Antibodies, kits and testing methods for testing peanut allergens

Abstract

To provide improved means for detecting peanut allergens remaining in food or food processing equipment.SOLUTION: Provided is a test kit for quantitatively or qualitatively detecting peanut allergens remaining in food or food processing equipment by immunological assay, the test kit comprising: an anti-Ara h3 antibody or an antigen-binding fragment thereof (anti-Ara h3 antibody, and the like) having heavy-chain CDR1 to 3 and light-chain CDR1 to 3 consisting of a predetermined amino acid sequence, or an anti-Ara h3 antibody or an antigen-binding fragment thereof (anti-Ara h3 competitive antibody, and the like) that competes with the anti-Ara h3 antibody, and the like for the same or overlap epitope in binding to the Ara h3 protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to techniques for detecting peanut allergens, particularly Ara h3 protein, contained in food or remaining in food processing equipment. [Background technology]

[0002] Food allergies are a condition in which the body recognizes certain proteins (food allergens) contained in ingested food as foreign substances, and through hypersensitive immunological mechanisms, various symptoms such as skin itching, hives, and coughing are caused. In severe cases, it can lead to loss of consciousness, low blood pressure, and shock symptoms, which can be life-threatening. It is estimated that 1-2% of the total population (about 10% if limited to infants) suffer from food allergies. The Food Sanitation Act requires or recommends that if a food contains ingredients that contain food allergens, that information be displayed on the food to prevent food allergy patients from ingesting foods that contain the food allergen that is the cause of the allergy, especially processed foods that are difficult to identify by appearance. Currently, eight items, "eggs, milk, wheat, shrimp, crab, peanuts, walnuts" (those with a high incidence of onset) and "buckwheat" (which often causes severe symptoms and can be life-threatening), are designated as "specific ingredients" for which labeling is mandatory, while 21 items, "abalone, squid, salmon roe, orange, cashew nuts, kiwi fruit, beef, sesame, salmon, mackerel, soybeans, chicken, bananas, pork, matsutake mushrooms, peaches, yams, apples, gelatin, almonds" (those that have been reported to cause allergies with a certain frequency in the past), are designated as "substances equivalent to specific ingredients" for which labeling is recommended. The Consumer Affairs Agency (formerly the Ministry of Health, Labor and Welfare) conducts a survey every three years to grasp the nationwide situation of food allergies, and reviews the list of specific ingredients based on the results of the survey.

[0003] In order to operate the above-mentioned food allergen system, food manufacturers and public testing organizations use kits to check whether foods containing food allergens are included as raw materials in processed products or whether they remain in food processing equipment. Since allergic symptoms to peanuts tend to be severe, there is a demand for kits that can detect peanut allergens with even greater reliability.

[0004] Generally, the techniques for detecting specific raw materials contained in processed products or remaining in food processing equipment include the ELISA method, which uses an antigen-antibody reaction as a screening test, and the PCR method, which uses a DNA amplification reaction as a confirmatory test. Of these, the ELISA method uses an antibody that recognizes a protein (allergen) specific to peanuts to check whether peanuts are included as an ingredient in processed products.

[0005] The peanut-specific proteins targeted for detection based on antigen-antibody reactions include peanut allergens Ara h1, Ara h2, and Ara h3, which are the main causes of peanut allergy. Ara h1 is a 7s globulin family protein with a molecular weight of 63 kDa, which accounts for approximately 12-16% of the total protein in peanut extracts and causes sensitization in 35-95% of allergic patients. Ara h2 is a 2s globulin family protein with a molecular weight of 20 kDa, which not only causes systemic allergy, but also enhances trypsin inhibitor activity several times when treated at high temperatures, thereby inhibiting the digestion of Ara h1 and further enhancing allergic activity. Ara h3 is a 11s globulin family protein with a molecular weight of 58 kDa, which accounts for approximately 19% of the total protein in peanut extracts and causes systemic allergy. Other peanut allergens are also known, including Ara h4, Ara h5, Ara h6, and Ara h7.

[0006] As a means for detecting peanuts in food by antigen-antibody reaction targeting such peanut-specific proteins, for example, a detection method by ELISA or the like using an anti-Ara h1 protein monoclonal antibody that recognizes the peanut allergens native Ara h1 protein and heat-denatured Ara h1 protein, and a kit for this purpose have been proposed (Patent Document 1).

[0007] In addition, Bali Peanuts from Indonesia are a special variety of peanut that does not contain Ara h1, but still contain other peanut-derived allergens (Ara h2, Ara h3, etc.), and it has been reported that the incidence of allergies is no different from that of ordinary peanuts (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2009-271091 A (Patent No. 5043077) [Non-patent literature]

[0009] [Non-Patent Document 1] Peanut varieties with reduced Ara h 1 content indicating no reduced allergenicity, Mol. Nutr. Food Res. 2010, 54, 381-387 Summary of the Invention [Problem to be solved by the invention]

[0010] Detection kits or methods using monoclonal antibodies targeting Ara h1 protein have problems in that the detection sensitivity is poor when the concentration of Ara h1 protein in a food sample is low, and on the other hand, when the concentration of Ara h1 protein in a food sample is high, false negatives occur and detection is impossible. In addition, when a food is produced using Ara h1-deficient peanuts, peanut allergens cannot be detected by detection kits or methods using monoclonal antibodies targeting Ara h1 protein, which is a major problem for patients with allergies to Ara h2 and Ara h3 proteins. Furthermore, the content of Ara h1, the main allergen protein in peanuts, increases or decreases when heated, making it difficult to detect peanuts with Ara h1 test kits.

[0011] An object of the present invention is to provide an improved means for detecting peanut allergens in foods based on immunological assays. [Means for solving the problem]

[0012] The present inventors have unexpectedly found that, in a kit or method for detecting peanut allergens based on immunological assay, when Ara h3 protein is detected using an anti-Ara h3 antibody, particularly an anti-Ara h3 antibody having a specific CDR, the detection sensitivity is superior even when the allergen protein in a food sample is at a low concentration, and the allergen protein in a food sample can be detected without false negative even when the allergen protein is at a high concentration, and preferably, the reactivity to various food allergens other than peanut allergens is sufficiently suppressed, and food cross-reactivity is low, and thus the present invention has been completed. Such characteristics are observed when an anti-Ara h3 antibody having a specific CDR is used as at least one of the capture antibody for Ara h3 protein (antibody for solid-phase immobilization in ELISA, antibody for immobilization in immunochromatography) and the detection antibody for the captured Ara h3 protein (antibody for enzyme labeling in ELISA, antibody for color labeling in immunochromatography). In addition, when anti-Ara h3 antibodies are used, there is an advantage that peanut allergens contained in foods can be detected even if the foods are produced using Ara h1-deficient peanuts.

[0013] That is, the present invention includes at least the following: [1] A heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:1; A heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:2, and A heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:3, and A light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:4; A light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:5, and Light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:6 An anti-Ara h3 antibody or an antigen-binding fragment thereof (hereinafter referred to as "first antibody, etc."), A heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:7; A heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:8, and A heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:9, and A light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10; A light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and Light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:12 An anti-Ara h3 antibody or an antigen-binding fragment thereof (hereinafter referred to as "second antibody, etc."), [2] An anti-Ara h3 antibody or antigen-binding fragment thereof (hereinafter referred to as the "first competing antibody, etc.") that competes with the first antibody, etc. for the same or overlapping epitope in binding to Ara h3 protein, or an anti-Ara h3 antibody or antigen-binding fragment thereof (hereinafter referred to as the "second competing antibody, etc.") that competes with the second antibody, etc. for the same or overlapping epitope. [3] A test kit for quantitatively or qualitatively detecting peanut allergens remaining in food or food processing equipment by an immunological assay, comprising: A testing kit comprising at least one selected from the group consisting of the first antibody etc. and the second antibody etc. described in Item 1, and the first competing antibody etc. and the second competing antibody etc. described in Item 2. [4] Item 4. The test kit according to Item 3, wherein the immunological measurement method is ELISA or immunochromatography, and the kit comprises the first antibody or the first competing antibody or the second antibody or the second competing antibody, for enzyme labeling in ELISA or color labeling in immunochromatography. [5] Item 4. The testing kit according to Item 3, wherein the immunological measurement method is ELISA or immunochromatography, and the kit comprises the first antibody or the first competing antibody or the second antibody or the second competing antibody for solid-phase immobilization in ELISA or for immobilization in immunochromatography. [6] the immunoassay is ELISA or immunochromatography; (1) The second antibody or the second competing antibody, etc., is used for enzyme labeling in ELISA or color labeling in immunochromatography, and the second antibody or the second competing antibody, etc., is used for solid-phase immobilization in ELISA or immobilization in immunochromatography. (2) The first antibody or the first competing antibody, etc., are used for enzyme labeling in ELISA or color labeling in immunochromatography, and the second antibody or the second competing antibody, etc., are used for solid-phase immobilization in ELISA or immobilization in immunochromatography, or (3) The testing kit according to Item 3, comprising the first antibody, etc. or the first competing antibody, etc. for enzyme labeling in ELISA or color labeling in immunochromatography, and an anti-Ara h3 antibody or an antigen-binding fragment thereof other than the first antibody, etc., the second antibody, etc., the first competing antibody, etc., and the second competing antibody, etc. for solid-phase immobilization in ELISA or immobilization in immunochromatography. [7] Item 4. The testing kit according to Item 3, further comprising an extraction reagent. [8] Item 8. The test kit according to Item 7, wherein the extraction reagent is an extraction reagent using a shaking method or a high-speed shear / agitation method without heating. [9] (1) extracting peanut protein remaining in food or in food processing equipment; and (2) quantitatively or qualitatively detecting Ara h3 protein in the obtained extract by an immunological assay using at least one antibody selected from the group consisting of the first antibody or the like and the second antibody or the like described in Item 1 and the first competing antibody or the like and the second competing antibody or the like described in Item 2; A method for testing for peanut allergens in food, comprising:

