Test kit and test method for walnut protein

Abstract

To provide new means for detecting walnut protein in food.SOLUTION: A test kit for walnut protein in food comprises (A) an antibody or antigen-binding fragment thereof (first antibody, etc.) specific for legmin B like protein (first walnut protein) and / or an antibody or antigen-binding fragment thereof (a second antibody, etc) specific for Jug r 6 (secondary walnut protein), and (B) an extraction reagent.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a technique for detecting walnut proteins in food. [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, seven items, "eggs, milk, wheat, shrimp, crab" (those with a high incidence rate) and "buckwheat, peanuts" (those that often cause severe symptoms and are 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, walnuts, 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 food allergen system described above, food manufacturers and public testing institutions use kits to check whether processed products contain foods that contain food allergens as ingredients. In recent years, the number of patients with allergies to "walnuts" has been rapidly increasing, and there are plans to add walnuts to the list of specified ingredients (mandatory labeling), so there is an urgent need to develop a kit that can detect walnuts in processed products.

[0004] Generally, there are two detection techniques for specific raw materials contained in processed foods: 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 specific to walnuts to detect whether walnuts are included as an ingredient in processed foods.

[0005] The first candidates for the walnut-specific proteins targeted for detection based on the antigen-antibody reaction are the walnut allergens Jug r 1 to 8, which are the main cause of walnut allergy. As a means for detecting "walnut" in food by antigen-antibody reaction targeting such walnut-specific proteins, for example, a method for detection by ELISA using a polyclonal antibody targeting the walnut allergen Jug r 1 (2S-albumin) protein and a kit therefor have been proposed (Non-Patent Document 1). In addition, as a means for detecting specific raw materials other than walnut, for example, a method for detecting "soybean" in food using a polyclonal antibody against the undenatured form of soybean allergen (Gly m Bd 30K protein) and a polyclonal antibody against the heat-denatured form in combination has been proposed (Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2008-258130 A (Patent No. 4866190) [Non-patent literature]

[0007] [Non-Patent Document 1] Sakai et al., Journal of AOAC international, 93(4), pp.1255-1261, 2010 Summary of the Invention [Problem to be solved by the invention]

[0008] As a screening test to check whether a food contains walnuts as an ingredient, the only test methods and kits that have been put into practical use so far are those that detect one of the walnut allergens, Jug r 1 protein. Furthermore, there are no practical means to detect walnuts in food by targeting proteins contained in walnuts other than the walnut allergen. If a walnut protein other than the walnut allergen is detected in a food, it means that the food contains walnuts as an ingredient, and there is a high probability that it also contains a walnut allergen (there is a risk of developing a walnut allergy).

[0009] An objective of the present invention is to provide a new means for detecting walnut proteins in foods. [Means for solving the problem]

[0010] The present inventors have found that two types of proteins contained in walnuts that have not been utilized so far (collectively referred to as "walnut proteins of the present invention") can be used to prepare and use antibodies against these proteins (collectively referred to as "antibodies of the present invention"). The first walnut protein discovered by the present invention is "legmin B like protein", and the second walnut protein is "Jug r 6" (vicilin). The antibodies of the present invention that specifically bind to these proteins have low cross-reactivity with specified ingredients (seven items that are required to be labeled) (i.e., have high specificity for the above two types of walnut proteins of the present invention), and can also react with the target proteins of the present invention that have been denatured to a certain extent and are extracted from processed products. Therefore, the present invention has been completed. It has been found that the antibodies of the present invention are suitable as a means for testing whether foods contain walnuts as ingredients.

