Antibodies, kits and testing methods for testing walnut allergens

Abstract

To provide means improved in cross-reactivity in order to detect walnut allergens in foods based on immunoassay.SOLUTION: The present invention provides: an anti-walnut legumin B-like protein antibody or an antigen-binding fragment thereof, which has heavy chains CDR1, CDR2, CDR3 and light chains CDR1, CDR2, CDR3 consisting of specific sequences; a testing kit containing the antibody or the like for quantitatively or qualitatively detecting walnut allergens remaining in food or food processing devices by immunoassay; and a testing method for walnut allergens in food.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a technique for detecting walnut allergens, particularly walnut legmin B-like protein, contained in food or remaining in food processing equipment. [Background technology]

[0002] Food allergies occur when the body recognizes specific proteins (food allergens) contained in ingested food as foreign substances, triggering an oversensitive immunological mechanism that can cause a variety of symptoms, including itchy skin, hives, and coughing. In severe cases, the condition can become life-threatening, including loss of consciousness, low blood pressure, and shock. It is estimated that 1-2% of the population suffers from food allergies (approximately 10% if limited to infants). To prevent food allergy sufferers from consuming foods containing the causative food allergen, especially processed foods that are difficult to identify visually, the Food Sanitation Act requires or recommends that foods containing ingredients containing food allergens be labeled as such. Currently, eight items, "eggs, milk, wheat, shrimp, crab, peanuts, and walnuts" (items with a high incidence of allergies) and "buckwheat" (item that often causes severe symptoms and can be life-threatening), are designated as "specified ingredients" for which labeling is mandatory, while 21 items, "abalone, squid, salmon roe, oranges, cashew nuts, kiwi fruit, beef, sesame, salmon, mackerel, soybeans, chicken, bananas, pork, matsutake mushrooms, peaches, yams, apples, gelatin, and almonds" (items that have been reported to cause allergies with a certain frequency in the past), are designated as "items equivalent to specified 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 understand the nationwide situation of food allergies, and reviews specified ingredients, etc. based on the results of the survey.

[0003] In order to operate the food allergen system described above, food manufacturers and public testing institutions use kits to check whether foods containing food allergens are included as ingredients in processed products or whether they remain in food processing equipment. Because allergic symptoms to walnuts tend to be severe, there is a demand for kits that can detect walnut allergens with even greater reliability.

[0004] Generally, there are two detection methods for specific raw materials contained in processed products or remaining in food processing equipment: 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 definitive test. Of these, the ELISA method uses an antibody that recognizes a protein (allergen) specific to walnuts to detect whether walnuts are included as an ingredient in processed products.

[0005] Walnut allergens Jug r 1-8, which are the main cause of walnut allergy, are the first candidates for walnut-specific proteins to be targeted in detection based on antigen-antibody reactions. For example, Non-Patent Document 1 proposes an ELISA detection method and kit for detecting "walnuts" in food using a polyclonal antibody targeting the walnut allergen Jug r 1 (2S albumin) protein as a means for detecting "walnuts" in food through antigen-antibody reactions. For a long time, the only practical means for detecting walnuts in food based on antigen-antibody reactions were test methods and test kits that detect the above-mentioned Jug r 1 protein.

[0006] However, in recent years, Patent Document 1 has proposed a test method and test kit for detecting walnuts in food using antibodies that target two types of proteins contained in walnuts that have not previously been utilized: legmin B-like protein and Jug r 6 (vicilin), demonstrating the possibility of commercializing test methods and test kits that detect walnut allergens other than Jug r 1 protein.

[0007] When detecting a target protein (food allergen) using an antibody, cross-reactivity, i.e., the ability of the antibody to bind to proteins not intended as targets, can affect the determination of whether the target protein is present in a food product. For example, Non-Patent Document 2 examines the cross-reactivity between various food allergens and describes that an ELISA using a polyclonal antibody targeting the walnut Jug r 1 (2S albumin) protein demonstrated cross-reactivity with pecan nuts. The cross-reactivity between walnut and pecan nuts is due to the extremely high homology (over 95%) between the amino acid sequences of proteins contained in them, such as 2S globulin (Jug r 1), 7S globulin (Jug r 2, vicilin-like globulin), and 11S globulin (Jug r 4). Therefore, conventional testing methods and kits for detecting walnut allergens in food have been problematic in that they can yield positive results even when the food contains pecan protein but not walnut protein, or negative results when the food contains both walnut protein and pecan protein (when a combination of antibodies that react only to walnut protein and antibodies that react to both walnut protein and pecan protein are used). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2022-157032 [Non-patent literature]

[0009] [Non-Patent Document 1] Sakai et al., Journal of AOAC international, 93(4), pp.1255-1261, 2010 [Non-patent document 2] Ministry of Health, Labour and Welfare Science Research Grant, Research Project to Promote Food Safety and Security, "Research on the Development of Testing Methods for Allergens in Foods," FY2006 General and Co-research Report (H17-Food-011), April 2007 Summary of the Invention [Problem to be solved by the invention]

[0010] As mentioned above, there is room for improvement in kits and methods for detecting walnut allergens in foods based on immunoassays, due to the cross-reactivity of the antibodies used with walnut proteins and pecan nut proteins.

[0011] The present invention aims to provide an improved means for detecting walnut allergens in foods based on immunoassays with respect to cross-reactivity. [Means for solving the problem]

[0012] The inventors have unexpectedly discovered that when walnut legumin B-like protein is detected using an anti-walnut legumin B-like protein antibody having a specific CDR in a kit or method for detecting walnut allergens based on immunological assays, cross-reactivity with pecan nut allergens can be sufficiently suppressed, leading to the completion of the present invention.

