the amino-terminal side and bind to the patient's IgE antibodies; sequence number 624 was identified as a sequence that contains a region adjacent to epitope 40 on the carboxyl-terminal side and binds to the patient's IgE antibodies.

[0919] Regarding SEQ ID NO: 311, amino acids 1 to 3 were identified as being particularly important for binding to IgE antibodies of allergic patients. Even when amino acids 4 to 15 of SEQ ID NO: 311 were substituted with alanine, the binding to IgE antibodies of allergic patients was not affected.

[0920] (41) Epitope 41

[0921] In epitope 41 (SEQ ID NO: 312), sequence numbers 627, 630, 636, 637 (amino acid sequence: ELL), 641, and 642 were identified as regions important for binding to the patient's IgE antibody. Sequence numbers 631 and 632 were also identified as sequences that are included in the region adjacent to epitope 41 on the carboxyl terminal side and bind to the patient's IgE antibody.

[0922] Regarding sequence number 312, it was identified that amino acids at positions 6, 7, 9 to 14 are particularly important for binding to IgE antibodies of allergic patients, and amino acid position 8 is an amino acid that has an influence on binding to IgE antibodies of allergic patients. Even if amino acids at positions 1 to 5 and 15 of sequence number 312 are substituted with alanine, the binding to IgE antibodies of allergic patients is not affected.

[0923] In addition, it was identified that the amino acids at positions 6 and 12 are particularly important for binding to IgE antibodies of allergic patients for SEQ ID NO: 312, and the amino acids at positions 1 and 7 are also amino acids that have an influence on binding to IgE antibodies of allergic patients. In this case, even if the amino acids at positions 2 to 5, 8 to 11, and 13 to 15 of SEQ ID NO: 312 are substituted with alanine, the binding to IgE antibodies of allergic patients will not be affected.

[0924] Furthermore, it was identified that amino acids at positions 3, 6, 7, and 13 are also particularly important for binding to IgE ant...

Claims

1. A polypeptide that specifically binds to an IgE antibody of an allergy patient, which is any one of the following: (1β) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 272 to 295 and 319 to 525; (2β) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 296 to 303 and 526 to 552; (3β) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 304 to 311 and 553 to 625; (4β) A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 312 to 318 and 626 to 682.

2. The polypeptide according to claim 1, wherein The number of amino acid residues is 500 or less.

3. A diagnostic kit for allergy, comprising at least one of the polypeptides according to claim 1 or 2.

4. A composition for diagnosing allergy, comprising at least one of the polypeptides according to claim 1 or 2 as an antigen.

5. A method for providing an index for diagnosing an allergic reaction of a subject, comprising the following steps: (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting the binding of IgE antibodies in a sample obtained from the subject to the antigen; (iii) when the binding of the subject's IgE antibody to the antigen is detected, providing an indication that the subject has an allergic reaction; Here, the antigen is at least one of the polypeptides described in claim 1 or 2.

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

7. The pharmaceutical composition according to claim 6, which is used for treating allergies.

8. A detector for determining whether an antigen is present in an object, characterized in that: It comprises an antibody that binds to at least one of the polypeptides of claim 1 or 2.

9. A detector for determining whether an antigen is present in an object, characterized in that: It contains any of the following primers: (a) a primer comprising a portion of a base sequence of a nucleic acid encoding the polypeptide of claim 1 or 2 and / or its complementary strand; or (b) a primer which is a portion of at least one of the base sequences shown in SEQ ID NOs: 46, 58, 105, 151, 236, 250 or 261; and / or A primer that is a portion of a sequence complementary to at least one of the base sequences shown in SEQ ID NOs: 46, 58, 105, 151, 236, 250, or 261.

10. A raw material or processed product, characterized in that: Its antigen is removed or reduced, and the antigen is at least one of the polypeptides described in claim 1 or 2.

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

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