[0011] That is, in one aspect, the present invention provides the following invention. [1] A testing kit for walnut proteins in food, comprising (A) an antibody specific to a first walnut protein (hereinafter referred to as the "first antibody") and / or an antibody specific to a second walnut protein (hereinafter referred to as the "second antibody") for quantitatively or qualitatively detecting legmin B like protein (hereinafter referred to as the "first walnut protein") and / or Jug r 6 (hereinafter referred to as the "second walnut protein") by immunological assay, and (B) an extraction reagent. [2] Item 2. The testing kit according to Item 1, wherein the immunological measurement method is ELISA, and the first antibody, etc. and / or the second antibody, etc. comprise a first antibody, etc. and / or a second antibody, etc. for immobilization, and a first antibody, etc. and / or a second antibody, etc. for enzyme labeling. [3] Item 2. The testing kit according to Item 1, wherein the immunological measurement method is immunochromatography, and the first antibody etc. and / or the second antibody etc. include a first antibody etc. and / or a second antibody etc. for color labeling. [4] Item 4. The testing kit according to any one of Items 1 to 3, wherein the extraction reagent contains an alkyl sulfate as a surfactant. [5] Item 5. The testing kit according to any one of items 1 to 4, wherein the extraction reagent contains a mercaptoalkanol and / or a sulfite as a reducing agent. [6] Item 6. The testing kit according to any one of Items 1 to 5, wherein the extraction is carried out by a shaking method or a high-speed shear / agitation treatment method. [7] (1) extracting a first walnut protein and / or a second walnut protein from a food product; and (2) A step of quantitatively or qualitatively detecting the first walnut protein and / or the second walnut protein by an immunological assay using a first antibody specific to the first walnut protein and / or a second antibody specific to the second walnut protein. A method for testing walnut protein in food, comprising: [8] Item 8. The testing method according to Item 7, wherein the immunological measurement method is ELISA, and the first antibody etc. and / or the second antibody etc. comprise a first antibody etc. and / or a second antibody etc. for immobilization, and a first antibody etc. and / or a second antibody etc. for enzyme labeling. [9] Item 8. The testing method according to Item 7, wherein the immunological measurement method is immunochromatography, and the first antibody etc. and / or the second antibody etc. include a first antibody etc. and / or a second antibody etc. for color labeling.

[10] Item 10. The method according to any one of items 7 to 9, further comprising a step of purifying the extracted first walnut protein and / or the second walnut protein between steps (1) and (2).

[11] Item 11. The testing method according to any one of Items 7 to 10, wherein the extraction reagent contains an alkyl sulfate as a surfactant.

[12] Item 12. The testing method according to any one of items 7 to 11, wherein the extraction reagent contains a mercaptoalkanol and / or a sulfite as a reducing agent.

[13] Item 13. The method according to any one of items 7 to 12, wherein the extraction is carried out by a shaking method.

[0012] 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. Effect of the Invention

[0013] According to the present invention, it is possible to test for the presence of walnuts in foods by adding new target proteins to the detection targets. [Brief description of the drawings]

[0014] [Figure 1] Figure 1 shows (A) the results of SDS-PAGE and (B) the results of Western blotting of the extracts using each extraction reagent in [1-1] of Example 1. The lane numbers correspond to the extract Nos. in Table 1, and M represents a molecular weight marker. [Diagram 2] 2 shows (A) the results of SDS-PAGE and (B) the results of Western blotting of the products extracted using each extraction reagent in [2-1] of Example 2. Lane numbers 1 to 4 correspond to the extraction reagent numbers in Table 2, 5 represents recombinant Jug r 1, and M represents a molecular weight marker. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] -Testing kit- The "test kit" of the present invention is a kit for testing walnut proteins in food, and includes (A) a first antibody etc. specific to the first walnut protein and / or a second antibody etc. specific to the second walnut protein for quantitatively or qualitatively detecting the first walnut protein and / or the second walnut protein by immunological measurement, and (B) an extraction reagent. Hereinafter, the first walnut protein and the second walnut protein are collectively referred to as "first / second walnut proteins", and the first antibody etc. specific to the first walnut protein and the second antibody etc. specific to the second walnut protein are collectively referred to as "first / second antibodies etc."

[0016] (A) First / second antibodies etc. (first / second walnut proteins) In the present invention, the "first walnut protein" refers to "legmin B like protein" and the "second walnut protein" refers to "Jug r 6" (vicilin). In the present invention, the "first antibody, etc." refers to an antibody specific to the first walnut protein (anti-legmin B like protein antibody) or an antigen-binding fragment thereof, and the "second antibody, etc." refers to an antibody specific to the second walnut protein (anti-Jug r 6 antibody) or an antigen-binding fragment thereof. Examples of the "antigen-binding fragment" include Fab, Fab', F(ab')2, Fv, , scFv, dsFv, diabody (bispecific antibody), nanobody, etc. In this specification, the "first walnut protein" and the "second walnut protein" can be read as "legmin B like protein" and "Jug r 6", respectively. In addition, unless otherwise specified, in this specification, "antibody" can be read as "antigen-binding fragment" or "antibody, etc.", that is, an embodiment using an "antibody" can be replaced with an embodiment using an "antigen-binding fragment" or "antibody, etc."

