US20210322543A1 - Egg allergy antigen - Google Patents

Egg allergy antigen Download PDF

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US20210322543A1
US20210322543A1 US16/305,470 US201716305470A US2021322543A1 US 20210322543 A1 US20210322543 A1 US 20210322543A1 US 201716305470 A US201716305470 A US 201716305470A US 2021322543 A1 US2021322543 A1 US 2021322543A1
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amino acid
protein
seq
acid sequence
antigen
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Kayoko Matsunaga
Akiko Yagami
Masashi Nakamura
Yuji Aoki
Erika Kondo
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Hoyu Co Ltd
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Hoyu Co Ltd
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Assigned to HOYU CO., LTD. reassignment HOYU CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: AOKI, YUJI, KONDO, Erika, MATSUNAGA, Kayoko, NAKAMURA, MASASHI, YAGAMI, Akiko
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/304—Foods, ingredients or supplements having a functional effect on health having a modulation effect on allergy and risk of allergy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/465—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates from birds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/775—Apolipopeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the present invention relates to a novel antigen of an egg allergy.
  • the present invention also relates to a kit, a composition, and a method for diagnosing an egg allergy.
  • the present invention also relates to a pharmaceutical composition comprising the antigen and an egg or a processed product of an egg in which the antigen is eliminated or reduced, or a bird which lays or has hatched from the egg.
  • the present invention also relates to a method for producing a processed product of an egg in which the antigen is eliminated or reduced.
  • the present invention also relates to a tester for determining the presence or absence of an egg antigen in an object of interest.
  • IgE antibodies specific to particular antigens are produced. Physiological consequences caused by interaction between such IgE antibodies and such particular antigens elicit allergic reactions.
  • the term “antigen” refers in its broad sense to a food or a food material which causes an allergic symptom and in its narrow sense to a protein contained in a food or a food material to which a specific IgE antibody binds (also referred to as the following, an allergenic component).
  • antigen reagents are commonly prepared simply by grinding a candidate allergenic food, food material or the like (Patent Literature 1). For this reason, the only case where conventional allergy tests have permitted detection of a positive allergic reaction is when the allergen component contained in a conventional antigen reagent is present in an amount exceeding a threshold that allows determination of a positive reaction for binding to an IgE antibody and the diagnostic efficiency has been too low to be considered to be sufficient.
  • Non Patent Literature 1 The allergens set forth in Table 1 below are currently known as egg allergens (Non Patent Literature 1).
  • allergen components are suggested to exist in foods and food materials which are allergen candidates, and test kits for such allergen components have been commercialized. However, while it is necessary to exhaustively identify allergen components in order to enhance the reliability of allergy tests, the patient detection rate by the measurement of such allergen components is far insufficient. Identification of novel allergens in eggs is very important not only for increasing the precision of diagnostic agent, but also for determining targets of low allergenic foods, low allergenic food materials, and therapeutic agents.
  • the present invention provides novel antigens of an allergy to egg.
  • the present invention also provides methods and kits for diagnosing allergy to egg.
  • the present invention also provides a pharmaceutical composition comprising the antigen and an egg or a processed product of an egg in which the antigen is eliminated or reduced, or a bird which lays or has hatched from the egg.
  • the present invention further provides testers for determining the presence or absence of an egg antigen in an object of interest.
  • the present inventors had made intensive studies to identify causative antigens of an allergy to egg. As a result, the inventors succeeded in identifying novel antigens to which an IgE antibody in the serum of an egg-allergic patient specifically binds. The present invention has been completed based on this finding.
  • the present invention can be as defined below.
  • kits for diagnosing an allergy to an egg comprising, as an antigen, at least one of proteins defined below in any of the following (1) to (3):
  • (1A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 2;
  • (1A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2;
  • (1A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 1;
  • (1A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 1;
  • (1A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1; or
  • (2A) a protein comprising a C-terminal portion of Vitellogenin-3 or a variant thereof, which is defined below in any of (2A-a) to (2A-e) which is an antigen for the egg allergy:
  • (2A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 12;
  • (2A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 12; or
  • (2A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12; or
  • (3A) A protein comprising a middle portion of Vitellogenin-1 or a C-terminal portion of lipovitelin-1 or a variant thereof, which is defined below in any of (3A-a) to (3A-e) which is an antigen for the egg allergy:
  • (3A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 18;
  • (3A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 18;
  • (3A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 17;
  • (3A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 17; or
  • (3A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 17; or
  • a composition for diagnosing an allergy to an egg comprising, as an antigen, at least one of proteins as defined above in any of (1) to (3) of [1].
  • a method for providing an indicator for diagnosing an allergy to an egg in a subject comprising the steps of:
  • a pharmaceutical composition comprising at least one of proteins as defined above in any of (1) to (3) of [1].
  • a tester for determining the presence or absence of an egg antigen in an object of interest comprising an antibody that binds to at least one of proteins as defined above in any of (1) to (3) of [1].
  • a tester for determining the presence or absence of an antigen for an egg allergy in an object of interest comprising a primer having a nucleotide sequence complementary to at least one of the nucleotide sequences set forth in SEQ ID NO: 1, 12, or 17.
  • kits for diagnosing an egg allergy comprising, as an antigen, at least one protein defined in any one of the following (4) to (10):
  • (4A) a protein comprising Vitellogenin-3 or a variant thereof, which is defined below in any of (4A-a) to (4A-e) which is an antigen for the egg allergy:
  • (4A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 47;
  • (4A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 47;
  • (4A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 46;
  • (4A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 46; or
  • (4A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 46; or
  • (5A) a protein comprising a Vitellogenin-2 precursor or a variant thereof, which is defined below in any of (5A-a) to (5A-e) which is an antigen for the egg allergy:
  • (5A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 58;
  • (5A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 58; or
  • (6A) a Vitellogenin-2 precursor or a variant thereof, which is defined below in any of (6A-a) to (6A-e) which is an antigen for the egg allergy:
  • (6A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 105;
  • (6A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 105; or
  • (6A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 105; or
  • (7A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 152;
  • (7A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 152;
  • (7A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 151;
  • (7A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 151; or
  • (7A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 151; or
  • (8A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 236;
  • (8A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 236; or
  • (8A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 236; or
  • (9A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 251;
  • (9A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 250;
  • (9A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 250; or
  • (9A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 250; or
  • (10) (10A) a Vitellogenin-2 precursor or a variant thereof, which is defined below in any of (10A-a) to (10A-e) which is an antigen for the egg allergy:
  • (10A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 261;
  • composition for diagnosing an allergy to an egg comprising, as an antigen, at least one of proteins as defined above in any of (4) to (10) of [9].
  • a method for providing an indicator for diagnosing an allergy to an egg in a subject comprising the steps of:
  • a pharmaceutical composition comprising at least one of proteins as defined above in any of (4) to (10) of [9].
  • a tester for determining the presence or absence of an egg antigen in an object of interest comprising an antibody that binds to at least one of proteins as defined above in any of (4) to (10) of [9].
  • a tester for determining the presence or absence of an antigen for an egg allergy in an object of interest comprising a primer having a nucleotide sequence complementary to at least one of the nucleotide sequences set forth in SEQ ID NO: 46, 58, 105, 151, 236, 250, or 261.
  • a method for producing a processed product of an egg in which an antigen is eliminated or reduced comprising the step of confirming that the antigen is eliminated or reduced in the process of producing the processed product, wherein the antigen is at least one of the proteins as defined in any of (1) to (3) of [1] or at least one of the proteins as defined in any of (4) to (10) of [9].