[10] Item 9. The testing method according to Item 8, wherein the immunological measurement method is ELISA or immunochromatography, and the first antibody or the first competing antibody or the second antibody or the second competing antibody or the like is used for enzyme labeling in ELISA or color labeling in immunochromatography.

[11] Item 9. The testing method according to Item 8, wherein the immunological measurement method is ELISA or immunochromatography, and the first antibody or the first competing antibody or the second antibody or the second competing antibody or the like is used for solid-phase immobilization in ELISA or for immobilization in immunochromatography.

[12] the immunoassay is ELISA or immunochromatography; (1) Using the second antibody or the second competing antibody for enzyme labeling in ELISA or color labeling in immunochromatography, and the second antibody or the second competing antibody for solid-phase immobilization in ELISA or immobilization in immunochromatography, (2) Using the first antibody or the first competing antibody for enzyme labeling in ELISA or color labeling in immunochromatography, and the second antibody or the second competing antibody for solid-phase immobilization in ELISA or immobilization in immunochromatography, or (3) The testing method according to Item 9, wherein the first antibody, etc. or the first competing antibody, etc. is used for enzyme labeling in ELISA or color labeling in immunochromatography, and an anti-Ara h3 antibody or an antigen-binding fragment thereof other than the first antibody, etc., the second antibody, etc., the first competing antibody, etc., and the second competing antibody, etc. is used for solid-phase immobilization in ELISA or immobilization in immunochromatography.

[13] Item 10. The method according to item 9, wherein the extraction in step (1) is carried out using an extraction reagent.

[14] Item 14. The testing method according to Item 13, wherein the extraction using the extraction reagent is performed by a shaking method or a high-speed shear / agitation method without heating.

[0014] In addition, a person skilled in the art would be able to convert (reinterpret) the matters relating to the invention described in each of the above paragraphs into matters relating to inventions in other categories based on the technical idea of ​​the present invention, the matters described in this specification, and common technical knowledge.

[0015] For example, the invention described in [3] above can be converted into "use of at least one selected from the group consisting of the first antibody, etc. and the second antibody, etc. described in Item 1 and the first competing antibody, etc. and the second competing antibody, etc. described in Item 2, in the manufacture of a test kit for quantitatively or qualitatively detecting peanut allergens remaining in food or in food processing equipment by immunological assay."

[0016] Furthermore, for example, the invention described in [9] above can be converted into "use of a method for testing a peanut allergen in food, comprising at least one selected from the group consisting of the first antibody, etc. and the second antibody, etc. described in Item 1 and the first competing antibody, etc. and the second competing antibody, etc. described in Item 2, the method comprising: (1) extracting peanut protein remaining in food or in food processing equipment; and (2) quantitatively or qualitatively detecting Ara h3 protein in the obtained extract by an immunological assay using at least one selected from the group consisting of the first antibody, etc., the second antibody, etc., the first competing antibody, etc., and the second competing antibody, etc." Effect of the Invention

[0017] The test kit and test method of the present invention make it possible to detect peanut allergens with higher reliability than ever before, whether the peanut allergen is present in a food at a low or high concentration, whether the food is heated, and, in a preferred embodiment, even in the presence of other food allergens. The antibody of the present invention is suitable for carrying out such a test kit and test method. [Brief description of the drawings]

[0018] [Figure 1] Figure 1 shows the results of Western blotting for SDS-PAGE described in Test Example 1. [Left] Western blotting for Ara h1 protein using clone "1-5" (anti-Ara h3 antibody) and a control antibody (anti-Ara h1 antibody). [Right] Western blotting for Ara h3 protein using clone "1-5" and a control antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] In this specification, the anti-Ara h3 antibody in the first embodiment (clone "2-106" in the Examples) is referred to as the "first antibody", its antigen-binding fragment is referred to as the "first fragment", and they are collectively referred to as the "first antibody, etc."; the anti-Ara h3 antibody in the second embodiment (clone "1-5" in the Examples) is referred to as the "second antibody", its antigen-binding fragment is referred to as the "second fragment", and they are collectively referred to as the "second antibody, etc."

[0020] In this specification, an anti-Ara h3 antibody that competes with a first antibody, etc. for the same or overlapping epitope is referred to as a "first competing antibody," its antigen-binding fragment is referred to as a "first competing fragment," and they are collectively referred to as a "first competing antibody, etc."; an anti-Ara h3 antibody that competes with a second antibody, etc. for the same or overlapping epitope is referred to as a "second competing antibody," its antigen-binding fragment is referred to as a "second competing fragment," and they are collectively referred to as a "second competing antibody, etc."

[0021] In this specification, the "first antibody," "second antibody," "first competing antibody," and "second competing antibody" are collectively referred to as "anti-Ara h3 antibodies of the present invention," the "first fragment," "second fragment," "first competing fragment," and "second competing fragment" are collectively referred to as "anti-Ara h3-binding fragments of the present invention," and the "first antibody, etc.," "second antibody, etc.," "first competing antibody, etc.," and "second competing antibody, etc." are collectively referred to as "anti-Ara h3 antibodies of the present invention, etc.."

[0022] In this specification, anti-Ara h3 antibodies other than the anti-Ara h3 antibodies of the present invention (e.g., clones "2-21" or "1-3" in the Examples) are referred to as "other anti-Ara h3 antibodies", and their antigen-binding fragments are referred to as "other anti-Ara h3 binding fragments", and they may be collectively referred to as "other anti-Ara h3 antibodies, etc."

[0023] In this specification, when the term "antibody" is used in an appropriate context, the term can be appropriately replaced with "antigen-binding fragment" or "antibody, etc." as a general term for "antibody" and "antigen-binding fragment," i.e., an embodiment using an "antibody" can be replaced with an embodiment using an "antigen-binding fragment" or "antibody, etc."

[0024] -antibody- In the first embodiment of the present invention, the anti-Ara h3 antibody or antigen-binding fragment thereof (such as the first antibody) has a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3, as well as a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6.

[0025] In a second embodiment of the present invention, an anti-Ara h3 antibody or an antigen-binding fragment thereof (e.g., second antibody) has a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 9, as well as a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 12.

[0026] The anti-Ara h3 antibody of the present invention has heavy chain CDR1-3 and light chain CDR1-3 consisting of the amino acid sequences represented by the specific sequence numbers described herein as three complementarity determining regions (CDRs) in the heavy chain and light chain, respectively. The antibody can be prepared in the same manner as a general antibody, etc., by combining with appropriate framework regions (FR), i.e., heavy chain FR1-4 located before and after the heavy chain CDR1-3 and light chain FR1-4 located before and after the light chain CDR1-3, and grafting the heavy and light chains in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus to construct a variable region, and further combining with the first to third constant regions of the heavy chain and the light chain constant region to construct the full-length heavy and light chains.

[0027] The anti-Ara h3 antibody of the present invention is capable of inhibiting IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG 2a , IgG 2b Although it may be any class (subclass) of IgM (IgG1, IgG2, IgG3, IgG4), it is typically IgG (IgG1, IgG 2a , IgG 2b Each class (subclass) of antibodies can be constructed with corresponding heavy and light chains, for example, IgG1, IgG2, and IgG4. 2a , IgG 2b The IgG, IgG3, and IgG4 subclasses can be constructed from γ1, γ2, γ3, γ4, or γ5 heavy chains and λ or κ light chains, respectively.