[0017] In the test kit of the present invention, the first antibody, etc. and the second antibody, etc. may be used either alone or both. For example, in the immunoassay method of the embodiment described below, as an antibody for capturing the first / second walnut protein (the first / second antibody, etc. for solid phase in ELISA, the first / second antibody, etc. for immobilization in immunochromatography, hereinafter collectively referred to as "capture antibody, etc."), either the first antibody, etc. or the second antibody, etc. may be used to capture and detect either the first walnut protein or the second walnut protein, respectively, or the first antibody, etc. and the second antibody, etc. may be used in combination to capture and detect both the first walnut protein and the second walnut protein. Furthermore, when either the first antibody, etc. or the second antibody, etc. is used as the capture antibody, etc., either the first antibody, etc. or the second antibody, etc. may be used as the antibody, etc. for detecting the captured first walnut protein or second walnut protein (the first / second antibody, etc. for enzyme labeling in ELISA, the first / second antibody, etc. for color labeling in immunochromatography, hereinafter collectively referred to as "detection antibody, etc."). When the first antibody, etc. and the second antibody, etc. are used in combination as the capture antibody, etc., the first antibody, etc. and the second antibody, etc. may also be used in combination as the detection antibody, etc.

[0018] The "first walnut protein" is a protein contained in walnuts or processed products containing walnuts as an ingredient, which is (A11) a protein that can be isolated as a protein of about 30 kDa by extraction with an extraction reagent containing a surfactant and SDS polyacrylamide gel electrophoresis (SDS-PAGE), or (A12) a protein that can be isolated as a protein of about 19 kDa by extraction with an extraction reagent containing a surfactant and a reducing agent and SDS-PAGE. The first walnut protein (A12) is presumed to be a protein generated by fragmentation of the SS bond contained in the first walnut protein (A11) by cleavage with a reducing agent. The first walnut protein (A12) is also understood to be contained in walnuts or processed products containing walnuts as an ingredient in a state that constitutes a part of the first walnut protein (A11) before fragmentation.

[0019] The "second walnut protein" is a protein contained in walnuts or processed products containing walnuts as an ingredient, and can be isolated as a protein of approximately 50 kDa by extraction using an extraction reagent containing a surfactant and SDS-PAGE.

[0020] In addition, the above-mentioned "extraction reagent" in relation to the definition of "first / second walnut protein" itself can be the same as the extraction reagent (B) contained in the test kit of the present invention, for example, an embodiment containing the same surfactant and / or reducing agent. The details of the extraction reagent (B), surfactant, reducing agent, etc. will be described later.

[0021] The first / second antibody etc. may be an antibody or an antigen-binding fragment thereof that can recognize and bind to the first / second walnut protein with the specificity required for its application. It is preferable that the first / second antibody etc. does not bind to various proteins contained in foods and food allergens other than walnuts (at least, it binds only to an extent that does not affect the results of the test method of the present invention), but it is acceptable for the antibody to bind (cross-react) to a certain extent with proteins other than the first / second walnut protein depending on the application and the degree, or to non-specifically adsorb to proteins or other substances that are unavoidable due to the nature of the antibody.

[0022] The first / second antibodies may be polyclonal or monoclonal depending on the application and embodiment of the test kit. Polyclonal and monoclonal antibodies differ in specificity (whether they react only with the first / second walnut proteins and do not react (cross) with other proteins), reproducibility (whether there are differences in test results depending on the manufacturing lot), stability (whether the antibody's binding to the antigen is lost due to the antibody immobilization treatment, labeling treatment, etc. in the kit), etc., so that the preferred antibody may vary depending on the application and embodiment of the test kit, and it is possible to select whether to use a polyclonal antibody or a monoclonal antibody, or to use both in combination, taking this into consideration.