  • antigen derived from an egg wherein the antigen is at least one of the proteins as defined in any of (1) to (3) of [1] or at least one of the proteins as defined in any of (4) to (10) of [9] and causative of an egg allergy.
  • the present invention can provide novel antigens of an allergy to egg. Since the novel antigens (allergen components) that trigger an egg allergy were identified in the present invention, it is possible to provide highly sensitive methods and kits for diagnosing an egg allergy, pharmaceutical compositions comprising such an antigen, eggs or processed products of eggs in which such an antigen is eliminated or reduced, or birds that lay or have hatched from the eggs, methods for producing a processed product of an egg in which the antigen is eliminated or reduced, and testers for determining the presence or absence of an egg antigen in an object of interest.
  • FIG. 1 is a photograph of a gel showing a protein electrophoretic pattern in two-dimensional electrophoresis of proteins contained in egg yolk of a chicken egg (raw egg).
  • the bands at the left of the photograph are bands of molecular weight markers.
  • the numbers on the left side of the photograph are molecular weights of respective molecular weight markers (KDa).
  • KDa molecular weight markers
  • FIG. 2 is a photograph of an immunoblot of a two-dimensional electrophoretic pattern of proteins contained in egg yolk of a chicken egg (raw egg) stained using serum of Egg-allergic patient 1.
  • FIG. 3 is a photograph of a gel of a two-dimensional electrophoretic pattern of proteins contained in egg yolk of a chicken egg (raw egg). Spots 1, 2, and 3 that have specifically reacted with the serum of Egg-allergic patient 1 are indicated with respective surrounding boxes.
  • FIG. 4 is a photograph of an immunoblot of a two-dimensional electrophoretic pattern of proteins contained in egg yolk of a chicken egg (raw egg) stained using serum of Egg-allergic patient 2. Spots 4 to 8 that have specifically reacted with the serum of Egg-allergic patient 2 are indicated with respective surrounding boxes.
  • FIG. 5 is a photograph of an immunoblot of a two-dimensional electrophoretic pattern of proteins contained in egg yolk of a chicken egg (raw egg) stained using serum of Egg-allergic patient 3. Spots 9 and 10 that have specifically reacted with the serum of Egg-allergic patient 3 are indicated with respective surrounding boxes.
  • FIG. 6 is a photograph of an immunoblot of a two-dimensional electrophoretic pattern of proteins contained in egg yolk of a chicken egg (raw egg) stained using serum of Egg-allergic patient 4. Spot 11 that has specifically reacted with the serum of Egg-allergic patient 4 is indicated with a surrounding box.
  • the “allergy” refers to the state in which, when a certain antigen enters the body of a living individual sensitized to said antigen, the living individual shows a hypersensitive reaction detrimental to him/her.
  • IgE antibodies specific to antigens are produced. IgE antibodies bind to mast cells or basophils.
  • an antigen specific to such an IgE antibody enters again the body of a patient with an allergic disease, said antigen combines with the IgE antibody bound to mast cells or basophils, and the IgE antibody crosslinks said antigen on the cell surface, resulting in physiological effects of IgE antibody-antigen interaction.
  • physiological effects include release of histamine, serotonin, heparin, eosinophil chemotactic factors, leucotrienes, or the like. These released substances provoke an allergic reaction resulting from the combination of an IgE antibody with particular antigens. Such allergic reactions caused by particular antigens occur through the aforementioned pathway.
  • the “egg” refers to an egg of a bird, preferably a chicken egg.
  • the “allergy to egg” refers to the state in which an individual has an allergic reaction caused by proteins, etc. present in egg which act as an antigen.
  • the allergy to egg can produce an allergic reaction upon contact with, or consumption of, an antigen present in egg.
  • allergic reactions caused by consumption of foods are particularly referred to as “food allergies”.
  • the allergy to egg may be a food allergy.
  • the “antigen” refers to a substance that provokes an allergic reaction, and is also referred to as an “allergen component”.
  • the antigen is preferably a protein.
  • the “protein” refers to a molecule having a structure in which naturally occurring amino acids are joined together by peptide bond.
  • the number of amino acids present in a protein is not particularly limited, but proteins having about 2 to 50 amino acids joined together by peptide bond are in some cases called “peptides”. In the case where amino acids can form different enantiomers, the amino acids are understood to form an L-enantiomer, unless otherwise indicated.
  • the amino acid sequences of proteins or peptides as used herein are represented by one-letter symbols of amino acids in accordance with standard usage and the notational convention commonly used in the art. The leftward direction represents the amino-terminal direction, and the rightward direction represents the carboxy-terminal direction.
  • Proteins contained in egg were analyzed by the aforementioned technique to identify causative antigens of an allergy to egg.
  • raw chicken eggs were separated into egg yolk and egg white and the egg proteins contained in each of them were subjected to two-dimensional electrophoresis under the conditions described below.
  • the electrophoresis in the first dimension was isoelectric focusing, which was performed using isoelectric focusing gels with a gel-strip length of 5 to 10 cm and a gel pH range of 3 to 10.
  • the isoelectric focusing was performed using the IPG gels, Immobiline Drystrip (pH3-10NL), produced by GE Healthcare Bio-Sciences Corporation (hereinafter abbreviated as “GE”).
  • the electrophoresis system used was IPGphor produced by GE.
  • the maximum current of the electrophoresis system was limited to 75 ⁇ A per gel strip.
  • the voltage program adopted to perform the first-dimensional isoelectric focusing was as follows: (1) a constant voltage step was performed at a constant voltage of 300 V until the volt-hours reached 750 Vhr (the current variation width during electrophoresis for 30 minutes before the end of this step was 5 ⁇ A); (2) the voltage was increased gradually to 1000 V for 300 Vhr; (3) the voltage was further increased gradually to 5000 V for 4500 Vhr; and then (4) the voltage was held at a constant voltage of 5000 V until the total Vhr reached 12000.
  • the electrophoresis in the second dimension was SDS-PAGE, which was performed using polyacrylamide gels whose gel concentration at the distal end in the direction of electrophoresis was set to 3 to 6% and whose gel concentration at the proximal end was set to a higher value than that at the distal end. More specifically, the SDS-PAGE was performed using NuPAGE 4-12% Bris-Tris Gels (IPG well, Mini, 1 mm) produced by Life Technologies. The electrophoresis system used was XCell SureLock Mini-Cell produced by Life Technologies. The electrophoresis was run at a constant voltage of 200 V for about 45 minutes using an electrophoresis buffer composed of 50 mM MOPS, 50 mM Tris base, 0.1% (w/v) SDS and 1 mM EDTA.
  • the antigens of Spots 1 to 3 in the gel in which the chicken egg proteins have been separated by two-dimensional electrophoresis under the conditions described above have been revealed to exhibit specific binding to IgE antibodies from an egg-allergic patient diagnosed to be have food-dependent exercise-induced anaphylaxis ( FIG. 3 ).
  • the antigens of Spots 4 to 11 have been revealed to exhibit specific binding to IgE antibodies from other egg-allergic patients ( FIGS. 4 to 6 ).
  • the mass spectroscopic data obtained for spot 1 (SEQ ID NOs: 3-11) on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, a C-terminal portion of Vitellogenin-3 (amino acid sequence: SEQ ID NO: 2, encoding nucleotide sequence: SEQ ID NO: 1) was identified.