[0013] That is, the present invention includes at least the following items. [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-legmin B-like protein antibody or an antigen-binding fragment thereof (hereinafter referred to as "first antibody, etc."), which has the following structure: 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 A walnut anti-legumin B-like protein antibody or an antigen-binding fragment thereof (hereinafter referred to as "second antibody, etc."), having the following structure: [2] An anti-walnut legumin B-like protein antibody or its antigen-binding fragment (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 walnut legumin B-like protein, or an anti-walnut legumin B-like protein antibody or its antigen-binding fragment (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 walnut allergens remaining in food or food processing equipment by immunoassay, A testing kit comprising at least one antibody or the like 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. [4] 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 or the like for enzyme labeling in ELISA or for 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 or the like for solid-phase immobilization in ELISA or for immobilization in immunochromatography. [6] the immunoassay is ELISA or immunochromatography; (1) 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. (2) The second antibody or the second competing antibody, etc., is used for enzyme labeling in ELISA or color labeling in immunochromatography, and the first antibody or the first competing antibody, etc., is used for solid-phase immobilization in ELISA or immobilization in immunochromatography, or (3) The second antibody or the second competing antibody, etc., for use in enzyme labeling in ELISA or color labeling in immunochromatography, and an anti-walnut legumin B-like protein antibody or its antigen-binding fragment other than the first antibody or the second antibody, etc., the first competing antibody or the second competing antibody, etc., for use in solid-phase immobilization in ELISA or immobilization in immunochromatography, Item 3. The testing kit according to Item 3. [7] Item 4. The testing kit according to Item 3, further comprising an extraction reagent. [8] Item 4. The testing kit according to Item 3, wherein the extraction reagent contains an alkyl sulfate as a surfactant. [9] Item 4. The test kit according to Item 3, wherein the extraction reagent contains mercaptoalkanol and / or sulfite as a reducing agent.

[10] Item 4. The testing kit according to Item 3, wherein the extraction reagent is an extraction reagent using a shaking method or a high-speed shear / agitation treatment method.

[11] (1) extracting residual walnut protein from food or food processing equipment; and (2) quantitatively or qualitatively detecting walnut legmin B-like protein in the obtained extract by immunoassay using at least one antibody selected from the group consisting of the first antibody, etc. and the second antibody, etc. described in Item 1 and the first competitive antibody, etc. and the second competitive antibody, etc. described in Item 2; A method for testing for walnut allergens, including:

[12] Item 12. The testing method according to Item 11, wherein the immunological measurement 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.

[13] Item 12. The testing method according to Item 11, 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.

[14] the immunoassay is ELISA or immunochromatography; (1) 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. (2) The second antibody or the second competing antibody, etc., for enzyme labeling in ELISA or color labeling in immunochromatography, and the first antibody or the first competing antibody, etc., for solid-phase immobilization in ELISA or immobilization in immunochromatography, are used; or (3) The second antibody or the second competing antibody is used for enzyme labeling in ELISA or color labeling in immunochromatography, and an anti-walnut legumin B-like protein antibody or an antigen-binding fragment thereof other than the first antibody, the second antibody, the first competing antibody, and the second competing antibody is used for solid-phase immobilization in ELISA or immobilization in immunochromatography. Item 11. The testing method described in Item 11.

[15] Item 12. The testing method according to Item 11, further comprising a step of purifying the extracted walnut legumin B-like protein between steps (1) and (2).

[16] Item 12. The testing method according to Item 11, wherein the extraction in step (1) is carried out using an extraction reagent containing an alkyl sulfate as a surfactant.

[17] Item 12. The testing method according to Item 11, wherein the extraction in step (1) is carried out using an extraction reagent containing a mercaptoalkanol and / or a sulfite as a reducing agent.

[18] Item 12. The testing method according to Item 11, wherein the extraction in step (1) is carried out by a shaking method or a high-speed shearing and stirring method.

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

[0015] For example, the invention described in [3] above can be translated as "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 a method for quantitatively or qualitatively detecting walnut allergens remaining in food or food processing equipment by immunological assay," or "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 walnut allergens remaining in food or food processing equipment by immunological assay."

[0016] Furthermore, for example, the invention described in

[11] above can be converted into "an antibody, etc., for use in a method for testing walnut allergens in food, the antibody, etc. being at least one antibody, etc. selected from the group consisting of the first antibody, etc. and the second antibody, etc. described in Item 1 and the first competitive antibody, etc. and the second competitive antibody, etc. described in Item 2, the testing method comprising: (1) a step of extracting walnut protein remaining in food or in food processing equipment; and (2) a step of quantitatively or qualitatively detecting walnut legumin B-like protein in the obtained extract by an immunological assay using at least one antibody, etc. selected from the group consisting of the first antibody, etc., the second antibody, etc., the first competitive antibody, etc. and the second competitive antibody, etc." [Effects of the Invention]

[0017] The test kit and test method of the present invention suppress cross-reactivity with pecan nut proteins in foods, enabling detection of walnut allergens with higher reliability than conventional methods. The antibodies and the like of the present invention are suitable for carrying out such test kits and test methods. DETAILED DESCRIPTION OF THE INVENTION

[0018] In the present invention, "walnut legumin B-like protein" refers to the protein disclosed as "legmin B-like protein" in Japanese Patent Publication No. 2022-157032 (the aforementioned Patent Document 1), which is contained in walnuts or processed products containing walnuts as raw materials and can be isolated as a protein of approximately 30 kDa by extraction with an extraction reagent containing a surfactant and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or as a protein of approximately 19 kDa by extraction with an extraction reagent containing a surfactant and a reducing agent and SDS-PAGE. Protein (A12) is presumed to be a protein generated by fragmentation of the SS bond contained in protein (A11) cleaved by a reducing agent. Protein (A12) is also understood to be contained in walnuts or processed products containing walnuts as a raw material in a state that constitutes a part of protein (A11) before fragmentation.

[0019] In this specification, the anti-walnut legumin B-like protein antibody in the first embodiment (clone "SJR2-51" 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-walnut legumin B-like protein antibody in the second embodiment (clone "SJR3-115" 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-walnut legumin B-like protein antibody that competes with a first antibody, etc. for the same or overlapping epitope is referred to as a "first competing antibody," and 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-walnut legumin B-like protein antibody that competes with a second antibody, etc. for the same or overlapping epitope is referred to as a "second competing antibody," and 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-walnut legumin B-like protein antibodies of the present invention," the "first fragment," "second fragment," "first competing fragment," and "second competing fragment" are collectively referred to as "anti-walnut legumin B-like protein 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-walnut legumin B-like protein antibodies, etc. of the present invention."