[0028] The anti-Ara h3 binding fragment of the present invention can be prepared by using heavy chain CDR1-3 and light chain CDR1-3 consisting of amino acid sequences represented by specific SEQ ID NOs described herein, and the variable and constant regions of the heavy and light chains containing them. Examples of "antigen-binding fragments" include Fab, Fab', F(ab')2, Fv (a polypeptide in which a single heavy chain variable region and a single light chain variable region are bound), scFv (single-chain Fv), and dsFv (double-chain Fv).

[0029] In one embodiment of the present invention, the first antibody etc. comprises a heavy chain variable region (CDR and FR) consisting of an amino acid sequence having at least 80%, for example, 85% or more, 90% or more, or 95% or more sequence identity (homology) to the amino acid sequence shown in SEQ ID NO:13.

[0030] In one embodiment of the present invention, the first antibody etc. comprises a light chain variable region (CDR and FR) consisting of an amino acid sequence having at least 80%, for example, 85% or more, 90% or more, or 95% or more sequence identity (homology) to the amino acid sequence represented by SEQ ID NO:15.

[0031] In one embodiment of the present invention, the second antibody etc. comprises a heavy chain variable region (CDR and FR) consisting of an amino acid sequence having at least 80%, for example, 85% or more, 90% or more, or 95% or more sequence identity (homology) to the amino acid sequence represented by SEQ ID NO:17.

[0032] In one embodiment of the present invention, the second antibody etc. comprises a light chain variable region (CDR and FR) consisting of an amino acid sequence having at least 80%, for example, 85% or more, 90% or more, or 95% or more sequence identity (homology) to the amino acid sequence represented by SEQ ID NO:19.

[0033] In the heavy chain variable region and / or light chain variable region of the first antibody etc. or second antibody etc. having sequence identity within the above-mentioned range, the difference in amino acid residues (substitution, deletion, insertion, addition, etc.) occurs in the FR other than the CDR, and it is preferable that the amino acid sequence of the CDR is maintained. On the other hand, the first competing antibody etc. or second competing antibody etc. provided in one aspect of the present invention may comprise a heavy chain variable region and / or a light chain variable region consisting of an amino acid sequence having sequence identity (homology) within the above-mentioned range to a variable region (CDR and FR) contained in a heavy chain or light chain consisting of an amino acid sequence represented by a specific SEQ ID NO, and the difference in amino acid residues (substitution, deletion, insertion, addition, etc.) may occur in one or both of the CDR and the FR.

[0034] The CDRs and FRs in the antibodies and the like of the present invention are sites identified by general methods in the technical field, for example, numbering methods such as Kabat, Chothia, IMGT, Aho, etc. In one embodiment of the present invention, the CDRs and FRs are sites identified by the CHOTHIA ANTIBODY NUMBERING method.

[0035] The sequence identity (homology) values ​​in this specification are values ​​determined by well-known and commonly used methods for so-called alignment in the present technical field, and can be calculated using publicly available tools such as BLASTN, BLASTp (see the US National Center for Biotechnology Information (NCBI) website), ClustalW2 (see the European Bioinformatics Institute website), ALIGN, etc., with default parameters, or with parameters customized by selecting an appropriate algorithm as necessary. In one embodiment of the present invention, the sequence identity (homology) values ​​are values ​​calculated with default parameters using BLASTN.

[0036] The anti-Ara h3 antibody etc. of the present invention can be produced by utilizing a polynucleotide encoding the amino acid sequence of the desired anti-Ara h3 antibody etc. according to a general method. A typical production method is outlined as follows. A DNA encoding the desired polynucleotide is synthesized and cloned into an expression vector. The expression vector is transfected into an appropriate host cell, and the host cell is cultured under appropriate conditions to cause the desired anti-Ara h3 antibody etc. to be secreted into the culture supernatant. The culture supernatant is collected, and the anti-Ara h3 antibody etc. contained therein is isolated and purified.

[0037] In a further aspect of the first embodiment of the present invention, the anti-Ara h3 antibody or antigen-binding fragment thereof (first competing antibody, etc.) is an anti-Ara h3 antibody or antigen-binding fragment thereof that competes for the same or overlapping epitope as the first antibody, etc. in binding to Ara h3 protein. The amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of the first competing antibody, etc., and further the amino acid sequences of the heavy chain FR1-4 and light chain FR1-4 as necessary, may be amino acid sequences obtained by modifying the amino acid sequence of the first antibody, etc. of the present invention, or may be amino acid sequences obtained without relying on the amino acid sequence of the first antibody, etc. of the present invention.

[0038] In a further aspect of the second embodiment of the present invention, the anti-Ara h3 antibody or antigen-binding fragment thereof (second competing antibody, etc.) is an anti-Ara h3 antibody or antigen-binding fragment thereof that competes for the same or overlapping epitope as the second antibody, etc. in binding to Ara h3 protein. The amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of the second competing antibody, etc., and further the amino acid sequences of the heavy chain FR1-4 and light chain FR1-4 as necessary, may be amino acid sequences obtained by modifying the amino acid sequence of the second antibody, etc. of the present invention, or may be amino acid sequences obtained without relying on the amino acid sequence of the second antibody, etc. of the present invention.

[0039] The first competing antibody etc. or the second competing antibody etc. that competes with the first antibody etc. or the second antibody etc. for the same or overlapping epitope may be any antibody capable of inhibiting the binding (interaction) between the first antibody etc. or the second antibody etc. and the Ara h3 protein to any detectable extent, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. Such a first competing antibody etc. or second competing antibody etc. can be selected from candidate clones without excessive trial and error by a general method known to those skilled in the art as so-called "epitope binning", in which the first antibody etc. or the second antibody etc. is used as a reference antibody etc. The binding (interaction) between two specific molecules is generally determined by the binding affinity (K D), i.e., the ratio of the dissociation rate to the association rate (Koff / Kon). D can be measured using, for example, a measuring device "Biacore" that utilizes surface plasmon resonance. The first competitive antibody etc. or the second competitive antibody etc. preferably has properties that can be evaluated as being equivalent to or superior to the first antibody etc. or the second antibody etc. in the application of detecting peanut allergens in food.

[0040] -Testing kit- The "test kit" of the present invention is a test kit for quantitatively or qualitatively detecting peanut allergens remaining in food or food processing equipment by immunological measurement, and comprises at least one anti-Ara h3 antibody, etc. of the present invention, i.e., at least one selected from the group consisting of the first antibody, etc., the second antibody, etc., the first competing antibody, etc. and the second competing antibody, etc. of the present invention.

[0041] The "immunological assay" is not particularly limited as long as it is a method that enables quantitative or qualitative detection of Ara h3 protein by utilizing the reaction between Ara h3 protein and anti-Ara h3 antibody, etc., and various known methods can be used. In addition, test kits corresponding to various immunological assay methods are known and commercially available, and the test kit of the present invention can be manufactured in accordance with known test kits, except for changing to use the specific antibody of the present invention.

[0042] In one embodiment of the present invention, the immunological measurement method is ELISA (Enzyme-Linked Immunosorbent Assay). ELISA is capable of quantitatively detecting various proteins with high accuracy, and is a suitable measurement method for detecting food allergens from processed foods that have been heated or otherwise processed, and complies with the Consumer Affairs Agency guidelines.

[0043] Anti-Ara h3 antibodies etc. for ELISA include, for example, anti-Ara h3 antibodies etc. for immobilization and anti-Ara h3 antibodies etc. for enzyme labeling. Anti-Ara h3 antibodies etc. for immobilization are intended to be immobilized on the surface of a member for performing ELISA, for example, the bottom of a plate (well). Anti-Ara h3 antibodies etc. for enzyme labeling are intended to bind to the Ara h3 protein captured by the immobilized antibody and then react with a substrate to develop a color with an intensity according to the amount of Ara h3 protein. The anti-Ara h3 antibody, etc. for enzyme labeling may be an anti-Ara h3 antibody, etc. directly labeled with an enzyme, i.e., an anti-Ara h3 antibody, etc. to which an enzyme (e.g., horseradish peroxidase (HRP)) has already been covalently bound to the antibody, etc. itself (e.g., via a linker); however, it may also be an anti-Ara h3 antibody, etc. that is indirectly labeled with an enzyme, such as an anti-Ara h3 antibody, etc. bound to biotin, which can be further reacted with an enzyme bound to streptavidin to complete an enzyme-labeled anti-Ara h3 antibody, etc., as exemplified in the procedure described below in relation to the "test method."