[0023] In one embodiment of the present invention, the test kit contains polyclonal antibodies as the first and second antibodies. The first and second walnut proteins derived from foods such as processed foods are considered to be denatured to a certain degree as the first and second walnut proteins during the manufacturing and storage processes, or during the extraction process when an extraction reagent containing a solubilizing agent is used for extraction from foods, and to be a mixture containing various denatured first and second walnut proteins. Such denatured first and second walnut proteins may have epitopes at sites (amino acid sequences) different from those of the undenatured natural first and second walnut proteins. If the first and second antibodies contained in the test kit are monoclonal antibodies, they may only react with some of the denatured first and second walnut proteins contained in foods such as processed foods, and the detection sensitivity of walnut proteins may be insufficient. Therefore, by using polyclonal antibodies corresponding to various epitopes, a test kit can be manufactured that has a certain detection sensitivity for the denatured first and second walnut proteins contained in various foods such as processed foods (preventing the detection sensitivity from decreasing depending on the food).

[0024] In one embodiment of the present invention, the detection kit includes both polyclonal and monoclonal antibodies as the first / second antibodies. For example, in an immunoassay of an embodiment described below, a monoclonal antibody can be used as a capture antibody for the first / second walnut protein (the first / second antibody for solid-phase immobilization in ELISA, the first / second antibody for immobilization in immunochromatography), and a polyclonal antibody can be used as a detection antibody for the captured first / second walnut protein (the first / second antibody for enzyme labeling in ELISA, the first / second antibody for gold colloid labeling or latex particle labeling in immunochromatography). By using a monoclonal antibody as a capture antibody, the specificity for the first / second walnut protein can be increased, and by using a polyclonal antibody as a detection antibody, the captured first / second walnut protein can be detected with high sensitivity.

[0025] The first / second antibody can be in a form suitable for quantitatively or qualitatively detecting the first / second walnut protein by immunological assay, for example by various modifications or alterations, particularly antigen-binding fragments.

[0026] The "immunological assay" is not particularly limited as long as it is a method that allows the first / second walnut protein to be quantitatively or qualitatively detected by utilizing the reaction between the first / second walnut protein and the first / second antibody, etc., and various known methods can be used. In addition, test kits corresponding to various immunological assay methods are also 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 one of the present invention as the extraction reagent.

[0027] 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.

[0028] The first / second antibodies etc. for ELISA include, for example, the first / second antibodies etc. for immobilization and the first / second antibodies etc. for enzyme labeling. The first / second antibodies etc. for immobilization are intended to be immobilized on the surface of the member for performing ELISA, for example, the bottom of a plate (well). The first / second antibodies etc. for enzyme labeling are intended to bind to the first / second walnut protein captured by the immobilized antibody, and then react with a substrate to develop a color with an intensity according to the amount of the first / second walnut protein. The first / second antibody, etc. for enzyme labeling may be a first / second antibody, etc. directly labeled with an enzyme, i.e., a first / second antibody, etc. to which an enzyme has already been covalently bound to the antibody, etc. itself (e.g., via a linker); however, it may also be a first / second antibody, etc. that will be indirectly labeled with an enzyme, such as a first / second antibody, etc. bound to biotin, which can complete the enzyme-labeled first / second antibody, etc. by further reacting with an enzyme bound to streptavidin, as exemplified in the procedure described below in relation to the "test method."

[0029] 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 time with a simple operation.

[0030] The first / second antibodies, etc. for immunochromatography include, for example, the first / second antibodies, etc. for immobilization and the first / second antibodies, etc. for color-producing labels (e.g., gold colloid labels, latex particle labels, platinum particle labels). The first / second antibodies, etc. for color-producing labels are generally the first / second antibodies, etc., that are bound to color-producing labels in advance and are included in a predetermined position (sample drop portion) of the 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 the immobilized antibodies, etc. in a state of forming a complex with the first / second walnut protein. The first / second antibodies, etc. for immobilization are generally the first / second antibodies, etc., that are included in a predetermined position (test line) of the test strip, and are antibodies for capturing the complex between the first / second walnut protein that has been moved by capillary action and the first / second antibodies, etc. for color-producing labels.

[0031] The test kit of the present invention includes at least the extraction reagent and the first / second antibodies of the present invention, and may include any other reagents, components, and other contents according to the embodiment, as necessary. In the case of an ELISA kit, for example, a plate with wells, a diluent for preparing a solution of the target protein, a washing solution for washing the plate (well) after each step, an enzyme reaction stopping solution in ELISA, and an instruction manual describing the procedure of the test method by BSA ELISA for protecting the extracted protein from degradation can be included in the contents of the kit together with the extraction reagent and the first / second antibodies of the present invention. In the case of an immunochromatography kit, for example, a test strip capable of developing various solutions and reagents by capillary action, a diluent for preparing a solution of the target protein, and an instruction manual describing the procedure of the test method by immunochromatography can be included in the contents of the kit together with the extraction reagent and the first / second antibodies of the present invention.