[0022] In this specification, anti-walnut legumin B-like protein antibodies other than the anti-walnut legumin B-like protein antibodies of the present invention (for example, clone "SJR2-2" in the Examples) are referred to as "other anti-walnut legumin B-like protein antibodies," and their antigen-binding fragments are referred to as "other anti-walnut legumin B-like protein binding fragments," and they are sometimes collectively referred to as "other anti-walnut legumin B-like protein 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." In other words, 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-walnut legumin B-like protein antibody or its antigen-binding fragment (first antibody, etc.) 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 the second embodiment of the present invention, the anti-walnut legumin B-like protein antibody or its antigen-binding fragment (second antibody, etc.) 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-walnut legumin B-like protein antibodies of the present invention have heavy chain CDR1-3 and light chain CDR1-3, each 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. These CDRs can be prepared in the same manner as general antibodies, by combining them with appropriate framework regions (FRs), i.e., heavy chain FR1-4 located before and after heavy chain CDR1-3 and light chain FR1-4 located before and after light chain CDR1-3, and grafting them in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus of each heavy chain and light chain, to construct variable regions, and then combining them with the first to third constant regions of the heavy chain and the constant region of the light chain to construct the full-length heavy and light chains.

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

[0028] The anti-walnut legumin B-like protein-binding fragment of the present invention can be produced by using heavy chain CDR1-3 and light chain CDR1-3 consisting of the amino acid sequences represented by specific SEQ ID NOs described herein, or 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 the variable region of a single heavy chain and the variable region of a single light chain are bound), scFv (single-chain Fv), and dsFv (dual-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 set forth 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 set forth in 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 shown in 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 set forth in SEQ ID NO: 19.

[0033] In the heavy chain variable region and / or light chain variable region of a first antibody, etc. or a second antibody, etc., having sequence identity within the above-mentioned range, differences in amino acid residues (substitutions, deletions, insertions, additions, etc.) occur in FRs other than CDRs, and it is preferable that the amino acid sequences of the CDRs are 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 predetermined SEQ ID NO:, and the differences in amino acid residues (substitutions, deletions, insertions, additions, etc.) may occur in either or both of the CDRs and FRs.

[0034] The CDRs and FRs in the antibodies and the like of the present invention are regions identified by methods commonly used in the technical field, such as numbering methods by Kabat, Chothia, IMGT, Aho, etc. In one embodiment of the present invention, the CDRs and FRs are regions identified by the Chothia Antibody Numbering method.

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

[0036] The anti-walnut legumin B-like protein antibodies of the present invention can be produced using a polynucleotide encoding the amino acid sequence of the desired anti-walnut legumin B-like protein antibody, etc., according to standard techniques. A typical production method is outlined as follows: DNA encoding the desired polynucleotide is synthesized and cloned into an expression vector. The expression vector is transfected into suitable host cells, and the host cells are cultured under appropriate conditions to secrete the desired anti-walnut legumin B-like protein antibodies, etc., into the culture supernatant. The culture supernatant is collected, and the anti-walnut legumin B-like protein antibodies, etc. contained therein are isolated and purified.

[0037] In a further aspect of the first embodiment of the present invention, the anti-walnut legumin B-like protein antibody or antigen-binding fragment thereof (first competing antibody, etc.) is an anti-walnut legumin B-like protein antibody or antigen-binding fragment thereof that competes with the first antibody, etc. for the same or overlapping epitope in binding to walnut legumin B-like protein. The amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 of the first competing antibody, etc., and further the amino acid sequences of heavy chain FR1-4 and light chain FR1-4 as needed, 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-walnut legumin B-like protein antibody or antigen-binding fragment thereof (second competing antibody, etc.) is an anti-walnut legumin B-like protein antibody or antigen-binding fragment thereof that competes with the second antibody, etc. for the same or overlapping epitope in binding to walnut legumin B-like protein. The amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 of the second competing antibody, etc., and further the amino acid sequences of heavy chain FR1-4 and light chain FR1-4 as needed, 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 independently of the amino acid sequence of the second antibody, etc. of the present invention.

[0039] The first or second competing antibody, etc., which competes with the first or second antibody, etc., for the same or overlapping epitope, may be any antibody capable of inhibiting the binding (interaction) between the first or second antibody, etc., and walnut legumin B-like 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 or second competing antibody, etc., can be selected from candidate clones without excessive trial and error by a common technique known to those skilled in the art as so-called "epitope binning," in which the first or 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 such as "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 those of the first antibody, etc. or the second antibody, etc., when used to detect walnut allergens in foods.

[0040] -Testing kit- The "test kit" of the present invention is a test kit for quantitatively or qualitatively detecting walnut allergens remaining in food or food processing equipment by immunological assay, and comprises at least one anti-walnut legumin B-like protein 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 competitive antibody, etc. and the second competitive 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 walnut legumin B-like protein by utilizing the reaction between walnut legumin B-like protein and anti-walnut legumin B-like protein antibody, etc., and various known methods can be used. In addition, test kits corresponding to various immunological assays are known and commercially available, and the test kit of the present invention can also be produced in accordance with known test kits, except for using the specific antibody of the present invention.

[0042] In one embodiment of the present invention, the immunological measurement method is an enzyme-linked immunosorbent assay (ELISA). ELISA is capable of quantitatively detecting various proteins with high accuracy, is a suitable method for detecting food allergens in processed foods that have been heated, etc., and complies with the Consumer Affairs Agency guidelines.