  • the antigen in spot 1 can be any of (1A-a) to (1A-e) and (1B) as defined below:
  • the proteins of (1A-a) to (1A-e) and (1B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 20 to 40 kDa, preferably around 30 to 35 kDa and an isoelectric point of 4.0 to 10.0, preferably an isoelectric point of 4.0 to 9.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
  • the mass spectroscopic data obtained for spot 2 (SEQ ID NOs: 9-11, 14, 16) on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, a C-terminal portion of Vitellogenin-3 (amino acid sequence: SEQ ID NO: 13, encoding nucleotide sequence: SEQ ID NO: 12) was identified.
  • the antigen in spot 2 can be any of (2A-a) to (2A-e) and (2B) as defined below:
  • a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 9-11, 13, and 16, preferably a protein comprising at least 2, 3, 4, or all sequences of the amino acid sequences.
  • the amino acid sequence of any of SEQ ID NOs: 9-11, 13, 14, 16 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.
  • the proteins of (2A-a) to (2A-e) and (2B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 30 to 60 kDa, preferably around 35 to 40 kDa and an isoelectric point of 5.0 to 10.0, preferably 6.0 to 9.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
  • the mass spectroscopic data obtained for spot 3 (SEQ ID NOs: 19-45) on a mass spectrometer was analyzed by comparing the data against the UniProt protein data, and as a result, a middle portion of Vitellogenin-1 or a C-terminal portion of Lipovitelin-1 (amino acid sequence: SEQ ID NO: 18, encoding nucleotide sequence: SEQ ID NO: 17) generated by N-terminal truncation of the protein was identified.
  • the antigen in spot 3 can be any of (3A-a) to (3A-e) and (3B) as defined below:
  • a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 22-31, 33-35, 37, 39, 40, 42, 43, and 45, preferably, a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or all sequences of the amino acid sequences.
  • the amino acid sequence of any of SEQ ID NOs:18-45 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.
  • the proteins of (3A-a) to (3A-e) and (3B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 35 to 60 kDa, preferably around 40 to 50 kDa and an isoelectric point of 5.0 to 10.0, preferably 6.0 to 9.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
  • the mass spectroscopic data (SEQ ID NOs: 48-57) obtained for spot 4 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, the antigen in question was identified as Vitellogenin-3 (amino acid sequence: SEQ ID NO: 47, encoding nucleotide sequence: SEQ ID NO: 46).
  • the antigen in spot 4 can be any of (4A-a) to (4A-e) and (4B) as defined below:
  • a protein comprising at least one amino acid sequence 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 sequences of the amino acid sequences.
  • the amino acid sequence of any of SEQ ID NOs:47-57 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.
  • the proteins of (4A-a) to (4A-e) and (4B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 20 to 50 kDa, preferably around 25 to 40 kDa, more preferably around 30 to 35 kDa and an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, more preferably 4.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
  • the mass spectroscopic data (SEQ ID NOs: 60-104) obtained for spots 5 and 6 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, the antigen in question was identified as Vitellogenin-2 precursor (amino acid sequence: SEQ ID NO: 59, encoding nucleotide sequence: SEQ ID NO: 58).
  • the antigen in spots 5 and 6 can be any of (5A-a) to (5A-e) and (5B) as defined below:
  • a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 60, 63, 65, 68, 69, 71, 73, 75-79, 81, 83-88, 91-94, 96-99, and 101-103, preferably, a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or all sequences of the amino acid sequences.
  • the amino acid sequence of any of SEQ ID NOs:59 to 104 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.
  • the proteins of (5A-a) to (5A-e) and (5B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 80 to 260 kDa, preferably around 90 to 200 kDa, more preferably around 110 to 160 kDa and an isoelectric point of 1.0 to 8.0, preferably 2.0 to 7.0, more preferably 3.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
  • the mass spectroscopic data (SEQ ID NOs: 107-150) obtained for spot 7 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, the antigen in question was identified as Vitellogenin-2 precursor (amino acid sequence: SEQ ID NO: 106, encoding nucleotide sequence: SEQ ID NO: 105).
  • the antigen in spot 7 can be any of (6A-a) to (6A-e) and (6B) as defined below:
  • a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 106, 108, 110, 111, 113, 114, 116, 117, 120-124, 126-131, 133, 134, 136-138, 140-143, 145-147, and 149, preferably, a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31, or all sequences of the amino acid sequences.
  • the amino acid sequence of any of SEQ ID NOs: 106-150 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.
  • the proteins of (6A-a) to (6A-e) and (6B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 110 to 300 kDa, preferably around 130 to 280 kDa, more preferably around 160 to 260 kDa and an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, more preferably 4.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
  • the mass spectroscopic data (SEQ ID NOs: 153-235) obtained for spot 8 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, the antigen in question was identified as Apolipoprotein B precursor (amino acid sequence: SEQ ID NO: 152, encoding nucleotide sequence: SEQ ID NO: 151).
  • the antigen in spot 8 can be any of (7A-a) to (7A-e) and (7B) as defined below:
  • the proteins of (7A-a) to (7A-e) and (7B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 160 to 550 kDa, preferably around 180 to 400 kDa, more preferably around 200 to 300 kDa and an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, more preferably 4.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
  • the mass spectroscopic data (SEQ ID NOs: 238-249) obtained for spot 9 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, the antigen in question was identified as Apolipoprotein B precursor (amino acid sequence: SEQ ID NO: 237, encoding nucleotide sequence: SEQ ID NO: 236).
  • the antigen in spot 9 can be any of (8A-a) to (8A-e) and (8B) as defined below:
  • a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 237-239, 241, 242, 244, and 246-249; preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, or all sequences of the amino acid sequences.
  • the amino acid sequence of any of SEQ ID NOs: 237-249 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.
  • the proteins of (8A-a) to (8A-e) and (8B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 20 to 50 kDa, preferably around 25 to 45 kDa, more preferably around 30 to 40 kDa and an isoelectric point of 7.0 to 11.0, preferably 8.0 to 10.0, more preferably 9.0 to 10.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
  • the mass spectroscopic data (SEQ ID NOs: 252-260) obtained for spot 10 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, Apolipoprotein B precursor (amino acid sequence: SEQ ID NO: 251, encoding nucleotide sequence: SEQ ID NO: 250) was identified was identified.
  • the antigen in spot 10 in the present invention can be any of proteins selected from the group consisting of the proteins as defined below in (9A-a) to (9A-e), and (9B):
  • a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 251-254, 256, 257, and 260; preferably a protein comprising at least 2, 3, 4, 5, 6, or all sequences of the amino acid sequences.
  • the amino acid sequence of any of SEQ ID NOs: 251-260 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids;
  • the proteins of (9A-a) to (9A-e) and (9B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 20 to 50 kDa, preferably 25 to 40 kDa, more preferably 30 to 40 kDa and an isoelectric point of 7.0 to 11.0, preferably 8.0 to 10.0, more preferably 9.0 to 10.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
  • the mass spectroscopic data (SEQ ID NOs: 263-271) obtained for spot 11 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, Vitellogenin-2 precursor (amino acid sequence: SEQ ID NO: 262, encoding nucleotide sequence: SEQ ID NO: 261) was identified.
  • the antigen in spot 11 in the present invention can be any of proteins selected from the group consisting of the proteins as defined below in (10A-a) to (10A-e) and (10B):
  • a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 262-265, 268, and 269; preferably a protein comprising at least 2, 3, 4, 5, or all sequences of the amino acid sequences.
