JP2015178461A - Buckwheat allergen specific monoclonal antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a buckwheat allergen specific monoclonal antibody that can be applied for detection of a variety of buckwheat strains and avoids cross-reaction with seed plants other than buckwheat, and provide a buckwheat allergen specific monoclonal antibody which can detect buckwheat allergens included in not-heated raw materials and food products as well as buckwheat allergens in food products after heat denaturation.SOLUTION: The monoclonal antibody recognizes an amino acid sequence included in a specific region of BWp16 as an epitope.

Description

  The present invention relates to a buckwheat allergen BWp16 specific monoclonal antibody.

  The present invention relates to a buckwheat allergen-specific monoclonal antibody that can be applied to the detection of buckwheat allergen BWp16 contained in raw materials and foods containing various buckwheat and does not exhibit antigen cross-reactivity with seed plants other than buckwheat.

  In recent years, the number of patients with food allergies in Japan has been increasing, and in order to prevent health damage caused by foods containing allergens, the labeling of foods containing allergens has been institutionalized in April 2001 due to the amendment of the Food Sanitation Law It was done. In particular, seven items such as wheat, buckwheat, egg, milk, peanuts, shrimp and crab are designated as “specific raw materials” and are not only products for general consumers, but also allergic substances in foods for commercial and processed food ingredients. The labeling has become mandatory.

Buckwheat allergy has a high risk of causing serious allergic symptoms including anaphylaxis and should be taken with caution.
The major proteins of buckwheat are the storage proteins 13S globulin and 2S albumin, and the allergens are digested in comparison with the 76 kDa allergen (13S globulin), 24 kDa allergen (13S globulin β subunit), and 24 kDa allergen, which are high in content. A 16 kDa allergen (a type of 2S albumin, hereinafter referred to as BWp16) that exhibits high resistance to an enzyme (pepsin) has been reported.

  Since buckwheat is a cross-pollinated plant, it is easy to cross with other varieties and there is no original species. Therefore, there are no clear varieties, and for convenience, they are classified with the name of the area where they are cultivated (for example, Kitawase Soba, Hitachi Aki Soba).

As a conventional method for detecting buckwheat allergens, the mainstream is a method for detecting crude buckwheat protein or 76 kDa allergen and 24 kDa allergen having a high content.
For example, Patent Document 1 proposes an IgG fraction or polyclonal antibody that uses a 70 to 500 kDa protein fraction of buckwheat as an antigen as a buckwheat component detection antibody, and a test kit that uses these antibodies.
Patent Document 2 proposes a method for detecting buckwheat allergen using monoclonal antibodies to native buckwheat crude protein, heat-denatured buckwheat crude protein, buckwheat major protein 24 kDa allergen and 76 kDa allergen protein.
Kits using these detection methods are commercially available as immunoassay kits.

However, these crude buckwheat proteins, the 76 kDa allergen and the 24 kDa allergen are subject to the detection method, which has a problem in that it may cross-react with proteins derived from other seed plants in foods and exhibit false positives. It was.
False positives have been accepted in the conventional method because of the patient's request that it is necessary to reliably detect buckwheat allergen in the sample. However, in order to prevent the decline in production efficiency, such as the occurrence of production loss and the necessity of cleaning the production line, as the demand for detecting the contamination of buckwheat allergens at the production site increases with the mandatory labeling of specific raw materials, Development of a detection method that is highly specific for buckwheat allergen that reduces the occurrence of false positives is desired, and in order to achieve this, a buckwheat allergen-specific monoclonal antibody that does not exhibit antigen cross-reactivity with seed plants other than buckwheat Is desired.

  In addition, from the viewpoint of ensuring versatility of the method for detecting buckwheat allergen, a buckwheat allergen-specific monoclonal antibody that can similarly detect buckwheat allergens in unheated raw materials and foods and buckwheat allergens in foods after heat denaturation is desired. .

Japanese Patent No. 3799290 Japanese Patent No. 5043078

Molecular cloning of cDNA, recombinant protein expression and characterization of a buckwheat 16-kDa major allergen.Int Arch Allergy Immunol. 2006; 140 (1): 73-81.

It is an object of the present invention to provide a buckwheat allergen-specific monoclonal antibody that can be applied to detection of various buckwheat and does not exhibit antigen cross-reactivity with seed plants other than buckwheat.
Another object of the present invention is to provide a buckwheat allergen-specific monoclonal antibody that can similarly detect buckwheat allergens contained in non-heated raw materials and foods and buckwheat allergens in foods after heat denaturation.

  The present inventors pay attention to buckwheat allergen BWp16 and recognize a specific region of recombinant BWp16 having an amino acid sequence common to various buckwheat and having an amino acid sequence having low homology with 2S albumin of seed plants other than buckwheat. It has been found that the monoclonal antibody is applicable to the detection of various buckwheat and does not exhibit antigen cross-reactivity with seed plants other than buckwheat. In addition, since the monoclonal antibody of the present invention recognizes an epitope regardless of the three-dimensional structure of buckwheat allergen BWp16, it similarly detects buckwheat allergens in unheated raw materials and foods and buckwheat allergens in foods after heat denaturation. Is possible.