[0032] The kit of the present invention may contain, as necessary, antibodies other than the first / second antibodies, etc. For example, the kit of the present invention may further contain, as the above-mentioned capture antibodies, etc. and detection antibodies, etc., antibodies targeting proteins other than the first / second walnut proteins, such as antibodies targeting walnut allergen proteins, or antibodies targeting proteins other than walnut allergen proteins that are different from the first / second walnut proteins.

[0033] (B) Extraction reagent The "extraction reagent" in the present invention refers to a reagent (solution) that is generally used to extract various proteins in food. Extraction reagents are generally used to extract various food allergens, and various extraction reagents are publicly known, and the present invention can use the same extraction reagents.

[0034] 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.

[0035] 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).

[0036] 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.

[0037] 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 20" (polyoxyethylene sorbitan monolaurate), "Tween 40" (polyoxyethylene sorbitan monopalmitate), "Tween 60" (polyoxyethylene sorbitan monostearate), and "Tween 80" (polyoxyethylene sorbitan monooleate).

[0038] 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.

[0039] 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.).

[0040] 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.

[0041] 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.

[0042] 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.

[0043] In the description of this specification, "antibody" or "antibody, etc." (antibodies and antigen-binding fragments thereof) can be further expanded to read as various known "detection molecules" capable of binding to proteins (and can be replaced in embodiments). "Detection molecules" other than antibodies and antigen-binding fragments (antibodies, etc.) include, for example, aptamers, receptors, antimicrobial peptides or other peptides. As with embodiments based on antigen-antibody reactions using antibodies, etc., the present invention can also be implemented by embodiments based on specific reactions using detection molecules.

[0044] -Testing method- The "test method" of the present invention is a method for detecting walnut proteins in food by targeting the first / second walnut proteins, and includes (1) a step of extracting the first / second walnut proteins in the food (referred to herein as the "extraction step"), and (2) a step of quantitatively or qualitatively detecting the extracted first / second walnut proteins by an immunological assay using antibodies or the like specific to the first / second walnut proteins (first / second antibodies, etc.) (referred to herein as the "detection step").

[0045] 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 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.

[0046] 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.

[0047] 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 a sample (in the present invention, the first / second walnut protein in a food) and an antibody that binds to the target protein. 1) A solution of the target protein is added to a well of a plate containing an immobilized antibody, and the immobilized antibody etc. is brought into contact with the target protein, causing binding through 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 of the solution to calculate the amount of target protein in the sample.

[0048] 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 in a sample (in this invention, the first / second walnut protein in a food) and an antibody that binds to the target protein. 1) A sample solution containing a target protein is dropped onto a designated area (sample drop area) on a test strip containing a color-labeled antibody, etc., and the color-labeled antibody, etc. is brought into contact with the target protein, causing them to bind through 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 specified 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 target protein was contained in the sample or not, it suggests that there was an abnormality in the development of the sample solution (retesting will be necessary).

[0049] ·Extraction process The "extraction process" can be carried out in the same manner as a general extraction process that is carried out as a process for extracting various proteins in foods using an extraction reagent, and can further be appropriately modified as necessary to suit the present invention.

[0050] The food to be subjected to the extraction step can be finely divided or emulsified to 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 a finely divided process may be performed on the food before the extraction step, and the obtained finely divided food may be mixed with the extraction reagent of the present invention, or it may be performed on a mixture of the food and the extraction reagent of the present invention, and the extraction process may be performed simultaneously with the finely divided process. The conditions for the finely divided process (time, temperature, rotation speed (rpm), centrifugal force (×g), etc.) can be appropriately adjusted depending on the selected method for finely divided process and the processing 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 subjected to a process, such as a defatting process, in consideration of the extraction of the first / second walnut protein and / or detection after extraction, as necessary.