  • the amino acid sequence of any of SEQ ID NOs:262 to 271 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.
  • the proteins of (10A-a) to (10A-e) and (10B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 15 to 40 kDa, preferably 17 to 35 kDa, more preferably 20 to 30 kDa and an isoelectric point of 6.0 to 11.0, preferably 7.0 to 10.0, more preferably 8.0 to 10.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
  • the antigen proteins of (1) to (3) and (4) to (10) as defined above also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.
  • the antigen proteins of (1) to (3) and (4) to (10) as defined above are antigens for an egg allergy.
  • deletion, substitution, insertion or addition of one or several amino acids in relation to amino acid sequence, it is meant that in an amino acid sequence of interest, one or several amino acids (e.g., 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2%, or 1% of amino acids with respect to the total length of the amino acid sequence) are deleted, one or several amino acids are substituted by any other amino acids, any other amino acids are inserted, and/or any other amino acids are added.
  • amino acids e.g., 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2%, or 1% of amino acids with respect to the total length of the amino acid sequence
  • substitution is preferably conservative substitution.
  • the “conservative substitution” refers to the substitution of a certain amino acid residue by a different amino acid residue having similar physicochemical characteristics, and can be any type of substitution as long as it does not substantially change the characteristics of the structure of the original sequence for example, any type of substitution is acceptable as long as any substituted amino acids do not disrupt the helical structure of the original sequence or other secondary structures that characterize the original sequence.
  • the following gives examples of separate groups of amino acid residues that are conservatively substitutable with each other, but substitutable amino acid residues are not limited to the examples given below.
  • Group A leucine, isoleucine, valine, alanine, methionine
  • Group B aspartic acid, glutamic acid
  • Group C asparagine
  • glutamine Group D lysine
  • arginine Group E serine
  • threonine Group F phenylalanine, tyrosine
  • one member belonging to one of the aforementioned groups can be replaced with a member belonging to any other group.
  • amino acids of group B, D or E as listed above may be substituted by those of any other group.
  • cysteines may be deleted or substituted by any other amino acids to prevent them from being folded into a protein in its tertiary structure.
  • any amino acids may be substituted in consideration of the hydropathy scales of amino acids, which are a measure of the hydrophilic and hydrophobic properties of amino acids (J. Kyte and R. Doolittle, J. Mol. Biol., Vol. 157, p. 105-132, 1982).
  • the percent identity between two amino acid sequences can be determined by visual inspection and mathematical calculation.
  • the percent identity can be determined using a computer program. Examples of such computer programs include BLAST and ClustalW. In particular, various conditions (parameters) for identity searches with the BLAST program are described in Altschul, et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997), and are publicly available on the websites of the National Center for Biotechnology Information (NCBI) and DNA Data Bank of Japan (DDBJ) (Altschul, et al., BLAST Handbook, Altschul, et al., NCB/NLM/NIH Bethesda, Md. 20894). Also, the percent identity can be determined using a genetic information processing software program, such as GENETYX Ver. 7 (Genetyx Corporation), DINASIS Pro (Hitachi Software Engineering Co., Ltd.), or Vector NTI (Infomax Inc.).
  • deletion, substitution, insertion or addition of one or several nucleotides in relation to nucleotide sequence, it is meant that in a nucleotide sequence of interest, one or several nucleotides (e.g., 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1%, of nucleotides with respect to the total length of the nucleotide sequence) are deleted, one or several nucleotides are substituted by any other nucleotides, any other nucleotides are inserted, and/or any other nucleotides are added. It is preferable that such a nucleotide deletion, substitution, insertion or addition should not give rise to a frame shift in an amino acid coding sequence.
  • the percent identity between two nucleotide sequences can be determined by visual inspection and mathematical calculation.
  • the percent identity can be determined using a computer program.
  • sequence comparison computer programs include the BLASTN program available on the website of the National Library of Medicine (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al. (1990) J. Mol. Biol. 215: 403-10), and it can be determined by using default parameters.
  • under stringent conditions means that hybridization takes place under moderately or highly stringent conditions.
  • the moderately stringent conditions can be easily determined by those having ordinary skill in the art on the basis of, for example, the length of DNA. Basic conditions are described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3rd ed., ch. 6-7, Cold Spring Harbor Laboratory Press, 2001.
  • the moderately stringent conditions include hybridization under the conditions of preferably 1 ⁇ SSC to 6 ⁇ SSC at 42° C. to 55° C., more preferably 1 ⁇ SSC to 3 ⁇ SSC at 45° C. to 50° C., most preferably 2 ⁇ SSC at 50° C.
  • a temperature around 5 to 15° C. lower than the foregoing should be adopted. Washing is also carried out under the conditions of 0.5 ⁇ SSC to 6 ⁇ SSC at 40° C. to 60° C. In the process of hybridization and washing, generally 0.05% to 0.2% SDS, preferably about 0.1% SDS, may be added.
  • the highly stringent conditions can be easily determined by those having ordinary skill in the art on the basis of, for example, the length of DNA.
  • the highly stringent (high stringent) conditions include hybridization and/or washing at a higher temperature and/or a lower salt concentration than those adopted under the moderately stringent conditions.
  • hybridization is carried out under the conditions of 0.1 ⁇ SSC to 2 ⁇ SSC at 55° C. to 65° C., more preferably 0.1 ⁇ SSC to 1 ⁇ SSC at 60° C. to 65° C., most preferably 0.2 ⁇ SSC at 63° C.
  • Washing is carried out under the conditions of 0.2 ⁇ SSC to 2 ⁇ SSC at 50° C. to 68° C., more preferably 0.2 ⁇ SSC at 60 to 65° C.
  • Antigens may be obtained by separating and purifying them from eggs (preferably chicken eggs) using a combination of protein purification methods well known to those skilled in the art. Also, antigens may be obtained by expressing them as recombinant proteins using a genetic recombination technique well known to those skilled in the art and by separating and purifying them using protein purification methods well known to those skilled in the art.
  • Exemplary protein purification methods include: solubility-based purification methods such as salt precipitation and solvent precipitation; purification methods based on molecular weight difference, such as dialysis, ultrafiltration, gel filtration and SDS-PAGE; charge-based purification methods such as ion exchange chromatography and hydroxylapatite chromatography; specific affinity-based purification methods such as affinity chromatography; purification methods based on hydrophobicity difference, such as reverse-phase high-performance liquid chromatography; and purification methods based on isoelectric point difference, such as isoelectric focusing.
  • solubility-based purification methods such as salt precipitation and solvent precipitation
  • purification methods based on molecular weight difference such as dialysis, ultrafiltration, gel filtration and SDS-PAGE
  • charge-based purification methods such as ion exchange chromatography and hydroxylapatite chromatography
  • specific affinity-based purification methods such as affinity chromatography
  • purification methods based on hydrophobicity difference such as reverse-phase high
  • Preparation of a protein by a genetic recombination technique is carried out by preparing an expression vector comprising an antigen-encoding nucleic acid, introducing the expression vector into appropriate host cells by gene transfer or genetic transformation, culturing the host cells under suitable conditions for expression of a recombinant protein, and recovering the recombinant protein expressed in the host cells.
  • the “vector” refers to a nucleic acid that can be used to introduce a nucleic acid attached thereto into host cells.