That is, the present invention is as follows.
(1) SEQ ID NO: 12,
A monoclonal antibody having the peptide represented by SEQ ID NO: 13 or SEQ ID NO: 14 as an antigen,
Recognizing the amino acid sequence contained in SEQ ID NO: 30 as an epitope;
A monoclonal antibody characterized by binding to buckwheat BWp16.
(2) The monoclonal antibody according to (1) above, which recognizes the amino acid sequence contained in SEQ ID NO: 31 as an epitope.
(3) The monoclonal antibody according to (2) above, wherein the amino acid sequence contained in SEQ ID NO: 32 is recognized as an epitope.
(4) The monoclonal antibody according to (3) above, wherein the amino acid sequence contained in SEQ ID NO: 33 is recognized as an epitope.
(5) SEQ ID NO: 1: Xaa1 Glu Ala Leu Xaa2 Arg Xaa3 Glu Gly Glu Gly Cys Lys Ser Glu Glu Ser Cys Met Arg
SEQ ID NO: 2: Met Xaa4 Glu Met Asp Asp Glu Cys Val Cys or SEQ ID NO: 3: Met Val Glu Asn Xaa5 Lys Gly Arg Ile
(In the sequence, Xaa1 to Xaa5 are any naturally occurring amino acids.)
The monoclonal antibody according to (4) above, wherein the amino acid sequence contained in is recognized as an epitope.
(6) SEQ ID NO: 1: Xaa1 Glu Ala Leu Xaa2 Arg Xaa3 Glu Gly Glu Gly Cys Lys Ser Glu Glu Ser Cys Met Arg
SEQ ID NO: 2: Met Xaa4 Glu Met Asp Asp Glu Cys Val Cys or SEQ ID NO: 3: Met Val Glu Asn Xaa5 Lys Gly Arg Ile
(In the sequence, Xaa1 is Ala or Glu, Xaa2 is Ser or Lys, Xaa3 is Val or Ile, Xaa4 is Lys or Arg, and Xaa5 is Gln.)
The monoclonal antibody according to (5) above, wherein the amino acid sequence contained in is recognized as an epitope.
(7) The monoclonal antibody according to any one of (1) to (6), which does not cross-react with seed plant-derived proteins other than buckwheat.
(8) A hybridoma identified by the receipt number NITE AP-01791, a hybridoma identified by the receipt number NITE AP-01792, a hybridoma identified by the accession number NITE AP-01793, and a hybridoma identified by the receipt number NITE AP-01794 The monoclonal antibody according to any one of the above (1) to (7), which is an antibody produced by a hybridoma selected from the group consisting of hybridomas specified by accession number NITE AP-01795.
(9) A method for detecting buckwheat in a specimen using the monoclonal antibody according to any one of (1) to (8).
(10) A buckwheat detection kit comprising the monoclonal antibody according to any one of (1) to (8).

A buckwheat allergen-specific monoclonal antibody that can be applied to detection of various buckwheat and does not show antigen cross-reactivity with seed plants other than buckwheat by using a monoclonal antibody that recognizes an amino acid sequence contained in a specific region of BWp16 of the present invention. Can be provided. That is, by using the buckwheat allergen-specific monoclonal antibody of the present invention, various buckwheat in food can be detected, and false positives due to the presence of seed plants other than buckwheat can be suppressed.
In addition, since the present invention can recognize an epitope regardless of the difference in the three-dimensional structure of BWp16, it can detect the buckwheat allergen contained in non-heated raw materials and foods and the buckwheat allergen in foods after heat denaturation in the same manner. .

The result of having examined the homology of BWp16 amino acid sequence is shown. The higher-order structure (3 patterns) of BWp16 is shown. The measurement result of the antibody titer of the anti-BWp16 antiserum by ELISA method is shown. The result of having confirmed the cross-reactivity of the monoclonal antibody by WB method is shown. The expression map of recombinant BWp16 is shown. The result of having confirmed the reactivity to each split BWp16 of a monoclonal antibody by WB method is shown. The result of having confirmed the reactivity to each split BWp16 of a monoclonal antibody by WB method is shown. The recognition area of the divided BWp16 is illustrated.

(Buckwheat)
In the present invention, buckwheat is an edible cereal belonging to the genus Buckwheat. There is no native buckwheat and it is easy to cross with other varieties because of cross-pollination. Therefore, there are no varieties that are clearly classified by gene, and are usually classified with the name of the area cultivated for convenience. Examples include, but are not limited to, Chinese mancans, Chinese northern middle grains, American mancans, Brazilian products, Kitawase, Hitachi autumn buckwheat, and the like.

(BWp16)
BWp16 in the present invention is an allergen protein of about 16 kDa contained in buckwheat and has pepsin resistance. Non-patent document 1 reports that BWp16 belongs to the storage protein 2S albumin family, and cDNA encoding BWp16 identified from Japanese buckwheat (Non-patent document 1).
Currently, the allergen registration database Allergome (http://www.allergome.org.) Contains two BWp16 amino acid sequences identified from Japanese buckwheat, respectively, buckwheat allergen Flag e2 (SEQ ID NO: 4), Flag e (Fag e 2.0101 is an amino acid sequence of 149 residues, and Flag e2 does not include a signal peptide, and amino acid sequences 23 to 149 of Fage 2.0101 are registered as 2.0101 (SEQ ID NO: 5). The amino acid sequence of 127 residues corresponding to the amino acid sequence of ## STR00001 ## In Flag e 2.0101, position 138 is Leu (L), whereas in Flag e 2, it is Met (M). BWp16 is not limited to the complete amino acid sequence, but is naturally occurring It includes mutants in various buckwheat.