[0044] In one embodiment of the present invention, the immunological measurement method is immunochromatography, which is a measurement method capable of qualitatively detecting various proteins in a relatively short period of time with a simple operation.

[0045] Anti-Ara h3 antibodies etc. for immunochromatography include, for example, anti-Ara h3 antibodies etc. for immobilization and anti-Ara h3 antibodies etc. for color-producing labeling (e.g., gold colloid labeling, latex particle labeling, platinum particle labeling). Anti-Ara h3 antibodies etc. for color-producing labeling are generally anti-Ara h3 antibodies etc. that are bound to color-producing labels in advance and are included in a predetermined position (sample dropping portion) of a test strip, and are antibodies that react with a substrate to produce color to indicate the presence of a target protein after being captured by an immobilized antibody etc. in a state of forming a complex with Ara h3 protein. Anti-Ara h3 antibodies etc. for immobilization are generally anti-Ara h3 antibodies etc. that are included in a predetermined position (test line) of a test strip, and are antibodies for capturing a complex between Ara h3 protein that has moved by capillary action and anti-Ara h3 antibodies etc. for color-producing labeling.

[0046] Such anti-Ara h3 antibodies for immunological assays, such as for ELISA or immunochromatography, may be a combination of the anti-Ara h3 antibodies of the present invention, or may be a combination of the anti-Ara h3 antibodies of the present invention and other anti-Ara h3 antibodies. The anti-Ara h3 antibodies may be used in combination with an antibody or an antigen-binding fragment thereof that can recognize and bind to the Ara h3 protein with the specificity required for the purpose of use, i.e., the purpose of the test kit. It is preferable that the anti-Ara h3 antibodies do not bind to various proteins contained in foods and food allergens other than peanuts (at least, the binding is limited to a degree that does not affect the test results according to the present invention), but it is acceptable for the antibodies to bind to a certain degree (cross-react) with proteins other than the Ara h3 protein or to non-specifically adsorb to proteins or other substances that are unavoidable due to the nature of the antibody, depending on the purpose and level.

[0047] Here, the Ara h3 protein in foods such as processed foods may contain variously denatured Ara h3 proteins, for example, aggregates of native Ara h3 proteins and variously denatured Ara h3 proteins, depending on the food manufacturing process and storage process (e.g., the presence or absence and processing conditions of kneading, molding, pressurization, heating, drying, enzyme treatment, freezing, thawing, etc.) and the extraction process from the food (specimen) (e.g., the presence or absence and processing conditions of extraction treatment using an extraction reagent containing a solubilizing agent). Therefore, it is preferable that the anti-Ara h3 antibody, etc. is an embodiment that can bind to various Ara h3 proteins so as to have a certain detection sensitivity for the aggregate of various Ara h3 proteins in foods (preventing the detection sensitivity from decreasing due to foods).

[0048] From such a viewpoint, in one embodiment of the present invention, the test kit can include an anti-Ara h3 antibody or the like that can specifically bind to both native Ara h3 protein and denatured Ara h3 protein (e.g., heat-denatured Ara h3 protein). In such applications, the anti-Ara h3 antibody or the like of the present invention has excellent detection sensitivity for both native Ara h3 protein and Ara h3 protein that has been denatured in various ways by heating or the like. It is preferable that other anti-Ara h3 antibodies or the like used in combination with the anti-Ara h3 antibody or the like of the present invention can also specifically bind to both native Ara h3 protein and denatured Ara h3 protein.

[0049] In a first aspect of the present invention, the test kit comprises a first antibody, etc. or a first competing antibody, etc., or a second antibody, etc. or a second competing antibody, etc., as an anti-Ara h3 antibody, etc. for enzyme labeling in ELISA or an anti-Ara h3 antibody, etc. for color labeling in immunochromatography.

[0050] In a second aspect of the present invention, the test kit comprises a first antibody, etc. or a first competing antibody, etc., or a second antibody, etc. or a second competing antibody, etc., as an anti-Ara h3 antibody, etc. for solid-phase ELISA or an anti-Ara h3 antibody, etc. for solid-phase immunochromatography.

[0051] The first and second aspects of the present invention can be combined so that the test kit comprises a first antibody, etc., or a first competing antibody, etc., or a second antibody, etc., or a second competing antibody, etc., as an anti-Ara h3 antibody, etc. for enzyme labeling in ELISA or an anti-Ara h3 antibody, etc. for color-labeling in immunochromatography, and comprises a first antibody, etc., or a first competing antibody, etc., or a second antibody, etc., or a second competing antibody, etc., as an anti-Ara h3 antibody, etc. for solid-phase ELISA or an anti-Ara h3 antibody, etc. for solid-phase immunochromatography. On the other hand, in the first aspect of the present invention, other anti-Ara h3 antibodies, etc. (e.g., clones "2-21" or "1-3" in the Examples) may be included as anti-Ara h3 antibodies, etc. for solid-phase ELISA or anti-Ara h3 antibodies, etc. for solid-phase immunochromatography, and in the second aspect of the present invention, other anti-Ara h3 antibodies, etc. may be included as anti-Ara h3 antibodies, etc. for enzyme labeling in ELISA or anti-Ara h3 antibodies, etc. for color labeling in immunochromatography.

[0052] In one preferred embodiment of the present invention (corresponding to Example 2 described below), the test kit contains a first antibody, etc. or a first competing antibody, etc. as an anti-Ara h3 antibody, etc. for enzyme labeling in ELISA or an anti-Ara h3 antibody, etc. for color labeling in immunochromatography, and contains a second antibody, etc. or a second competing antibody, etc. as an anti-Ara h3 antibody, etc. for solid-phase immobilization in ELISA or an anti-Ara h3 antibody, etc. for immobilization in immunochromatography.

[0053] In one preferred embodiment of the present invention (corresponding to Example 1 described below), the test kit contains a second antibody, etc. or a second competing antibody, etc. as an anti-Ara h3 antibody, etc. for enzyme labeling in ELISA or an anti-Ara h3 antibody, etc. for color labeling in immunochromatography, and also contains a second antibody, etc. or a second competing antibody, etc. as an anti-Ara h3 antibody, etc. for solid-phase immobilization in ELISA or an anti-Ara h3 antibody, etc. for immobilization in immunochromatography.

[0054] In one preferred embodiment of the present invention (corresponding to Example 3 described below), the test kit contains a first antibody, etc. or a first competing antibody, etc. as an anti-Ara h3 antibody, etc. for enzyme labeling in ELISA or an anti-Ara h3 antibody, etc. for color labeling in immunochromatography, and contains another anti-Ara h3 antibody, etc. (for example, clone "2-21" in the Examples) as an anti-Ara h3 antibody, etc. for solid-phase immobilization in ELISA or an anti-Ara h3 antibody, etc. for immobilization in immunochromatography.

[0055] As for other anti-Ara h3 antibodies, depending on the application and embodiment of the test kit, any one of monoclonal antibodies or antigen-binding fragments thereof (referred to as "monoclonal antibodies, etc." in this specification) may be used, or two or more types of monoclonal antibodies, etc. may be used in combination, or polyclonal antibodies or antigen-binding fragments thereof (referred to as "polyclonal antibodies, etc." in this specification) may be used. Monoclonal antibodies, etc. and polyclonal antibodies, etc. differ in specificity (whether they react only with Ara h3 protein and do not react (cross) with other proteins), reproducibility (whether there are differences in test results depending on the manufacturing lot), stability (whether the binding to the antigen is lost due to the solid-phase treatment, labeling treatment, etc. of the antibody in the kit), etc., so that the preferred antibody may vary depending on the application and embodiment of the test kit, and therefore, taking this into consideration, it is possible to select whether to use monoclonal antibodies, etc. or polyclonal antibodies, etc., or to use both in combination.

[0056] In one embodiment of the present invention, the test kit includes polyclonal antibodies as other anti-Ara h3 antibodies. In a further embodiment of the present invention, the detection kit includes both polyclonal antibodies and monoclonal antibodies as anti-Ara h3 antibodies. For example, in an immunological measurement method of an embodiment described below, a monoclonal antibody can be used as a capture antibody for Ara h3 protein (antibody for solid-phase immobilization in ELISA, antibody for immobilization in immunochromatography), and a polyclonal antibody can be used as a detection antibody for the captured Ara h3 protein (antibody for enzyme labeling in ELISA, antibody for color labeling in immunochromatography). By using a monoclonal antibody as a capture antibody, the specificity for Ara h3 protein can be increased, and by using a polyclonal antibody as a detection antibody, the captured Ara h3 protein can be detected with high sensitivity.