  • the “expression vector” is a vector that can induce the expression of a protein encoded by a nucleic acid introduced therethrough.
  • Exemplary vectors include plasmid vectors and viral vectors. Those skilled in the art can select an appropriate expression vector for the expression of a recombinant protein depending on the type of host cells to be used.
  • the “host cells” refers to cells that undergo gene transfer or genetic transformation by a vector.
  • the host cells can be appropriately selected by those skilled in the art depending on the type of the vector to be used.
  • the host cells can be derived from prokaryotes such as E. coli .
  • the antigen of the present invention may be designed to contain an N-terminal methionine residue in order to facilitate the expression of a recombinant protein in the prokaryotic cells.
  • the N-terminal methionine can be cleaved from the recombinant protein after expression.
  • the host cells may be cells derived from eukaryotes, such as single-cell eukaryotes like yeast, plant cells and animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, murine cells or insect cells) or silkworm.
  • eukaryotes such as single-cell eukaryotes like yeast
  • plant cells and animal cells e.g., human cells, monkey cells, hamster cells, rat cells, murine cells or insect cells
  • silkworm e.g., silkworm.
  • Gene transfer or genetic transformation of an expression vector into host cells can be carried out as appropriate by following a technique known to those skilled in the art.
  • Those skilled in the art can make possible the expression of a recombinant protein by selecting suitable conditions for the expression of the recombinant protein as appropriate depending on the type of host cells and culturing the host cells under the selected conditions. Then, those skilled in the art can homogenize the host cells having the expressed recombinant protein, and separate and purify an antigen expressed as the recombinant protein from the resulting homogenate by using an appropriate combination of such protein purification methods as mentioned above.
  • the present invention provides a method for providing an indicator for diagnosing an allergy to an egg in a subject, the method comprising the steps of:
  • the sample obtained from a subject is a solution containing an IgE antibody, as collected from the subject.
  • solutions include blood, saliva, sputum, snivel, urine, sweat, and tear.
  • the sample obtained from the subject may be subjected to pretreatment for increasing the concentration of an IgE antibody in the sample before being contacted with an antigen.
  • the pretreatment of a sample may involve, for example, collection of the serum, plasma from the blood.
  • a Fab portion that is a binding portion with the antigen may be purified.
  • the step (i) mentioned above is carried out by contacting an IgE antibody present in the serum obtained from a subject with an antigen.
  • the IgE antibody may be the IgE antibody itself or mast cells or the like to which the IgE antibody is bound.
  • Detection of contact and binding between the sample obtained from a subject and an antigen can be carried out by using a known method. Examples of such methods that can be used include detection by ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, or immunochromatography. These are all techniques for detecting binding between an antigen and an IgE antibody from a subject by contacting and binding the IgE antibody from a subject with the antigen, allowing an enzymatically labelled secondary antibody to act on the IgE antibody specifically bound to the antigen, and adding an enzyme substrate (generally, chromogenic or luminescent reagent) to detect an enzymatic reaction product.
  • an enzyme substrate generally, chromogenic or luminescent reagent
  • a method for detecting a fluorescently labeled secondary antibody can be used. Also, detection by a measurement method that permits the analysis of binding between an antigen and an IgE antibody, such as surface plasmon resonance (SPR), can be used. A plurality of antigen-specific IgE antibodies may be mixed.
  • SPR surface plasmon resonance
  • the antigen may be provided as an isolated antigen in a state immobilized on a carrier.
  • the steps (i) and (ii) mentioned above can be carried out using ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance, or the like.
  • the step (i) mentioned above is carried out by contacting the sample obtained from a subject with an antigen-immobilized surface.
  • the isolated antigen may be obtained by separating and purifying it from egg (preferably chicken egg) using a combination of protein purification methods well known to those skilled in the art, or by preparing it using a genetic recombination technique.
  • the antibody may be in an immobilized state.
  • the antigen may be in a state unfixed on a carrier.
  • the steps (i) and (ii) mentioned above can be carried out by flow cytometry or the like and the presence of the antigen bound to an antibody may be confirmed using a laser beam.
  • Examples include basophil activation test (BAT) and the like.
  • BAT basophil activation test
  • HRT histamine release test in which it is examined whether histamine is released or not by bringing an antigen further in contact with hemocytes in a sample.
  • the antigen may be detected by immunoblotting in a state separated by two-dimensional electrophoresis.
  • the two-dimensional electrophoresis is a technique for separating a protein sample by performing isoelectric focusing in the first dimension and performing SDS-PAGE (SDS-polyacrylamide gel electrophoresis) in the second dimension.
  • the conditions for two-dimensional electrophoresis are not particularly limited as long as the conditions permit the separation of the antigen of the present invention.
  • the conditions for two-dimensional electrophoresis as described above in the subsection titled “Identification of antigens” can be adopted.
  • the electrophoresis conditions may be defined by reference to the descriptions in PTLs 1 to 4 mentioned above.
  • two-dimensional electrophoresis can be carried out under the conditions that satisfy at least one selected from the group consisting of the following requirements:
  • the isoelectric focusing gels used in the first dimension should have a gel-strip length of 5 to 10 cm and a gel pH range of 3 to 10, and the pH gradient of the gels in the direction of electrophoresis should be as follows: where the gel-strip length up to pH 5 is taken as “a”, that length from pH 5 to 7 as “b”, and that length above pH 7 as “c”, the relations “a ⁇ b” and “b>c” are satisfied; (B) in the case of (A), when the total gel-strip length is taken as 1, “a” should be in the range of 0.15 to 0.3, “b” should be in the range of 0.4 to 0.7, and “c” should be in the range of 0.15 to 0.3; (C) in the first dimensional isoelectric focusing, a constant voltage step should be performed by applying a constant voltage ranging from 100 V to 600 V per gel strip containing a sample, and after the electrophoresis variation width during electrophoresis for 30 minutes falls within the range of 5
  • the aforementioned antigens (1) to (3) and (4) to (10) are antigens that specifically bind to IgE antibodies from patients with allergy to egg. Therefore, when binding between an IgE antibody from a subject and the antigen is detected, an indicator of the fact that the subject is allergic to egg is provided.
  • the present invention further provides a kit for diagnosing an allergy to an egg, comprising at least one of the aforementioned antigens (1) to (3) and (4) to (10).
  • the diagnosis kit of this invention may be used in the aforementioned method for providing an indicator for diagnosing an allergy to an egg or in a diagnosis method as described later.
  • the diagnosis kit of this invention may comprise not only the at least one of the aforementioned antigens (1) to (3) and (4) to (10), but also an anti-IgE antibody labeled with an enzyme and a chromogenic or luminescent substrate serving as a substrate for the enzyme. Also, a fluorescent-labeled anti-IgE antibody may be used.
  • the antigen may be provided in a state immobilized on a carrier.
  • the diagnosis kit of this invention may also be provided together with instructions on the procedure for diagnosis or a package containing said instructions.
  • the diagnosis kit includes a companion diagnostic agent for an egg allergy.
  • the companion diagnostic agent is used to analyze the reactivity of a pharmaceutical product for the purpose of identifying a patient in which the pharmaceutical product is expected to work or a patient at risk for serious side effects from the pharmaceutical product, or optimizing a therapy using the pharmaceutical product.
  • optimizing a therapy include determination of the usage and the volume, decision of stopping the administration, determination of the allergen component to be used for the induction of immune tolerance, and the like.