[0043] Anti-walnut legumin B-like protein antibodies and the like for ELISA include, for example, solid-phase anti-walnut legumin B-like protein antibodies and enzyme-labeled anti-walnut legumin B-like protein antibodies and the like. Solid-phase anti-walnut legumin B-like protein antibodies and the like are intended to be solidified on the surface of a component for ELISA, such as the bottom of a plate (well). Enzyme-labeled anti-walnut legumin B-like protein antibodies and the like bind to the walnut legumin B-like protein captured by the solid-phased antibody, and then react with a substrate to develop a color with an intensity corresponding to the amount of walnut legumin B-like protein. Anti-walnut legumin B-like protein antibodies, etc. for enzyme labeling may be anti-walnut legumin B-like protein antibodies, etc. that are directly labeled with an enzyme, i.e., anti-walnut legumin B-like protein antibodies, 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), but they may also be anti-walnut legumin B-like protein antibodies, etc. that are indirectly labeled with an enzyme, such as biotin-bound anti-walnut legumin B-like protein antibodies, etc. that can be further reacted with an enzyme bound to streptavidin to complete enzyme-labeled anti-walnut legumin B-like protein antibodies, etc., as exemplified in the procedure described below in relation to the "test method."

[0044] In one embodiment of the present invention, the immunological assay is immunochromatography, which is a simple and easy-to-use assay method capable of qualitatively detecting various proteins in a relatively short time.

[0045] Anti-walnut legumin B-like protein antibodies etc. for immunochromatography include, for example, anti-walnut legumin B-like protein antibodies etc. for immobilization and anti-walnut legumin B-like protein antibodies etc. for colorimetric labeling (e.g., gold colloid labeling, latex particle labeling, platinum particle labeling). Anti-walnut legumin B-like protein antibodies etc. for colorimetric labeling are generally anti-walnut legumin B-like protein antibodies etc. that have been pre-bound to a colorimetric label and are contained in a predetermined position (sample dropping section) on a test strip; they are antibodies that are captured by the immobilized antibody etc. in a complex with walnut legumin B-like protein, and then react with a substrate to produce a color that indicates the presence of the target protein. The anti-walnut legumin B-like protein antibody, etc. for immobilization is generally an anti-walnut legumin B-like protein antibody, etc. that is contained in a predetermined position (test line) on the test strip, and is an antibody for capturing the complex between the walnut legumin B-like protein that has migrated by capillary action and the anti-walnut legumin B-like protein antibody, etc. for color-labeling.

[0046] Such anti-walnut legumin B-like protein antibodies, etc. for immunological assays, such as ELISA or immunochromatography, may be a combination of the anti-walnut legumin B-like protein antibodies, etc. of the present invention, or a combination of the anti-walnut legumin B-like protein antibodies, etc. of the present invention with other anti-walnut legumin B-like protein antibodies, etc. Anti-walnut legumin B-like protein antibodies, etc. may be used in combination with antibodies or antigen-binding fragments thereof that can recognize and bind to walnut legumin B-like protein with the specificity required for the application, i.e., the intended use of the test kit. In the present invention, the anti-walnut legumin B-like protein antibodies, etc. must not exhibit cross-reactivity with pecan nut protein, i.e., they must bind to walnut legumin B-like protein but not to pecan nut protein (at least, they must bind only to an extent that does not affect the test results of the present invention). It is further preferable that anti-walnut legumin B-like protein antibodies, etc. do not bind to various other proteins (food allergens) contained in foods (at least, bind to an extent that does not affect the test results of the present invention), but depending on the use and purpose, it is acceptable for them to bind to a certain extent (cross-react) or to non-specifically adsorb to proteins or other substances, which is unavoidable due to the nature of the antibody.

[0047] Here, the walnut legumin B-like protein in foods such as processed foods may contain variously denatured walnut legumin B-like proteins, for example, aggregates of native walnut legumin B-like protein and variously denatured walnut legumin B-like proteins, depending on the food manufacturing and storage processes (e.g., whether or not kneading, molding, pressurizing, heating, drying, enzyme treatment, freezing, thawing, etc., and the processing conditions) and on the extraction process from the food (specimen) (e.g., whether or not extraction treatment using an extraction reagent containing a solubilizing agent is performed and the processing conditions). Therefore, it is preferable that the anti-walnut legumin B-like protein antibody, etc., be an embodiment that can bind to various walnut legumin B-like proteins, so as to have a certain detection sensitivity for the various walnut legumin B-like protein aggregates in foods (preventing a decrease in detection sensitivity depending on the food).

[0048] From this perspective, in one embodiment of the present invention, the test kit can comprise an anti-walnut legumin B-like protein antibody, etc., that is capable of specifically binding to both native walnut legumin B-like protein and denatured walnut legumin B-like protein (e.g., heat-denatured walnut legumin B-like protein). In such applications, the anti-walnut legumin B-like protein antibody, etc. of the present invention has excellent detection sensitivity for both native walnut legumin B-like protein and walnut legumin B-like protein that has been denatured in various ways by heating, etc. It is preferable that other anti-walnut legumin B-like protein antibodies, etc. used in combination with the anti-walnut legumin B-like protein antibody, etc. of the present invention, are also capable of specifically binding to both native walnut legumin B-like protein and denatured walnut legumin B-like protein.

[0049] In a first aspect of the present invention, the test kit comprises a first antibody or a first competing antibody, or a second antibody or a second competing antibody, as an anti-walnut legumin B-like protein antibody for enzyme labeling in ELISA or an anti-walnut legumin B-like protein antibody for colorimetric labeling in immunochromatography.

[0050] In a second aspect of the present invention, the test kit comprises a first antibody or a first competing antibody, or a second antibody or a second competing antibody, as an anti-walnut legumin B-like protein antibody or ... for immobilization in 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-walnut legumin B-like protein antibody, etc. for enzyme labeling in ELISA or an anti-walnut legumin B-like protein antibody, etc. for color-labeling in immunochromatography, and a first antibody, etc. or a first competing antibody, etc., or a second antibody, etc. or a second competing antibody, etc., as an anti-walnut legumin B-like protein antibody, etc. for solid-phase ELISA or an anti-walnut legumin B-like protein antibody, etc. for solid-phase immunochromatography. On the other hand, in the first aspect of the present invention, other anti-walnut legumin B-like protein antibodies, etc. (for example, clone "SJR2-2" in the Examples) may be included as anti-walnut legumin B-like protein antibodies, etc. for solid-phase ELISA or anti-walnut legumin B-like protein antibodies, etc. for solid-phase immunochromatography, and in the second aspect of the present invention, other anti-walnut legumin B-like protein antibodies, etc. may be included as anti-walnut legumin B-like protein antibodies, etc. for enzyme labeling in ELISA or anti-walnut legumin B-like protein antibodies, etc. for colorimetric labeling in immunochromatography.