[0051] Extraction can also be performed (shaking method) by mixing the food, which has been pulverized or not, with the extraction reagent of the present invention as described above, and then shaking the mixture. The shaking conditions (time, temperature, shaking speed or rotation speed (rpm), etc.) can be appropriately adjusted 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 and less than 24 hours (e.g., overnight). The shaking temperature is usually room temperature, but may be a heated temperature if necessary.

[0052] Recovery process In the present invention, after performing high-speed shearing / stirring or shaking using an extraction reagent, the resulting treated product can be further centrifuged to recover the supernatant, or filtered to recover the filtrate (referred to as the "recovery step" in this specification). The supernatant or filtrate obtained by the recovery step contains the first / second walnut protein extracted from the food. The treatment conditions for the recovery process by centrifugation, filtration, etc. (time, temperature, centrifugation rotation speed (rpm) or centrifugal force (×g), filter pore size for filtration, etc.) can be appropriately adjusted depending on the selected recovery method and the treatment device used, etc.

[0053] ·Refining process In the present invention, a step of purifying the first / second walnut protein contained in the obtained recovery liquid (referred to as a "purification step" in this specification) can be further carried out after the extraction step and recovery step. By using an appropriate means such as gel filtration chromatography, typified by SDS-PAGE, or ion exchange chromatography, the first / second walnut protein can be isolated and purified from a mixture of the first / second walnut protein and other proteins contained in the recovery liquid. The treatment conditions for the purification treatment such as gel filtration chromatography and ion exchange chromatography can be appropriately adjusted depending on the selected purification treatment method and the treatment device used.

[0054] The first / second walnut proteins contained in the recovery liquid obtained in the recovery step may be proteins that are physically and / or chemically different from the natural first / second walnut proteins, that is, proteins that are denatured to a certain degree, depending on the type of food from which they are extracted, for example, whether the processed food has been subjected to a manufacturing process or storage process (with or without and under what conditions such processing as kneading, molding, pressurization, heating, drying, enzyme treatment, freezing, thawing, etc.). Alternatively, the recovered first / second walnut proteins may be a mixture of the first / second walnut proteins that are denatured to various degrees.

[0055] -Extraction processing product- In another aspect, the present invention provides an extraction product comprising an extraction reagent and the solubilized first / second walnut proteins derived from a food. Such an extraction product includes those obtained by an extraction step carried out as step (1) of the testing method of the present invention, as described below, and those obtained by an extraction step carried out as step (1) of the antibody production method of the present invention.

[0056] Before the supernatant is separated as the extraction liquid by filtration, the extraction product of the present invention may further contain substances other than the extraction reagent and the solubilized first / second walnut proteins, such as food as an extraction residue.

[0057] In addition, in conventional technology, the first / second walnut proteins contained in the extraction product are a mixture containing various denatured first / second walnut proteins according to the embodiment of the food, which have been isolated from food at least for the purpose of using them as detection targets in testing for walnut proteins in foods and for the purpose of producing antibodies, and it is difficult to generally identify their substance composition (for example, the composition of the denatured first / second walnut proteins contained in the mixture, i.e., how they are denatured and in what proportions).

[0058] ―Method for producing antibodies, etc.― The "antibody etc. production method" of the present invention is a method for producing antibodies etc. (first / second antibodies etc.) specific to the first / second walnut proteins, and includes (1) a step of extracting the first / second walnut proteins in food (corresponding to the "extraction step" in the above-mentioned "testing method"), and (2) a step of producing antibodies (first / second antibodies etc.) specific to the first / second walnut proteins using the extracted first / second walnut proteins (referred to in this specification as the "antibody etc. production step").

[0059] The extraction step in the antibody production method, etc., can be carried out, similarly to the "extraction step" in the test method, for example, using an extraction reagent as described above, but can be modified (e.g., scaled up) as necessary to make it suitable for antibody production.

[0060] Antibody production process The antibody etc. production process is a process of producing antibodies etc. (first / second antibodies etc.) specific to the first / second walnut proteins using the purified products of the first / second walnut proteins obtained at least after the extraction process described above, and usually after a purification process that follows the extraction process. Once the first / second walnut proteins in food can be extracted and obtained, the first / second antibodies or their antigen-binding fragments can be obtained in the same manner as in the conventional process of producing antibodies etc. specific to food allergen proteins, except that the first / second walnut proteins are used, and further modified as necessary to suit the present invention.