  • the present invention further provides a composition for diagnosing an allergy to an egg, comprising at least one of the aforementioned antigens (1) to (3) and (4) to (10).
  • the diagnosis composition of this invention can be used in a diagnosis method as described below.
  • the diagnosis composition of this invention may further comprise a pharmaceutically acceptable carrier and/or additives commonly used with the antigen of this invention depending on the need.
  • the present invention provides a method for diagnosing an allergy to an egg in a subject, the method comprising:
  • the present invention provides a method for diagnosing an allergy to an egg in a subject, the method comprising administering to the subject at least one of the aforementioned antigens (1) to (3) and (4) to (10).
  • This method may be performed in the form of a skin test characterized by applying the antigen onto the skin.
  • Examples of the skin test include various forms of tests, such as: a prick test in which a diagnosis composition is applied onto the skin and then a tiny prick to such an extent as not to provoke bleeding is made in the skin to allow an antigen to penetrate the skin, thereby observing a skin reaction; a scratch test in which a diagnosis composition is applied onto the skin and then the skin is lightly scratched to observe a reaction; a patch test in which a diagnosis composition in the form of cream, ointment, etc. is applied onto the skin to observe a reaction; and an intracutaneous test in which an antigen is administered intracutaneously to observe a reaction.
  • a prick test in which a diagnosis composition is applied onto the skin and then a tiny prick to such an extent as not to provoke bleeding is made in the skin to allow an antigen to penetrate the skin, thereby observing a skin reaction
  • a scratch test in which a diagnosis composition is applied onto the skin and then the skin is lightly scratched to observe a reaction
  • a patch test
  • the subject of interest is diagnosed as having an allergy to an egg.
  • the amount of the antigen to be applied to the skin in such tests can be, for example, not more than 100 ⁇ g per dose.
  • the antigen protein to be used in the load test may be a protein that has been expressed and purified and may be a protein that has been expressed in a food and a food material, for example, rice-based vaccine produced by transforming rice with a gene of a cedar pollen antigen to express the antigen protein therein.
  • the present invention provides at least one of the aforementioned antigens (1) to (3) and (4) to (10), intended for use in the diagnosis of an allergy to an egg.
  • the present invention encompasses the provision of at least one of the aforementioned antigens (1) to (3) and (4) to (10) mixed with a known antigen.
  • the present invention provides use of at least one of the aforementioned antigens (1) to (3) and (4) to (10) for the production of a diagnostic agent for an allergy to an egg.
  • the present invention provides a pharmaceutical composition comprising at least one of the aforementioned antigens (1) to (3) and (4) to (10).
  • the aforementioned pharmaceutical composition is used for the treatment of an allergy to an egg.
  • the present invention also provides a method for treating an allergy to an egg, the method comprising administering at least one of the aforementioned antigens (1) to (3) and (4) to (10) to a patient in need of a treatment for an allergy to an egg.
  • the present invention provides at least one of the aforementioned antigens (1) to (3) and (4) to (10), intended for use in the treatment for an allergy to an egg.
  • the present invention provides use of at least one of the aforementioned antigens (1) to (3) and (4) to (10) for the production of a therapeutic agent for an allergy to an egg.
  • a hyposensitization therapy aiming to induce immunological tolerance by administering an antigen to a patient is often adopted.
  • the at least one of the aforementioned antigens (1) to (3) and (4) to (10) can be used as an active ingredient for a hyposensitization therapy for an allergy to an egg.
  • the antigen protein to be used in the hyposensitization therapy may be a protein that has been expressed and purified and may be a protein that has been expressed in a food and a food material, for example, rice-based vaccine produced by transforming rice with a gene of a cedar pollen antigen to express the antigen protein therein.
  • the pharmaceutical composition of the present invention can be administered by common administration routes.
  • common administration routes include oral, sublingual, percutaneous, intracutaneous, subcutaneous, intravascular, intranasal, intramuscular, intraperitoneal, and intrarectal administrations.
  • the pharmaceutical composition of the present invention can be used as a pharmaceutical composition to which a commonly used pharmaceutically acceptable adjuvant or excipient or any other additives (e.g., stabilizer, solubilizer, emulsifier, buffer, preservative, colorant) are added by a conventional method together with the antigen of this invention depending on the need.
  • a commonly used pharmaceutically acceptable adjuvant or excipient or any other additives e.g., stabilizer, solubilizer, emulsifier, buffer, preservative, colorant
  • the dosage form of the pharmaceutical composition can be selected by those skilled in the art as appropriate depending on the administration route.
  • the pharmaceutical composition can be in the form of, for example, tablet, capsule, troche, sublingual tablet, injection, intranasal spray, poultice, solution, cream, lotion, or suppository.
  • the administration dose, frequency and/or period of the pharmaceutical composition of this invention can be selected by a physician as appropriate depending on the administration route and the patient's condition and characteristics such as age and body weight.
  • the pharmaceutical composition may be administered to an adult patient at a dose of not more than 100 ⁇ g per dose.
  • the administration interval can be, for example, once daily, once weekly, twice weekly, once per three months or so.
  • the administration period can be, for example, several weeks to several years.
  • the pharmaceutical composition may be administered in such a manner that the dose is increased in incremental steps over the administration period.
  • the present invention provides a tester comprising an antibody for at least one of the aforementioned antigens (1) to (3) and (4) to (10).
  • the antibody can be prepared by a conventional method.
  • the antibody may be prepared by immunizing a mammal such as rabbit with one of the aforementioned antigens (1) to (3) and (4) to (10).
  • the antibody may be an IgE antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (e.g., Fab, F(ab′) 2 , Fab′).
  • the antibody may be provided in a form bound to a carrier.
  • the type of the carrier is not particularly limited as long as it is usable for detection of binding between an antibody and an antigen. Any given carrier known to those skilled in the art can be used.
  • Examples of a method for determining the presence or absence of an antigen include the following:
  • the present invention encompasses a tester for determining the presence or absence of an antigen for an egg allergy in an object of interest, comprising a primer having a nucleotide sequence complementary to a part of at least one of the nucleotide sequences set forth in SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250, or 261.
  • the primer has a nucleotide sequence complementary to preferably 12 bases, 15 bases, 20 bases, or 25 bases in the sequence of a 3′ terminal or middle portion of a part of at least one sequence of the nucleotide sequences set forth in, for example, SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250, or 261.
  • the tester comprises a poly A tail-complementarity primer.
  • the tester including the primer may further comprise a primer comprising a nucleotide sequence of a 5′ terminal portion of at least one sequence of the nucleotide sequences set forth in SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250, or 261, preferably a nucleotide sequence consisting of 12 bases, 15 bases, 20 bases, 25 bases.
  • the presence or absence of the antigen is determined by amplifying cDNA by polymerase chain reaction (PCR) including RT-PCR with DNA or mRNA obtained from an egg or a bird as a template and the complementary primer and comparing the amplified cDNA sequence(s) with SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250, or 261.
  • PCR polymerase chain reaction
  • Examples of the method of the amplification by PCR include RACE and the like.
  • the antigen is determined to be present when the comparison between the amplified cDNA and SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250, or 261 indicates the presence of one or more point mutations encoding the same amino acid, or when the amino acid sequence encoded by the amplified cDNA has 70% or more, preferably 80, 90, 95, 98, or 99% or more identity with an amino acid sequence of SEQ ID NO: 2, 13, 18, 47, 59, 106, 152, 237, 251 or 262, even if the nucleotide sequence of the amplified cDNA is modified from a nucleotide sequence of SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250, or 261 by insertion, deletion, substitution, or addition of one or more nucleotides.