[0052] In one preferred embodiment of the present invention (corresponding to Example 3 described below), the test kit comprises a first antibody or a first competitive antibody as an anti-walnut legumin B-like protein antibody for enzyme labeling in ELISA or an anti-walnut legumin B-like protein antibody for color labeling in immunochromatography, and a second antibody or a second competitive antibody as an anti-walnut legumin B-like protein antibody for solid-phase ELISA or an anti-walnut legumin B-like protein antibody for immobilization in immunochromatography.

[0053] In one preferred embodiment of the present invention (corresponding to Example 2 described below), the test kit comprises a second antibody or a second competing antibody as an anti-walnut legumin B-like protein antibody for enzyme labeling in ELISA or an anti-walnut legumin B-like protein antibody for colorimetric labeling in immunochromatography, and comprises a first antibody or a first competing antibody as an anti-walnut legumin B-like protein antibody for solid-phase ELISA or a walnut legumin B-like protein antibody for immobilization in immunochromatography.

[0054] In one preferred embodiment of the present invention (corresponding to Example 1 described below), the test kit comprises a second antibody or a second competing antibody as an anti-walnut legumin B-like protein antibody for enzyme labeling in ELISA or an anti-walnut legumin B-like protein antibody for color labeling in immunochromatography, and comprises another anti-walnut legumin B-like protein antibody (for example, clone "SJR2-2" in the Examples) as an anti-walnut legumin B-like protein antibody for solid-phase ELISA or an anti-walnut legumin B-like protein antibody for immobilization in immunochromatography.

[0055] The "other anti-walnut legumin B-like protein antibodies, etc." may be any one of monoclonal antibodies or antigen-binding fragments thereof (referred to herein as "monoclonal antibodies, etc.") or a combination of two or more monoclonal antibodies, etc., or a polyclonal antibody or antigen-binding fragment thereof (referred to herein as "polyclonal antibodies, etc.") depending on the application and embodiment of the test kit. Monoclonal antibodies, etc. and polyclonal antibodies, etc. differ in specificity (whether they react only with walnut legumin B-like protein and do not react (cross-react) with other proteins), reproducibility (whether test results vary depending on the production lot), stability (whether binding to the antigen is lost due to treatment such as immobilization or labeling of the antibody in the kit), etc., and therefore the preferred antibody may vary depending on the application and embodiment of the test kit. Taking this into consideration, it is possible to select whether to use a monoclonal antibody, etc. or a polyclonal antibody, etc., or a combination of both.

[0056] In one embodiment of the present invention, the testing kit includes a polyclonal antibody or the like as the other anti-walnut legumin B-like protein antibody or the like. In a further embodiment of the present invention, the detection kit includes both a polyclonal antibody or the like and a monoclonal antibody or the like as the anti-walnut legumin B-like protein antibody or the like. For example, in the immunoassay method of the embodiment described below, a monoclonal antibody or the like can be used as a capture antibody for walnut legumin B-like protein (an antibody for solid-phase immobilization in ELISA, an antibody for immobilization in immunochromatography), and a polyclonal antibody or the like can be used as a detection antibody for the captured walnut legumin B-like protein (an antibody for enzyme labeling in ELISA, an antibody for color labeling in immunochromatography). Using a monoclonal antibody or the like as a capture antibody increases the specificity for walnut legumin B-like protein, and using a polyclonal antibody or the like as a detection antibody allows for highly sensitive detection of the captured walnut legumin B-like protein.

[0057] The anti-walnut legumin B-like protein antibodies, etc. used in the present invention, particularly anti-walnut legumin B-like protein antibodies, etc. other than the anti-walnut legumin B-like protein antibodies, etc. of the present invention whose CDR amino acid sequences have been identified, can be produced as polyclonal antibodies, etc. or monoclonal antibodies, etc., in the same manner as antibodies against other food allergens, according to conventional methods, generally using a food allergen (in the present invention, walnut legumin B-like protein) that has been extracted from food and usually further purified.

[0058] Common methods for producing polyclonal antibodies include methods using immunized animals and phage display. The procedure for methods using immunized animals is outlined below. An appropriate amount of purified target protein (in this invention, walnut legumin B-like protein) is mixed with an adjuvant, if necessary, to form an immunogen, and this immunogen is injected (immunized) into an animal (rabbit, guinea pig, goat, sheep, rat, mouse, chicken, etc.) to produce antibodies in the animal's blood. 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 (in this invention, walnut legumin B-like protein) is immobilized, and further purification by gel filtration chromatography, if necessary, to obtain polyclonal antibodies. The procedure for phage display is outlined below. An antibody gene is introduced into a bacteriophage, and a protein consisting of linked H- and L-chain variable regions is expressed (displayed) on the bacteriophage coat protein. The resulting antibody phage library is used to select antibodies with affinity for the target protein (in this invention, walnut legumin B-like protein). The bacteriophage producing the antibody are infected into Escherichia coli, allowed to grow, and then recovered. The antibodies contained therein are purified to obtain polyclonal antibodies.