[0057] The anti-Ara h3 antibodies, etc. used in the present invention, particularly anti-Ara h3 antibodies, etc. other than the anti-Ara h3 antibodies, etc. of the present invention in which the amino acid sequence of the CDR is specified, can be produced as polyclonal antibodies, etc. or monoclonal antibodies, etc. in accordance with conventional methods, like antibodies against other food allergens, generally using a food allergen (in the present invention, Ara h3 protein) that has been extracted from food and usually further purified.

[0058] Polyclonal antibodies are generally produced by a method using immunized animals or a phage display method. The procedure of the method using immunized animals is as follows: An appropriate amount of purified target protein (Ara h3 protein in the present invention) is mixed with an adjuvant as necessary to prepare an immunogen, and the immunogen is injected (immunized) into an animal (rabbit, guinea pig, goat, sheep, rat, mouse, chicken, etc.), thereby producing antibodies in the blood of the animal. After repeated immunization at appropriate intervals and times, blood (plasma, serum) is collected and the antibodies contained therein are purified, for example, by affinity chromatography using an affinity column on which the target protein (Ara h3 protein in the present invention) is immobilized, and further purified by gel filtration chromatography as necessary, to obtain polyclonal antibodies. The procedure of the phage display method is as follows: An antibody gene is introduced into a bacteriophage, and a protein in which the variable regions of the H chain and L chain are linked is expressed (presented) on the coat protein of the bacteriophage. The resulting antibody phage library is used to select antibodies that have affinity for the target protein (Ara h3 protein in this invention). The bacteriophage that produces the antibody is infected into E. coli, grown, and then harvested. The antibody contained therein is purified to obtain a polyclonal antibody.

[0059] A method using a hybridoma is generally used as a method for producing a monoclonal antibody. The outline of the method using a hybridoma is as follows. As in the case of producing a polyclonal antibody, a purified product of a target protein (Ara h3 protein in the present invention) is injected into an animal as an immunogen multiple times to produce an antibody. Alternatively, according to the "DNA immunization method", an expression vector (plasmid) containing a gene of a target protein is injected into an animal multiple times to express the target protein in the body, thereby producing an antibody. B cells are collected from the spleen of the immunized animal and fused with myeloma cells (immortalized cancer cells) to produce a hybridoma (fused cell). Among the hybridomas, those that produce an antibody with excellent binding affinity and specificity to the target protein (Ara h3 protein in the present invention) are selected (screened). The hybridoma is cultured to produce a single antibody in the culture supernatant. The culture supernatant is collected and the antibody contained therein is purified to obtain a monoclonal antibody. The first and second antibodies of the present invention are also antibodies obtained by the method using hybridomas as described above.

[0060] After obtaining an anti-Ara h3 antibody (polyclonal or monoclonal), its amino acid sequence can be determined, or various antigen-binding fragments (polyclonal or monoclonal) can be produced according to a conventional method. The amino acid sequences of the first and second antibodies of the present invention, particularly the amino acid sequences of the heavy chain CDR1-3 and the light chain CDR1-3, respectively, were also identified by isolating total RNA from a hybridoma, cloning the cDNA, and performing DNA sequencing according to a conventional method.

[0061] The anti-Ara h3 antibody, etc., can be in a form suitable for quantitatively or qualitatively detecting the Ara h3 protein by immunological measurement, for example, can be modified or altered in various ways. If necessary, the anti-Ara h3 antibody, etc., can be humanized according to a general method. The amino acid sequence of a suitable template human antibody for humanization (particularly the framework regions of the heavy chain variable region and the light chain variable region) can be obtained by using a suitable database, etc.

[0062] The test kit of the present invention may contain, as necessary or to the extent that the effect of the present invention is not inhibited, antibodies, etc. other than anti-Ara h3 antibodies, etc. For example, the test kit of the present invention may further contain, as the above-mentioned capture antibodies, etc. and detection antibodies, etc., antibodies, etc. targeting proteins other than Ara h3 (e.g., peanut allergens other than Ara h3, or food allergens other than peanut allergens).

[0063] The test kit of the present invention includes at least anti-Ara h3 antibody, etc., but may include any content such as an extraction reagent, other reagents, and components, etc., as necessary depending on the embodiment. In the case of an ELISA kit, for example, a plate with wells, a diluent for preparing a solution of a target protein, a washing solution for washing the plate (well) after each step, an enzyme reaction stopping solution in ELISA, BSA for protecting the extracted protein from degradation, an instruction manual describing the procedure of the test method by ELISA, etc., can be included in the contents of the kit together with anti-Ara h3 antibodies for immobilization and enzyme labeling, etc., and an extraction reagent. In the case of an immunochromatography kit, for example, a test strip that can develop various solutions and reagents by capillary action, a diluent for preparing a solution of a target protein, an instruction manual describing the procedure of the test method by immunochromatography, etc., can be included in the contents of the kit together with anti-Ara h3 antibodies for immobilization and color labeling, etc., and an extraction reagent.

[0064] "Extraction reagent" refers to a reagent (solution) that is generally used to extract various proteins in food, such as food allergens. Various types of extraction reagents are known, and similar extraction reagents can be used in the present invention.

[0065] The extraction reagent is generally a solution prepared by adding a "solubilizing agent" to a buffer solution having an appropriate pH as an agent for extracting various proteins in food.

[0066] Examples of buffer solutions for preparing the extraction reagent include Tris buffer, phosphate buffer, citrate buffer, EDTA buffer, HEPES buffer, and acetate buffer. Examples of buffer solutions in the present invention are preferably Tris buffer and phosphate buffer. The pH of the buffer solution is generally 4.5 to 8.0 (a pH range that can occur in vivo), for example, 6.0 to 8.0. Those skilled in the art can prepare a buffer solution of a desired concentration and a desired pH by using an appropriate amount of a suitable compound (adding and dissolving each of a plurality of compounds in water in an appropriate amount).

[0067] Examples of solubilizing agents for preparing the extraction reagent include surfactants, chaotropic agents, and reducing agents. The extraction reagent may contain any one of these agents alone or a combination of multiple agents.

[0068] Examples of surfactants include anionic surfactants, cationic surfactants, and nonionic surfactants. Examples of anionic surfactants include alkyl sulfates and alkylbenzene sulfonates. The "alkyl" in alkyl sulfates, alkylbenzene sulfonates, and the like is, for example, dodecyl, decyl, nonyl, and octyl. The "salt" in alkyl sulfates, alkylbenzene sulfonates, and the like is, for example, sodium salt, potassium salt, and ammonium salt. A specific example of an alkyl sulfate is sodium dodecyl sulfate (SDS), and a specific example of an alkylbenzene sulfonate is sodium dodecylbenzene sulfonate. Specific examples of cationic surfactants include hexadecylpyridinium chloride, hexadecyltrimethylammonium bromide, and hexadecyltrimethylammonium chloride. Specific examples of nonionic surfactants include "Tween (registered trademark) 20" (polyoxyethylene sorbitan monolaurate), "Tween (registered trademark) 40" (polyoxyethylene sorbitan monopalmitate), "Tween (registered trademark) 60" (polyoxyethylene sorbitan monostearate), and "Tween (registered trademark) 80" (polyoxyethylene sorbitan monooleate).

[0069] When the extraction reagent contains a surfactant, the concentration of the surfactant can be appropriately adjusted taking into consideration the type of surfactant and its action and effect, and the presence, type and concentration of other components (e.g., chaotropic agents, reducing agents) in the extraction reagent as necessary. For example, when the extraction reagent contains an anionic surfactant such as SDS or other surfactant as a surfactant, the lower limit of the concentration can be, for example, 0.005, 0.01 or 0.05% (w / v), and the upper limit can be, for example, 5.0, 3.0 or 2.0% (w / v), and these upper and lower limits can be combined in any way.

[0070] Examples of "chaotropic agents" include urea, formamide, and salts containing anions or cations with a salting-in effect (e.g., guanidine hydrochloride containing a guanidinium ion, sodium chloride containing a sodium ion, potassium chloride containing a potassium ion, etc.).

[0071] When the extraction reagent contains a chaotropic agent, the concentration of the chaotropic agent can be appropriately adjusted taking into consideration the type of chaotropic agent and its action and effect (influence on the action and effect of the present invention), and, if necessary, the presence, type and concentration of other components (e.g., surfactant, reducing agent) in the extraction reagent. For example, when the extraction reagent contains urea as a chaotropic agent, the lower limit of its concentration can be, for example, 0.01, 0.05 or 0.1 (w / v), and the upper limit can be, for example, 10, 5.0 or 3.0% (w / v), and these upper and lower limits can be combined arbitrarily.