  • the aforementioned tester is used to determine the presence or absence of an antigen in foods or in products of interest in a food material (egg or bird) or a food production line.
  • the tester may also be used for quality inspection of production lines and pre-shipment products by manufacturers, or may be used for self-checking of the presence or absence of an antigen in a food, food material of interest by consumers.
  • the present invention provides an egg, a processed product of an egg, or a bird which lays or has hatched from the egg in which at least one of the aforementioned antigens (1) to (3) and (4) to (10) is eliminated or reduced.
  • the method for eliminating or reducing the antigen of the present invention in an egg, a processed product of an egg, or a bird which lays or has hatched from the egg is not limited.
  • the elimination or reduction of the antigen may be conducted by any method, as long as the method permits the elimination or reduction of the antigen.
  • the egg of the present invention whose antigen is eliminated or reduced may be obtained by preparing an egg in which the expression of the antigen of the present invention is knocked out, using a gene knock-out technique.
  • the gene knock-out technique there can be used any methods known to those skilled in the art.
  • Oishi, et al. (Scientific Reports, Vol. 6, Article number: 23980, 2016, doi:10.1038/srep23980) describes that the genome editing technique CRISPER/Cas9 is applied to chicken primordial germ cells to obtain individual animals deficient in ovomucoid gene.
  • the egg devoid of the antigen of this invention may also be obtained by using the same technique as above.
  • a bird or an egg in which the antigen of the present invention is eliminated or decreased may be obtained by mating by artificial insemination with a bird or an egg that does not contain the antigen or contains a low content of the antigen.
  • the artificial mating of birds or eggs can be performed by a conventional method.
  • An antigen of the present invention may be the artefact that an antigen of the present invention assumed the removal or a reduced egg raw material as for the removal or the reduced processed products of egg.
  • a treatment for removing or reducing the antigen of this invention is performed before or after preparation of a processed product of egg.
  • Examples of a method of eliminating or decreasing the antigen of the present invention in a processed product made from a common egg include methods for eliminating the protein component in a food or a food material, such as elution with a high pressure treatment and a neutral salt solution and hot steam and methods for hydrolysis, denaturation, or amino acid modification (chemical modification or elimination of side chains) by heat treatment and acid treatment.
  • the bird in which the antigen of the present invention is eliminated or reduced may be a bird that has hatched and grown from an egg in which the aforementioned antigen of the present invention is eliminated or reduced.
  • the birds include birds at growth stages of nestling, juvenile, young bird, adult bird and old bird.
  • the bird means an animal belonging to Ayes, preferably a chicken.
  • the present invention provides a method for producing a processed product of an egg in which an antigen is eliminated or reduced, comprising the step of confirming that the antigen is eliminated or reduced in the process of producing the processed product, wherein the antigen is at least one of the aforementioned antigens (1) to (3) and (4) to (10).
  • the step of confirming that the antigen is eliminated or reduced in the process of producing the processed product of an egg in which an antigen is eliminated or reduced may be performed by examining whether the antigen is contained or not by the method described in the section of “Tester” above.
  • the method for producing a processed product of an egg in which an antigen is eliminated or reduced may be performed by the method described in the section “Antigen-free food and the like” above.
  • Proteins contained in egg were investigated using a two-dimensional electrophoresis method described below.
  • Raw chicken eggs were separated into egg white and egg yolk.
  • the extraction and the purification of proteins contained in the egg white and the egg yolk were performed as follows.
  • a solubilization agent was added to the egg white to extract proteins and then water or a urea buffer was added to obtain a protein extract.
  • the composition of the urea buffer is as follows.
  • MCLI Mesmalian Lysis Buffer
  • the precipitation procedure was repeated twice using a 2D-CleanUP Kit (produced by GE).
  • the collected liquid protein extract was precipitated by adding TCA (trichloroacetic acid) thereto and the precipitated product produced by this procedure (TCA-precipitated product) was collected.
  • TCA-precipitated product the precipitated product produced by this procedure
  • the second round of precipitation the TCA-precipitated product collected above was further precipitated by adding acetone thereto and the precipitated product (sample) produced by this procedure was collected.
  • First-dimensional isoelectric focusing gel strips (Immobiline Drystrip IPG gels (pH3-10NL); produced by GE) were immersed in 140 ⁇ L of the foregoing sample solution for first-dimensional isoelectric focusing (sample solution for swelling) and impregnated with the solution at room temperature overnight.
  • An electrophoresis tray was filled with silicone oil.
  • Filter paper moisten with water was positioned at both ends of the gel strips impregnated with the sample, and the gel strips were set in the electrophoresis tray such that the gel strips were covered with silicone oil. Electrodes were placed on the gel strips with the filter paper intervening therebetween.
  • the maximum current of the isoelectric focusing system was set to 75 ⁇ A per gel strip, and the first-dimensional isoelectric focusing was carried out according to the following voltage program: (1) a constant voltage step was performed at a constant voltage of 300 V until the volt-hours reached 750 Vhr (the current variation width during electrophoresis for 30 minutes before the end of this step was 5 ⁇ A); (2) the voltage was increased gradually to 1000 V for 300 Vhr; (3) the voltage was further increased gradually to 5000 V for 4500 Vhr; and then (4) the voltage was held at a constant voltage of 5000 V until the total Vhr reached 12000.
  • the gel strips were taken out of the isoelectric focusing system, immersed in an equilibration buffer containing a reducing agent, and shaken at room temperature for 15 minutes.
  • the constituents of the equilibration buffer containing the reducing agent are as mentioned below.
  • the equilibration buffer containing the reducing agent was removed, and then the gel strips were immersed in an equilibration buffer containing an alkylating agent and shaken at room temperature for 15 minutes to obtain SDS-equilibrated gels.
  • the constituents of the equilibration buffer containing the alkylating agent are as mentioned below.
  • the XCell SureLock Mini-Cell electrophoresis system produced by Life Technologies was used.
  • the second-dimensional electrophoresis gels used were NuPAGE 4-12% Bis-Tris Gels produced by Life Technologies.
  • an electrophoresis buffer composed of the following constituents was prepared and used.
  • an agarose solution for gel adhesion was used in this example, which was prepared by dissolving 0.5% (w/v) Agarose S (produced by Nippon Gene Co., Ltd.) and a moderate amount of BPB (bromophenol blue) in the electrophoresis buffer.
  • SDS-PAGE wells were washed well with the electrophoresis buffer, and then the buffer used for the washing was removed. Next, the washed wells were charged with the fully dissolved agarose solution for gel adhesion. Next, the SDS-equilibrated gel strips were immersed in agarose and closely adhered to second-dimensional electrophoresis gels using tweezers. After it was confirmed that agarose was fully fixed with the gels being closely adhered to each other, electrophoresis was performed at a constant voltage of 200 V for about 45 minutes.
  • the gels were fluorescently stained with SYPRO Ruby (produced by Life Technologies).
  • an airtight container to be used was washed well in advance with 98% (v/v) ethanol.
  • the electrophoresed second-dimensional electrophoresis gel strips were taken out of the SDS-PAGE system, placed onto the washed airtight container, and treated twice by immersion in 50% (v/v) methanol and 7% (v/v) aqueous solution containing acetic acid for 30 minutes. Then, a further immersion treatment was done for 10 minutes, with the solution being replaced by water.