[0059] A common method for producing monoclonal antibodies is to use hybridomas. The hybridoma method is outlined as follows: As with the production of polyclonal antibodies, a purified target protein (in this invention, walnut legumin B-like protein) is injected multiple times into an animal as an immunogen to produce antibodies. Alternatively, in accordance with "DNA immunization," an expression vector (plasmid) containing the target protein gene is injected multiple times into an animal to express the target protein in the body, thereby producing antibodies. B cells are collected from the spleen of the immunized animal and fused with myeloma cells (immortalized cancer cells) to produce hybridomas (fused cells). Hybridomas that produce antibodies with excellent binding affinity and specificity for the target protein (in this invention, walnut legumin B-like protein) are selected (screened). The hybridomas are cultured to produce a single antibody in the culture supernatant. The culture supernatant is collected, and the antibodies contained therein are purified to obtain monoclonal antibodies. The first and second antibodies of the present invention are also antibodies obtained by the method using hybridomas as described above.

[0060] After an anti-walnut legumin B-like protein antibody (polyclonal or monoclonal) is obtained, its amino acid sequence can be determined, or various antigen-binding fragments (polyclonal or monoclonal) can be produced according to standard methods. The amino acid sequences of the first and second antibodies of the present invention, particularly the heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 thereof, were also determined by standard methods, by isolating total RNA from hybridomas, cloning the cDNA, and performing DNA sequencing.

[0061] Anti-walnut legumin B-like protein antibodies and the like can be made in a form suitable for quantitatively or qualitatively detecting walnut legumin B-like protein by immunological assay, for example, by various modifications or alterations. If necessary, anti-walnut legumin B-like protein antibodies and the like can be humanized according to standard techniques. The amino acid sequences of suitable template human antibodies for humanization (particularly the framework regions of the heavy chain variable region and the light chain variable region) can be obtained by using an appropriate database, for example.

[0062] The test kit of the present invention may contain, as needed, or to the extent that the effects of the present invention are not impaired, antibodies, etc. other than anti-walnut legumin B-like protein antibodies, etc. For example, the test kit of the present invention may further contain, as the capture antibodies, etc. and detection antibodies, etc. described above, antibodies, etc. that target proteins other than walnut legumin B-like protein (e.g., walnut allergens other than walnut legumin B-like protein, or food allergens other than walnut allergens).

[0063] The test kit of the present invention contains at least an anti-walnut legumin B-like protein antibody, etc., but may also contain optional components such as an extraction reagent and other reagents and components, as needed depending on the embodiment. For ELISA kits, for example, a plate with wells, a diluent for preparing a target protein solution, a washing solution for washing the plate (well) after each step, an enzyme reaction stop solution for ELISA, BSA for protecting the extracted protein from degradation, and an instruction manual describing the ELISA test method procedure can be included in the kit contents along with the anti-walnut legumin B-like protein antibodies for immobilization and enzyme labeling, and the extraction reagent. For immunochromatography kits, for example, a test strip capable of developing various solutions and reagents by capillary action, a diluent for preparing a target protein solution, and an instruction manual describing the immunochromatographic test method procedure can be included in the kit contents along with the anti-walnut legumin B-like protein antibodies for immobilization and color-labeling, and the extraction reagent.

[0064] "Extraction reagent" refers to a reagent (solution) that is commonly 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 extraction reagents include Tris buffer, phosphate buffer, citrate buffer, EDTA buffer, HEPES buffer, and acetate buffer. Preferred buffer solutions for use in the present invention include 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 pH by using appropriate amounts of suitable compounds (by adding and dissolving appropriate amounts of multiple compounds in water).

[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, or octyl. Examples of the "salt" in alkyl sulfates, alkylbenzene sulfonates, and the like is, for example, sodium salt, potassium salt, or 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 adjusted appropriately taking into consideration the type of surfactant and its functional effects, as well as the presence, type, and concentration of other components (e.g., chaotropic agents, reducing agents) in the extraction reagent, if necessary. For example, when the extraction reagent contains an anionic surfactant such as SDS or other surfactant as the 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 as desired.

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

[0071] When the extraction reagent contains a chaotropic agent, the concentration of the chaotropic agent can be adjusted appropriately taking into consideration the type of chaotropic agent and its effect (its influence on the effect of the present invention), and the presence, type, and concentration of other components (e.g., surfactants, reducing agents) in the extraction reagent as needed. 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 as desired.

[0072] Examples of reducing agents include mercaptoalkanols and / or sulfites. Examples of mercaptoalkanols include thioglycerol, 2-mercaptoethanol, and dithiothreitol. Examples of sulfites include alkali metal sulfites, and examples of the alkali metals include sodium and potassium. A specific example of a sulfite is sodium sulfite.

[0073] When the extraction reagent contains a reducing agent, the concentration of the reducing agent can be adjusted appropriately, taking into account the type and effect of the reducing agent, and the presence, type, and concentration of other components (e.g., surfactants, chaotropic agents) in the extraction reagent, as needed. 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 in any combination. 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 in any combination.

[0074] -Inspection method- The "testing method" of the present invention is a method for testing walnut allergens remaining in food or food processing equipment, and comprises: (1) a step of extracting walnut protein remaining in food or food processing equipment (referred to herein as the "extraction step"), and (2) a step of quantitatively or qualitatively detecting walnut legumin B-like protein in the obtained extract by an immunological assay using at least one anti-walnut legumin B-like protein 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 competitive antibody, etc., and the second competitive antibody, etc. of the present invention (referred to herein as the "detection step").

[0075] The "food" may be walnuts themselves as a raw material (e.g., unglazed walnuts), or a processed product that contains walnuts as a raw material (or is the subject of testing). 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 heat 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 form: solid, semi-solid, jelly, liquid, or emulsion.

[0076] Detection process The "immunological assay" 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 (in this invention, walnut legumin B-like protein) in a food (sample) and an antibody that binds to the target protein. 1) A sample solution prepared from food (specimen) is added to the wells of a plate equipped with immobilized antibodies, and the target protein contained in the sample solution is brought into contact with the immobilized antibodies, 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, resulting in binding via the biotin-streptavidin reaction, thereby forming a third complex. 4) After washing the wells, a substrate solution (coloring agent) is added, and the enzyme in the third complex reacts with the substrate to develop color. 5) After the color reaction is 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 (solution concentration) 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 assay in the detection step is immunochromatography. Immunochromatography can be performed, for example, by the following procedure using a target protein (in this invention, walnut legumin B-like protein) 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 (development area) on the test strip, and the first complex, etc. also moves 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 is contained in the sample solution. 4) When unreacted colloidal gold-labeled antibodies, etc., reach a predetermined area containing anti-immunoglobulin antibodies (the control line area, downstream of the test line) 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, it suggests that there was an abnormality in the development of the sample solution (retesting will be necessary).