[0072] The reducing agent may, for example, be a mercaptoalkanol and / or a sulfite. The mercaptoalkanol may, for example, be thioglycerol, 2-mercaptoethanol, or dithiothreitol. The sulfite may, for example, be an alkali metal sulfite, and the alkali metal may, for example, be sodium or potassium. A specific example of the sulfite may be sodium sulfite.

[0073] When the extraction reagent contains a reducing agent, the concentration of the reducing agent can be appropriately adjusted taking into consideration the type of reducing agent and its action effect, and the presence, type and concentration of other components (e.g., surfactant, chaotropic agent) in the extraction reagent as necessary. When the extraction reagent contains thioglycerol as a reducing agent, the lower limit of its concentration can be, for example, 0.001, 0.005, 0.01 or 0.02% (w / v), and the upper limit can be, for example, 10, 5.0, 3.0, 2.0 or 1.0% (w / v), and these upper and lower limits can be combined arbitrarily. When the extraction reagent contains 2-mercaptoethanol or dithiothreitol as a reducing agent, the lower limit of its concentration can be, for example, 0.05, 0.1 or 0.5% (w / v), and the upper limit can be, for example, 5.0, 2.0 or 1.0% (w / v), and these upper and lower limits can be combined arbitrarily.

[0074] -Testing method- The "testing method" of the present invention is a method for testing peanut allergens remaining in food or food processing equipment, and comprises: (1) a step of extracting peanut protein remaining in food or food processing equipment (referred to herein as the "extraction step"); and (2) a step of quantitatively or qualitatively detecting Ara h3 protein in the obtained extract by an immunological assay using at least one anti-Ara h3 antibody, etc. of the present invention, i.e., at least one selected from the group consisting of the first antibody, etc., the second antibody, etc., the first competing antibody, etc., and the second competing antibody, etc. of the present invention (referred to herein as the "detection step").

[0075] The "food" may be peanuts themselves as a raw material (e.g., raw peanuts, dried peanuts, roasted peanuts, boiled peanuts), or a processed product that contains peanuts as a raw material (or is to be tested for whether it is). The form of the food (processed product) is not particularly limited, but may be, for example, a processed product that is manufactured through a process carried out under heating and / or pressure conditions, which generally tends to insolubilize allergen proteins and make them difficult to extract. The food may be, for example, in any of the following forms: solid, semi-solid, jelly, liquid, or emulsified liquid.

[0076] Detection process The "immunological measurement method" is not particularly limited as long as it can detect proteins such as food allergens extracted from food, and can be selected from various known methods.

[0077] In one embodiment of the present invention, the immunological measurement method in the detection step is ELISA. ELISA can be performed, for example, by the following procedure using a target protein (Ara h3 protein in the present invention) in a food (sample) and an antibody that binds to the target protein. 1) A sample solution prepared from food (specimen) is added to a well of a plate equipped with a solid-phase antibody, and the target protein contained in the sample solution is brought into contact with the solid-phase antibody, etc., and bound by an antigen-antibody reaction to form a first complex. 2) After washing the well, a solution of biotin-conjugated antibody is added, and the target protein in the first complex is brought into contact with the biotin-conjugated antibody, etc., and bound by an antigen-antibody reaction to form a second complex. 3) After washing the well, a solution of streptavidin-conjugated enzyme is added, and the biotin-conjugated antibody, etc. in the second complex is brought into contact with the streptavidin-conjugated enzyme, and bound by the biotin-streptavidin reaction to form a third complex. 4) After washing the well, a substrate solution (color developer) is added to react the enzyme in the third complex with the substrate to develop color. 5) After the color reaction has stopped, the absorbance at a specified wavelength is measured using a plate reader. 6) Prepare a standard curve graph from the absorbance obtained by measuring the standard solution separately, and read the amount of target protein (concentration of the solution) from the absorbance measured in 5) and the standard curve graph. Multiply this by the dilution factor used when preparing the sample solution to calculate the amount of target protein in the specimen.

[0078] In one embodiment of the present invention, the immunological measurement method in the detection step is immunochromatography. Immunochromatography can be performed, for example, by the following procedure using a target protein (Ara h3 protein in the present invention) in a food (sample) and an antibody that binds to the target protein. 1) A sample solution prepared from food (specimen) is dropped onto a designated area (sample drop area) on a test strip containing a color-labeled antibody, etc., and the target protein contained in the sample solution is brought into contact with the color-labeled antibody, etc., and bound by an antigen-antibody reaction to form a first complex. 2) The sample solution containing the first complex and unreacted color-labeled antibody, etc. spreads over a predetermined area (spreading area) on the test strip, and the first complex, etc. also move due to capillary action. 3) When the first complex reaches a predetermined site (test line) containing immobilized antibodies, etc., and is captured by the immobilized antibodies, etc., a colored line (e.g., a reddish-purple line caused by gold colloid) appears due to the color-producing label. The appearance of the test line indicates that the target protein was contained in the sample solution. 4) When unreacted colloidal gold-labeled antibodies, etc. reach a predetermined area (control line area, downstream of the test line) containing anti-immunoglobulin antibodies and are captured by the anti-immunoglobulin antibodies, a reddish purple line caused by colloidal gold appears. If the control line does not appear, regardless of whether the sample solution contained the target protein or not, it suggests that there was an abnormality in the development of the sample solution (retesting will be necessary).

[0079] ·Extraction process The "extraction process" can be carried out in the same manner as a general extraction process that is usually carried out using an extraction reagent as a process for extracting various proteins in foods, and can further be appropriately modified as necessary to suit the present invention.

[0080] The food specimen to be subjected to the extraction step can be prepared by finely pulverizing or emulsifying the food into a homogeneous state by high-speed shearing and stirring using a food cutter, a mill, a mixer, a homogenizer, etc., depending on the form of the food (high-speed shearing and stirring method). Such finely pulverizing and other treatments may be performed on the food before the extraction step, and the obtained finely pulverized food and the extraction reagent may be mixed, or may be performed on a mixture of the food and the extraction reagent, and the extraction treatment may be performed simultaneously with the finely pulverizing and other treatments. The treatment conditions for finely pulverizing and other treatments (time, temperature, rotation speed (rpm) or centrifugal force (×g), etc.) can be appropriately adjusted depending on the selected method of finely pulverizing and other treatments and the treatment device used, and taking into consideration the effects of the present invention. In addition, the food to be subjected to the extraction step may be further treated, as necessary, for example, by defatting, etc., in consideration of the extraction of Ara h3 protein and / or detection after extraction.

[0081] The food, which may or may not be micronized as described above, may be mixed with an extraction reagent, and then the mixture may be shaken to perform extraction (shaking method). The shaking conditions (time, temperature, shaking rate or rotation rate (rpm), etc.) may be appropriately adjusted according to the selected shaking method and the processing device used, and taking into consideration the effects of the present invention. The shaking time is usually 12 hours or more and less than 24 hours (for example, overnight). The shaking temperature is usually room temperature, but may be a heated temperature if necessary. In the present invention, low concentrations of Ara h3 protein can be detected with high sensitivity, and high concentrations of Ara h3 protein can be detected without false negatives, even without heating in the extraction step (shaking treatment).

[0082] When testing for allergens remaining in food processing equipment, for example, prepare a cotton swab moistened with PBS or saline, wipe the designated areas of the food processing equipment with the swab, and suspend the dirt adhering to the swab in a diluted buffer solution that has been dispensed in advance (e.g., 1 mL) into a test tube, etc., and the resulting suspension can be used as a sample solution equivalent to an "extract." EXAMPLES

[0083] Hereinafter, the embodiments of the present invention will be more specifically disclosed through the examples, but the technical scope of the present invention is not limited to the embodiments disclosed as examples. Those skilled in the art can understand that, in order to adapt to the intended use and effects of the present invention, the embodiments disclosed as examples can be expanded or modified into various other embodiments, or, if necessary, the technical features of the prior art (known inventions) can be further combined, taking into consideration the technical ideas of the present invention and the contents of this specification and drawings as a whole. Matters necessary to implement the present invention other than those described in this specification can be appropriately taken into consideration of the technical common knowledge and prior art in the technical field to which the present invention belongs.

[0084] [Example] Preparation of anti-Ara h3 monoclonal antibody Raw peanuts were ground into powder, which was then stirred in hexane for defatting, and then stirred in acetone. Stirring in hexane and stirring in acetone were repeated three more times (for a total of four times), and then air-dried to obtain a raw peanut powder sample (sample A).