[0061] The first / second antibodies, etc. to be prepared may be polyclonal antibodies, etc., or monoclonal antibodies, etc., depending on the purpose of the antibody production process (e.g., depending on the embodiment of the testing kit of the present invention in which the prepared antibodies are used).

[0062] In one embodiment of the present invention, in the antibody etc. production step, polyclonal and / or monoclonal antibodies are produced in order to produce the above-mentioned embodiment of the present invention's testing kit. By using the (mixture of) denatured first / second walnut proteins obtained in the extraction step, polyclonal antibodies (antibody mixture) and / or monoclonal antibodies suitable for detecting the denatured first / second walnut proteins contained in foods such as processed foods can be obtained that correspond to more diverse epitopes than when the undenatured first / second walnut proteins are used, or correspond to epitopes that the undenatured first / second walnut proteins do not have.

[0063] 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 (first / second walnut 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 (first / second walnut 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 (the first and second walnut proteins 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.

[0064] A method using hybridomas is generally used to produce monoclonal antibodies. The outline of the method using hybridomas is as follows. In the same manner as in the case of producing polyclonal antibodies, an immunogen is injected into an animal to produce 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). From the hybridomas, those that produce antibodies with excellent binding affinity and specificity to the target protein (in this invention, the first / second walnut proteins) 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.

[0065] After the first / second antibody is obtained, its amino acid sequence can be determined, and various antigen-binding fragments (polyclonal or monoclonal) can be produced according to standard methods.

[0066] -Antibodies, etc.- In another aspect, the present invention provides antibodies and antigen-binding fragments thereof (first / second antibodies, etc.) specific to the first / second walnut proteins derived from food, which are obtained by the method of the present invention for producing antibodies, etc.

[0067] The antibodies etc. of the present invention are obtained (e.g. as immunogens) using a mixture containing various denatured first / second walnut proteins according to the embodiment of the food, which in conventional technology have never been isolated from food, at least for the purpose of using them as detection targets in testing for walnut proteins in food, and it is difficult to generally specify their composition as a substance (e.g. the composition of polyclonal antibodies, i.e. what amino acid sequences the antibodies contain). EXAMPLES

[0068] 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.

[0069] [Example 1] Second walnut protein and second antibody [1-1] Extraction of second walnut protein Unglazed walnuts were crushed and stirred in hexane and then in acetone, and the stirring in hexane and then in acetone was repeated three more times (for a total of four times), followed by air drying to obtain defatted walnuts.

[0070] 1 g of unheated or heated defatted walnuts was mixed with 19 mL of specimen extract from "FASTKIT ELISA Ver.III" (Nippon Ham Co., Ltd.) and shaken overnight at room temperature to prepare the extract shown in Table 1. The resulting extract was centrifuged at room temperature (3000 x g, 20 minutes), the supernatant was filtered, and the filtrate was subjected to SDS-PAGE. Western blotting was then performed using one of the secondary antibodies obtained as described below in [1-2].

[0071] The results are shown in Figure 1. In Figure 1(A), a band is observed at approximately 50 kDa in SDS-PAGE for each of the extraction reagents No. 1 to 4. In Figure 1(B), the protein contained in that band is a protein that reacts with a second antibody prepared separately, that is, a second walnut protein. The second walnut protein was identified as Jug r 6.

[0072] [Table 1]

[0073] [1-2] Preparation of secondary antibodies A separate SDS-PAGE was performed in the same manner as in [1-1] above, and the resulting band of approximately 50 kDa was excised, and the protein (second walnut protein) was isolated and purified. Following standard procedures, the resulting purified protein was administered to mice for immunization, and the produced polyclonal antibody (anti-second walnut protein antibody) was then recovered from the serum. In addition, using the resulting polyclonal antibody, a monoclonal antibody was also produced according to standard procedures. Multiple types of polyclonal and monoclonal antibodies were obtained.

[0074] [Example 2] First walnut protein and first antibody [2-1] Extraction of the first walnut protein (A11) Unglazed walnuts were crushed and stirred in hexane and then in acetone, and the stirring in hexane and then in acetone was repeated three more times (for a total of four times), followed by air drying to obtain defatted walnuts.