(Monoclonal antibody)
The monoclonal antibody of the present invention has a recombinant BWp16 peptide represented by BWp16-1 (SEQ ID NO: 12), BWp16-2 (SEQ ID NO: 13) or BWp16-3 (SEQ ID NO: 14) as an antigen, It corresponds to the amino acid sequence of positions 23-149 of Flag e 2.0101 among the amino acid sequences of BWp16, and positions 34, 50, 62, 66, 70, 72, 92, 110 and 131 in Flag 2.010. It is a monoclonal antibody characterized by recognizing as an epitope the amino acid sequence contained in the sequence (SEQ ID NO: 30), which is any amino acid naturally occurring, and binding to buckwheat BWp16.
Of the amino acid sequence of buckwheat BWp16, it corresponds to the amino acid sequence at positions 66-149 of Flag 2.010, and the amino acids at positions 66, 70, 72, 92, 110 and 131 in Flag 2.010 are natural. An amino acid sequence included in the sequence (SEQ ID NO: 31) which is an arbitrary amino acid present in E. coli, as an epitope, and among amino acid sequences of buckwheat BWp16, amino acid sequence of amino acid sequence 66-131 of Fage 2.0101 The amino acid sequence included in the sequence (SEQ ID NO: 32) in which amino acids at positions 66, 70, 72, 92, 110, and 131 in Flag 2.010 are naturally occurring amino acids is recognized as an epitope. Of the amino acid sequence of buckwheat BWp16, It corresponds to the amino acid sequence of amino acid sequence 66-114 and is included in the sequence (SEQ ID NO: 33) in which amino acids at positions 66, 70, 72, 92 and 110 in Flag 2.010 are naturally occurring amino acids. It is preferable in this order to recognize the amino acid sequence as an epitope.
More preferably, the monoclonal antibody of the present invention comprises
SEQ ID NO: 1: Xaa1 Glu Ala Leu Xaa2 Arg Xaa3 Glu Gly Glu Gly Cys Lys Ser Glu Glu Ser Cys Met Arg
SEQ ID NO: 2: Met Xaa4 Glu Met Asp Asp Glu Cys Val Cys or SEQ ID NO: 3: Met Val Glu Asn Xaa5 Lys Gly Arg Ile
The amino acid sequence contained in is recognized as an epitope.
In the sequence, Xaa1 to Xaa5 are any naturally occurring amino acids. Xaa1 is preferably Ala or Glu, more preferably Ala. Xaa2 is preferably Ser, Lys, Arg or Asn, more preferably Ser or Lys, and most preferably Ser. Xaa3 is preferably Val or Ile, more preferably Val. Xaa4 is preferably Lys or Arg, more preferably Lys. Xaa5 is preferably Gln.
The monoclonal antibody of the present invention recognizes, as an epitope, a sequence consisting of preferably 5 or more, more preferably 6 or more amino acids in the amino acid sequence contained in each of SEQ ID NOs: 30 to 33 and 1 to 3. .
The class of the monoclonal antibody of the present invention includes IgA, IgD, IgE, IgG and IgM, preferably IgG.

(Immune method)
The animal used for producing the antibody is not particularly limited, and examples thereof include mice, rats, humans, rabbits and chickens. Mice are preferably used because of the ease of production. In addition, the monoclonal antibody of the present invention is a hybridoma prepared by cell fusion of antibody-producing cells and myeloma cells collected from an animal immunized with BWp16, cultured in a medium, or administered into an abdominal cavity of an animal, and allowed to grow in ascites. And then collected from the culture or ascites.

(Production of hybridoma)
The anti-BWp16 antibody-producing hybridoma, for example, immunizes a BALB / c mouse with BWp16, fuses the antibody-producing cells of the immunized mouse and mouse myeloma cells by a conventional method, and screens with an immunofluorescent staining pattern Thus, an anti-BWp16 monoclonal antibody-producing hybridoma can be produced. Examples of the antibody-producing cells include spleen cells, lymph node cells, B-lymphocytes and the like obtained from animals immunized with BWp16 or a composition containing the same. Examples of animals to be immunized include mice, rats, rabbits and horses. Immunization is performed, for example, by administering BWp16 alone or together with an appropriate adjuvant to the animal subcutaneously, intramuscularly or intraperitoneally 1 to 3 times / month for 1 to 6 months. Separation of antibody-producing cells is performed by collecting from the immunized animal 2 to 4 days after the final immunization. As myeloma cells, those derived from mice and rats can be used. The antibody-producing cells and the myeloma cells are preferably derived from the same species.