[0085] 1g of raw peanut powder was mixed with 50ml of Tris-HCl buffer and shaken for 30 minutes to obtain an extract. The extract was centrifuged at 4°C (3000×g, 20 minutes) and the supernatant was filtered to obtain a filtrate. The filtrate was purified with an anion exchanger (column) to obtain an Ara h3 protein solution. A portion of the Ara h3 protein solution was used as an unheated sample, and a portion of the Ara h3 protein solution was heated at 100°C for 10 minutes to be used as a heated sample. The unheated sample and the heated sample were mixed in equal amounts and used as the immunization antigen.

[0086] According to a general immunization method, the above-mentioned immunization antigen (a mixture of equal amounts of unheated and heated samples) was injected into mice to produce antibodies. B cells were collected from the spleen of the immunized animal and fused with myeloma cells (immortalized cancer cells) to produce hybridomas (fusion cells). From the hybridomas, those that produce antibodies with excellent binding affinity and specificity to the target protein (Ara h3 protein, in this example, both native Ara h3 protein and heat-denatured Ara h3 protein) were selected (screened). The hybridomas were cultured to produce a single antibody in the culture supernatant. The culture supernatant was collected, and the antibodies contained therein were purified (adsorbed and eluted) using a Protein G column to obtain monoclonal antibodies.

[0087] The anti-Ara h3 monoclonal antibodies obtained as described above included those that were confirmed to specifically bind to both the native Ara h3 protein purified product and the heat-denatured Ara h3 protein purified product (data not shown). Among these specific anti-Ara h3 antibodies, four clones "2-106", "1-5", "2-21" and "1-3" were selected and used as immobilized anti-Ara h3 antibodies for preparing membrane antibodies or color-labeled anti-Ara h3 antibodies for preparing gold colloid-labeled antibodies in the test strips in the following Examples (and Reference Examples). In addition, an antibody against the color-labeled anti-Ara h3 antibody (anti-mouse IgG antibody) prepared according to a general method was also used.

[0088] Among the four specific anti-Ara h3 antibodies, the amino acid sequences of the heavy and light chains of each of the clones "2-106" and "1-5" were sequenced according to a general method. More specifically, the procedure is as follows: total RNA was isolated from each of the hybridomas of the clones "2-106" and "1-5" using a kit, cDNA was synthesized using a kit, and the nucleotide sequence was identified using a sequencer after amplification by PCR using a specific primer.

[0089] The amino acid sequences of the CDRs of the sequenced clones "2-106" and "1-5", the variable regions including the CDRs and FRs, and the heavy and light chains composed of the variable and constant regions are shown in Table 1. The CDRs and other sites were identified by CHOTHIA ANTIBODY NUMBERING in accordance with a general method.

[0090] [Table 1-1] [Table 1-2] [Table 1-3]

[0091] [Manufacturing example] Manufacturing of immunochromatography kit for detecting peanut allergen (Ara h3 protein) The immunochromatography kit was manufactured in the following procedure, following the manufacturing method of a commercially available kit for detecting peanut allergens (or other food allergens) by conventional immunochromatography. First, the four specific clones were used as anti-Ara h3 antibodies for color labeling, and four types of gold colloid-labeled antibodies were prepared by binding them to gold colloids according to a general method. Meanwhile, the four specific clones were also used as four types of anti-Ara h3 antibodies for immobilization. In general components for test strips including a sample dropping section, a reagent-containing section, a development section, an absorption pad, etc., a test strip was manufactured in which one of the four types of gold colloid-labeled antibodies was placed in the reagent-containing section, one of the four types of anti-Ara h3 antibodies for immobilization was placed at the test line appearance position, and an antibody against the anti-Ara h3 antibody for color labeling (anti-mouse IgG antibody, common) was placed at the control line appearance position.

[0092] The outline of the thus prepared Examples 1 to 10 and Reference Example is shown in Table 2 below. The test strips of these Examples and Reference Examples were used to construct an immunochromatography kit for detecting peanut protein (allergen) by targeting Ara h3 protein, and were used in the following Test Examples. The results of Test Example 3 are also shown in the table, and the details will be described later.

[0093] [Table 2]

[0094] [Test Example 1] Detection of peanut allergen (Ara h3 protein) by immunochromatography In this test example, a test was performed to detect high and low concentrations of peanut allergens using the immunochromatography kit of Example 2 obtained by the above manufacturing example, i.e., a kit using clone "2-106" (the first antibody of the present invention) as the anti-Ara h3 antibody for color labeling and clone "1-5" (the second antibody of the present invention) as the anti-Ara h3 antibody for immobilization, and a conventional commercially available kit for detecting peanut allergens (hereinafter referred to as the "control product") in which an anti-Ara h1 monoclonal antibody (hereinafter referred to as the "control antibody") is used as the color labeling antibody and the immobilization antibody.

[0095] It was confirmed by Western blotting on SDS-PAGE performed in the same manner as in the above preparation example that clones "2-106" and "1-5" (anti-Ara h3 antibodies) bind to Ara h3 protein but not to Ara h1 protein, and that the control antibody (anti-Ara h1 antibody) binds to Ara h1 protein but not to Ara h3 protein. The results for clone "1-5" are shown in Figure 1. Similar results were obtained for clone "2-106" (not shown). It was also confirmed that clones "2-106" and "1-5" do not substantially bind to other food allergens (milk, wheat, buckwheat, egg, crustacean, soybean, walnut, macadamia nut, cashew nut, hazelnut, pecan nut, pistachio, coconut) (not shown).

[0096] [1-1] Tests using high-concentration samples To each of 1 g of raw peanut powder sample (sample a) and peanut butter sample (sample b), 19 mL of sample extract (prepared by adding 1 volume each of the first and second extracts to 8 volumes of purified water and stirring) contained in the control product was added, thoroughly stirred, and then heated in a boiling water bath for 10 minutes. Each solution was filtered through filter paper, and the obtained filtrate (undiluted solution) was used as the test sample (test sample a1, test sample b1).

[0097] Test sample a1 and test sample b1 were dropped onto the sample dropping section of the test strip of the kit of Example 2. After 15 minutes, if a reddish purple line was observed at the test line appearance position and the control line appearance position, it was judged as positive, and if a reddish purple line was not observed at the test line appearance position and only at the control line appearance position, it was judged as negative.

[0098] Similarly, test sample a1 and test sample b1 were dropped onto the sample dropping section of the test strip of the control kit. After 15 minutes, if a reddish purple line was observed at the test line appearance position and the control line appearance position, it was judged as positive, and if a reddish purple line was not observed at the test line appearance position and only at the control line appearance position, it was judged as negative.

[0099] The results are shown in the table below. The control kit gave negative (-) results for both samples a1 and a2, but these are considered to be false negatives due to the high protein concentrations. In contrast, the kit of Example 2 can give positive (+) results even for samples with high protein concentrations, such as these.

[0100] [Table 3]

[0101] [1-2] Tests using low-concentration samples The concentration of Ara h3 protein contained in the test sample a1 (undiluted filtrate) obtained in [1-1] above was quantified by the BCA method, and then the sample was serially diluted to prepare test samples a2 and a3 having Ara h3 protein concentrations of 500 ng / mL and 100 ng / mL, respectively.

[0102] Test sample a2 and test sample a3 were dropped onto the sample dropping section of each test strip of the kit of Example 2 and the control kit. If reddish purple lines were observed at the test line appearance position and the control line appearance position after 15 minutes, it was judged as positive, and if no reddish purple line was observed at the test line appearance position and only at the control line appearance position, it was judged as negative.

[0103] The results are shown in the table below. The control kit gave a positive (+) result for sample a2, but the line was faint, and a negative (-) result for sample a3. In contrast, the kit of Example 2 has good sensitivity and can give a positive (+) result even in samples with low protein concentrations, such as these.

[0104] [Table 4]

[0105] [Test Example 2] Test using an extracted sample by stirring without heating [2-1] Preparation of powder samples Raw peanuts were ground into powder, which was then stirred in hexane for defatting, and then stirred in acetone. Stirring in hexane and stirring in acetone were repeated three more times (for a total of four times), and then air-dried to obtain a raw peanut powder sample.

[0106] [2-2] Extraction process 38 mL of extraction buffer (FASTKIT Slim, Nippon Ham Co., Ltd.) was added to 2 g of raw peanut powder, and the operation was repeated 3 times for 30 to 60 seconds at room temperature using a homogenizer or the like. The obtained sample was centrifuged at 3000 x g or more at 4 ° C for 20 minutes, and the supernatant was filtered. The filtrate was diluted 10 times with dilution buffer (FASTKIT Slim, Nippon Ham Co., Ltd.) to obtain high-concentration sample c1 without heating. The high-concentration sample c1 was diluted with dilution buffer to a concentration of Ara h3 protein of 25 ng / mL to obtain low-concentration sample c2 without heating.