[0075] 1 g of defatted walnuts was mixed with 19 mL of each extraction reagent shown in Table 2 prepared according to a conventional method, and shaken overnight at room temperature. The resulting extract was centrifuged at room temperature (3000×g, 20 minutes), the supernatant was filtered, and the filtrate was subjected to SDS-PAGE. Western blotting was then performed using one of the primary antibodies obtained by [2-2] below.

[0076] The results are shown in Table 2 and Figure 2. In Figure 2(A), when extraction reagents No. 2 and 3 were used, a band was observed at approximately 30 kDa in SDS-PAGE. In Figure 2(B), the protein contained in that band was found to be the protein that reacted with the separately prepared first antibody, i.e., the first walnut protein. The first walnut protein was identified as a legmin B-like protein.

[0077] [Table 2]

[0078] [2-2] Preparation of the first antibody Similarly to [2-1] above, SDS-PAGE was performed separately, and the resulting band of approximately 30 kDa was excised, and the protein (protein 1) was isolated and purified. Following standard procedures, the resulting purified protein was administered to mice for immunization, and the produced polyclonal antibody (anti-walnut protein 1 antibody) was then recovered from the serum. Furthermore, using the resulting polyclonal antibody, a monoclonal antibody was also produced according to standard procedures. Multiple types of polyclonal and monoclonal antibodies were obtained.

Claims

1. A kit for testing walnut proteins in food, comprising (A) an antibody or its antigen-binding fragment specific to a first walnut protein (hereinafter referred to as the "first antibody, etc.") and / or an antibody or its antigen-binding fragment specific to a second walnut protein (hereinafter referred to as the "second antibody, etc.") for quantitatively or qualitatively detecting legmin B like protein (hereinafter referred to as the "first walnut protein") and / or Jug r 6 (hereinafter referred to as the "second walnut protein") by immunological assay, and (B) an extraction reagent.

2. 2. The testing kit according to claim 1, wherein the immunological measurement method is ELISA, and the first antibody etc. and / or the second antibody etc. comprise a first antibody etc. and / or a second antibody etc. for immobilization, and a first antibody etc. and / or a second antibody etc. for enzyme labeling.

3. The testing kit according to claim 1 , wherein the immunological measurement method is immunochromatography, and the first antibody etc. and / or the second antibody etc. include a first antibody etc. and / or a second antibody etc. for color labeling.

4. The test kit according to any one of claims 1 to 3, wherein the extraction reagent contains an alkyl sulfate as a surfactant.

5. The test kit according to any one of claims 1 to 4, wherein the extraction reagent contains mercaptoalkanol and / or sulfite as a reducing agent.

6. The test kit according to any one of claims 1 to 5, wherein the extraction reagent is a reagent for extraction performed by a shaking method or a high-speed shearing and stirring treatment method.

7. (1) extracting legmin B like protein (hereinafter referred to as "first walnut protein") and / or Jug r 6 (hereinafter referred to as "second walnut protein") from a food; and (2) A step of quantitatively or qualitatively detecting the first walnut protein and / or the second walnut protein by an immunological assay using an antibody or its antigen-binding fragment specific to the first walnut protein (hereinafter referred to as the "first antibody, etc.") and / or an antibody or its antigen-binding fragment specific to the second walnut protein (hereinafter referred to as the "second antibody, etc."). A method for testing walnut protein in food, comprising:

8. 8. The testing method according to claim 7, wherein the immunological measurement method is ELISA, and the first antibody etc. and / or the second antibody etc. comprise a first antibody etc. and / or a second antibody etc. for solid-phase immobilization, and a first antibody etc. and / or a second antibody etc. for enzyme labeling.

9. The testing method according to claim 7 , wherein the immunological assay is immunochromatography, and the first antibody etc. and / or the second antibody etc. include a first antibody etc. and / or a second antibody etc. for color-developing labeling.

10. The method for testing described in any one of claims 7 to 9, further comprising a step of purifying the extracted first walnut protein and / or the second walnut protein between steps (1) and (2).

11. The method according to any one of claims 7 to 10, wherein the extraction is carried out using an extraction reagent containing an alkyl sulfate as a surfactant.

12. The method according to any one of claims 7 to 11, wherein the extraction is carried out using an extraction reagent containing a mercaptoalkanol and / or a sulfite as a reducing agent.

13. The method according to any one of claims 7 to 12, wherein the extraction is carried out by a shaking method.

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