  Examples of the monoclonal antibody of the present invention include an anti-BWp16 protein monoclonal antibody 1A12 produced by a hybridoma (BWp16-1A12, accession number NITE AP-01791) and an anti-BWp16 produced by a hybridoma (BWp16-34D9, accession number NITE AP-01792). Anti-BWp16 protein monoclonal antibody produced by anti-BWp16 protein monoclonal antibody 34F9 produced by hybridoma (BWp16-34F9, receipt number NITE AP-01793) or hybridoma (BWp16-35E2, receipt number NITE AP-01794) produced by protein monoclonal antibody 34D9 Anti-B produced by antibody 35E2 or hybridoma (BWp16-41D4, receipt number NITE AP-01795) The p16 protein monoclonal antibody 41D4 can be mentioned, and these hybridomas are listed on January 23, 2014 (the date of receipt), the National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation (National Institute of Technology and Technology) (Chiba Prefecture 292-0818) Deposited with Kisarazu City Kazusa Kamashika 2-5-8).

(Seed plants other than buckwheat)
In the present invention, “seed plants other than buckwheat” means all seed plants that have been cross-reactive with a conventional buckwheat allergen detection method. Examples thereof include nuts such as cashew nuts, walnuts, almonds, and peanuts, beans such as red beans, black beans, soybeans, and bean, cereals such as sesame, rice, wheat, and barley, and vegetables such as tomatoes.

(Cross reaction)
The monoclonal antibody of the present invention preferably does not cross-react with seed plant-derived proteins other than buckwheat. In the present invention, “seed plant-derived protein other than buckwheat” means a protein naturally contained in seed plants other than buckwheat, and “does not cross-react with seed plant-derived protein other than buckwheat”. This means that the monoclonal antibody recognizes a protein derived from a seed plant other than buckwheat and does not react. For example, in a normal WB (Western blot) method, 1-15 μg / 10 μl of a seed plant protein extract other than buckwheat is separated as a protein by SDS-PAGE gel, and the separated protein is transferred to a membrane. This means that no positive band is detected when the monoclonal antibody is reacted as a primary antibody and immunostained with an appropriate labeled secondary antibody.

(How to detect buckwheat)
In the buckwheat detection method of the present invention, “buckwheat” to be detected includes raw grains and grains, as well as those processed by threshing, heating, drying, pulverization and the like. Samples include a wide range of food ingredients, pre-processed food such as heat, and post-processed food such as heat. The processing method and the degree of processing are not limited, including those that may be mixed.
The buckwheat detection method of the present invention is not particularly limited as long as it is an immunological detection method using the monoclonal antibody of the present invention. For example, biosensors such as sandwich ELISA, competition method, SPR, and immunochromatography can be mentioned. The usage form of the antibody is not limited to the case of using the whole antibody, and fragments such as F (ab ′) 2 and Fab can also be used.

(Buckwheat detection kit)
The buckwheat detection kit of the present invention may be any kit as long as it contains the monoclonal antibody of the present invention as a constituent component. The antibodies included in the kit may exist individually or in the form of a mixture, or may be bound to a solid support or in free form. Furthermore, such a kit may contain a labeled antibody, a carrier, a washing buffer, a sample diluent, an enzyme substrate, a reaction stop solution, a marker (target) polypeptide as a purified standard substance, instructions for use, etc. it can.

  EXAMPLES Examples will be shown below for specifically explaining the present invention, but the present invention is not limited to the following examples.

1. Confirmation of amino acid sequence of various buckwheat BWp16 (1) Preparation of various buckwheat DNA Washing of common buckwheat (Chinese mancan, Chinese northern middle grain, American mancan, Brazilian, Kitawase, Hitachi autumn buckwheat) with 0.1% SDS solution. After freezing overnight at −80 ° C., it was crushed in a mortar. DNA was extracted from the obtained powder using DNeasy Plant Mini Kit (Qiagen).

(2) Gene sequence of target protein BWp16 According to the method described in Non-Patent Document 1, the gene of BWp16 was amplified from DNA by PCR, and the sequence was confirmed.

PCR Primer sequence BWp16-R: 5'- AATTACACAAAATACCGATTTCCTCTC-3 '
BWp16-F: 5'-AAAATGAAGCTCTTCATCATCCTAGCA-3 '

(3) Characteristic of BWp16 amino acid sequence BWp16 DNA sequence of 6 kinds of buckwheat is converted to amino acid sequence and registered as buckwheat allergen in allergen registration database Allergome (http://www.allergome.org.) The amino acid sequence of Fage 2.0101, and six kinds of Genso buckwheat obtained (Chinese mancan (SEQ ID NO: 6), Chinese northern middle grain (SEQ ID NO: 7), American mancan (SEQ ID NO: 8), Brazilian (SEQ ID NO: 9) As a result of comparing the homology of Kitawase (SEQ ID NO: 10) and Hitachi Akisoba (SEQ ID NO: 11)), 9, 10, 34, 50, 62, 66 from the N-terminal side of Flag 2.010 , 70, 72, 92, 110 and 131) (FIG. 1, amino acids in white letters). In FIG. 1, X represents a mixture of a plurality of types of amino acids.
The combinations of the 66th, 70th, 72th, 92nd and 110th amino acids at the 66th to 114th positions from the N-terminal side of Flag 2.010 had 7 patterns shown in Table 1-1 below. Further, Fage e2.0101, Fag e2, and BWp16 of each buckwheat took the pattern indicated by ◯ in Table 1-2.