[0107] [2-3] Evaluation The high concentration c1 and low concentration c2 samples obtained were each dropped in an amount of 100 μL onto the sample dropping section of the test strip of the kit of Example 2, and evaluated. The results are shown in the table below.

[0108] [Table 5]

[0109] [Test Example 3] Evaluation of the kits of Examples 1 to 10 and Reference Example [3-1]Reactivity to Ara h3 protein A low-concentration sample c2 with an Ara h3 protein concentration of 25 ng / mL was prepared without heating by the same procedure as in [2-2] of the above-mentioned Test Example 2, and was dropped onto the sample dropping section of the test strip of each Example and Reference Example kit. After a certain period of time, the reactivity to Ara h3 protein was evaluated according to the following criteria based on the color development of the test line.

[0110] [3-2] Food cross-reactivity Samples were prepared from foods other than peanuts (eggs, milk, wheat, buckwheat, crustaceans, soybeans, walnuts, almonds, sesame, cashew nuts, non-glutinous rice, red beans, green peppers, and black peppers) and dropped onto the sample drop section of the test strip of each Example and Reference Example kit. After a certain period of time, the reactivity of each food to food allergens was evaluated according to the following criteria based on the color of the test line. The results are also shown in Table 2 (see above).

[0111] <Evaluation criteria> ++: A dark reddish purple line appears at the test line location (indicating strong reactivity). +: A reddish purple line appears at the test line location (reactivity confirmed). ±: A faint reddish purple line appears where the test line appears (reactivity is difficult to confirm but cannot be denied). -: No reddish purple line appears at the test line location (no reactivity observed). *It was confirmed that a line appeared at the control line appearance location in both cases.

[0112] With regard to reactivity to Ara h3 protein, all of the kits (antibody pairs) of Examples 1 to 10, in which at least one of the first antibody (clone 2-106) or the second antibody (clone 1-5) of the present invention was used as the anti-Ara h3 monoclonal antibody for immobilization or color-developing labeling, were sufficient (evaluation A, ++ or +), and in particular, Examples 2 and 4 had strong reactivity to Ara h3 protein (++). In contrast, the kit (antibody pair) of the Reference Example, in which neither the first antibody (clone 2-106) nor the second antibody (clone 1-5) of the present invention was used as the anti-Ara h3 monoclonal antibody for immobilization or color-developing labeling, had insufficient reactivity to Ara h3 protein (evaluation C, ±). On the other hand, with regard to food cross-reactivity, the kits (antibody pairs) of Examples 1 to 3 were excellent in that they showed no cross-reactivity with any foods other than peanuts that were tested (rating A), whereas the kits (antibody pairs) of Examples 4 and 5 and the Reference Example showed cross-reactivity with one of the foods other than peanuts that were tested, which was somewhat unsatisfactory (rating B), and the other Examples showed cross-reactivity with two or more of the foods other than peanuts that were tested, which was unsatisfactory (rating C).

[0113] From the above results, it can be said that an immunochromatography kit using at least one of the first antibody, etc. or the second antibody, etc. of the present invention (or at least one of the first competitive antibody, etc. or the second competitive antibody, etc.) as an anti-Ara h3 monoclonal antibody for immobilization or color-developing labeling, or a similar kit for ELISA (and other immunological measurement methods), should be included in the detection kit of the present invention from the viewpoint of being able to detect even low concentrations of Ara h3 protein. Among them, an immunochromatography kit using at least one of the first antibody, etc. or the second antibody, etc. of the present invention (or at least one of the first competitive antibody, etc. or the second competitive antibody, etc.) as an anti-Ara h3 monoclonal antibody for immobilization or color-developing labeling in an embodiment such as Examples 1 to 3, or a similar kit for ELISA (and other immunological measurement methods), can be said to be particularly excellent as a detection kit of the present invention from the viewpoint of food tolerance reactivity.

Claims

1. A heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:1; A heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:2, and A heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:3, and A light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:4; A light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:5, and Light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:6 or an anti-Ara h3 antibody or an antigen-binding fragment thereof (hereinafter referred to as "first antibody, etc."), A heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:7; A heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:8, and A heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:9, and A light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10; A light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and Light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:12 An anti-Ara h3 antibody or an antigen-binding fragment thereof (hereinafter referred to as "second antibody, etc."),

2. An anti-Ara h3 antibody or antigen-binding fragment thereof that competes with the first antibody, etc. for the same or overlapping epitope in binding to Ara h3 protein (hereinafter referred to as the "first competing antibody, etc."), or an anti-Ara h3 antibody or antigen-binding fragment thereof that competes with the second antibody, etc. for the same or overlapping epitope (hereinafter referred to as the "second competing antibody, etc.").

3. A test kit for quantitatively or qualitatively detecting peanut allergens remaining in food or food processing equipment by an immunological assay, comprising: A testing kit comprising at least one selected from the group consisting of the first antibody etc. and the second antibody etc. described in claim 1 and the first competing antibody etc. and the second competing antibody etc. described in claim 2.

4. The test kit according to claim 3, wherein the immunological measurement method is ELISA or immunochromatography, and the kit comprises the first antibody or the first competing antibody or the second antibody or the second competing antibody for enzyme labeling in ELISA or color labeling in immunochromatography.

5. The test kit according to claim 3, wherein the immunological measurement method is ELISA or immunochromatography, and the kit comprises the first antibody, etc., or the first competing antibody, etc., or the second antibody, etc., or the second competing antibody, etc., for solid-phase immobilization in ELISA or for immobilization in immunochromatography.

6. the immunoassay is ELISA or immunochromatography; (1) The second antibody or the second competing antibody, etc., for use in enzyme labeling in ELISA or color labeling in immunochromatography, and the second antibody or the second competing antibody, etc., for use in solid-phase immobilization in ELISA or immobilization in immunochromatography, (2) The first antibody or the first competing antibody, etc., are used for enzyme labeling in ELISA or color labeling in immunochromatography, and the second antibody or the second competing antibody, etc., are used for solid-phase immobilization in ELISA or immobilization in immunochromatography, or (3) The testing kit according to claim 3, comprising the first antibody, etc. or the first competing antibody, etc. for enzyme labeling in ELISA or color labeling in immunochromatography, and an anti-Ara h3 antibody or an antigen-binding fragment thereof other than the first antibody, etc., the second antibody, etc., the first competing antibody, etc., and the second competing antibody, etc. for solid-phase immobilization in ELISA or immobilization in immunochromatography.

7. The test kit according to claim 3 , further comprising an extraction reagent.

8. 8. The test kit according to claim 7, wherein the extraction reagent is an extraction reagent obtained by a shaking method or a high-speed shear / agitation treatment method without heating.

9. (1) extracting peanut protein remaining in food or in food processing equipment; and (2) A step of quantitatively or qualitatively detecting Ara h3 protein in the obtained extract by an immunological assay using at least one antibody selected from the group consisting of the first antibody or the like and the second antibody or the like according to claim 1 and the first competing antibody or the like and the second competing antibody or the like according to claim 2. A method for testing for peanut allergens in food, comprising:

10. The testing method according to claim 9, wherein the immunological measurement method is ELISA or immunochromatography, and the first antibody or the first competing antibody or the second antibody or the second competing antibody is used for enzyme labeling in ELISA or for color labeling in immunochromatography.

11. The testing method according to claim 9, wherein the immunological measurement method is ELISA or immunochromatography, and the first antibody or the first competing antibody or the second antibody or the second competing antibody is used for solid-phase immobilization in ELISA or for immobilization in immunochromatography.

12. the immunoassay is ELISA or immunochromatography; (1) The second antibody or the second competing antibody, etc., for enzyme labeling in ELISA or color labeling in immunochromatography, and the second antibody or the second competing antibody, etc., for solid-phase immobilization in ELISA or immobilization in immunochromatography are used. (2) The first antibody or the first competing antibody is used for enzyme labeling in ELISA or color labeling in immunochromatography, and the second antibody or the second competing antibody is used for solid-phase immobilization in ELISA or immobilization in immunochromatography, or (3) The testing method according to claim 9, wherein the first antibody, etc. or the first competing antibody, etc. is used for enzyme labeling in ELISA or color labeling in immunochromatography, and an anti-Ara h3 antibody or an antigen-binding fragment thereof other than the first antibody, etc., the second antibody, etc., the first competing antibody, etc., and the second competing antibody, etc. is used for solid-phase immobilization in ELISA or immobilization in immunochromatography.

13. The testing method according to claim 9 , wherein the extraction in the step (1) is carried out using an extraction reagent.

14. The testing method according to claim 13, wherein the extraction using the extraction reagent is carried out by a shaking method or a high-speed shear / stirring method without heating.

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