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

[0080] Food samples to be subjected to the extraction step can be prepared by pulverizing or emulsifying them to a homogeneous state (high-speed shearing and stirring method) using a food cutter, blender, mixer, homogenizer, or other device, depending on the type of food. Such pulverization or other treatment may be performed on the food prior to the extraction step, and the resulting pulverized food may then be mixed with an extraction reagent. Alternatively, this treatment may be performed on a mixture of the food and extraction reagent, and the extraction process may be performed simultaneously with the pulverization or other treatment. The pulverization or other treatment conditions (e.g., time, temperature, rotation speed (rpm), centrifugal force (×g)) can be adjusted appropriately depending on the selected pulverization or other treatment method and the processing device used, as well as taking into account the effects of the present invention. Furthermore, the food to be subjected to the extraction step may, if necessary, be further treated, for example, by defatting, to allow for the extraction and / or post-extraction detection of walnut legumin B-like protein.

[0081] Extraction can also be performed (shaking method) by mixing a food, which may or may not have been pulverized as described above, with an extraction reagent and then shaking the mixture. Shaking conditions (time, temperature, shaking rate or rotation speed (rpm), etc.) can be adjusted appropriately depending on the shaking method selected and the processing device used, and taking into consideration the effects of the present invention. The shaking time is usually 12 hours or more but less than 24 hours (e.g., overnight). The shaking temperature is usually room temperature, but may be heated if necessary. In the present invention, low concentrations of walnut legumin B-like protein can be detected with high sensitivity, and high concentrations of walnut legumin B-like 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 suspension can be used as a sample solution equivalent to an "extract." [Example]

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

[0084] [Example of production] Production of anti-walnut legumin B-like protein monoclonal antibody The unglazed walnuts were ground into powder and stirred in hexane and then in acetone to defatted them. The stirring in hexane and then in acetone was repeated three more times (a total of four times), and then air-dried to obtain defatted walnuts.

[0085] One gram of defatted walnuts was mixed with 19 mL of 100 mM Tris-HCl buffer and shaken overnight at room temperature. The resulting extract was centrifuged at room temperature (3000 × g, 20 minutes), and the supernatant was filtered. The filtrate was used as a crude walnut extract. This crude walnut extract contains walnut legumin B-like protein.

[0086] Following standard immunization techniques, the above-mentioned immunizing antigen (crude walnut extract) was injected into mice to induce antibody production. B cells were collected from the spleens of the immunized animals and fused with myeloma cells (immortalized cancer cells) to produce hybridomas (fused cells). From the hybridomas, those producing antibodies with excellent binding affinity and specificity to the target protein (in this example, walnut legumin B-like protein) were selected (screened). The hybridomas were cultured, and a single antibody was produced in the culture supernatant. The culture supernatant was collected, and the antibody contained therein was purified (adsorbed and eluted) using a Protein G column to obtain a monoclonal antibody.

[0087] Among the anti-walnut legumin B-like protein monoclonal antibodies obtained as described above, some were confirmed to have relatively strong binding affinity to walnut legumin B-like protein (data not shown). Of these specific anti-walnut legumin B-like protein antibodies, five clones, "SJR2-51," "SJR3-115," "SJR2-2," "SJR2-55," and "SJR3-12," were selected and used in the test strips in the following Examples and Comparative Examples as immobilized anti-walnut legumin B-like protein antibodies for preparing membrane antibodies or as colorimetrically labeled anti-walnut legumin B-like protein antibodies for preparing gold colloid-labeled antibodies. Furthermore, an antibody (anti-mouse IgG antibody) against the colorimetrically labeled anti-walnut legumin B-like protein antibody, prepared according to a standard method, was also used.

[0088] Of the five specific anti-walnut legumin B-like protein antibodies, the heavy and light chain amino acid sequences of clones "SJR2-51" and "SJR3-115" were sequenced according to standard procedures. More specifically, total RNA was isolated from the hybridomas of clones "SJR2-51" and "SJR3-115" using a kit, cDNA was synthesized using the kit, and the cDNA was amplified by PCR using specific primers. The nucleotide sequence was then identified using a sequencer.

[0089] The amino acid sequences of the CDRs of the sequenced clones "SJR2-51" and "SJR3-115," the variable regions containing the CDRs and FRs, and the heavy and light chains composed of the variable and constant regions are shown in Table 1. The CDR and other regions were identified by CHOTHIA ANTIBODY NUMBERING according to standard methods.

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

[0091] [Manufacturing example] Manufacturing of an immunochromatography kit for detecting walnut allergens (walnut legumin B-like protein) The immunochromatography kit was manufactured according to the manufacturing method of conventional commercially available kits for detecting walnut allergens (or other food allergens) by immunochromatography, as follows: First, using each clone indicated as "colloidal gold-conjugated antibody" in Table 2, colloidal gold-conjugated antibodies were prepared by conjugating them to colloidal gold according to a standard method. Test strips were prepared using standard components for test strips, including a sample drop section, a reagent-containing section, a development section, and an absorbent pad. One of the colloidal gold-conjugated antibodies was placed in the reagent-containing section, while each clone indicated as "membrane antibody" in Table 2 was placed where the test line appeared. Furthermore, an antibody against the anti-walnut legumin B-like protein antibody (anti-mouse IgG antibody, common) for the colloidal gold-conjugated antibody was placed where the control line appeared.

[0092] The test strips of Examples 1 to 3 and Comparative Examples 1 and 2 thus prepared constituted an immunochromatography kit for detecting walnut proteins (allergens) targeting walnut legumin B-like protein, and were used in the following test examples. The results of the test examples are also shown in the tables, and details of these will be described later.