As mentioned above, there are various mutations in BWp16 contained in buckwheat. Therefore, the target is “Chinese mancans” (cultivated products of varieties of mancans) that contain many mutation patterns and account for more than 60% of domestic distribution. Analyzed by narrowing down.
As a result, 5 types of BWp16 amino acid sequences are included in Chinese mancan, and the mutations in the BWp16 amino acid sequences are limited to those shown in FIG. 1, and all the mutations are substituted with amino acids that can take amino acids. When the body was simulated on a computer and the higher order structure was estimated using analysis software (GENETYX), it was found that the predicted higher order structure of the protein was aggregated into the three patterns shown in FIG.

(4) Selection of BWp16 buckwheat gene sequence A protein (2S albumin) corresponding to BWp16 in other plants (eg, peanuts) having a low homology with the amino acid sequence is selected, and BWp16-1, BWp16-2 and BWp16 -3.
Table 2 below shows which positions are mutated and how compared to the amino acid sequence of Flag 2.010.

2. Preparation of recombinant BWp16 protein (1) Preparation of E. coli vector for expression of BWp16 The amino acid sequences of the three types of BWp16 selected as described above, namely BWp16-1, BWp16-2 and BWp16-3, were respectively expressed as His tag or GST tag. For expression as a fusion protein, the gene sequence encoding each amino acid sequence was inserted into a vector with a corresponding tag sequence, pET-22b (+) (Novagen) or pGEX-6P (GE Healthcare Japan).
This plasmid was introduced into Escherichia coli BL21 (DE3) Rosetta gami2 (Novagen) to isolate a target colony.

(2) Expression and purification of recombinant BWp16 using Escherichia coli The above-mentioned Escherichia coli was pre-cultured overnight at 37 ° C. in 2 × YT medium (Trypton, Yeast Extract, NaCl). Subsequently, the culture solution was diluted in fresh same medium and cultured at 37 ° C. for 30 minutes, and IPTG (Isopropyl β-D-1-thiogalactopylanoside) was added at a final concentration of 1 mM to induce protein expression at 16 ° C. Cultured for 16 hours. The cells were collected from the culture solution by centrifugation, frozen at −80 ° C., and sonicated to obtain a centrifugal supernatant. Recombinant BWp16-1, BWp16-2, and BWp16-3 were subjected to nickel sepharose purification (GE Healthcare Japan) or glutathione sepharose (SIGMA) using the tag as an index.
Each concentration of purified recombinant BWp16-1, BWp16-2 and BWp16-3 was calculated by Bradford Protein Assay (Bio-Rad).

3. Preparation of anti-BWp16 monoclonal antibody (1) Preparation of hybridoma Three types of His-tagged recombinant BWp16-1, BWp16-2 and BWp16-3 and adjuvant (Gibco) were mixed and emulsified, and BALB / c mice (Japan SLC) Were injected 5 times every 10 days. Three days after the final injection, blood was collected from the tail vein, the serum was diluted to 102,400 times, and the antibody titer was measured by ELISA. An increase in antibody titer was confirmed (color development was confirmed at a dilution of 100,000 times or more, see FIG. 3). The spleen of a mouse was excised and fused with a mouse myeloma cell to prepare an antibody-producing hybridoma. .

(2) Selection of hybridomas 10 days after cell fusion, cell supernatants were collected in order from clones in which the hybridomas were grown on a surface area of two-thirds or more of the wells on a 96-well cell plate.
(A) ELISA method A mixture of GST-fused recombinant BWP16-1, BWp16-2 and BWp16-3 was immobilized on a 96-well ELISA plate. After washing and blocking (5% skim milk-PBST), the hybridoma cell supernatant was reacted as a primary antibody. After washing, it was reacted as a secondary antibody with an HRP-labeled anti-mouse IgG antibody (Invitrogen). After washing, color development was detected with a TMB substrate. Absorbance was measured at 650 nm. As a result, 140 clones with a measured value of 2.5 or more were selected.
(B) WB method A mixture of GST-fused recombinant BWP16-1, BWp16-2 and BWp16-3 was transferred to a PVDF membrane (GE Healthcare Japan) using SDS-PAGE gel (Wako). After blocking the membrane (5% skim milk-TBST), the cell supernatant of the selected hybridoma was reacted as a primary antibody. After the membrane was washed, HRP-labeled anti-mouse IgG antibody was reacted as a secondary antibody. The membrane was washed, reacted with ECL Western Blotting Detection Reagents (GE Healthcare Japan), and chemiluminescence was detected with an X-ray film (Fujifilm). As a result, 20 clones with strong chemiluminescence were selected.