  • the second-dimensional electrophoresis gel strips were immersed in 40 mL of SYPRO Ruby and shaken at room temperature overnight.
  • FIG. 1 illustrates the result of two-dimensional electrophoresis of proteins contained in the egg yolk (the result of the egg white is not illustrated).
  • Molecular weight marker bands are found at the left of the photograph of the gel. The positions of the bands indicate particular molecular weights (KDa).
  • Identification of antigens by immunoblotting was carried out by taking all the steps up to the step of “Second-dimensional SDS-PAGE” as described above in Example 1, followed by the steps of “Transfer to membrane”, “Immunoblotting” and “Analysis” as described below.
  • Transfer to membrane was done using the following transfer system and transfer buffer.
  • Transfer system XCell SureLock Mini-Cell and XCell II Blot Module (produced by Life Technologies)
  • Transfer buffer NuPAGE Transfer Buffer (X20) (produced by Life Technologies), used in a form diluted 20-fold with milliQ water.
  • proteins in the two-dimensional electrophoresis gels were transferred to a membrane (PVDF membrane) according to the following procedure.
  • the PVDF membrane was immersed in 100% methanol followed by milliQ water, and then moved into the transfer buffer to hydrophilize the PVDF membrane.
  • Immunoblotting of the membrane was carried out using, as a primary antibody, the serum from a patient with an egg allergy (patient 1) or the serum from a non-egg-allergic subject.
  • This egg-allergic patient is a patient diagnosed as food-dependent exercise-induced anaphylaxis (FDEIA) by chicken egg.
  • FDEIA food-dependent exercise-induced anaphylaxis
  • the transferred membrane was shaken in a 5% skim milk/PBST solution (a PBS buffer containing 0.1% Tween 20 nonionic surfactant) at room temperature for one hour.
  • a 5% skim milk/PBST solution a PBS buffer containing 0.1% Tween 20 nonionic surfactant
  • the membrane was left to stand in a solution of 5% primary antibody serum in 5% skim milk/PBST at room temperature for one hour.
  • the membrane was washed with a PBST solution (5 min. ⁇ 3 times).
  • the membrane was left to stand in a 1:5000 dilution of the secondary antibody, anti-human IgE-HRP (horseradish peroxidase), with a 5% skim milk/PBST solution at room temperature for one hour.
  • the membrane was washed with a PBST solution (5 min. ⁇ 3 times).
  • the membrane was left to stand in Pierce Western Blotting Substrate Plus (produced by Thermo Fisher Scientific) for 5 minutes.
  • the membrane obtained through the foregoing series of treatments was subjected to fluorescent image scanning on Typhoon 9500 (produced by GE).
  • the immunoblot obtained with the serum from the egg-allergic patient was compared with that obtained with the control serum from the non-egg-allergic subject. Three spots that are different from those with the serum of the non-egg-allergic patient and different from the known chicken egg allergen proteins were detected on an immunoblot of the proteins contained in raw egg yolk with the serum of the egg-allergic patient ( FIG. 2 ).
  • the molecular weights and isoelectric points of the three spots are as follows ( FIG. 3 ).
  • Spot 1 Molecular weight 20 to 40 kDa, pI 4.0 to 10.0
  • Spot 2 Molecular weight 30 to 60 kDa, pI 5.0 to 10.0
  • Spot 3 Molecular weight 35 to 60 kDa, pI 5.0 to 10.0
  • the amino acid sequences of the antigens that form the three protein spots of Example 2 were identified by mass spectroscopy.
  • Spot 1 The amino acid sequences set forth in SEQ ID NOs: 3 to 11
  • Spot 2 The amino acid sequences set forth in SEQ ID NOs: 9 to 11, 14, 16 Spot 3: The amino acid sequences set forth in SEQ ID NOs: 19 to 45
  • spots were identified as the following proteins by the analysis of the mass data obtained from the mass spectrometer for spots 1 and 2 at NCBI and for spot 3 with UniProt.
  • the Identification of antigens was performed by immunoblotting using the serum of three different egg-allergic patients (Patient 2, Patient 3, Patient 4), in the same manner as in Example 2. Spots that are different from those with the serum of the non-egg-allergic patient and different from the known chicken egg allergen proteins were detected on immunoblots of the proteins contained in raw egg yolk with the serums of the egg-allergic patients ( FIG. 4 for Patient 2, FIG. 5 for Patient 3, FIG. 6 for Patient 4). Specifically, Spots 4 to 8, Spots 9 and 10, and Spot 11 were respectively detected for patient 2, Patient 3, and Patient 4.
  • the molecular weights and isoelectric points of Spots 4 to 11 are as follows ( FIGS. 4 to 6 ).
  • Example 4 The identification of the amino acid sequences of antigens producing spots 4 to 11 in Example 4 by the mass spectrometry was performed in the same manner as in Example 3.
  • Spot 4 The amino acid sequences set forth in SEQ ID NOs: 48 to 57 Spots 5, 6: The amino acid sequences set forth in SEQ ID NOs: 60 to 104 Spot 7: The amino acid sequences set forth in SEQ ID NOs: 107 to 150 Spot 8: The amino acid sequences set forth in SEQ ID NOs: 153 to 235 Spot 9: The amino acid sequences set forth in SEQ ID NOs: 238 to 249 Spot 10: The amino acid sequences set forth in SEQ ID NOs: 252 to 260 Spot 11: The amino acid sequences set forth in SEQ ID NOs: 263 to 271
  • spots were identified as the following proteins by the analysis of the mass data obtained from the mass spectrometer for the spots at NCBI.
  • a Vitellogenin-2 precursor (amino acid sequence: NCBI accession number NP_001026447.1, encoding nucleotide sequence: GenBank accession number NM_001031276.1) (amino acid sequence: SEQ ID NO: 106, encoding nucleotide sequence: SEQ ID NO: 105)
  • Spot 8 An Apolipoprotein B precursor (amino acid sequence: NCBI accession number NP_001038098.1, encoding nucleotide sequence: GenBank accession number NM_001044633.1) (amino acid sequence: SEQ ID NO: 152, encoding nucleotide sequence: SEQ ID NO: 151)
  • Spot 9 An Apolipoprotein B precursor (amino acid sequence: NCBI accession number NP_001038098.1, encoding nucleotide sequence: GenBank accession number NM_001044633.1) (amino acid sequence: 237, encoding nucleot
  • Spot 10 An Apolipoprotein B precursor (amino acid sequence: NCBI accession number NP_001038098.1, encoding nucleotide sequence: GenBank accession number NM_001044633.1) (amino acid sequence: SEQ ID NO: 251, encoding nucleotide sequence: SEQ ID NO: 250)
  • Spot 11 A Vitellogenin-2 precursor (amino acid sequence: NCBI accession number NP_001026447.1, encoding nucleotide sequence: GenBank accession number NM_001031276.1) (amino acid sequence: SEQ ID NO: 262, encoding nucleotide sequence: SEQ ID NO: 261).
  • the present invention can provide a novel antigen of an egg allergy, a method for diagnosing an egg allergy and a kit for diagnosing an egg allergy, a pharmaceutical composition comprising the antigen, an egg, a processed product of an egg, or a bird which lays or has hatched from the egg in which the antigen is eliminated or reduced, and a method for producing a processed product of an egg in which the antigen is eliminated or reduced.
  • the present invention can further provide testers for determining the presence or absence of an egg antigen in an object of interest.

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