[0093] [Table 2]

[0094] [Test Example] Evaluation of the kits of Examples 1 to 3 and Comparative Examples 1 and 2 [1] Reactivity to walnut protein (walnut legumin B-like protein) 38 mL of a pre-prepared extraction buffer (FASTKIT Slim, Nippon Ham Co., Ltd.) was added to 2 g of unroasted walnut powder, and the mixture was homogenized at room temperature for 30–60 seconds three times. The resulting sample was centrifuged at 3000 × g or higher at 4°C for 20 minutes, and the supernatant was filtered. The filtrate (raw extract) was diluted 10-fold with dilution buffer (FASTKIT Slim, Nippon Ham Co., Ltd.) to prepare a high-concentration sample without heating. The concentration of the high-concentration sample was quantified using the Biuret method, and then diluted to a total walnut protein concentration of 25 ng / mL to prepare a low-concentration sample without heating. The low-concentration walnut sample thus prepared was dispensed into the sample dispenser of the test strip in each example and comparative kit. After a certain period of time, the reactivity to walnut legumin B-like protein was evaluated based on the color development of the test line according to the following criteria.

[0095] [2] Cross-reactivity with pecan nut proteins Following the method for preparing the walnut sample described in [1] above, a high-concentration sample was prepared from pecan nuts by diluting the extract stock solution 10 times, and the sample was dropped onto the sample drop-in section of the test strip of each Example and Comparative Example kit. After a certain period of time, the reactivity of each food to food allergens was evaluated based on the color development of the test line according to the following criteria. The concentration of pecan nut protein in the high-concentration pecan nut sample is generally several hundred μg / mL, which is significantly higher than the 25 ng / mL concentration of walnut protein in the low-concentration walnut sample described in [1] above.

[0096] <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). w+: 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). *In both cases, it was confirmed that a line appeared at the control line appearance position.

[0097] The results of reactivity with walnut legumin B-like protein and pecan nut protein are also shown in Table 2. The kits (antibody pairs) of Examples 1 to 3, which use at least one of the first antibody (clone "SJR2-51") or the second antibody (clone "SJR3-115") of the present invention as the anti-walnut legumin B-like protein monoclonal antibody for immobilization or colorimetric labeling, exhibited sufficient reactivity with walnut legumin B-like protein (++ or +), but showed no reactivity with pecan nut protein (-). In contrast, the kits (antibody pairs) of Comparative Examples 1 and 2, which do not use the first antibody (clone "SJR2-51") or the second antibody (clone "SJR3-115") of the present invention, showed a certain degree of reactivity with walnut legumin B-like protein (w+ or ±), but also showed cross-reactivity with pecan nut protein (+ or ±).

[0098] These results show that immunochromatography kits, or similar kits for ELISA (and other immunological assays), that use 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 anti-walnut legumin B-like protein antibodies for immobilization or color labeling are particularly excellent kits for detecting walnut allergens, in that cross-reactivity with pecan nuts is sufficiently suppressed; in other words, the anti-walnut legumin B-like protein antibodies, etc. of the present invention are suitable antibodies for producing such excellent kits.

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 an anti-walnut legmin B-like protein antibody or an antigen-binding fragment thereof (hereinafter referred to as "first antibody, etc."), having the following structure: 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-walnut legumin B-like protein antibody or an antigen-binding fragment thereof (hereinafter referred to as "second antibody, etc."), having the formula:

2. An anti-walnut legumin B-like protein antibody or its antigen-binding fragment (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 walnut legumin B-like protein, or an anti-walnut legumin B-like protein antibody or its antigen-binding fragment (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 walnut allergens remaining in food or food processing equipment by immunoassay, A testing kit comprising at least one antibody 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 or the like for enzyme labeling in ELISA or for color labeling in immunochromatography.

5. The testing 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 or the like for solid-phase immobilization in ELISA or for immobilization in immunochromatography.

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

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

8. 4. The test kit according to claim 3, wherein the extraction reagent contains an alkyl sulfate as a surfactant.

9. 4. The test kit according to claim 3, wherein the extraction reagent contains a mercaptoalkanol and / or a sulfite as a reducing agent.

10. 4. The test kit according to claim 3, wherein the extraction reagent is an extraction reagent obtained by a shaking method or a high-speed shearing and stirring treatment method.

11. (1) extracting walnut protein remaining in food or food processing equipment; and (2) A step of quantitatively or qualitatively detecting walnut legmin B-like protein in the obtained extract by an immunological assay using at least one antibody selected from the group consisting of the first antibody and the second antibody described in claim 1 and the first competitive antibody and the second competitive antibody described in claim 2. A method for testing for walnut allergens in food, including:

12. The testing method according to claim 11, 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 for color labeling in immunochromatography.

13. The testing method according to claim 11, 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.

14. the immunoassay is ELISA or immunochromatography; (1) 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. (2) The second antibody or the second competing antibody, etc., for enzyme labeling in ELISA or color labeling in immunochromatography, and the first antibody or the first competing antibody, etc., for solid-phase immobilization in ELISA or immobilization in immunochromatography, are used; or (3) The second antibody or the second competitive antibody is used for enzyme labeling in ELISA or color labeling in immunochromatography, and an anti-walnut legumin B-like protein antibody or an antigen-binding fragment thereof other than the first antibody, the second antibody, the first competitive antibody, and the second competitive antibody is used for solid-phase immobilization in ELISA or immobilization in immunochromatography. The inspection method according to claim 11.

15. The testing method according to claim 11, further comprising a step of purifying the extracted walnut legumin B-like protein between steps (1) and (2).

16. The testing method according to claim 11, wherein the extraction in step (1) is carried out using an extraction reagent containing an alkyl sulfate as a surfactant.

17. 12. The testing method according to claim 11, wherein the extraction in step (1) is carried out using an extraction reagent containing a mercaptoalkanol and / or a sulfite as a reducing agent.

18. The testing method according to claim 11, wherein the extraction in step (1) is carried out by a shaking method or a high-speed shearing and stirring method.

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