(3) Large-scale preparation of hybridoma and purification of anti-BWP16 monoclonal antibody Pristane was injected into the abdominal cavity of BALB / c mice, and one week later, an antibody-producing hybridoma suspension (2 × 10 ⁶ cells / 200 μl) was injected into the abdominal cavity of the mice. did. After confirming the expansion in the abdominal cavity, ascites was collected with a syringe with a needle. The monoclonal antibody was purified from this ascites fluid using ammonium sulfate precipitation and a protein G column (GE Healthcare Japan). The concentration of the purified monoclonal antibody was calculated by the following calculation formula.
Concentration (mg / ml) = Absorbance 280 nm value / 1.35

4). Confirmation of monoclonal antibody (1) Measurement of detection sensitivity by ELISA method Recombinant BWp16 mixture (BWp16-1, BWp16-2 and BWp16-3) is solid-phased at a concentration of 1 mg / ml in a 2-fold dilution series on a 96-well ELISA plate. Turned into. After washing, blocking (5% skim milk-PBST) was added to the plate. Next, the monoclonal antibody was subjected to a primary antibody reaction in a 2-fold dilution series from a concentration of 1 mg / ml. After washing, a secondary antibody reaction was performed with an HRP-labeled anti-mouse IgG antibody. After washing, color development was detected with a TMB substrate. Absorbance was measured at 650 nm. As a result, a positive reaction with a measured value of 0.05 or more was determined, and the minimum protein concentration of the recombinant BWp16 mixture was defined as the detection limit value (sensitivity) (Table 3).

(2) Confirmation of crossability by WB method Various buckwheat (Hitachi autumn buckwheat, Chinese northern middle grain, Kitawase, Chinese mancan) and seeds of other plants (cashew nut, walnut, almond, peanut, sesame, rice, tomato, red beans , Black beans, soybeans and bean beans) were washed with 0.1% SDS solution, freeze-dried, and ground in a mortar. To this powder, an extract (4% SDS, 8M Urea, 20% Glycerol, 0.25M Tris-HCl (pH 6.8), 5% 2-Mercaptoethanol) was added, and after stirring with a rotator (room temperature, 2 hours), Centrifugation (10,000 × g, 20 minutes, room temperature) supernatant was collected to obtain an extract. The extract (1 μg / 10 μl as a protein was separated by SDS-PAGE gel, and the separated protein and recombinant BWp16 mixture (BWp16-1, BWp16-2 and BWp16-3) were transferred to a PVDF membrane. The membrane was blocked ( 5% skim milk-TBST), and the prepared monoclonal antibody was reacted.After washing the membrane, the HRP-labeled anti-mouse IgG antibody was reacted as a secondary antibody.The membrane was washed and reacted with ECL Western Blotting Detection Reagents. And chemiluminescence was detected with an X-ray film.

The results are shown in FIG. “No cross-reactivity (−)” indicates that each monoclonal antibody clone did not react with anything other than buckwheat. “Cross-reactive (+)” indicates that it reacted with something other than buckwheat.
Monoclonal antibody clone no. Any of 1-2, 6-8 and 10 recognized proteins separated from various buckwheat.
Monoclonal antibody clone no. 2 has low detection sensitivity compared to other monoclonal antibody clones, and also crosses many non- buckwheat plant-derived proteins (cashew nuts, almonds, peanuts, sesame seeds, tomatoes, red beans, black beans, soybeans, and bean beans). It showed reactivity.
Clone No. of a monoclonal antibody showing high reactivity with the recombinant BWp16 mixture. 1 and no. No. 10 showed a cross-reaction with some plant-derived proteins other than buckwheat (No. 1: almond, sesame and red beans, No. 10: cashew nuts).
Monoclonal antibody clone no. 6-8 showed no cross-reactivity for all plant-derived proteins studied except buckwheat.

5. Confirmation of subclass Using the culture supernatant of the hybridoma as a specimen, it was measured with an Iso Strip Mouse Monoclonal Antibody Isotyping Kit (Roche). This reagent identified the subclass of mouse antibodies. The results are shown in Table 3.

6). Confirmation of recognition region of monoclonal antibody (1) Preparation of divided recombinant E. coli vector for expression of recombinant BWp16 Three regions were determined from the entire sequence of BWp16 gene so that the encoded amino acid sequence somewhat overlapped (as amino acid sequences 23aa-65aa , 55aa-107aa and 99aa-149aa encoding positions), and recombinant proteins having amino acid sequences encoded by the divided gene sequences were designated as BWp16-A, BWp16-B and BWp16-C, respectively (FIG. 5). In order to express the protein of each region as a GST tag fusion protein, it was inserted into a vector pGEX-6P (GE Healthcare Japan) with a corresponding tag sequence.

  In addition, the amino acid sequence coding for the position coding 66aa-149aa as the amino acid sequence of the BWp16 gene that does not overlap with the region encoding BWp16-A and overlaps with the region encoding BWp16-B and BWp16-C. Five regions were determined so as to be somewhat overlapped (positions coding for 66aa-90aa, 86aa-105aa, 101aa-109aa, 115aa-136aa, 132aa-149aa as amino acid sequences), and the amino acid sequences encoded by the divided gene sequences The recombinant proteins possessed were BWp16-a, BWp16-b, BWp16-c, BWp16-d and BWp16-e, respectively (FIG. 5). In order to express the protein of each region as a GST tag fusion protein, it was inserted into a vector pGEX-6P (GE Healthcare Japan) with a corresponding tag sequence.

BWp16-A, BWp16-B, BWp16-C, BWp16-a, BWp16-b, BWp16-c, BWp16-d, and BWp16-e have multiple amino acid sequence patterns in each divided region. In this case, a protein for each pattern was prepared. For example, BWp16-A has 1 mutation since no mutation is present in the region, BWp16-B has 2 mutations, and BWp16-C has 3 mutations in the region.
Table 4 below shows which position and how it is mutated by comparing each split recombinant BWp16 with the amino acid sequence of Flag 2.010.

These plasmids were introduced into Escherichia coli BL21 (DE3) Rosetta gami2 (Novagen) to isolate target colonies.
The PCR Primer sequence for obtaining the gene sequence encoding each split recombinant BWp16 is as follows.
(A) BWp16-A
BWp16-AR: 5'-GGGGGATCCAGAGATGAAGGCTTCGATTTAGGC-3 '
BWp16-AF: 5'-CCCGAATTCGTATTTGTCGTCGAGTATATCCATGGC-3 '
(B) BWp16-B1
BWp16-B1-R: 5'-GGGGGATCCATAGCCATGGATATACTCGACGAC-3 '
BWp16-B1-F: 5'-CCCGAATTCCTCAACCATCATCTTCATCCACTC-3 '
(C) BWp16-B2
BWp16-B2-R: 5'- GGGGGATCCATAGCCATGGATATACTCGATGAC-3 '
BWp16-B2-F: 5'- CCCGAATTCCTCAACCATCATCTTCATCCACTC-3 '
(D) BWp16-C1
BWp16-C1-R: 5'-GGGGGATCCGTTTGTGAGTGGATGAAGATGATGG-3 '
BWp16-C1-F: 5'- CCCGAATTCTTACACAAAATACCGATTTCCTCT-3 '
(E) BWp16-C2
BWp16-C2-R: 5'-GGGGGATCCGTTTGTGAGTGGATGAAGATGATGG-3 '
BWp16-C2-F: 5'- CCCGAATTCTTACACAAAATACCGATTTCCTCT-3 '
(F) BWp16-C3
BWp16-C3-R: 5'-GGGGGATCCGTTTGTGAGTGGATGAAGATGATGG-3 '
BWp16-C3-F: 5'- CCCGAATTCTTACACAAAATACCGATTTCCTCT-3 '
(G) BWp16-a1
BWp16-a1-R: 5'-GGGGGATCCGCAGAAGCTTTGAGTAGGGTTG-3 '
BWp16-a1-F: 5'-CCCGAATTCCGCCACACAGCATCCTCTCA-3 '
(H) BWp16-a2
BWp16-a2-R: 5'-GGGGGATCCGAAGAAGCTTTGAAGAGGATTGAAGG-3 '
BWp16-a2-F: 5'-CCCGAATTCCGCCACACAGCATCCTCTCA-3 '
(I) BWp16-b1
BWp16-b1-R: 5'-GGGGGATCCGGATGCTGTGTGGCGATGAAG-3 '
BWp16-b1-F: 5'-CCCGAATTCCATCTTCATCCACTCACAAACAC-3 '
(J) BWp16-b2
BWp16-b2-R: 5'-GGGGGATCCGGATGCTGTGTGGCGATGAGG-3 '
BWp16-b2-F: 5'-CCCGAATTCCATCTTCATCCACTCACAAACAC-3 '
(K) BWp16-c
BWp16-cR: 5'-GGGGGATCCGAGTGGATGAAGATGATGGTTG-3 '
BWp16-cF: 5'-CCCGAATTCAATCAACCTTTCACCAATACGCCC-3 '
(L) BWp16-d1
BWp16-d1-R: 5'-GGGGGATCCGGTGAAAGGTTGATTAAGGAGGG-3 '
BWp16-d1-F: 5'-CCCGAATTCACTAAGCCCACACTTACTAGG-3 '
(M) BWp16-d2
BWp16-d2-R: 5'-GGGGGATCCGGTGAAAGGTTGATTAAGGAGGG-3 '
BWp16-d2-F: 5'-CCCGAATTCACTAAGCCCACACTTACCAGG-3 '
(N) BWp16-e1
BWp16-e1-R: 5'-GGGGGATCCAAGTGTGGGCTTAGTGAAATGG-3 '
BWp16-e1-F: 5'-CCCGAATTCCACAAAATACCGATTTCCTCTCG-3 '
(O) BWp16-e2
BWp16-e2-R: 5'-GGGGGATCCAAGTGTGGGCTTAGTGAATTGG-3 '
BWp16-e2-F: 5'-CCCGAATTCCACAAAATACCGATTTCCTCTCG-3 '

(2) Expression and Purification of Recombinant Recombinant BWp16 Using E. coli The above E. coli was precultured overnight at 37 ° C. in 2 × YT medium (Trypton, Yeast Extract, NaCl). Subsequently, the culture solution was diluted in fresh same medium and cultured at 37 ° C. for 30 minutes, and IPTG (Isopropyl β-D-1-thiogalactopylanoside) was added at a final concentration of 1 mM to induce protein expression at 16 ° C. Cultured for 16 hours. The cells were collected from the culture solution by centrifugation, frozen at −80 ° C., and sonicated to obtain a centrifugal supernatant. These recombinants were subjected to glutathione sepharose purification (SIGMA). The concentration of the purified split recombinant BWp16 was calculated by Bradford (Bio-Rad).

(3) Confirmation of recognition region by WB method On SDS-PAGE gel, 0.5 μg / 1 lane of each recombinant BWp16 was run, and the electrophoretic protein was transferred to a PVDF membrane. After blocking the membrane (5% skim milk-TBST), the prepared monoclonal antibodies (clone Nos. 1, 2, 6, 7, 8, and 10) were reacted. After the membrane was washed, HRP-labeled anti-mouse IgG antibody was reacted as a secondary antibody. The membrane was washed, reacted with ECL Western Blotting Detection Reagents, and chemiluminescence was detected with an X-ray film.
The results of WB are shown in FIGS. 6A and 6B.
As shown in FIG. 1, 2, 6, 7, 8 and 10 all showed the same level of reaction to BWp16-1, BWp16-2 and BWp16-3. This result indicates that these clones can recognize epitopes regardless of their three-dimensional structure, and the same is true for buckwheat allergens in unheated raw materials and foods and buckwheat allergens in heat-denatured foods. It was suggested that it could be detected.

Table 5 summarizes the recombinant BWp16 of each division recognized by each monoclonal antibody clone.
(Displays as + when antibody is bound, and as-when not reacting)
FIG. 7 illustrates the specific region of BWp16 recognized by each monoclonal antibody clone by combining these results with the divided expression map of recombinant BWp16.
The epitope of BWp16 recognized by each monoclonal antibody clone was shown to be within the specific region shown in Table 6 below.

Each monoclonal antibody clone reacted with various buckwheat having mutations at 11 sites (9th, 10th, 34th, 50th, 62th, 66th, 70th, 72th, 92th, 110th and 131st from the N-terminal side of Flag 2.0101). In addition, it reacted with any of the recombinant BWp16 proteins including the 6 mutations shown in Table 2 (66th, 70th, 72th, 92th, 131st and 138th from the N-terminal side of Flag 2.010). .
Therefore, the monoclonal antibody of the present invention is an antibody that recognizes specific regions 66aa-85aa containing mutations in 66aa, 70aa and 72aa, specific regions 91aa-100aa containing mutations in 92aa, and specific regions 106aa-114aa containing two mutations in 110aa. It was shown that.

Claims (10)

SEQ ID NO: 12,
A monoclonal antibody having the peptide represented by SEQ ID NO: 13 or SEQ ID NO: 14 as an antigen,
Recognizing the amino acid sequence contained in SEQ ID NO: 30 as an epitope;
A monoclonal antibody characterized by binding to buckwheat BWp16.   The monoclonal antibody according to claim 1, wherein the amino acid sequence contained in SEQ ID NO: 31 is recognized as an epitope.   The monoclonal antibody according to claim 2, wherein the amino acid sequence contained in SEQ ID NO: 32 is recognized as an epitope.   The monoclonal antibody according to claim 3, wherein the amino acid sequence contained in SEQ ID NO: 33 is recognized as an epitope. SEQ ID NO: 1: Xaa1 Glu Ala Leu Xaa2 Arg Xaa3 Glu Gly Glu Gly Cys Lys Ser Glu Glu Ser Cys Met Arg
SEQ ID NO: 2: Met Xaa4 Glu Met Asp Asp Glu Cys Val Cys or SEQ ID NO: 3: Met Val Glu Asn Xaa5 Lys Gly Arg Ile
(In the sequence, Xaa1 to Xaa5 are any naturally occurring amino acids.)
The monoclonal antibody according to claim 4, wherein the amino acid sequence contained in is recognized as an epitope. SEQ ID NO: 1: Xaa1 Glu Ala Leu Xaa2 Arg Xaa3 Glu Gly Glu Gly Cys Lys Ser Glu Glu Ser Cys Met Arg
SEQ ID NO: 2: Met Xaa4 Glu Met Asp Asp Glu Cys Val Cys or SEQ ID NO: 3: Met Val Glu Asn Xaa5 Lys Gly Arg Ile
(In the sequence, Xaa1 is Ala or Glu, Xaa2 is Ser or Lys, Xaa3 is Val or Ile, Xaa4 is Lys or Arg, and Xaa5 is Gln.)
The monoclonal antibody according to claim 5, wherein the amino acid sequence contained in is recognized as an epitope.   The monoclonal antibody according to any one of claims 1 to 6, which does not cross-react with protein derived from seed plants other than buckwheat.   Hybridoma identified by receipt number NITE AP-01791, hybridoma identified by receipt number NITE AP-01792, hybridoma identified by accession number NITE AP-01793, hybridoma identified by receipt number NITE AP-01794, accession number The monoclonal antibody according to any one of claims 1 to 7, which is an antibody produced by a hybridoma selected from the group consisting of hybridomas specified by NITE AP-01795.   A method for detecting buckwheat in a sample using the monoclonal antibody according to any one of claims 1 to 8.   A buckwheat detection kit comprising the monoclonal antibody according to any one of claims 1 to 8.